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Caveat when using ADC(2) for studying the photochemistry of carbonyl-containing molecules†

Several electronic-structure methods are available to study the photochemistry and photophysics of organic molecules. Among them, ADC(2) stands as a sweet spot between computational efficiency and accuracy. As a result, ADC(2) has recently seen its number of applications booming, in particular to unravel the deactivation pathways and photodynamics of organic molecules. Despite this growing success, we demonstrate here that care has to be taken when studying the nonradiative pathways of carbonyl-containing molecules, as ADC(2) appears to suffer from a systematic flaw.

caveat_when_using_adc(2)_for_studying_the_photochemistry_of_carbonyl-containing_molecules†

<!>Conflicts of interest

<p>The theoretical description of photophysics and photochemistry of molecules and materials has matured enough that nowadays most experimental observations can be successfully interpreted and sometimes predicted fully in silico. Ab initio calculations have contributed substantially to understanding the molecular mechanisms behind the human and animal vision,1 optimizing solar cells2 and light-emitting devices,3 designing molecular motors and rotors,4 dyes5 or photochemical switches,6 understanding complex photobiological,7 atmospheric8 or interstellar9 phenomena, etc. The processes underlying all these applications involve the interplay and interconversion between ground and excited electronic states.</p><p>Molecular electronic states are rigorously obtained as the solutions of the time-independent electronic Schrödinger equation. While an exact solution of this Schrödinger equation is an unattainable goal for nearly all molecular systems, a number of approximate electronic-structure methods has been developed and used to study realistic molecular systems. The ground state of a standard closed-shell organic molecule is typically well described by a single closed shell electronic configuration, using popular electronic-structure methods like Kohn-Sham density-functional theory (DFT) or Møller-Plesset perturbation theory up to second-order (MP2). In more challenging situations, one needs to resort to so-called multireference approaches, where the ground-state wavefunction is described by a linear combination of multiple electronic configurations with sizable weights. Along with the ground state, excited states can also be obtained with methods like extended multi-state complete active space second-order perturbation theory (XMS-CASPT2) and multi-reference configuration interaction singles and doubles (MR-CISD).10,11 Such multireference methods often require the careful selection of key orbitals forming an active space, and are truly applicable only to relatively small molecular systems due to their computational burden. However, they allow for an accurate description of conical intersections (CIs) and their branching space–key ingredients for the understanding of nonradiative processes.</p><p>Substantially cheaper than multireference methods, strategies based on a single reference or on DFT have gained a large popularity in the computations of excited states. Linear-response time-dependent density-functional theory (TDDFT), an extension of DFT to excited states, is perhaps the most popular method for excited states nowadays. TDDFT (and its Tamm-Dancoff approximation, TDA) has become a convenient tool to support, predict, or interpret the experimental data in molecular spectroscopy,12 and can be combined with excited-state molecular dynamics simulations.13 Over the years, different failures of standard TDDFT (and underlying DFT) approximations have been documented for certain types of excited states (e.g. charge-transfer states, doubly-excited states), meaning that the use of TDDFT requires careful benchmarks with respect to higher levels of theory.14 Importantly, gaining knowledge in the failures of TDDFT approximations has also contributed to a better and safe use of this efficient electronic-structure method.</p><p>A commonly employed single-reference method is the algebraic diagrammatic construction of second order, ADC(2), an extension of MP2 to excited states.15 ADC(2) is not as computationally affordable as TDDFT, but also not as computationally cumbersome as multireference approaches. It shares the black-box nature of TDDFT, which makes it an appealing approach for wide applications in spectroscopy and molecular dynamics. Just like in TDDFT, the accuracy of ADC(2) critically depends on the quality of the ground-state reference–situations where MP2 fails to describe the ground electronic state are detrimental for the description of the excited states. Nevertheless, ADC(2) appears to be a more reliable method than TDDFT, granting a balanced description of excited-state energies for standard organic molecules, with a relatively low mean error around 0.2 eV.15 While the development of ADC(2) method dates back to 1982,16 its wide applications to photochemistry and photophysics are not older than a decade. Among relevant studies, Tuna et al.17 showed that ADC(2) does not properly describe the topology of conical intersections between the ground (S0) and the first excited (S1) singlet state (while multireference methods do), yet it can still predict reasonable crossing energies and geometries. Plasser et al.18 investigated the application of different single-reference methods in excited-state molecular dynamics of adenine, placing ADC(2) as a serious competitor to the commonly-accepted TDDFT for nonadiabatic dynamics. Following these reports, an explosion of studies employing ADC(2) in excited-state dynamics appeared in the literature,19–33 also mapping the reaction paths between the Franck-Condon (FC) geometry and the electronic states crossings,28–45 and calculating absorption properties of functional molecules.46,47 The community has gained a large confidence with ADC(2), to the point where many studies employing it do not contain systematic comparisons with high-level multireference methods.19,24,26–29,38–42 This is understandable though considering that the latter studies involve middle-sized or bigger molecular systems. For these systems ADC(2) is an ace in the hole, appearing as an ideal compromise between accuracy and computational efficiency.</p><p>In this communication, we highlight what appears to be a systematic issue of ADC(2) in describing some electronic states of molecule bearing a carbonyl group, which concerns a large class of molecules like chromophores, nucleobases, or atmospheric volatile organic compounds. Such systems possess characteristic low-lying singlet excited state of n(O)π*(C <svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata> Created by potrace 1.16, written by Peter Selinger 2001-2019 </metadata><g transform="translate(1.000000,15.000000) scale(0.017500,-0.017500)" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg> O) character (shortened by nπ* in the following), where n(O) is the lone pair orbital associated to the oxygen of the carbonyl group and π*(CO) refers here to an unoccupied π-type orbital localized either fully on the carbonyl functional group or partially when delocalization is possible. We start our investigation by examining the ADC(2) excited-state dynamics of small molecules bearing carbonyl moiety. Fig. 1 shows the time evolution of the ground (solid lines) and excited (dashed lines) state electronic energies following the molecular dynamics initiated by a nπ* photoexcitation. The electronic states were computed with the spin-component-scaled (SCS) variant of MP2/ADC(2), which was recently shown to improve the potential energy surfaces,48 combined with a def2-SVP basis set (abbreviated SVP in the following, see ESI† for the full computational details and a comparison between SCS-ADC(2) and ADC(2)–in the following we will note SCS-MP2/ADC(2) to denote the combination of SCS-MP2 for S0 and SCS-ADC(2) for the excited states). Inspecting the time trace of the electronic energies for five different carbonyl-bearing molecules, namely formaldehyde, (anti-)acrolein, pyrone, 2-hydroperoxy-propanal (2-HPP), and oxalyl fluoride, reveals a common feature: all the systems appear to possess an easily accessible nonradiative decay channel via a S1/S0 crossing, achieved by an ultrafast elongation of the CO bond. Interestingly, a similar nonradiative pathway showed up in the excited-state dynamics of the nucleobase thymine, accounting for 61% of radiationless decay in gas phase,49 and 54% in water.26 The decay channel associated with CO elongation was also identified in a guanine derivative,32 though it was argued that other deactivation mechanisms would prevail. Interestingly, all these studies employed ADC(2) method. As such, a nonradiative decay channel mediated by the CO elongation appears to be a general and well-established feature in excited-state dynamics of carbonyl-containing molecules. Can it really be the case? In fact, more systematic studies of cytosine33,50 and guanine31 derivatives photophysics cast some shadow of doubt on these observations. In these studies, ADC(2) predictions were compared to the results from a high-level multireference method, finding some conspicuous discrepancies. What does that mean for the molecules shown in Fig. 1? We will show in the following that such an easily-accessible deactivation pathway is an artefact of the MP2/ADC(2) method that end users should be aware of.</p><p>To highlight the artificial nature of these nonradiative pathways, we optimized the S0 minimum geometry–the Franck-Condon (FC) point–with SCS-MP2/SVP and the S1/S0 crossing point (S1/S0 CP) with SCS-MP2/ADC(2)/SVP. We then interpolated geometries in internal coordinates between these two critical points and computed electronic energies both with SCS-MP2/ADC(2) and XMS-CASPT2/SVP (see ESI† for additional details on these calculations, active space, SCS-ADC(2) vs. ADC(2), and influence of the basis set). Surprisingly, XMS-CASPT2 shows an entirely different behavior when the CO bond is extended beyond 1.4 Å. The intersection observed between S0 and S1 (nπ*) at the SCS-MP2/ADC(2) level is not present at the XMS-CASPT2 for all molecules. Any attempt to locate the crossing at XMS-CASPT2 level did not lead to a structure resembling the S1/S0 CP of SCS-MP2/ADC(2). In other words, SCS-MP2/ADC(2) suggests the presence of an easily accessible S1/S0 CP, while XMS-CASPT2 strongly indicates that the crossing is not easily accessible–whether it is much higher in energy or does not exist at all. Importantly, such a discrepancy between the two methods is systematic for all five molecules. We also note that ADC(2) shows the same artificial crossings (see ESI†).</p><p>Let us now focus on the case of formaldehyde to gain deeper insights in this issue. Fig. 3 shows the same electronic-energy profiles as in Fig. 2 but now for five different methods: TDA-PBE0/SVP (grey), SCS-ADC(2)/SVP (black), XMS(2)-CASPT2(2/2)/SVP (red), XMS(2)-CASPT2(4/3)/SVP (blue) and MR-CISD(4/3)/SVP (green). For details about these methods see ESI.† The high-level methods XMS(2)-CASPT2(4/3) and MR-CISD both agree on the absence of an intersection at the SCS-MP2/ADC(2) S1/S0 CP geometry. The D1 diagnostic (dashed orange) measures the degree of multiconfigurational character for the MP2 ground state. The quick D1 surge beyond the recommended limit value of 0.0451 (and even the less conservative value of 0.1 proposed by others52) indicates that the S0 state acquires a multiconfigurational character along the pathway. In other words, the single-reference wavefunction that serves as reference for perturbation theory is no more adequate for large D1 values. Interestingly, the PBE0 ground state appears to have an incorrect shape in comparison to high-level methods, but as the S1 electronic energy is also raising steeply along the profile, the S1/S0 CP is (fortuitously) avoided. The artificial S1/S0 CP of SCS-MP2/ADC(2) can be explained by the combination of two recurring factors for all molecules bearing a carbonyl group: (i) the MP2 ground state is not adequate and overestimates the S0 energy when stretching the CO bond and (ii) the curvature of the nπ* state along the CO stretching coordinate is too small. The poor description of S1 can be correlated with an increasing contribution of doubly-excited configurations along the interpolation pathway (see Fig. S2 in the ESI†). Such contributions cannot be optimally described by ADC(2) since the treatment of double excitations in ADC(2) is very limited.15 Finally, the point (ii) resonates with earlier findings that ADC(2) was inaccurate in describing the nπ* state of cytosine,48 (even if SCS-ADC(2) was shown to perform better), while it has been pointed out that ADC(2) performs less accurately when describing nπ* transition, yielding too low frequencies associated with the carbonyl stretching.53 Another study also showed that ADC(2) already overestimates the CO bond length of formaldehyde at its S1 minimum.54</p><p>Based on the considerations above, we could reproduce the S1/S0 CP using XMS-CASPT2 by employing a minimal active space constituted only by two electrons in n(O) and the π*(CO) (XMS(2)-CASPT2(2/2) in Fig. 3). Such a small active space does not include the π orbital, which is a key contributor to the multiconfigurational character of the S0 along the pathway, when the closed-shell configuration starts to strongly mix with a ππ* contribution (see Fig. S2 in the ESI†). As a result, XMS(2)-CASPT2(2/2) leads, as for MP2, to a poor description of the ground-state reference wavefunction on which perturbation theory is applied, ultimately leading to a failure of the method when reaching the S1/S0 CP region (in stark contrast with XMS(2)-CASPT2(4/3) and MR-CISD, both including the π orbital). Therefore, the failure of XMS-CASPT2 caused by the small active space appears to mimic that of SCS-ADC(2). The very same trends are systematically reproduced for the other four compounds, as depicted in Fig. S6 in the ESI.† Hence, our observations point towards (i) a too shallow S1(nπ*) along the elongation of the CO bond combined with (ii) a bad reference for MP2 destabilizing too rapidly the ground state to explain the fast and artificial decay observed in the excited-state dynamics. We note that the point (ii) resonates with earlier findings that ADC(2) tends to have energy gaps between S1 and S0 closing too rapidly even still far from the crossing region (e.g. ref. 55 and 56).</p><p>We note that we also investigated the case of the aforementioned thymine nucleobase. Our rational explanation for the failure of both ADC(2) and XMS(2)-CASPT2(2/2) is clearly confirmed for this additional molecule: both ADC(2) and XMS(2)-CASPT2(2/2) predict an artificial S1/S0 crossing, which is verily avoided when using a suitably high level of theory (Fig. S7, ESI†).</p><p>In summary, the electronic-structure method ADC(2) is a rising star to study the photochemistry and photophysics of organic molecules, but its pitfalls still need to be fully uncovered. The unexpected failures of ADC(2) for carbonyl-containing compounds were discussed in several case studies but the systematic nature of these errors in the context of S1/S0 crossing points has not been widely recognized. The recurrence of artificial S1/S0 crossings upon CO elongation closely resembles the predictions from multireference methods with an inadequate active space, which prevents the proper description of both ground and excited state, and leads to the unphysical nonradiative decay channels in molecular dynamics. Similar issues are expected for molecules bearing a CS group, based on the results presented in ref. 57. Considering the omnipresence of CO functional group and the increasing interest in using ADC(2) to study photochemical deactivation pathways, our current findings should serve as a warning bell for future research in the field.</p><!><p>There are no conflicts to declare.</p>

PubMed Open Access

Molecular characterization of sequence-driven peptide glycation

Peptide glycation is an important, yet poorly understood reaction not only found in food but also in biological systems. The enormous heterogeneity of peptides and the complexity of glycation reactions impeded large-scale analysis of peptide derived glycation products and to understand both the contributing factors and how this affects the biological activity of peptides. Analyzing time-resolved Amadori product formation, we here explored site-specific glycation for 264 peptides. Intensity profiling together with in-depth computational sequence deconvolution resolved differences in peptide glycation based on microheterogeneity and revealed particularly reactive peptide collectives. These peptides feature potentially important sequence patterns that appear in several established bio-and sensory-active peptides from independent sources, which suggests that our approach serves system-wide applicability. We generated a pattern peptide map and propose that in peptide glycation the herein identified molecular checkpoints can be used as indication of sequence reactivity.

molecular_characterization_of_sequence-driven_peptide_glycation

<!>OPEN<!>Results<!>Convergence of tryptone peptides and peptides with established activities into common sequences.<!>Discussion<!>Statistical analysis.

<p>Glycation presents a ubiquitous non-enzymatic post-translational modification 1,2 , which is formed by the reaction of amino compounds and reducing sugars. It refers to a complex reaction network and produces a multitude of heterogeneous reaction products, also known as Maillard reaction products (MRPs) or advanced glycation end products (AGEs) 3,4 . Glycation is a multifactorial reaction, which depends on the nature of the precursors and the reaction conditions, including time and concentration [5][6][7][8] . The Maillard reaction (MR) is one of the most common and essential reactions in food processing and determines color, flavor and taste of food. Further, its reaction products are known to affect human health 9,10 , and contribute to various pathologies, such as diabetes 11,12 . Here, glycation leads to molecular and cellular changes in a series of complicated events. Hyperglycemia drives glycation of lipids and proteins and development of vascular lesions by AGE engagement of the receptor for AGE (RAGE) in cells of the vessel wall [13][14][15][16] . Due to their broad relevance, thorough understanding of glycation reactions is indispensable.</p><p>As insights into the MR of amino acids continue to emerge, new models are needed to improve the understanding of peptide and protein glycation 17 . Many previous studies analyzing the health effects of glycation products and peptides point to their miscellaneous bioactivities and their potential as nutraceuticals and functional food ingredients [18][19][20][21][22][23] . Glycation induced alterations in the bioactivity and improvement of sensory attributes have been described for various peptide mixtures [24][25][26][27][28][29][30][31][32] . However, the specific peptides related to these changes remain largely uncharacterized and, even more important, the behavior of peptides in glycation reactions has barely been systematically analyzed and thus is unaccounted. Only a limited number of studies on peptide reactivity in the MR have been conducted and focused on synthetic peptides 26,33,34 or peptide derived MRPs in specific foods [35][36][37][38] . These approaches have revealed the relevance of both peptide length and composition in the MR and the importance of peptide glycation in various fields, not only including diverse food matrices but also biological systems and disease progression 10 . The information describing the general determinant factors of peptide glycation, however, remains elusive. Therefore, particular sequences and, thus classes of proteins that have preference to undergo glycation reactions and the final consequences, such as loss and gain of bioactivities, cannot be determined. Especially required are model systems for large-scale MRP characterization that enable general understanding of the influence of the amino acid composition, sequence and peptide length on glycation product formation.</p><!><p>1 Chair of Analytical Food Chemistry, Technical University Munich, Maximus-von-Imhof-Forum 2, 85354 Freising, Germany. 2 Research Unit Analytical BioGeoChemistry (BGC), Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany. 3 The Waltham Pet Science Institute, Mars Petcare UK, Waltham-on-the-Wolds, Leicestershire LE14 4RT, UK. * email: michelle.berger@tum.de; schmitt-kopplin@ helmholtz-muenchen. de To acquire a better understanding of site-specific peptide glycation the analysis of the Amadori product (AP), a relatively stable intermediate of the MR, and consecutive downstream reaction products MRPs is particularly well-suited. High-resolution mass spectrometry (MS) is a fast and highly sensitive method, which enables detection and identification of both early and advanced products of the MR 39,40 . Information on net chemical transformations and precursor reactivity in such systems can be gained by non-targeted experiments and generation of mass difference networks 41,42 . Collision-induced dissociation (CID) after electrospray ionization (ESI) MS has been successfully applied for peptide derived AP analysis [43][44][45] . However, non-targeted large-scale analysis and interpretation of peptide derived MRPs is limited. Only a few studies have gained insight into peptide reactivity and the influence of sequence microheterogeneity 33,34,[46][47][48] . This includes glycation based on accessibility of the N-terminus and catalytic effects in some short-chain peptides. Here, we report that the combination of highresolution ESI quadrupole time of flight (QTOF) MS, bioinformatics and multivariate statistics enables a deep and molecular-level investigation of complex peptide systems. Using this combinatorial method for large-scale AP analysis, we characterized the reaction behavior of 264 casein-derived peptides in the MR and used this data to gain insight into sequence-dependent differences in AP formation profiles and, thus, peptide reactivity. Furthermore, we discovered potentially relevant glycation-patterns and demonstrate system-wide applicability of this study to various food-peptide sources by in silico sequence mapping. Database search serves as a reference for investigation of bioactive and sensory-active peptide reaction behavior. This approach may be amendable to practically any type of glycation system, and it allows exploration at various levels of information, from the influence of the peptide composition to the role of specific sequence-patterns in peptide glycation.</p><!><p>A time-resolved analysis of peptide dependent Amadori product formation. To study the reaction behavior of peptides in glycation, we heated complex model systems containing glucose (2.7-54 mg/mL) and tryptone at 95 °C for 2, 4, 6 and 10 h, respectively. Compared to an in silico tryptic protein digest, tryptone provides approximately four times more free peptides with increased diversity (Fig. 1a). A vast number of non-enzymatic cleavage sites generates many diverse peptides (Supplementary Fig. 1) with partially overlapping amino acid sequences. This enables characterization of site-specific microheterogeneity and, ultimately, identification of specific sequence patterns that promote glycation. Apart from that, C-and N-terminal amino acids in tryptone peptides comprise a much greater diversity than enzymatic digests can cause, which becomes apparent by comparison with an in silico tryptic casein digest (Fig. 1b and Supplementary Fig. 2a). Unlike tryptone, there is a bias toward lysine-(Lys) and arginine-(Arg) containing peptides for tryptic casein digestion (Supplementary Fig. 2b) and poor enzymatic cleavage of certain protein regions (Supplementary Fig. 3) may lead to the preclusion of particularly relevant peptides. Even alternative proteases or sequential digestion would only provide a marginally increased total number and diversity of peptides compared to trypsin 49 . With the applied method, we could nearly completely cover the casein protein sequences (Fig. 1c).</p><p>With this extensive dataset in hand we first explored the concentration-and time-dependent reaction behavior of this diverse pool of peptides based on the formation of the corresponding APs. The AP is a relatively stable intermediate of the early MR 50 , which is formed via condensation between the amino compound and the reducing sugar, and subsequent rearrangement 11 . The number of hexose residues coupled to an amino acid or peptide was estimated by a mass increase of 162.0528 Da per attached monosaccharide (C 6 H 12 O 6 -H 2 O). Tandem MS was applied to obtain structural information, which allowed confirmation of the AP chemical structure of 47 amino compounds (Supplementary Table 1). Significant correlations (p-value ≤ 0.05) were observed across normalized AP intensity profiles, and APs clustered by the influence of sugar concentration but also reaction time (Fig. 2a). Interestingly, APs clustered that were formed from peptides with comparable amino acid sequences. Sequence similarity is highlighted by the suspended numbers indicating sequence groups, e.g. HLPLP and LHLPLP (Cluster 2, sequence group 16). Certain APs found in Cluster 1 and 2 seemed to form isomers, which caused them to also appear in Cluster 3. In Fig. 2b, the representative normalized mean intensity profiles for the three clusters are shown, and Supplementary Fig. 4 provides the individual normalized intensity curves for each AP, separately. The intensities of APs in Cluster 1 and 2 reached their maximum after two hours for all glucose concentrations and decreased with further reaction time (Fig. 2b). In contrast, Cluster 3 contains APs, which increased with time and reached maximum intensity after either six or ten hours of incubation. Further, enrichment of AP levels at different glucose concentrations was observed. The highest intensity of Cluster 1-APs was either detected at 5.4 or 27 mg/mL of glucose, whereas for APs in Cluster 2 (27-54 mg/mL) and 3 (54 mg/mL) higher sugar concentrations were required to reach their maximum. Figure 2c compares AP formation between different peptide lengths, demonstrating that nearly all amino acids and dipeptides were found in Cluster 3, whereas larger peptides www.nature.com/scientificreports/ did not show uniform normalized AP intensity profiles. Moreover, the percentage observed as an AP for each peptide length is displayed, but no general correlation between peptide reactivity and sequence length could be observed. Note, this calculation is based on the total number of peptides per length, so one length may appear to be glycated to a greater extent if a minor number of peptides was identified with this length. A higher proportion of APs derived from dipeptides compared to tripeptides resembles observations from previous studies 33,43 , which suggested decreasing reactivity with increasing peptide length. Here, we confirm and extend these observations by investigating a larger range of peptide chain lengths. Interestingly, longer-chain peptide sequences were not generally associated with reduced reactivity in early glycation reactions, e.g. when comparing penta-and hexapeptides or nona-and decapeptides. Even with these observations, it is difficult to comment on the influence of peptide length on glycation on a universal level. All APs significantly increased after 2 h (t-test, p-value ≤ 0.05); however, different glucose levels were required (Supplementary Table 1). Most (25) of the identified APs significantly increased at a glucose concentration of 2.7 mg/mL, while other peptides required higher glucose levels. Interestingly, different observations were made for highly similar peptides. For example, VPQLEIVPN required a glucose concentration of 27 mg/mL to increase, significantly, whereas for KVPQLEIVPN only 5.4 mg/mL of glucose were needed. Analogous behavior was observed for the APs of VAPFPE (27 mg/mL) and VAPFPEV (5.4 mg/mL).</p><p>Shedding light onto the role of peptide composition in glycation. AP analysis uniquely facilitates characterization of site-specific glycation, and our dataset provides insight into the highly complex and yet largely unknown reaction behavior of peptides in glycation. Previous studies explored peptide derived MRPs in particular foods providing limited information from a global prospect [35][36][37][38] . Others have investigated the reactivity of highly specific synthetic peptides depending on factors explored herein to some degree, such as peptide length (discussed above) and amino acid composition 33,34 . Here, we aspired to approach these research questions from a general level using a large reservoir of peptides and APs. Mapping glycated peptides onto casein proteins revealed that AP formation was observed for only 14% of α-S2-and 9% of κ-casein peptides (Supplementary Fig. 5), but APs related to α-S1-casein (29%) and ß-casein (45%) were detected to a larger extent (Fig. 3a). Analyzing the amino acid sequence of α-S2-and κ-casein peptide APs, we identified protein-specific peptides such as FLPYP (F 55 -P 59 of κ-casein) . The ability to profile glycosites at this scale provides opportunities to determine the relative susceptibility of peptide collectives with similar amino acid sequences to the early MR. Especially reactive peptide classes are captured here, as the protein heatmaps show the frequency that sequences co-occurred on glycated peptides . AP formation of peptides sharing certain amino acid sequences appeared to be favored, e.g. including peptides that originated from N 73 -V 92 and V 170 -V 173 of β-casein.</p><p>To examine the influence of the amino acid composition on peptide reactivity in the early phase of glycation, we calculated the contribution of each amino acid to AP formation, given as a percentage of the total observations in tryptone peptides. Figure 3b shows amino acids, e.g. glutamic acid (Glu) and leucine (Leu), that appeared equally in peptide APs from α-S1-(top) and β-casein (bottom). Other amino acids, such as proline (Pro) and histidine (His) showed considerable variations in their contribution to AP formation (Supplementary Fig. 6) depending on the source protein, meaning the overall peptide sequence, which fits with the known role of the microenvironment of amino acids in glycation. Importantly, a previous report described varying in vivo reactivity of lysine depending on its position in the albumin sequence and, thus, its neighboring amino acids 46 . Further, investigation of short-chain peptide model systems showed that AP formation is considerably influenced by the immediate chemical environment, hence, adjacent amino acids side chains in the peptide sequence 26,33 , which overall indicates that we may also have identified reactivity-sequence interrelation for peptide structures. We visualized the median percentage of each amino acid in APs to explore the effect of amino acid composition and microheterogeneity over all four casein proteins (Fig. 3c). Most amino acids showed a wide distribution of the values, again demonstrating that the type of amino acids that contribute to AP forming peptides can vary based on their immediate chemical environment. This presents a promising starting point to explore for sequencespecific glycation.</p><p>To dive into this intriguing facet of peptide glycation, we examined the location of amino acids relative to the reactive peptide N-termini . This was based on De Kok's hypothesis that the side chain carboxylic group of Glu catalyzes glycation of primary amino groups 33 , and on the suggestion of Lhiang Zhili and co-workers that Leu and isoleucine (Ile) promote AP formation 26 . As short-chain peptides were investigated in these studies, they describe the influence of directly neighboring amino acid side chains on N-terminal peptide glycation. Hence, we reasoned that our dataset could allow to explore the impact of both the N-terminal amino acid and the adjacent amino acid side chain across a large number of highly diverse peptide species. To detect amino acid overrepresentation at the mentioned positions, we generated sequence logos by comparing sequences between peptide APs and non-glycated peptides (Fig. 4a and Supplementary Fig. 7a). Here, amino acids enriched at certain positions in AP forming peptides are illustrated (relative abundance glycated − relative abundance non-glycated > 0). This analysis indicated preference for valine (Val), Ile and Leu at the first two positions of the amino acid sequence for glycated peptides (Fig. 4a). Indeed, the percentage of N-terminal Val was considerably increased for AP forming peptides compared to peptides, for which the corresponding AP could not be identified (Fig. 4b, top). An illustration of the absolute amino acid abundance can be found in Supplementary Fig. 7b. Interestingly, we further found substantially higher relative frequencies for Ile, Leu and Val next to the N-terminus in peptides also observed as an AP (Fig. 4b, bottom), echoing the result from the sequence logo. To account for preferred glycation of peptides with Ile, Leu or Val at the second sequence position, their summed relative frequency depending on AP detectability is shown in Fig. 4c. This result complies with previous findings that hypothesized that Ile and Leu promote N-terminal glycation based on pronounced hydrophobicity 51 (Ile 1.80; Leu 1.70) and polarizability 52,53 www.nature.com/scientificreports/ www.nature.com/scientificreports/ to Ile and Leu and was previously shown to exert a similar effect on the reactivity of the peptide N-terminus 26 . Furthermore, the percentage of aspartic acid (Asp), methionine (Met), phenylalanine (Phe) and Pro next to the N-terminus was considerably decreased in glycated peptides (Fig. 4b, top). We also observed that glycation of peptides was disfavored for Pro at the first two sequence positions (Fig. 4b and Supplementary Fig. 7a). A recent study on Pro containing dipeptides (Gly-Pro, Pro-Gly) suggested that its secondary amine may hinder Schiff base formation 54 . Nevertheless, we found that proline was frequently observed at the third and fifth sequence position of glycated peptides (Fig. 4a), thus raising the possibility of its involvement in increased peptide reactivity towards early glycation. This is supported by an increased relative abundance of proline at the same locations relative to the glycation site in AP forming peptides compared to peptides, for which the corresponding AP was not detected (Supplementary Fig. 7c and d).</p><p>Capturing relevant sequence patterns in peptide glycation. Large-scale peptide derived AP analysis enables us to identify potentially relevant glycation-patterns, and our dataset can provide an initial glimpse into this intriguing aspect of glycation. While others have explored the influence of the amino acid sequence based on di-and tripeptide glucose model systems 26,33 , we can now comment on trends across 264 peptides originating from four proteins. In enzymatic glycosylation the importance of the N-X-S/T sequon and the negative effect of Pro in X position has been shown, which may result from conformational changes 55 . However, relevant structural motifs in peptide glycation have not been identified. In this detailed analysis, we identified small regions of identical subsequences in casein proteins and, thus, the thereof arising peptides (length = 2, 3, and 4) using the amino acid one letter code (Fig. 5a, top). We mapped (non-) glycated peptides onto proteins (Fig. 5a, bottom right part) and across each other (Fig. 5a, bottom left part). This provided information on the degree of co-occurrence for sequence patterns on glycated peptides with a different overall amino acid sequence. This protocol allows to detect relevant glycation patterns that are anticipated to be important factors in determining the preference for early peptide glycation. Analysis of common sequence patterns in casein proteins revealed several sequence overlays. Figure 5b provides information on the degree of sequence similarity, from which it is evident that the number of shared sequences varied for different pattern lengths and pairs of proteins. No shared tetra-sequences were found for κ-casein. To explore sequence patterns of maximum length, tri-patterns were chosen for further investigation. Figure 5c captures the total frequency of tri-sequence patterns in the casein protein sequences and which percentage of these differentially located subsequences was covered by peptide APs. This analysis allows to identify how different patterns contribute to glycation of peptides with shared subsequences but different overall amino acid composition as they originate from different casein protein regions. Of the three P-E-V sequons in casein proteins (P 44 -V 46 of α-S1-casein and P 105 -V 107 of ß-casein, Fig. 3a; P 171 -V 173 of κ-casein, Supplementary Fig. 5), two appear as subsequences in peptide APs, as well as the alternated V-E-P (V 131 -P 133 of ß-casein, Fig. 3a). Several interesting cases where substructures highly similar to P-E-V contribute to AP formation are highlighted (P-E-L, V-L-N, V-P-N, and V-P-Q; Fig. 5c). These subsequences all share amino acids with a low dissimilarity score (D), which is based on 134 categories of activity and structure 56 , and are partially rearranged (Supplementary Table 2). For example, P-E-V and P-E-L only differ by a single amino acid with strong physicochemical similarity (D(Val, Leu) = 9), whereas in case of P-E-V and V-P-Q (D(Glu, Gln) = 14) also the sequence order was changed. By comparison, peptides that contain I-V-E, which shows more pronounced sequence variations (D(Pro, Ile) = 24 and sequence rearrangement), do not participate in AP formation. All of these patterns, which show strong contribution to AP formation, either comprise Glu (carboxylic group), glutamine (acid amide group) or asparagine (acid amide group). A catalytic effect of the carboxylic group on AP formation was previously hypothesized 33 , which resembles the here found promoting effect of Glu-containing sequence patterns on the early MR. P-Y-P and P-F-P contain amino acids with comparable properties. The substructures P-I-P, P-L-P and P-V-P feature pronounced physicochemical similarities as well 56 . All these patterns showed a high co-occurrence on peptide APs relative to their total abundance (displayed as percent in Fig. 5c), which indicates their contribution to glycation independent of the overall peptide composition, thus their origin in the source protein . In contrast, P-N-P (P 198 -V 200 of α-S1-casein) did not contribute to an AP (Fig. 3a) and shows pronounced differences in its amino acid characteristics.</p><p>A peptide-sequence pattern plot in Fig. 5d maps relevant sequence patterns to different (glyco-) peptides for which they could be observed. These peptides vary in their overall composition and peptide length. The map indicates, which patterns contribute to glycation on a peptide-level, and other peptide properties that considerably affect their reaction behavior. This analysis reveals several interesting trends, such as pronounced discrepancies in glycation of peptides with the same pattern and, perhaps most striking differences in AP formation of strongly related peptides. Small variations in the peptide amino acid sequence may cause (VVPPFLQPEV vs. VVVPPFLQPEV; YPFPGPI vs. VYPFPGPI) or not cause (VAPFPE vs. VAPFPEV; VYPFPGPI vs. VYPFPGPIN) differentiated behavior in the early MR. General correlation was not observed between AP formation and peptide physicochemical properties, expressed as hydrophobicity according to their retention time.</p><p>System-wide analysis of bioactive and sensory-active peptide glycation enabled by in silico sequence mapping. The complexity of glycation represents a great challenge for the identification of glycation patterns that are associated with the gain or loss of bioactivity and glycation induced changes in sensory attributes. A combination of bioinformatics and database search enabled to study the established sensory and bioactivities of peptides in our dataset and to evaluate their behavior in glycation. We matched 60 peptides (Supplementary Table 3) with reported bioactivities (Fig. 6a), which were included in databases compiled from literature 57,58 . While 36 peptides were exclusively found in milk, 24 peptides appeared in a variety of other food sources as well (Fig. 6b). We also found that these peptides of diverse chain lengths (Fig. 6c) cover various bioactivity categories (Supplementary Fig. 8), suggesting that tryptone models may facilitate inter-disciplinary investigation of peptide glycation. In our study, for 62% of the bioactive peptides the corresponding AP was detected (33% confirmed by MS/MS tandem experiment; Fig. 6d). Hence, approximately 34% of the 47 peptides, for which the AP was identified via MS2 fragmentation, were previously reported to be bioactive. Furthermore, we identified 25 peptides with sensory attributes 59 . Figure 6e illustrates the prevalence of AP detection for sensory-active peptides and the level of AP identification (MS and MS2). Approximately 24% of all sensoryactive peptides and 19% of the bitter peptides were observed as the corresponding AP on MS2 level. As noted by Shiyuan Dong and co-workers 32 , bitterness of MRPs was decreased compared to original casein peptides, and further reduced with heating time and glucose concentration. Xiaohong Lan et al. previously published that bitter soybean peptides below 1000 Da decreased 28.49% after reaction with xylose at 120 °C60 . In contrast to the reported experiment, digesting casein with trypsin would produce large peptides, through protein cleavage after arginine and lysine, which would lead to the loss of highly interesting bioactive peptides (Supplementary Fig. 9) and would not allow comprehensive investigation of their reaction behavior (Supplementary Fig. 10 and Supplementary Fig. 11). For example, using a tryptic casein digest, the analysis of the opioid peptides, e.g. YPF-PGPI and YPVEPF, for which N-glycation is known to have major consequences on the bioactivity of the parent www.nature.com/scientificreports/ peptide 61,62 , would not be possible. By comparison, in our experiment the large and diverse peptide spectrum of tryptone enabled us to widely predict bioactive peptide glycation.</p><!><p>Given that similarities in the amino acid sequence and sequence patterns may determine the reaction behavior of peptides, we reasoned our approach could provide insight into peptide reactivity from a systems level. To test this hypothesis, we searched peptides from our dataset as a pattern of bioactive and sensory-active peptide sequences reported in databases by substring matching [57][58][59] . First, we examined the num- www.nature.com/scientificreports/ ber of matches observed. In total, tryptone peptides were successfully mapped to 1172 unique bioactive peptide species (Supplementary Fig. 12). Even though caseins are major proteins in milk, a considerable number of common sequences between tryptone peptides and a plethora of peptides from other sources was found (Fig. 7a). Overall, we achieved 3046 sequence overlays with 675 bioactive milk peptides, 1350 overlays with 510 peptides from other sources, and 599 overlays with 174 sensory-active peptides (Supplementary Fig. 13). Importantly, not only small sequence commonalities were found as indicated by the length of the tryptone peptides mapped (Fig. 7a). Tryptone amino acid sequences, up to a length of thirteen amino acids, occurred on bioactive peptides from entirely different origins. Even larger sequences (n = 14) co-occurred in peptides with established sensory activity. The heatmap (Fig. 7a) visualizes classes of peptides delineated by the number of overlays per peptide length, showing increased sequence similarities for sensory-active peptides and fish. Or, in case of potato peptides we found a lower proportion of common di-and tri-sequences compared to other sources and no matches for larger peptides (n > 3) were observed. We note that calculating co-occurring sequences is straightforward and may provide information about glycation susceptibility of specific peptide classes from various protein origins. Supplementary Fig. 14 demonstrates that a broad variety of bioactivities is covered by the database peptides, to which tryptone peptides were successfully mapped. Other trends arise, such as the presence of a relatively high abundance of antihypertensive peptides. Note, this is expected because of the inordinate number of antihypertensive peptides in the databases used. The same number of dipeptides was successfully mapped to bioactive peptides independent of the source (Fig. 7b), and the number of tripeptides found as a subsequence was comparable as well. For larger tryptone peptides, approximately half of the peptides matched for milk were found to overlay with peptides, which were (also) found in other food sources. A striking feature of this analysis are the percentages of peptides in our dataset found as a subsequence (Fig. 7c). Considering the 264 peptides that could be mapped to a domain, 82% existed in bioactive peptides exclusively from milk and 47% in those from (milk and) other sources. Furthermore, 39% of tryptone peptides were successfully mapped to sensory-active peptides (Fig. 7c). Figure 7d depicts the percentage of matched tryptone peptides, for which the corresponding AP was identified. Up to 22% of the shorter substring peptides (n ≤ 6 amino acids) were detected as an AP by tandem MS experiments, while the majority of larger peptides appeared to be glycated. In total, 93% of the peptides detected as an AP are substructures of bioactive peptides. This represents a considerably larger proportion compared to other peptide groups, e.g. sensory-active peptides (Fig. 7d).</p><!><p>In all, here we present a straightforward approach to refine evaluation of peptide derived APs by using the power of high-resolution MS in combination with multivariate statistics and bioinformatics to access large-scale information about peptide reactivity in the MR and the influence of both reaction time and sugar concentration. Investigation of glucose-tryptone model systems enabled the most in-depth profiling of peptide APs to date. By comparison with a in silico tryptic casein-digest, we demonstrated considerable advantages of tryptone models, such as a notably larger coverage of (bioactive) peptides from various food sources. This strategy is amenable to virtually any type of MR model system or reactivity study with known reaction intermediates. Finally, the reaction behavior of 264 casein derived peptides was characterized by AP analysis from a single type of model system, which demonstrates that new models must be developed to unravel the glycation reaction network in its full complexity. Clearly, large-scale studies are needed to explore peptide glycation and its importance particularly in food but also biological systems and, thus, health.</p><p>A caveat of practically any MS-based experiment is that detectability can be affected by the type of ionization, analyte concentration as well as sample complexity. Thus, there may be a bias toward specific peptides and APs to consider in this dataset. Furthermore, Fig. 1c shows a high frequency of tryptone peptides from certain protein regions , which may arise from its production process and above-mentioned detectability issues. Our data interpretation, however, reflects on ubiquitous observations in the overall dataset, and not on specific peptide species . Despite this, we achieve in-depth characterization of a large reservoir of peptides and provide thorough information on peptide properties influencing AP formation. We relied on normalized AP intensity profiles for reaction behavior investigation, meaning there are limitations in the stability of this early reaction intermediate to consider. Greifenhagen et al. has noted pronounced susceptibility of the N-terminally acetylated Amadori peptide Ac-Ala-Lys-Ala-Ser-Ala-Ser-Phe-Leu-NH 2 toward oxidative degradation in aqueous model systems 63 . Loss of the Amadori compound of the endogenous opioid pentapeptide leucine-enkephaline (Tyr-Gly-Gly-Phe-Leu) was also noticed by Jakas and Horvat 64 , however, markedly slower degradation behavior was observed in this study. Different reaction conditions, such as concentration of catalytically active phosphate buffer and temperature, tend to affect AP stability, but also the amino acid sequence of the peptides. Even with this, all peptide APs detected in this dataset remained above the limit of detection at all time points.</p><p>While other studies have investigated glycation using a limited number of highly specific synthetic peptides, we could simultaneously study the reaction behavior of a large pool of casein peptides. We can also see similar trends to previous studies, such as the influence of both reaction time and sugar concentration 65,66 . In contrast, we can provide detailed information on how APs derived from specific peptide sequences are affected. We show that upon reaction time the bulk of peptides differentiated into three clusters, reaching maximum AP intensity at different time points and, thus that AP formation rates likely depend on the peptide structure. Peptides forming APs that peaked at early reaction time points and low glucose concentrations may represent more reactive precursors in glycation. A consecutive decrease in relative AP peak intensity may be attributed to further rearrangement and oxidative cleave reactions yielding heterogeneous AGEs 67 . Further, we show that there is no general correlation between peptide length and reactivity. More pronounced susceptibility of dipeptides compared to tripeptides toward glycation was seen in early studies of glycine (Gly) peptide model systems (GlyGly > GlyGlyGly) and in more miscellaneous synthetic peptide studies 26,33,34 . Similarly, we found higher proportions of glycated dipeptides www.nature.com/scientificreports/ than tripeptides. Although our observations are in congruence with previous studies, we are the first to investigate the impact of peptide length at this scale providing a new perspective on its influence on reactivity. We also show that there is not a general correlation between amino acid content and susceptibility towards the initial phase of glycation reactions. This suggests a strong contribution of other factors such as the amino acid sequence, thus, the amino acid microenvironment.</p><p>In peptide glycation, reaction behavior has been proposed to be driven by the amino acid sequence. An important role of the amino acid adjacent to the N-terminus has been suggested based on short-chain peptide model systems 26,33,34 . Here, we noticed strong preference of glycation for valine-starting peptides and noted more pronounced AP formation of peptides, which contain Ile, Leu and Val positioned next to the N-terminal amino acid. Further, we observed prevalence of Met, Phe, and especially Pro at the second sequence position in peptides, for which the corresponding AP could not be identified, which indicates that steric hindrance or conformational changes may prevent N-terminal glycation. Congruent observations were made for the N-X-S/T sequon, where Pro in the X position causes pronounced changes in conformation and, thus prevention of enzymatic glycosylation 55 . We show that neighboring Glu, for example, may not always exert a catalytic effect on N-terminal glycation as a result of its carboxylic side chain. Interestingly, APs from peptides containing Asp adjacent to the N-terminus were not observed, even though its structure is closely related to Glu. Depending on the amino acid sequence and the peptide N-terminus, differentiated effects of the neighboring amino acid may be observed 33 , and here, we can cover a broad range of diverse peptide properties. Such thorough and integrated characterization of peptide APs depending on the reaction conditions is necessary for a complete understanding of peptide glycation and its impact on food and biological systems. We further found two location sites near the N-terminus with increased relative abundance of Pro in AP forming peptides, namely the third and fifth position of the amino acid sequence.</p><p>We identified peptide collectives particularly prone to early glycation reactions by mapping APs to casein sequences and across each other. Furthermore, we leverage these reactive peptide species to provide information on potentially important tri-sequence patterns and propose that glycation patterns among many other factors promote peptide glycation, which is indicated by strong connectivity in glycation susceptibility and presence of specific sequence patterns. Even though N-terminal proline may inhibit Schiff base formation 54 , here, we established several proline-rich sequence patterns, which considerably triggered AP formation. Furthermore, Glu containing sequence patterns, such as P-E-V, may exert a catalytic effect towards early MR 33 . Depending on whether glycation is desired or not, peptides may be chosen, accordingly (discussed below). Overall, we provide a comprehensive set of molecular checkpoints for peptide reactivity estimation towards glycation.</p><p>Finally, we established system-wide applicability of tryptone model systems by mapping tryptone peptides to a plethora of bioactive and sensory-active peptides from various food sources. Depending on whether glycation of peptides is desired or not, we suggest that the amino acid sequences may be chosen, accordingly. For development of functional foods, health benefits must be preserved, thus for most bioactive peptides AP formation needs to be obviated. For example, for opioid peptides, the requirement of free N-terminal tyrosine was demonstrated and the loss of antihypertensive properties of casein peptides as a result of glycation was revealed in various model systems 29,30,68 . Conversely, if increased antioxidant potency 26,69 must be achieved, we suggest that the peptide species may be capitalized that are more prone to AP formation. The increased antioxidative properties of MRPs compared to their corresponding casein peptides has previously been determined 27,28,30 , and consequently targeted peptide glycation may enable to enhance the health benefits of peptides. Superior antioxidative properties have been established for MRPs derived from small peptides compared to larger peptide species 69 , which makes tryptone particularly suitable for identification of potential peptide candidates. In total, we observed APs from 47 amino compounds. For 34% of them, bioactivity was previously established and 93% were identified as a substructure of bioactive peptides, which suggests that bioactive peptides are particularly prone to glycation. For sensory-active peptides, others have observed reduced bitterness of MRPs compared to heated casein peptides alone, while antioxidative properties where increased 26 . Similar findings were reported for peptides from other sources 60,70,71 . Even more benefits for food production can thus be provided by choosing appropriate peptide candidates, such as enhanced sensory attributes offoods. To assess desired flavor improvement 71,72 , we reasoned that selection of peptides susceptible to early glycation may be promising. As bitter peptides cannot be employed above their bitter flavor threshold 72 , increased bioactivity accompanied concurrently by decreased bitterness may be desirable for the production of functional foods to improve health and enhance customer acceptance 23 . Taken together, our dataset allows to select suitable peptide candidates, given (1) a checklist for estimation of their reaction behavior in early glycation reactions according to the amino acid at the N-terminus, the adjacent sequence position and presence of relevant sequence patterns, and (2) screening for established sensory attributes and bioactivity.</p><p>Future studies are required to investigate a wider range of peptides from different proteins and, thus a broader variety of amino acid sequences to gain more global information on the relevance of amino acid composition and sequence patterns. Model systems prepared from highly specific synthetic peptides have provided valuable findings in previous studies, which suggests that targeted investigation of peptides, in particular with potentially relevant sequence patterns, may be promising for identification of peptides especially prone to peptide glycation. These strategies also present an opportunity for determination of peptides less susceptible toward glycation reactions. Investigation of changes in sensory attributes and bioactivity as a result of glycation will be a worthwhile endeavor in future experiments to gain the necessary refined information for systematic use of specific peptides and their glycation products as functional food ingredients. Chemical peptide structures were confirmed by peptide mapping in GeneData Expressionist Refiner MS 13.0 (GeneData GmbH, Basel, Switzerland) with an absolute m/z tolerance of 0.005 and 0.1 for the precursor and product ions, respectively, unspecific enzyme cleavages, a minimum peptide length of 1 AA, and no fixed or variable modifications. The fragmentation type was set to ESI CID/HCD. The peptide mapping was performed using a text file containing four AA sequences in FASTA format of bovine milk caseins including α-S1-, α-S2-, β-, and κ-casein. Top-down sequencing annotations for each of the four casein proteins were exported from Refiner MS module providing a list of the identified peptides along with their positions in the protein sequence they were successfully mapped to. Further processing was performed in R software (version 3.5.2). Amadori product precursor mass was calculated by a mass increase of 162.0528 Da. Amadori product precursor signals were computationally assigned by an algorithm within a mass tolerance of ± 10 ppm. Putatively assigned Amadori products with available MS2 spectra from our data were clustered according to their similarity in normalized intensity profiles using Pearson correlation. Product ion annotation was automatically performed in R software by in silico fragmentation 43,73 and manually validated. Monoisotopic mass tolerance was set to ± 0.005 Da for product ions. To separate false positive assignments, we excluded signals with a poor fit of the MS/MS spectrum to the in silico predicted fragments and maximum intensity in the tryptone control samples heated for 10 h. GeneData Expressionist Refiner MS 13.0 (GeneData GmbH, Basel, Switzerland) peptide mapping activity provides a consolidated score, which describes the average fit for each peptide across all MS2 spectra available. Consolidated scores of all peptides, for which the corresponding Amadori product (MS2 level) could be verified, were computed. The minimum consolidated score per peptide length was chosen as a threshold for peptide identification.</p><!><p>Pearson correlation coefficients were calculated in R software between intensity values for putatively assigned MS2 Amadori products (n ≥ 3, p ≤ 0.05). For this analysis, relative intensity values were used. Relative intensity values were calculated by normalizing intensity values to the maximum intensity value across all time points and for each Amadori product, respectively. Hierarchical clustering to provide the domain ordering was done using R software. Amadori product wise distances were calculated based on these correlations using the as.dist() function followed by hierarchical clustering using the hclust() function. To assess the importance of small sequence variations, pairwise two-sided t-tests were performed. Intensity values in model systems and control samples were compared. Significantly increased Amadori products (p ≤ 0.05) and relevant reaction conditions are reported in Supplementary Table 1. Multiple testing correction was performed using the Benjamini-Hochberg procedure.</p><p>Sequence grouping. The computational analysis of sequence groups was performed with the peptide single letter code using R software. To identify peptides with common sequences the grepl() and match() base functions were applied. Based on derived sequence commonalities, we assigned all peptides to sequence groups (Supplementary Table 1). Amino acids were not assigned to sequence groups. Sequence groups, for which no Amadori product was detected, were excluded in Supplementary Table 1.</p><p>Database search. Bioactive peptide database search was carried out using the Milk Bioactive Peptide Database (March 13, 2020) 57 and the BioPepDB database (March 13, 2020) 58 . Sensory peptide database search was</p>

Scientific Reports - Nature

Ir-Catalyzed Intermolecular Branch-Selective Allylic C-H Amidation of Unactivated Terminal Olefins

An efficient method for intermolecular branch-selective allylic C-H amidation has been accomplished via Ir(III) catalysis. The reaction proceeds through initial allylic C-H activation, supported by the isolation and crystallographic characterization of an allyl-Ir(III) intermediate, followed by a subsequent oxidative amidation with readily available dioxazolones as nitrenoid precursors. A diverse range of amides are successfully installed at the branched position of terminal alkenes in good yields and regioselectivities. Importantly, the reaction allows the use of amide-derived nitrenoid precursors avoiding problematic Curtius-type rearrangements.

ir-catalyzed_intermolecular_branch-selective_allylic_c-h_amidation_of_unactivated_terminal_olefins

<p>Due to the ubiquity of nitrogen-containing functionalities in both natural and synthetic bioactive molecules,1 C-N bond formation reactions are some of the most frequently used transformations in medicinal chemistry.2 In addition to classic strategies which usually require prefunctionalization, direct amination of C-H bonds could dramatically simplify synthetic routes, providing more straightforward disconnections for amine synthesis.3 A number of reports have recently demonstrated chelate-assisted C–H amination; however, these methods usually employ pre-installed coordinating directing groups that limit their general application.4 In contrast, allylic C-H amination only involves a simple alkene as the "directing group" which is not only prevalent in a variety of compounds but is also easily manipulated with a diverse range of robust methods.</p><p>Current strategies in allylic C-H amination utilize either C-H insertion by a metal nitrenoid5 or nucleophilic amination of allylmetal species generated via C-H activation.6 Despite these advances, controlling chemoselectivity and regioselectivity remains challenging in intermolecular allylic C-H amination reactions. For instance, terminal olefins that react with a metal-nitrenoid species suffer from poor chemoselectivity between aziridination of the alkene and desired C–H amination.5h,7 (Figure 1a(i)). An alternative strategy was independently reported by the White6d and Liu6e groups in 2008, demonstrating that linear amination could be achieved under Pd(II) catalysis (Figure 1a(ii)). The reaction is proposed to proceed through an allyl-Pd(II) intermediate that is generated via allylic C-H activation, followed by reductive quenching with a nitrogen nucleophile at the less hindered terminal position. Based on these initial reports, many modifications and improvements have been developed in recent years,8 offering various choices for the synthesis of linear allylic amines. On the other hand, the branch-selective allylic C-H amination of terminal olefins has been achieved through an intramolecular manner.5c,5e,5g,5i, 6b, 6g,9 Two important exceptions are Tambar's work employing a sulfurdiimide reagent followed by a Pd-catalyzed asymmetric [2,3]-rearrangement, and Hartwig's Pd-catalyzed oxidation followed by asymmetric Ir-catalyzed amination to deliver the branched amination product.10 Moreover, the majority of these examples involve the use of a nitrogen bearing a sulfonyl or carbamate group; amide-derived nitrenoids are more challenging due to the potential intervention of a Curtius-type rearrangement. 11 Herein, we report an intermolecular Ir(III)-catalyzed branch-selective allylic C-H amidation of unactivated terminal olefins.</p><p>We have recently reported the intramolecular Ir-catalyzed diamination of alkenyl hydroxamate esters wherein the transformation was proposed to partially proceed via Ir-nitrenoid species.12 We wondered whether Ir nitrenoids might have sufficient lifetime to allow intermolecular aziridination or amination of feedstock α-olefins. Inspired by previous reports of group 9 metal-catalyzed C-H amidations,3f together with our long-term interests in C-H functionalizations,13 the hypothesis was initially tested with [Cp*MCl2]2 as the pre-catalysts (M=Rh, Co, Ir), and either sulfonyl azides14 or dioxazolones15 as nitrene precursors. Cossy and coworkers have reported that Cp*Rh(III) promotes an intramolecular allylic C-H amidation reaction proceeding through an allyl-Rh(III) intermediate.6g We previously demonstrated that the allylic activation chemistry could be coupled with a C-C bond formation.16 More recently, Blakey and coworkers reported an intermolecular allylic C-H amidation of β-substituted styrenes that exhibits exclusive linear selectivity with allylbenzene.6i In sharp contrast, the allylic C-H amidation of 1a with [Cp*RhCl2]2 in combination with methyl dioxazolone 2a gives the branched amidation product 3a as the major isomer, albeit with a moderate yield and regioselectivity (Table 1, entry 1). We speculated that this was due to the oxidative nitrene formation and insertion process which favors the more electron-rich position. Both yield and regioselectivity were found to be significantly improved with [Cp*IrCl2]2 while [Cp*CoCl2]2 failed to produce any amidation products (entries 2, 3).</p><p>The higher reactivity of Cp*Ir(III) is consistent with the observations and computational arguments advanced by Baik and Chang.17 Additionally, the optimized conditions include a catalytic amount of AgNTf2 and LiOAc. We also found that the use of Li2CO3 instead of LiOAc produces 3a in 62% yield with only 10 mol% of AgNTf2 (see the Supporting Information). Cp*Ir(OAc)2, which was believed to be the reactive catalyst generated in situ, was ineffective without adding additional AgNTf2 (entry 5, 6). Taken together with the above observations, we suggest that AgNTf2 probably helps the dissociation of the acetate ligand (or the amide product) from iridium, which promotes the alkene coordination and further activation. It is also worth noting that the yield is dramatically reduced when CsOAc is used instead of LiOAc (entry 7). Given that CsOAc is much more soluble than LiOAc in DCE, this further suggests that excess acetate inhibits the reaction, supporting our assertion that a monoacetato-Ir is required. Moreover, tosyl azide was also tested. It was found to afford moderate yield of branched amidation product with excellent regioselectivity, when the reaction was heated to 80 °C (entry 11).</p><p>Having established the optimal conditions for branch-selective allylic amidation, we next sought to examine the scope of this transformation with various terminal alkenes. The reaction tolerates a broad range of functional groups, giving rise to various branched amidation products. As shown in Scheme 2, several commonly used oxygen, nitrogen or arene containing functional groups are compatible, affording corresponding amidation products in good yields and regioselectivities (3b-3h). Even substrates with susceptible functionalities, like –Br, –CN and –CO2H, participated smoothly with the standard condition to provide the desired products (3i-3k). Although a lower yield was observed with the alkene bearing a carboxylic acid group, the regioselectivity remains unaffected, giving the branched product 3k predominately. Despite precedent for Ir-catalyzed dehydrogenation of alkenes bearing homoallylic carbonyl groups under oxidative conditions,18 exclusive amidation of 4-phenyl-1-butene and ethyl 4-pentenoate was observed with no evidence of diene formation (3l, 3m). Although yield and regioselectivity are moderate with ethyl 4-pentenoate, the alternative conditions with TsN3 as the nitrene precursor leads to moderate yield and excellent regioselectivity (3m). Furthermore, substrates with homoallylic heteroatoms were found to decrease the reactivity. Moderate conversions and yields were obtained even with elevated temperature (3n, 3o). Additionally, sterically hindered allyl-cyclohexane was remarkably well-tolerated (3p). Allylarenes with electron- rich (-OMe) and electron-poor (-CF3) substituents were also examined. It was found that the regioselectivity decreased from >20:1 to 2.6:1 by changing the para- substituent from –OMe to –CF3 (3q-3s). Excellent regioselectivity could be restored with TsN3 as the nitrene precursor.</p><p>The generality of dioxazolone coupling partners were next explored. Primary, secondary and tertiary alkyl groups on the dioxazolone are tolerated with no significant decrease in reactivity (4b-4f). Additionally, the cyclopropane dioxazolone could also be utilized without detectable ring-opening (4d). Moreover, electron-donating and electron-withdrawing substituted phenyl dioxazolones are well tolerated (4g-4j). Even an alkenyl substituted dioxazolone can participate to afford 4k in moderated yield. In general, these aryl and alkenyl dioxazolones are slightly less reactive and require elevated temperature for full conversion. Under all these reaction conditions, the Curtius/Lossen-type rearrangement is not competitive5i,19 presumably because of a change in mechanism (vide infra).</p><p>Further mechanistic insight was gained from isotopic labeling studies and stoichiometric reactions. When subjecting 1aa-d2 to the optimized reaction conditions using acetic acid as the proton source, no deuterium leaching was observed at the allylic position of the amidation product. This indicates that the allylic C–H activation is irreversible under the standard reaction condition. Furthermore, a smaller kinetic isotopic effect from intermolecular competition reaction compared to the one from intramolecular reaction was observed (KIE= 1.9 vs 2.6). We speculate that the allylic C–H bond activation is probably not the sole rate-determining step.20 Stoichiometric studies were also performed. An allyl-Ir(III) complex 5 was trapped by an additional p-toluenesulfonamide ligand which stabilizes the intermediate for isolation. Interestingly, the subsequent amidation is only achieved with the assistance of AgNTf2, which delivers the desired product in high yield and regioselectivity. In this case, AgNTf2 might also act as a Lewis acid promoting the dissociation of the p-toluenesulfonamide ligand to regenerate the active species with an empty coordination site. Finally, stoichiometric reactions with 1ab-d1 were also conducted, leading to the same distribution between deuterated and protonated products of either the allyl-Ir(III) complex (PD/PH = 2.6) or the amidation product (PD/PH = 2.7) (see SI).</p><p>Based on the above observations, we hypothesize the following catalytic cycle (Scheme 5). The active catalyst is presumably the coordinatively unsaturated cationic monoacetato Cp*Ir(III) I which is generated from [Cp*IrCl2]2, AgNTf2, and LiOAc. Alkene coordination and irreversible metalation form allyl-Ir(III) species III. The oxidation of Ir(III) via N–O bond cleavage and CO2 extrusion produces the key allyl-Ir-nitrenoid species IV. Subsequent migratory insertion would install the desired amide bond at the internal position. Finally proto-demetalation regenerates the active catalyst and produces the amide product. Importantly, the allyl Ir intermediate III is more oxidizable than I or II and only undergoes oxidation/nitrene formation once the allyl moiety is in place. Thus, the Ir nitrenoid undergoes reductive elimination to form product faster than Curtius/Lossen-type rearrangement. This is consistent with Chang's observations in intramolecular C-H insertion with related Ir nitrenoids.5i</p><p>In summary, we have developed an intermolecular branch-selective allylic C-H amidation reaction via Ir(III) catalysis. The inner-sphere Ir-nitrenoid insertion after allylic C-H activation is key for prevention of undesired aziridination and achieving branched-selectivity. This opens a new pathway for branched functionalizations of terminal olefins. Further efforts at elucidating the mechanism and extending this chemistry are currently underway.</p>

PubMed Author Manuscript

Fluorogenic Probes for Imaging Cellular Phosphatase Activity

The ability to visualize enzyme activity in a cell, tissue, or living organism can greatly enhance our understanding of the biological roles of that enzyme. While many aspects of cellular signaling are controlled by reversible protein phosphorylation, our understanding of the biological roles of the protein phosphatases involved is limited. Here, we provide an overview of progress towards the development of fluorescent probes that can be used to visualize the activity of protein phosphatases. Significant advances include the development of probes with visible and near-IR excitation and emission profiles, which provides greater tissue and whole-animal imaging capabilities. In addition, the development of peptide-based probes has provided some selectivity for a phosphatase of interest. Key challenges involve the difficulty of achieving sufficient selectivity for an individual member of a phosphatase enzyme family and the necessity of fully validating the best probes before they can be adopted widely.

fluorogenic_probes_for_imaging_cellular_phosphatase_activity

Introduction<!>Fluorophores with excitation and emission wavelengths in the visible region of the spectrum<!>Peptide-based fluorophores<!>Near-IR fluorophores<!>Conclusions

<p>Reversible protein phosphorylation is widely used in biology to regulate protein-protein interactions, cellular signaling pathways, etc. [1]. The biological phosphorylation and dephosphorylation of serine, threonine and tyrosine residues has been extensively studied, but phosphorylation also occurs at histidine, lysine, arginine, aspartate, glutamate and cysteine residues [2]. While much less is known about these latter phospho sites, protein phosphorylation is important in both pathological and physiological processes [3–9]. The development of chemical probes to study the biological roles of the enzymes that dephosphorylate phosphoproteins is a topic of intense investigation [10–13].</p><p>Substrates that undergo a significant change in fluorescence upon dephosphorylation provide a convenient approach to monitoring phosphatase activity both in vitro and in living systems [14]. Such substrates can provide a highly sensitive readout of phosphatase activity. However, a significant challenge in the field is the development of probes with sufficient selectivity to report on the activity of only one family of phosphatases. Even more challenging is the development of probes that selectively report on the activity of a single phosphatase of interest. An ideal chemical probe for monitoring phosphatase activity in a cellular context would provide a large signal difference between the phosphorylated and dephosphorylated states and react selectively with one family of phosphatases or one specific phosphatase. Excitation and emission wavelengths in the visible region are preferable to UV-excitable probes, and probes with emission in the near-IR region of the spectrum provide additional advantages in tissue and whole animal imaging. In addition, an ideal probe must be thoroughly validated in vitro and in cells/tissues/animals such that its readout can confidently be correlated to activity of the enzyme(s) of interest. In this review, we have highlighted some recent work illustrating progress toward the development of phosphatase-reactive fluorescent probes that can be used to address key questions about phosphatase biology, with a focus on probes that have been used in cellular experiments. Significant contributions have been made in the field in recent years and are discussed here, along with key challenges that remain in the quest to develop an ideal probe.</p><!><p>The prototype fluorogenic probe for monitoring phosphatase activity is 6,8-difluoromethylumbelliferyl phosphate (DiFMUP, Figure 1) [15–17]. DiFMUP is an excellent substrate for many phosphatases, including alkaline phosphatases, acid phosphatases, tyrosine phosphatase and serine/threonine phosphatases. Recently, DiFMUP was reported as a sensitive, fluorogenic substrate for assaying histidine phosphatase activity as well [18]. While this substrate is not ideal for cellular applications because it can readily be hydrolyzed by most classes of phosphatases and would thus report on aggregate phosphatase activity levels rather than the activity of an individual family of phosphatases or a single family member, it does provide a facile in vitro assay for phosphatase activity, making it the substrate of choice for many applications [18–25].</p><p>A number of other fluorophores have been investigated for utility as fluorogenic phosphatase substrates in addition to the coumarin scaffold. The recently developed phosphorylated resorufin (pRES, Figure 1), for example, has several advantages over coumarin-based probes [26]. Specifically, the excitation and emission wavelengths are significantly red-shifted (λex = 560 nm, λem = 585 nm as compared to λex = 360 nm and λem = 455 nm for DiFMUP), resulting in lower background from cellular autofluorescence and less toxicity arising from high energy excitation. Notably, in addition to the large fluorescence increase observed upon dephosphorylation of pRES, a significant shift in absorbance results in a color change from orange to bright pink. The substrate pRES provides a facile colorimetric and fluorogenic assay for monitoring tyrosine and alkaline phosphatase activity. The fluorinated derivative, F2pRES, can also be used to monitor acid phosphatase activity. In addition to its utility for monitoring in vitro phosphatase activity, pRES was also shown to provide a sensitive readout of intracellular phosphatase activity [26]. There was no cellular autofluorescence observed at the wavelengths used in this experiment. Incubation of HeLa cells with 50 µM pRES for 10 min resulted in a significant accumulation of intracellular red fluorescence as a result of hydrolysis of pRES by intracellular phosphatases. It was proposed that the fluorescence observed arose primarily from tyrosine phosphatase catalyzed pRES hydrolysis, as pretreatment of the cells with pervanadate, a known tyrosine phosphatase inhibitor [27], significantly reduced the fluorescence observed. However, HeLa cells also express significant levels of alkaline phosphatase activity [28]. While pervanadate does not appreciably inhibit alkaline phosphatase, it does degrade to the potent alkaline phosphatase inhibitor vanadate [29], so it is difficult to rule out the possibility that alkaline phosphatase activity contributes to hydrolysis of pRES in HeLa cells.</p><p>Several other scaffolds have recently been investigated as fluorogenic substrates for protein phosphatase activity (Figure 1) [30–36]. These fluorophores all have red-shifted excitation and emission profiles as compared to DiFMUP, and one (TP-Phos [35]), can be excited using two-photon excitation at long wavelength, resulting in negligible cellular autofluorescence and minimal tissue damage. The probe TP-Phos also takes advantage of an auto-immolative linker that spontaneously releases the fluorophore upon dephosphorylation of an ortho-alkyl aryl phosphate. The addition of an ortho substituent adjacent to the aryl phosphate results in significant selectivity of the probe for alkaline phosphatase – tyrosine phosphatases, dual-specificity phosphatases and serine/threonine phosphatases were unable to hydrolyze this substrate [35]. All of the probes are excellent substrates for alkaline phosphatase in vitro and are not appreciably hydrolyzed by acid phosphatase, but the ability of tyrosine and serine/threonine phosphatases to hydrolyze these substrates was not reported (with the exception of TP-Phos, as described above). In cellular experiments, two of these probes (LP1 and Lyso-Phos) were suggested to localize in lysosomes [32,33], while the subcellular localization of the others was not investigated in detail. All of the probes are clearly processed by intracellular phosphatases and cell lines known to express high levels of alkaline phosphatase activity show the largest fluorescence increases when incubated with the probes. Nonetheless, there is a possibility that tyrosine phosphatase activity may contribute to the signal seen in several cell types. Most of the experiments used vanadate to inhibit intracellular phosphatase activity, which can inhibit both alkaline phosphatase and tyrosine phosphatases (although it is significantly more potent against alkaline phosphatase). In cellular experiments with TCF-ALP, levamisole, an alkaline phosphatase selective inhibitor, was also used and minimal fluorescence was observed in C2C12 cells pretreated with levamisole prior to incubation with TCF-ALP [31]. These data support the assertion that this probe reports primarily on alkaline phosphatase activity in this cell line.</p><!><p>To develop probes which show increased selectivity towards a variety of different phosphatase families, peptide-based probes can be used. The sequences of these peptides are often obtained from known physiological substrates of the phosphatase of interest or discovered de novo through the use of peptide library screening methods, and thus can provide significantly more selectivity as compared to small molecule fluorescent probes. Frequently, the fluorophore is incorporated within the peptide sequence, although examples where the fluorophore is placed on a peptide terminus have been reported [37,38].</p><p>The diversity of this strategy in developing probes for different phosphatase families is best exemplified by the C-Sox (cysteine sulfonamide-oxine) fluorophore, which is typically placed into the peptide sequence two residues away from the phosphoresidue (Figure 2a) [39]. Upon coordination of a Mg2+ ion between the phosphate hydroxyl groups and the C-Sox fluorophore, a strong fluorescent signal is observed which decreases as the probe is dephosphorylated due to loss of Mg2+ coordination. Using this method, probes to measure serine/threonine, tyrosine, histidine, and arginine phosphatase activity have been reported [40–43]. While the serine/threonine phosphatase targeted probe was hydrolyzed well by PP2A, significant background signal was observed in HeLa cells depleted of PP2A, indicating that some other cellular activity contributes to a loss of signal from this probe. Likewise, both the tyrosine and histidine phosphatase targeted probes showed some selectivity towards PTP1B and PHPT1 respectively in vitro, but a significant decrease in background fluorescence was observed in cells lacking PTP1B or PHPT1. These data suggest that the C-Sox probes could be processed by off-target cellular phosphatases or some other cellular process.</p><p>An alternative approach to the development of peptide-based phosphatase probes involves the use of a fluorogenic phosphotyrosine mimetic moiety in place of the native phosphoresidue. An example of this is the use of pCAP, an unnatural amino acid variant of the fluorogenic phosphatase substrate 4-methylumbelliferyl phosphate [44]. This fluorophore was used to produce a CD45 selective probe (SP1) based on the amino acid sequence surrounding tyrosine 394 of the kinase Lck, a known biological substrate of CD45 (Figure 2b) [45]. Screening of SP1 against a 7-member panel of cytosolic PTPs revealed some in vitro selectivity towards CD45. Monitoring SP1 hydrolysis in Jurkat T cells (CD45-expressing) and J45.01 T cells (CD45-null) revealed higher levels of fluorescence in the Jurkat cells, and partial knockdown of CD45 or introduction of a CD45 selective inhibitor showed a significant reduction in fluorescence, indicating that the probe is primarily hydrolyzed by CD45 in T cells. The probe was used to identify cell-permeable CD45 inhibitors via an in-cell screen [45]. In addition, SP1 was used to identify differences in CD45 activity in B cells isolated from patients with systemic lupus erythematosus as compared to healthy controls for the first time [46], providing a nice demonstration of the utility of a well-validated protein phosphatase targeted probe.</p><!><p>In order to move beyond in vitro analyses and cellular imaging, several groups have been developing fluorogenic phosphatase probes with excitation and emission wavelengths in the near-IR (NIR) region of the spectrum. With appropriate excitation and emission profiles and sensitivity, NIR probes have the potential to be used in tissue and whole animal imaging experiments. Two main scaffolds have been used for this purpose in recent work; a dihydroxanthene-hemicyanine scaffold and a cyanine dye scaffold (Figure 3a & 3b). The dihydroxanthene-based probes in Figure 3a have fairly similar excitation and emission profiles and serve as sensitive reporters of alkaline phosphatase activity in vitro [47–52]. These probes report on phosphatase activity in cells, although the selectivity for alkaline phosphatase over other phosphatase activity was not explored in detail. Generally, alkaline phosphatase rich HeLa cells showed significantly higher fluorescence when incubated with the probes than alkaline phosphatase poor HEK 293 cells [47,49,53]. Probes based on a cyanine dye scaffold localized in the mitochondria, consistent with similar reports in the literature [53,54]. In addition, the NIR probes can be used in animal imaging experiments. For example, the probes NALP, LET-3 and QcyP were used to visualize alkaline phosphatase overexpressing tumor xenograft in nude mice [49,50,53]. Upon intratumoral injection of probe, significant fluorescence was observed in the tumor region, while mice that received intratumoral injections of vanadate prior to probe displayed significantly lower fluorescence. These studies indicate that NIR probes could ultimately be useful for imaging phosphatase-rich tissues.</p><!><p>Several fluorogenic probes for monitoring intracellular phosphatase activity have been reported recently. Significant advances have been made in the development of probes with excitation and emission wavelengths in the visible and near-IR regions of the spectrum, providing greater compatibility with tissue and whole animal imaging. Peptide-based probes have been optimized to provide some selectivity for individual families of protein phosphatases and even for an individual member of an enzyme family. Despite these advances, significant challenges remain. The development and validation of selective substrates is particularly challenging. The selectivity of many of the probes described here for their intended phosphatase target(s) has not been established unambiguously. In addition, the peptide-based probes, which have the potential to be optimized for selectivity, utilize fluorophores that require UV excitation. Efforts to develop a well-validated, selective probe that can be excited with visible or near-IR irradiation are in progress and would be expected to have significant impact on the field.</p>

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TRAF6 and p62 inhibit amyloid \xce\xb2-induced neuronal death through p75 neurotrophin receptor

Amyloid \xce\xb2 (A\xce\xb2) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient\xe2\x80\x99s brain. A\xce\xb2 is known to bind p75 neurotrophin receptor (p75NTR) and mediates A\xce\xb2-induced neuronal death. Recently, we showed that NGF leads to p75NTR polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that A\xce\xb2 stimulation impaired the p75NTR polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75NTR on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75NTR polyubiquitination upon A\xce\xb2/NGF treatment. A\xce\xb2 significantly reduced NF-\xce\xbaB activity by attenuating the interaction of p75NTR with IKK\xce\xb2. p75NTR increased NF-\xce\xbaB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the A\xce\xb2-mediated inhibition of p75NTR polyubiquitination and restored neuronal cell survival.

traf6_and_p62_inhibit_amyloid_\xce\xb2-induced_neuronal_death_through_p75_neurotrophin_receptor

1. Introduction<!>2.1. Antibodies and reagents<!>2.2. Cell culture<!>2.3. Amyloid \xce\xb2 fragment (1\xe2\x80\x9340)<!>2.4. Immunoprecipitation and Western blotting analysis<!>2.5. Measurement of NF-\xce\xbaB activity<!>2.6. Measurement of cell viability<!>3.1. Amyloid \xce\xb2 inhibits the NGF-induced polyubiquitination of p75NTR<!>3.2. Amyloid \xce\xb2 impairs the interaction of p75NTR with TRAF6 and p62<!>3.3. p75NTR polyubiquitination was enhanced by TRAF6/p62 overexpression<!>3.4. TRAF6/p62 potentiates NGF-induced NF-\xce\xbaB activity

<p>Amyloid β protein (Aβ) is a peptide (39–43 amino acids) derived by the β- and γ-secretase cleavage of amyloid precursor protein (Sisodia and Tanzi, 2007). Aβ aggregates are the primary component of senile plaques found in the brains of individuals with Alzheimer disease (AD) (Selkoe, 2004). Overproduction of Aβ causes it to accumulate, which leads to early-onset familial AD (EOFAD) (Sisodia and Tanzi, 2007). Failure of the processes to remove Aβ from the brain causes late-onset AD (LOAD) (Whitfield, 2007). This failure might be due to diminished ability of microglial cells to clear Aβ, impairment of neprilysin and insulysin Aβ degrading proteases, and diminished perivascular and vascular drainage (Whitfield, 2007; Deane and Zlokovic, 2007; Huang et al., 2006; Marques et al., 2009; Nicoll et al., 2004).</p><p>p75 Neurotrophin receptor (p75NTR) is expressed in basal forebrain cholinergic neurons that undergo degeneration in AD (Dechant and Barde, 2002). The ligands for p75NTR are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The p75NTR can also bind to Aβ (Yaar et al., 1997; Yaar et al., 2002; Kuner et al., 1998), which mediates neuronal cell death (Coulson, 2006; Yaar et al., 2007; Rabizadeh et al., 1994). Recently, we showed that binding of NGF to p75NTR mediates neuronal cell survival (Geetha et al., 2012). In addition, polyubiquitination of p75NTR and interaction of tumor necrosis factor receptor-associated factor 6 (TRAF6) with p75NTR was NGF dependent (Geetha et al., 2012). TRAF6 functions as an ubiquitin ligase (Joazeiro and Weissman, 2000) and polyubiquitinates several substrates (Deng et al., 2000). The ubiquitin ligase activity of TRAF6 is dependent upon its polyubiquitination and oligomerization (Wang et al., 2001; Ea et al., 2004). p62 is found to interact with TRAF6 (Sanz et al., 2000). p62 induces polyubiquitination and oligomerization of TRAF6, thereby enhancing the ubiquitin ligase activity of TRAF6 (Wooten et al., 2005). The TRAF6/p62 complex ubiquitinates several substrates including ubiquitination of TrkA, leading to cell survival and differentiation (Geetha et al., 2005a); activation of NRIF, leading to apoptosis (Geetha et al., 2005b); activation of Unc-51-like kinase 1/2 to regulate filopodia extension and axon branching in sensory neurons (Zhou et al., 2007); and initiation of the proteasomal degradation of tau (Babu et al., 2005).</p><p>NF-κB activation requires the phosphorylation of IκBα by the IκB kinase (IKK), which leads to nuclear translocation of NF-κB. IκBα is tyrosine phosphorylated (Bui et al., 2001) and degraded on NGF stimulation (Arevalo et al., 2009); however, 800 nM Aβ blocked the phosphorylation and degradation of IκBα (Arevalo et al., 2009). Activation of NF-κB by NGF is predominantly mediated through the p75NTR receptor (Mamidipudi et al., 2002). Interaction of TRAF6 with p75NTR enhances the NF-κB activation (Khursigara et al., 1999). p62 functions as a scaffold for the activation of NF-κB by NGF (Wooten et al., 2001). In this study, we demonstrate that Aβ impaired p75NTR polyubiquitination, the interaction of TRAF6 and p62 with p75NTR, NF-κB activation and neuronal cell survival that is otherwise induced by NGF. However, overexpression of TRAF6/p62 restored p75NTR polyubiquitination, NF-κB activation and neuronal survival upon Aβ/NGF treatment.</p><!><p>p75NTR antibody was purchased from Millipore (Billerica, MA) and rabbit TRAF6 antibody for Western blotting was from Abcam (Cambridge, MA). Rabbit p75NTR, p62, Oct A, and ubiquitin antibodies were purchased from Santa Cruz Biotechnology (La Jolla, CA). IKKβ antibody was from Cell Signaling (Danvers, MA). NGF (2.5S) was obtained from Bioproducts for Science (Indianapolis, IN). Anti-rabbit IgG and anti-mouse IgG-HRP linked secondary antibodies were from GE Healthcare UK Ltd., and Enhanced Chemiluminescence (ECL) was from Thermo Scientific, IL. Amyloid β fragment (1–40), Protein A Sepharose beads, and all other reagents were obtained from Sigma–Aldrich Co. (St. Louis, MO).</p><!><p>The mouse hippocampal cell line HT-22 was a generous gift from Dr. David Schubert (The Salk Institute, La Jolla, CA) (Li et al., 1997). Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Sigma–Aldrich, Co., St. Louis, MO) supplied with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) as previously described (Suo et al., 2003). Cells were incubated in a 5% CO2 atmosphere at 37 °C. HT-22 cells were transfected using Lipofectamine™ 2000 transfection reagent (Invitrogen, Carlsbad, CA). The cells were deprived of serum in culture medium overnight at 37 °C before cell lysis.</p><!><p>The amino acid sequence of Aβ 1–40 is the human sequence (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-OH). The lyophilized peptide was initially dissolved in water (6 mg/ml). For maximal biological activity, it was further diluted with calcium-free PBS to 1 mg/ml and incubated at 37 °C for 4 days before adding to the culture media at the final desired concentration.</p><!><p>Cells were stimulated with NGF (100 ng/ml) for 10 min or with 10 or 20 μM amyloid β (1–40) for 24 h at 37 °C. To detect protein–protein interactions, the cells were lysed with Triton lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 10 mM NaF, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 2 μg/ml leupeptin and aprotinin) or SDS lysis buffer (Triton lysis buffer containing 1% SDS) to detect covalent interaction of ubiquitin and p75NTR (Geetha et al., 2005a). Protein concentrations were estimated using the Bradford procedure (Bio-Rad, Hercules, CA) using bovine serum albumin as a standard for all samples except for those that contained SDS where the DC assay was used (Bio-Rad, Hercules, CA). The cell lysates were incubated with 4 μg of primary antibody at 4 °C for 3 h. The immunoprecipitates were collected with agarose-coupled secondary antibody for 2 h at 4 °C and then were washed three times with lysis buffer. Samples were boiled in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and resolved on 7.5–12% SDS–PAGE gels, transferred onto nitrocellulose membranes, and analyzed by Western blotting with the appropriate antibodies.</p><!><p>HT-22 cells were transfected with TRAF6 and p62 or TRAF6ΔR and ASp62, which was followed by addition of 10 μM of amyloid β (1–40) for 24 h with or without 100 ng/ml NGF for 1 h. Nuclear extracts were prepared from the cells using a nuclear extract kit (Marlingen Biosciences Inc., Ijamsville, MD), and NF-κB activity was measured in the nuclear extracts using a NF-κB transcription factor microplate assay (Marlingen Biosciences Inc. Ijamsville, MD) according to the manufacturer's protocol.</p><!><p>HT-22 cells were transfected with TRAF6 and p62 or TRAF6ΔR and ASp62, which was followed by serum-deprivation and addition of 10 μM of amyloid β (1–40) with or without 50 ng/ml NGF for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide (MTT) assay. MTT is a tetrazolium salt that is reduced by metabolically viable cells to a colored, formazan salt. The MTT assay was performed with the CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI) according to the manufacturer's protocol. The results are represented as percentage of surviving neurons relative to control values (untreated serum-deprived cells, 100%).</p><!><p>We previously showed that NGF induces p75NTR polyubiquitination and leads to neuronal cell survival (Geetha et al., 2012). Aβ is known to bind p75NTR and induce cell death (Coulson, 2006; Yaar et al., 2007; Rabizadeh et al., 1994); therefore, we sought to investigate whether Aβ would block the polyubiquitination of p75NTR. HT-22 cells were treated either with 100 ng/ml NGF alone for 10 min or with the addition of 10 or 20 μM amyloid β (1–40) for 24 h at 37 °C or both. The p75NTR polyubiquitination was determined by immunoprecipitation with p75NTR antibody and Western blotting with anti-ubiquitin and anti-p75NTR. NGF treatment resulted in a strong signal indicating p75NTR polyubiquitination. Aβ treatment diminished p75NTR polyubiquitination in the absence or presence of NGF (Fig. 1). Interestingly, the p75NTR polyubiquitination induced by NGF was prevented by Aβ.</p><!><p>NGF stimulation promotes the interaction of p75NTR with TRAF6 and may lead to p75NTR ubiquitination (Geetha et al., 2012). TRAF6 in turn binds to p62 through its TRAF6 interacting domain (Sanz et al., 2000; Wooten et al., 2001). Since Aβ blocked the p75NTR polyubiquitination, we hypothesized that the binding of Aβ to p75NTR would also impair the interaction of p75NTR with TRAF6 and p62. HT-22 cells were treated with 10 or 20 μM amyloid β (1–40) for 24 h with or without NGF for 10 min. The cells were lysed in Triton lysis buffer and immunoprecipitated with p75NTR and Western blotted with p75NTR, TRAF6 and p62 antibodies. NGF stimulation resulted in a substantial increase in the interaction of both TRAF6 and p62 with p75NTR (Fig. 2A), which is expected when p75NTR is polyubiquitinated. However, the interaction of p75NTR with TRAF6 and p62 was abrogated when HT-22 cells were treated with amyloid β (1-40) alone or with NGF (Fig. 2A). These results corroborate the impaired p75NTR ubiquitination caused by Aβ shown in Fig. 1. Reciprocal immunoprecipitation with anti-TRAF6 or anti-p62 was performed and similar results were obtained as shown in Fig. 2B and C. The cell lysates were also Western blotted for the presence of p75NTR, TRAF6 and p62 (Fig. 2D).</p><!><p>TRAF6 is an E3 RING ubiquitin ligase that mediates the transfer of ubiquitin (Joazeiro and Weissman 2000) and directs synthesis of noncanonical K63-linked polyubiquitin chains (Deng et al., 2000). TRAF6 requires p62 for both oligomerization and polyubiquitination, and thereby enhances the E3 ubiquitin ligase activity of TRAF6 (Wooten et al., 2005). To verify whether TRAF6 and p62 are essential for p75NTR polyubiquitination upon NGF stimulation, HT-22 cells were transfected with a RING finger deletion mutant of TRAF6 (Flag-TRAF6ΔR) or antisense p62 (ASp62), which depletes the endogenous p62. Analysis of anti-p75NTR immunoprecipitates revealed that TRAF6ΔR and ASp62 blocked the NGF-induced polyubiquitination of p75NTR (Fig. 3A). The lysates were also Western blotted with p62 and Oct-A antibody to check the protein expression of the plasmids. These findings revealed that p75NTR polyubiquitination is TRAF6/p62 dependent.</p><p>Additional evidence that TRAF6 and p62 were essential for p75NTR polyubiquitination was obtained by overexpression of TRAF6 and p62 to restore the p75NTR polyubiquitination upon Aβ/NGF treatment (Fig. 3B). HT-22 cells were treated with 20 μM amyloid β (1–40) for 24 h followed by stimulation with 100 ng/ml NGF for 10 min. In lane 5, the cells were transfected with Flag-tagged WT-TRAF6 and WT-p62 and treated with Aβ/NGF. In lane 6, cells were transfected with Flag-tagged TRAF6ΔR and ASp62 and treated with Aβ/NGF. Cells were lysed in SDS lysis buffer and immunoprecipitated (IP) with p75NTR and Western blotted with ubiquitin or p75NTR antibody. NGF stimulation led to p75NTR polyubiquitination, but this was impaired by Aβ pretreatment as shown in Fig. 3B (lane 2 vs lane 4). Transfection of the cells with WT TRAF6 and p62 restored the p75NTR polyubiquitination otherwise inhibited by Aβ (Fig. 3B, lane 5), but transfection of the cells with the TRAF6 mutant delta RING finger and ASp62 did not (Fig. 3B, lane 6). Thus, while Aβ alone prevents polyubiquitination of p75NTR, the overexpression of TRAF6 and p62 can remediate the problem.</p><!><p>Aβ has been found to induce neuronal cell death through p75NTR (Sotthibundhu et al., 2008). Upon stimulation with NGF, TRAF6 binds to p75NTR and leads to NF-κB activation (Khursigara et al., 1999). ASp62 increased p75-mediated cell death and decreased NGF-induced NF-κB activation (Wooten et al., 2001). To determine how TRAF6/p62 contributes to the activity of NF-κB, HT-22 cells were transfected with WT-TRAF6 and p62 or with TRAF6ΔR and ASp62 and then treated with Aβ in presence or absence of NGF. Fig. 4A shows that Aβ treatment in presence or absence of NGF reduced NF-κB activity. Conversely, transfection with WT-TRAF6 and p62 followed by Aβ/NGF treatment enhanced the activity of NF-κB. However, TRAF6ΔR and ASp62 did not support NF-κB activity (Fig. 4A). These results indicate that the interaction of TRAF6 and p62 with p75NTR is essential for the activity of NF-κB.</p><p>Two IκB kinases (IKK) have been identified (IKKα and IKKβ) to phosphorylate IκBα (Mercurio et al., 1997; Karin, 1999; DiDonato et al., 1997; Regnier et al., 1997). NGF leads to the activation of both IKKα and IKKβ (Foehr et al., 2000). It has been previously shown that ASp62 impairs NGF-stimulated activation of IKKβ (Wooten et al., 2001). To examine if TRAF6/p62 would enhance the interaction of p75NTR with IKKβ upon Aβ/NGF treatment, HT-22 cells were treated with 10 μM Aβ with or without NGF. Cells were transfected with Flag-tagged WT-TRAF6 and p62 (Fig. 4B, lane 5) or with Flag-TRAF6ΔR and ASp62 (Fig. 4B, lane 6) and treated with Aβ/NGF. The cells were lysed and p75NTR was immunoprecipitated and Western blotted with anti-IKKβ or anti-p75NTR. NGF stimulation led to the interaction of IKKβ with p75NTR (Fig. 4B, lane 2), whereas the Aβ treatment with or without NGF blocked that interaction (Fig. 4, lane 3 and 4). Overexpression of WT-TRAF6 and p62 restored the recruitment of IKKβ with p75NTR that was otherwise impaired by Aβ (Fig. 4B, lane 5), but overexpression of TRAF6ΔR and ASp62 did not (Fig. 4B, lane 6). These results demonstrate that interaction of IKKβ with p75NTR upon NGF stimulation requires TRAF6 and p62.</p><p>NGF binding to p75NTR increases the neuronal survival (Geetha et al., 2012) and Aβ increases the neuronal death (Sotthibundhu et al., 2008). As TRAF6 and p62 are essential for NF-κB activation, we sought to determine whether TRAF6 and p62 promotes neuronal cell survival using the MTT assay. HT-22 neuronal cells are grown normally in DMEM containing 10% FBS, however deprivation of serum for 24 h induces cell death. NGF treatment protected HT-22 cells from death on serum deprivation, whereas Aβ significantly increased cell death (Fig. 4C). Overexpression of WT-TRAF6 and p62 followed by Aβ/NGF treatment protected serum-starved HT-22 cells significantly from cell death, whereas TRAF6ΔR and ASp62 were not neuroprotective (Fig. 4C). Taken together, these findings underscore the requirement for TRAF6 and p62 for p75NTR-mediated NF-κB activation and survival signaling (Fig. 4).</p><p>In conclusion, the findings reported here reveal that Aβ impairs (1) p75NTR polyubiquitination, (2) the interaction of p75NTR with TRAF6 and p62, (3) the NF-κB activity and neuronal survival otherwise induced by NGF. TRAF6 and p62 are essential for p75NTR polyubiquitination upon NGF stimulation. Overexpression of TRAF6/p62 restored the p75NTR polyubiquitination upon Aβ/NGF treatment. p75NTR activates NF-κB by recruiting TRAF6/p62 to mediate cell survival (Mamidipudi et al., 2002). Aβ reduced the NF-κB activity by blocking the interaction of p75NTR and IKKβ, but overexpression of WT-TRAF6 and p62 followed by Aβ/NGF treatment increased the NF-κB activity and neuronal survival. These findings suggest that TRAF6/p62 possesses the unique ability to reverse Aβ mediated inhibition of p75NTR polyubiquitination, NF-κB activity and neuronal survival.</p>

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Gold Redox Catalysis through Base Initiated Diazonium Decomposition toward Alkene, Alkyne and Allene Activation

The discovery of photo-assisted diazonium activation toward gold(I) oxidation greatly extended the scope of gold redox catalysis by avoiding the use of a strong oxidant. Some practical issues that limit the application of this new type of chemistry are the relative low efficiency (long reaction time and low conversion) and the strict reaction condition control that is necessary (degassing and inert reaction environment). Herein, an alternative photo-free condition has been developed through Lewis base induced diazonium activation. With this method, an unreactive Au(I) catalyst was used in combination with Na2CO3 and diazonium salts to produce a Au(III) intermediate. The efficient activation of various substrates, including alkyne, alkene and allene was achieved, followed by rapid Au(III) reductive elimination, which yielded the C-C coupling products with good to excellent yields. Relative to the previously reported photo-activation method, our approach offered greater efficiency and versatility through faster reaction rates and broader reaction scope. Challenging substrates such as electron rich/neutral allenes, which could not be activated under the photo-initiation conditions (<5% yield), could be activated to subsequently yield the desired coupling products in good to excellent yield.

gold_redox_catalysis_through_base_initiated_diazonium_decomposition_toward_alkene,_alkyne_and_allene

Introduction<!>Alkene activation<!>Allene activation<!>Alkyne activation<!>Critical Oxidation and Assessment of Ar-Au(III) carbophilicity<!>Conclusion<!>General Methods and Materials<!>General procedure of conditions for Au(I) catalyzed Oxy-/ Amino-arylation of alkenes or alkyne towards products 5, 6 and 8 or products 12 respectively<!>General procedure of conditions for Au(I) catalyzed Oxy-/ Amino-arylation of allenes towards product 10

<p>Gold redox catalysis has received far less attention compared to the well-established regime of carbophilic Au(I) Lewis acid catalysis. 1 This can be attributed to the high oxidation potential between Au(I) and Au(III),2 which creates a reliance on strong oxidants such as selectfluor or hypervalent iodine.3 Due to this dependence and the current effectiveness of other late transition metals, the development of Au-redox catalysis has, over the past decade, been greatly hampered. The dependence on strong oxidants was recently broken through photochemical activation of diazonium salts, pioneered by Glorius,4 Toste,5 Hashimi6 and others7 (Scheme 1A). In their studies, it has been clearly demonstrated that diazonium salts could serve as affective oxidants to promote gold oxidation under photo-activation conditions (with or without photo-sensitizer). 8 The utility of this work could be further demonstrated through low reaction temperature and the absence of nucleophilic transmetallating reagents, which have been necessary in previous reports utilizing strong sacrificial oxidants. Furthermore, these groups have demonstrated the power of harnessing the strong carbophilic nature of Au in conjunction with this new redox-model. These seminal reports have therefore established a solid foundation for the development of gold redox catalysis under milder conditions.</p><p>It has been generally established that photo activation promotes diazonium decomposition to generate an aryl radical.9 With regard to the oxidation of Au(I) under these conditions, mechanistic insight is still lacking, though it has been suggested that an aryl radical serves to initiate the Au(I) oxidation, eventually leading to the formation of Ar-Au(III) intermediates10 as shown in Scheme 1A. An alternative pathway could be a 2e-oxidative addition though this seems to be less likely in the case of photo-activation.</p><p>The Ar-Au(III) intermediate formed may then promote C-C multiple bond activation for some less reactive substrates such as internal alkynes and alkenes. Based on this paradigm, interesting transformations have been reported by combining alkene/alkyne activation, nucleophilic addition and Au(III) reductive elimination, leading to dual functionalization of unsaturated C-C synthons (Scheme 1B).11,12</p><p>Other than photo-initiation, the productive decomposition of diazonium salts has been accomplished using simple thermal or mildly basic conditions.13 In the case of base activation, it has been proposed in several reports that a nucleophilic activator, such as water, plays a critical role to facilitate the aryl-radical formation, via the formation of intermediate A followed by rapid loss of N2. A simple example of this is demonstrated in the case of diazene formation as seen in Scheme 1C, where the formation of intermediate A and B have been proposed. Radical intermediate B can undergo C-N homolytic bond cleavage that leads to nitrogen extrusion and the final aryl radical. 14 We currently believe that the interplay between the nucleophile and diazonium salt is critical in the base-mediated Au-redox system, as we have previously disclosed a Lewis-base assisted diazonium activation condition to achieve several cross coupling reactions.15 More specifically, C-C, C-Br, C-S and C-P bonds could be efficiently made through intermolecular coupling. 16 Notably, photo-activation conditions were less affective in that case, which validates the versatility and generality of our simple base-activation protocol. Herein, we establish that this activation mode is amenable to a host of synthetically valuable reactions and demonstrates a more robust (no solvent degassing, which is crucial for high performance using photoactivation) and more efficient (faster reactions in general) protocol relative to photo-activation conditions (Scheme 2). Furthermore, the conditions used in this report permit the activation of allene, which could not be tolerated under photo activation conditions. These factors further demonstrate the advantages of a chemical activation approach over photo activation in promoting gold redox catalysis.</p><p>While our interests lye in applying this chemical activation strategy towards new reaction development using Au-redox catalysis, we also want to assess its performance relative to the previously reported photo-activation strategy. To address each of those goals, we've chosen to investigate a broad range of substrates, which includes alkyne, allene, and alkene, as these can be activated in the presence of an electrophilic Au(I) or Au(III) catalyst. Throughout each functional group investigation, we have also gained insight towards the chemo and regioselectivity of the Au-catalysts generated under chemical activation of diazonium salts. We've also evaluated the functional group tolerance and the overall efficiency of each reaction under base activation. Upon optimal condition determination, we provide a head to head comparison of our studies relative to photo-activation conditions. Notably, all substrates give good to excellent yields under the chemical activation strategy, while significantly lower yields (sometimes <5%) were obtained using alternative photo-initiation conditions. These results highlighted the mild conditions and high efficiency of this activation strategy towards gold redox catalysis.</p><!><p>To initially evaluate the reaction scope of the proposed diazonium chemical activation method in promoting gold redox catalysis, we first focused on alkene substrates. Despite the practical advantages discussed above, one potential challenge of this activation strategy is competitive reaction pathways. As shown in Scheme 1C, it has been reported in literature that alkene alcohol 2 could react with diazonium 1 under mild chemical activation conditions to give product 3 through aryl radical addition to the alkene. To investigate potential competitive reaction pathways, 2a and diazonium 1a were reacted under various conditions as shown in Table 1.</p><p>The results suggested that the presence of base (NaHCO3) is crucial for diazonium decomposition, perhaps for the initiation of aryl radical formation. Interestingly, a gold catalyst significantly increased the rate of this process, which was consistent with the proposed mechanism involving aryl radical oxidation of gold(I). Subsequently, 5a was formed in good yield (78%) while using the chemical activation conditions. For this substrate, similar results were obtained when implementing a previously reported photo activation approach, though careful solvent degassing was absolutely critical.</p><p>To evaluate the interplay of catalyst and reagents for both the chemical and photo-activation conditions, several conditions deviating from the optimal ones were assessed. The results are summarized in Table 2. The desired product 5a was not observed without gold (entry 1), which confirmed the crucial role of the gold catalyst for this transformation. Under photo-activation conditions, solvent degassing was very important as demonstrated in previous reports (with or without photo-sensitizer). Significantly lower yields were observed without rigorous degassing (entries 2–3, yields in parenthesis). Notably, methanol solvent was important for the photoactivation approach. When switching solvent to acetonitrile, much lower yields were obtained when the optimal catalysts were present (entry 4).</p><p>When performing these reactions without Au-catalysts or activator (photo- or chemical-), low conversion was observed without the formation of 5a (Entry 5). However, when utilizing NaHCO3 as a base activator, complete conversion and good yield could be obtained (Entry 6). Under optimal chemical activation conditions, gold decomposition was evident over time. Aryl phosphonium salt was detected as the major product through 31P NMR monitoring (see SI), which supports the oxidation of Au(I) to Au(III) and subsequent reductive elimination for the P-C bond formation.17 These results confirmed that the synthetically appealing alkene dual functionalization could be achieved by simply using gold catalyst and base. To examine the generality of this reaction, we evaluated both oxy- and amino-arylation of alkene with this chemical activation strategy. The results are summarized in Table 3.</p><p>As expected, the chemical activation method worked well for both oxy- and amino-arylation reactions with much better performance than the photoactivation method. In all cases, the exo-trig cyclization was preferred over the endo-trig cyclization. Additionally, the 6- and 7-membered ring structures were successfully accessed in good yields with excellent regioselectivity via exclusive exo-cyclization (5b, 5c). This heightened regioselectivity was refreshing as traditional Au(I) catalysts have yielded regioisomer mixtures for similar substrates.18 Carboxylic acids were also suitable nucleophiles for this transformation, although products were obtained in slightly reduced yields (5h–5j, 5l). Some sugar derivatives (5k, 5l) were also prepared using this approach, which further extends the potential of this method towards the construction of chemically and biologically important frameworks.</p><p>The optimized chemical activation conditions could also be extended to the intramolecular aminoarylation as various pyrrolidine products were obtained in excellent yields (>90%, 6a-6d). The Cbz protected amine was not an effective nucleophile (6f), which suggests that the nucleophilicity of the amine is critical in this approach. However, it is not clear at this point whether this factor is most influential in the oxidation of Au(I) or the cyclization. Impressively, the reaction could also be applied to piperidine synthesis through a 6-exo cyclization with good yields and excellent regioselectivity being observed (6o, 6p). Extending this approach to the 7-exo cyclization was unsuccessful, likely due to lower reactivity of the alkene during the cyclization step (<10% yield). Overall, the results from alkene oxy- and amino-arylation demonstrated the high efficiency of the chemical activation strategy in promoting alkene dual-functionalization through gold redox catalysis.</p><p>Encouraged by the results obtained for alkene activation, we applied this chemical activation method in the arylative ring expansion reaction, which is another literature reported example of photo-initiated gold redox catalysis through alkene activation. The direct comparison of photo and chemical activation conditions is shown in Table 4.</p><p>Interestingly, the ring expansion of alkene 7a could not be achieved under laborious photo-activation conditions without the assistance of photocatalyst. With the addition of 2.5% Ru(bpy)3(PF6)2, ketone 8a was formed in good yield (76%). However, a significant reduction of yield was obtained if strict degassing and argon protection was not followed during the reaction (35%). In contrast, through chemical activation, much better yields (85%) were obtained with untreated solvent, which greatly highlighted the practical advantages of the chemical activation method.</p><p>Despite the competing radical addition side reaction leading to the formation of diazane 3a, this diazonium chemical activation approach was demonstrated to be an effective protocol in applying gold redox catalysis toward alkene activation. More importantly, these conditions were superior to the photo-activation conditions, as simple base activation was significantly more robust (no strict solvent degassing or argon protection) and efficient (faster reaction and better yields). Upon obtaining these results, we turned our focus on extending this chemical activation strategy toward other challenging substrates, such as allene and alkyne.</p><!><p>According to literature, allene is an under-represented precursor within mild Au-redox dual-functionalization strategies. To the best of our knowledge, the only reported example involving allene precursors is the cyclization of an allene ester using the combination of photocatalyst and gold catalyst reported by Shin and coworkers (Figure 1A). In that report, conjugated allene-esters were necessary for successful cyclization and coupling. Notably, the product formed through this transformation is an electron deficient alkene, which is relatively inert towards gold catalyst activation. Given the success of our chemical activation strategy, we were interested to determine if chemoselectivity would be an issue if electron rich-alkene products were formed.</p><p>First, to assess the feasibility of our approach with a simpler allene substrate, compounds 9a/9b were prepared and submitted to typical photo-activation conditions (2.5% Ru(pby)3(PF6)2). In this case, a very messy reaction was observed with 100% conversions of allene 9.</p><p>No desired product 10 was formed at all (Figure 1B). This result demonstrated the clear challenge in conducting gold redox catalysis with allene-containing substrates under photo-activation conditions. Notably, while photo-activation gave absolutely no product 10, the base promoted diazonium decomposition showed significantly better reactivity and good substrate scope (Figure 1C).</p><p>Interestingly, in contrast to alkene substrates where 5-exo oxy- or aminoarylation were dominant, 6-endo cyclization was primarily obtained in the case of allene substrates. This was expected based upon the typical Markovnikov selectivity observed in similar examples.19 For un-substituted allene (10h), 5-exo selectivity was obtained with slighted lower yield, likely due to the undesired side reaction of the alkene product. With intramolecular nitrogen nucleophiles, various aryl diazonium salts were investigated. Electron-deficient substituents generally gave better reactivity as reported in literature. Both aryl iodide (10c) and lactone (10f) were suitable, further highlighting the mild conditions of this method. 20 No further alkene functionalization was observed under this condition, which highlights the excellent chemoselectivity of this newly developed chemical activation protocol using PPh3AuCl and inorganic base.</p><p>Notably, due to the difficulty in allene synthesis, this transformation serves as a conceptual demonstration. We are currently working on reaction cascade development to generate an allene in-situ, followed by gold redox catalysis to produce complex organic frameworks from simple starting materials. However, as the first successful example of gold-redox catalysis using simple allene substrates in the absence of strong oxidants, we believe future reaction discovery is soon to follow.</p><!><p>In the case of alkyne substrates, 21 there were several immediate concerns. Based on our previous report, alkyne diazonium coupling could be achieved using a Au(I) catalyst under mildly basic conditions. Therefore, the use of terminal alkynes as substrates is likely incompatible under all Au-redox conditions. Furthermore, we had considered two separate modes of alkyne activation: 1) gold(I) activation of alkyne followed by vinyl-gold(I) oxidation or 2) gold(III) serves as the carbophilic Lewis acid catalyst for alkyne activation followed by reductive elimination. One recent successful example of gold redox chemistry was the alkyne hydroxyarylation recently reported by Hashmi and coworkers.2b In that example, an unreactive LAuCl gold(I) catalyst was used. Upon photo induced diazonium activation, Ar-Au(III) was formed and thus served as the active catalyst for alkyne activation. Again, strict solvent degassing and argon protection are required. Nevertheless, this pioneering work confirmed that Ar-Au(III) can be the active species toward alkyne activation.</p><p>Given the competing terminal alkyne diazonium coupling described above, we were interested in assessing whether the propargyl-acetate rearrangement could efficiently occur under either chemical or photoactivation conditions. Within this work by Zhang and coworkers, the mechanism was proposed to occur via Au(I) activation of an alkyne, rearrangement, vinyl gold(I) oxidation and reductive elimination from a transient Au(III)- species. 22 In this case, the strong oxidant selectfluor was required to promote gold oxidation. Notably, through the enlistment of cationic Au(I) catalysts, alkyne hydration was also difficult to avoid. Thus, we were interested in determining whether Ar-Au(III) can be applied to promote the propargyl ester activation followed by the productive reductive elimination. To test this hypothesis, both photo-activation and chemical activation conditions were applied for the reaction of substrate 11a.</p><p>As shown in Figure 2, under the photoactivation conditions (with or without photocatalyst), very messy reactions were observed. The hydrolysis product was obtained under these conditions, suggesting the formation of a cationic-Au species throughout the reaction. In contrast, cleaner transformations were observed under chemical activation conditions, giving the desired product 12a in good yield (78%). Notably, under this gold-redox condition, homo-dimerization byproducts were not observed, which rules out the formation of vinyl-Au(I) species as described in a Zhang's report. Some representative substrates were prepared to evaluate the reaction scope (Table 5).</p><p>Both aromatic and aliphatic substituted alkynes (R2 position) are suitable for this transformation under the chemical activation conditions. At the R1 position, aryl and alkyl groups were both compatible, highlighting the broad substrate scope of this method. The use of diazonium electrophiles instead of sacrificial oxidants and transmetallating species (such as boronic ester) permits greater substrate scope and higher efficiency in the aryl coupling partner. This result further establishes the significantly improved reactivity of this chemical activation condition over the alternative photo activation conditions.</p><!><p>Throughout each substrate we've investigated, the Au(I) resting state shows almost no ability to competitively activate alkene, alkyne or allene. This is also evidenced through the fact that no homo-dimerization products could be observed in any example, likely ruling out vinyl/alkyl-Au(I) intermediates. To substantiate this claim, we synthesized an Ar-Au(III) species to submit as a stoichiometric reagent in the model reaction described in Figure 3A.</p><p>The coordinately-saturated Au(III) complex E was generated by reacting a modified diazonium (ArN2Cl) with PPh3AuCl, as described in a previous literature procedure.10d A stoichiometric amount of complex E was reacted with alkene 2a (without diazonium) under modified conditions (see SI), giving the desired product 5a in quantitative yield. This indicates a retained π-acidity of the Ar-Au(III) intermediate and highlights an interesting preference for the tandem cyclization/coupling over the P-Ar reductive elimination demonstrated in previous reports.17 Reaction rate studies were then performed to analyze the effect of various catalyst permutations on the outcome of the reaction between 1a and diazonium 2a (Figure 3B). A difference in reaction rate was observed for different primary ligands with the order of reactivity being: (p-Me-C6H4)3PAuCl>PPh3AuCl>(p-CF3-C6H4)3PAuCl. This clearly suggests a critical role of the phosphine ligand in the oxidation step, which is likely the rate-determining step in this reaction. Notably, the stable Ar-Au(III) complex E could also catalyze the reaction with a similar reaction rate as the optimal catalyst PPh3AuCl. Interestingly, PPh3AuNTf2 offered a much slower reaction rate relative to PPh3AuCl, which may indicate that the Cl anion is also critical in the oxidation step. Furthermore, using DMSAuCl as catalyst gave the same cyclization product 3a albeit with lower yield (43% yield, 81% 1a conversion), likely caused by fast gold decomposition. Comprehensive mechanistic investigations are currently ongoing within our group.</p><!><p>Our group has revealed a Lewis-base assisted diazonium activation as an alternative strategy to achieve gold oxidation. Previous reports have required either strong oxidants or photocatalysts for Au-oxidation or substrate activation respectively. In our system, through oxidation of Au(I) using diazonium and simple inorganic bases, a more reactive Au(III) intermediate is formed in situ. This allows the productive activation of less reactive substrates such as alkenes, internal alkynes and electronically unbiased allenes. The efficiency of Au(III) reductive elimination affectively allowed us to circumvent the unwanted protodeauration en route to a number of synthetically useful products. Furthermore, high chemoselectivity could be observed when alkene products were formed in the case of allene and alkyne substrates. The absence of other homo-coupling products also allows us to exclude the formation of nucleophilic vinyl/alkyl-Au(I) species. Through tracking reaction rate profiles for a model reaction using different catalysts, we can deduce that the Au-oxidation is likely the rate-determining step. Future mechanistic investigations are currently underway and will be disclosed in due course. The extension of this methodology within more complex molecule design is also currently being pursued within our group.</p><!><p>All of the reactions dealing with air and/or moisture-sensitive reactions were carried out under an atmosphere of nitrogen using oven/flame-dried glassware. Unless otherwise noted, all commercial reagents and solvents were obtained from the commercial providers (Aldrich, Fisher and Oakwood companies), and used without further purification.</p><!><p>In a dried glass tube, alkenes or alkynes (0.2 mmol, 1 equiv), PPh3AuCl (0.014 mmol, 7 mol%), aryl diazonium (0.4 mmol, 2 equiv) and NaHCO3 (0.4 mmol, 2 equiv) were dissolved in CH3CN (0.6 mL). The reaction mixture was stirred at 60 °C for 1–3 h. After the reaction completed, the reaction mixture was directly submitted to flash column chromatography for purification and yield determination (5–15:1 Hexanes: Ethyl Acetate as eluent). See SI for specific conditions.</p><!><p>In a dried glass tube, allenes (0.2 mmol, 1 equiv), PPh3AuCl (0.014 mmol, 7 mol%), aryl diazonium (0.4 mmol, 2 equiv) and NaHCO3 (0.4 mmol, 2 equiv) were dissolved in CH3CN (0.6 mL). The reaction mixture was stirred at 50 °C for 1–3 h. After the reaction completed, the reaction mixture was directly submitted to flash column chromatography for purification and yield determination (5–8:1 Hexanes: Ethyl Acetate as eluent).</p>

PubMed Author Manuscript

Polaron-Adsorbate Coupling at the TiO2(110)-Carboxylate Interface

Understanding how adsorbates influence polaron behavior is of fundamental importance in describing the catalytic properties of TiO2. Carboxylic acids adsorb readily at TiO2 surfaces, yet their influence on polaronic states is unknown. Using UV photoemission spectroscopy (UPS), two-photon photoemission spectroscopy (2PPE), and density functional theory (DFT) we show that dissociative adsorption of formic and acetic acids has profound, yet different, effects on the surface density, crystal field, and photoexcitation of polarons in rutile TiO2(110). We also show that these variations are governed by the contrasting electrostatic properties of the acids, which impacts the extent of polaron–adsorbate coupling. The density of polarons in the surface region increases more in formate-terminated TiO2(110) relative to acetate. Consequently, increased coupling gives rise to new photoexcitation channels via states 3.83 eV above the Fermi level. The onset of this process is 3.45 eV, likely adding to the catalytic photoyield.

polaron-adsorbate_coupling_at_the_tio2(110)-carboxylate_interface

<!>Author Present Address<!>

<p>TiO2 is a versatile, low-cost material for a wide range of light-driven applications such as photovoltaics,1 water splitting,2 and organic photodegradation.3−8 It is well known that defects and their associated polarons have a large influence on the activity of these functions, behaving as charge transfer sites and electron traps.9−11</p><p>Carboxylic acids are ubiquitous at photocatalytic titania surfaces due to their high affinity for bonding to surface Ti atoms.12 Formic (HCOOH) and acetic (CH3COOH) acid represent the simplest carboxylic acid analogues. Their adsorption on TiO2 results in the formation of atomic-scale ordered overlayers at the ultrahigh vacuum (UHV), liquid and atmospheric interface, which can be observed by scanning tunneling microscopy.12−16 At the rutile TiO2(110) surface specifically, the dominant adsorption configuration of these acids consists of bidentate-bound carboxylates (RCOO–) at five-coordinate titanium atoms (Ti5c) along the [001] direction.17 This is accompanied by the protonation of bridging O (OHb) and the formation of a (2 × 1) majority phase adsorption structure (see Figure 1(a)). A minority carboxylate component is also present, which is a monodentate species oriented perpendicular to [001] and accounts for up to 1/3 of the interface.14,18−20 Formic and acetic acid adsorption saturates at ∼0.5 ML in UHV at 298 K, where a monolayer corresponds to the number of surface unit cells. The two terminations are denoted FA- and AA-R110, respectively.</p><p>(a) Rutile TiO2(110) model showing the majority phase (2 × 1) formate and OHb overlayer resulting from dissociative chemisorption of formic acid. An interstitial titanium atom (Tiint) is shown at position L1. Blue, red, brown, and white spheres represent Ti, O, C, and H, respectively. (b) Comparison of the dominant t2g → t2g transition in the 2PPE spectra of the Hp-R110, FA-R110, and AA-R110 terminations at a photon energy of 3.54 eV (p-[001], 350 nm). Incoherent (i) features are produced according to the equation E – EF = hνprobe + Eintermediate. Spectra were produced continuously at constant laser power with in situ gas-phase dosing. Peaks are isolated via the method described in SI Section S2. (c) Comparison of the BGS region in the UPS (He–I, 21.2 eV) spectra on the Hp-R110, FA-R110, and AA-R110 surfaces. Peaks are isolated via the method described in SI Section S2. (d) Bar chart showing the difference in energy (ΔE) between a surface (L1) and bulk (L3) Tiint in the clean, formate, and acetate termination of rutile TiO2(110), calculated with HSE06 DFT. A positive ΔE means that L1 is energetically less stable than L3. See details in Table S1.</p><p>Defects in rutile TiO2, namely surface oxygen vacancies (Ovac) and bulk interstitial titanium atoms (Tiint), give rise to excess electrons in localized polaronic states.21 The energy levels of the electron polarons represent what are commonly referred to as the band gap states (BGS) of reduced TiO2, which are detectable at ∼1.0 eV binding energy (BE) in UV photoelectron spectroscopy (UPS).22,23 Formally, the BGS are Ti 3dxy in character. This results from the Jahn–Teller splitting of Ti3d atomic states in the pseudo-octahedral crystal field of rutile, which gives rise to orbitals of t2g- and eg-like symmetry.10,24,25 Polarons at surface Ovac readily react with water to form OHb,9 resulting in a small increase in the UPS BGS signal without altering the BE.26 This indicates that OHb triggers deeper lying polarons to redistribute toward the surface, a mechanism that is also supported by density functional theory (DFT) calculations.26,27</p><p>Although electron polaronic states have been studied extensively, it is only recently that pump–probe studies have allowed access to their excited states. One technique employed is two-photon photoemission spectroscopy (2PPE).28−30 At reduced and hydroxylated TiO2(110) surfaces, 2PPE spectra are dominated by a t2g → t2g excitation feature where the excited state lies ∼2.6–2.8 eV above EF.28,30−32 The oscillator strength of this excitation is strongly dependent on the orientation of the electric field vector. This increases when the scattering plane is perpendicular to the [001] crystal azimuth, (p-[001]).28−30,32 In contrast, a weaker feature is observed when the scattering plane is parallel to the [001] azimuth, (s-[001]).29,30 Furthermore, it has been shown that water and methanol adsorption influences this channel, altering the orbital character and resulting in an enhancement of the t2g → t2g excitation oscillator strength.27,33−35 Despite these recent advances, the impact of carboxylates on electron polaronic states remains unknown. This understanding is potentially valuable for several technologies since carboxylates serve as the most important anchoring group for the functionalization of TiO2 surfaces. In this Letter, we describe a UPS, 2PPE, and DFT study that investigates the modification of electron polarons by carboxylates and their subsequent photoexcitation.</p><p>Features in 2PPE spectra are most commonly produced as a result of coherent (simultaneous two-photon excitation of an occupied state) or incoherent (two sequential one-photon excitations via an intermediate state) processes.36 At resonant photon energy conditions, optimal coherence between an initial and intermediate state energy results in an increase in the 2PPE intensity.36 In reduced and hydroxylated TiO2(110) (see SI for preparation methods), the resonant photon energy for the t2g → t2g excitation is known to be ∼3.54 eV (350 nm).28 In Figure 1(b), this resonance was monitored (p-[001], 3.54 eV, 350 nm) as a reduced rutile TiO2(110) sample (R-R110) partially hydroxylated in UHV (Hp-R110, ∼0.05 ML) and was sequentially exposed to gas-phase acid. This was performed in situ until the saturation level (∼0.5 ML) was reached. The increase of the 2PPE resonance via hydroxylation of Ovac has been discussed in prior work.27,28,30,31 Upon creation of FA- and AA-R110, we find that the dominant incoherent process is approximately 3× and 2× larger, respectively (taken by peak area). An example of spectral evolution throughout this experiment is also shown in the Supporting Information (SI) Figure S1. In a similar framework, the BGS is monitored via UPS (He–I, 21.2 eV) and increases by a factor of ∼1.4 following formate adsorption. Following acetate adsorption the BGS area is ∼0.9 times the size, consistent with previous measurements.37−39 This is represented in Figure 1(c). In Figures 1(b) and (c) the peaks are isolated by removing backgrounds and are fit with Gaussian distributions (see SI, Figure S2). The difference in trend between the UPS and 2PPE data for AA-R110 is likely due to the escape depth of the two techniques (∼1 and 5 nm, for UPS and 2PPE, respectively).40</p><p>To further understand these observations we carried out DFT calculations (see SI for methods) using the HSE06 hybrid functional,41 which describes polaronic states in TiO2 with good accuracy.30,32 Tiint defects were used as the source of excess electrons, and the location of Tiint was varied from the immediate subsurface (L1) to two (L2) and three (L3) layers below the surface in a (4 × 2) 6-trilayers slab. Previous DFT work showed that the most stable Tiint location changes upon water and methanol adsorption.27 Here, we determine the relative energies of Tiint at clean (C) TiO2(110) and at the surface covered by a (2 × 1) formate or acetate monolayer. The relative stabilities of different Tiint locations change significantly in the presence of a carboxylate monolayer. At the adsorbate-free surface, the most stable Tiint site is L2, which is 0.46 (0.11) eV more stable than L1 (L3). In contrast, at the formate-covered surface Tiint at L2 is only 0.08 eV more stable than at L1 (and 0.18 eV more stable than at L3). In the presence of an acetate monolayer, L2 and L3 are 0.09 and 0.10 eV more stable, respectively, than L1. The energetics of surface Tiint following carboxylate adsorption is summarized in Figure 1(d), which shows the energy (ΔE) difference between surface (L1) and bulk (L3) Tiint locations (see full details in SI, Table S1). Together, the UPS, 2PPE, and DFT results indicate that formate adsorption leads to the redistribution of polarons toward the surface of TiO2(110) through the mechanism of Tiint migration. The data also show that this effect is less pronounced in the acetate termination.</p><p>On FA-R110, polaron photoexcitation was further studied by rotating the electric field vector by 90° relative to the crystal azimuth (s-[001]). Figure 2(a) follows the 2PPE spectrum (3.54 eV, 350 nm) as formic acid is dosed directly onto R-R110, allowing contributions from initially reactive Ovac (∼0.1 ML) sites to be separated.19 At ∼0.1 ML coverage the spectrum largely resembles that of R-R110, evidencing a slight increase in the t2g → t2g feature, labeled feature 1. Following saturation of the Ti5c rows (∼0.5 ML), an additional feature, labeled feature 2, becomes clear. The apparent shift of feature 1 at this coverage is due to its convolution with feature 2. The inset shows the difference spectrum between the ∼0.5 ML coverage and ∼0.1 ML coverage, where the appearance of feature 2 is clear. The dependence of feature 2 on the ∼0.5 ML coverage of formate was additionally confirmed by inducing formate decomposition reactions (see SI, Figure S3).</p><p>(a) 2PPE spectra (hν = 3.54 eV, 350 nm, s-[001]) of a reduced rutile TiO2(110) sample (R-R110) taken continuously as formic acid is dosed in situ. The subscript number in the legend signifies the approximate ML coverage of the formate. Numbers in dashed boxes represent the position and label of the respective features. Feature 1 denotes the previously identified t2g → t2g transition. The inset shows the spectrum with 0.5 ML coverage minus that at 0.1 ML coverage. (b) 2PPE spectra of selected regions containing features 1 and 2, with photon energies (hν = 3.44–3.87 eV, 360–320 nm), s-[001]). Spectra are normalized to fit the figure window for clarity. (c) Plot of the photon energy dependence of the two fitted peaks in FA0.5-R110 (see Section S2 in the SI for the fitting procedure) given by the equation for the incoherent process, E – EF = hνprobe + Eintermediate. (d) Computed PDOS of Ti3+ states on FA-R110, with Tiint located at L1. Peaks in the conduction band are labeled to correspond to features 1 and 2. (e) Computed oscillator strengths for transitions from BGS to the conduction band in the same system considered in (d). Red [001], green [110], and blue [110] represent directions of transition dipole moments. Peaks in the oscillator strengths are labeled that coincide with features 1 and 2. (f) Scheme showing the transitions of features 1 and 2. Feature 2 represents a t2g → eg excitation. A red arrow represents transitions observed in R-, FA-, and AA-R110. A yellow arrow represents transitions observed in FA-R110 only.</p><p>The photon energy dependence of feature 2 was also examined and is shown in Figure 2(b). Feature 2 occurs close to the EF + 2hν maxima of the 2PPE spectra and has an onset hν of ∼3.45 eV (360 nm). 2PPE spectra with hν > 3.54 eV (350 nm) show that feature 2 becomes more prominent in the spectra as feature 1 is less resonant. It is also observed that feature 2 is visible at much higher photon energies compared to feature 1. Figure 2(c) shows the plot of final-state energy (E – EF) versus photon energy (eV). It is well understood that in these plots incoherent and coherent processes produce gradients of 1 and 2, respectively.36 Both features are produced via an incoherent process where the excited state lies ∼2.7 and 3.8 eV above EF for features 1 and 2, respectively (given by the y-intercepts). In both plots, photon energies are chosen so as to minimize overlap of the features.</p><p>DFT is again used to obtain insight into the origin of the observations. Figure 2(d) shows the partial density of states (PDOS) of excess electrons from Tiint at L1 of a TiO2(110) surface with a (2 × 1) formate overlayer. The distribution has been separated into individual d-orbital contributions. The excited state energies of features 1 and 2 are represented clearly by significant density of states in the Ti3+ conduction band having dxy and dz2 orbital character, respectively. Figure 2(e) shows the results of associated oscillator strength calculations for BGS to conduction band excitation. Peaks corresponding to both features are observed. The transition dipole moment for feature 1 lies in both the [001] and [11̅0] direction in this environment. In contrast, a transition dipole moment for feature 2 is present only in the [001] direction, explaining the observed polarization dependence. Features 1 and 2 therefore represent an excitation from occupied states t2g-like in character to unoccupied states of t2g- and eg-like character, respectively. A schematic of this excitation scheme is shown in Figure 2(f). Extended PDOS and oscillator strength calculations (Tiint L1–L3) showing the effects of carboxylates on polaron orbital energies are given in the Figure S4.</p><p>Adsorption of the carboxylates leads to a pronounced reduction in the workfunction (5.1, 4.4, and 4.2 eV for R-, FA-, and AA-R110, respectively), which has implications for the 2PPE spectra. Specifically, this results in an enlarged 2PPE spectral window and an increased scope to study lower energy photoexcitation processes. However, at higher photon energies lower energy 2PPE features are often imperceptible due to dominating coherent valence band contributions, as well as single-photon photoemission from states near EF. Figure 3(a) shows the 2PPE spectra of AA-R110 (p-[001], 3.75–3.35 eV, 330–370 nm). As expected in this orientation, a strong peak associated with feature 1 is present. At ∼5.2 eV above EF a broad feature is present that is unaffected by the shifting photon energy. This feature is also present in the 2PPE spectra (p-[001]) of FA-R110 (see Figure S5). We assign this distribution to Auger electrons, ejected from the BGS via the multiphoton excitation and recombination of valence band electrons. This feature also acts as a normalization point. The Auger feature is discussed in further detail inFigure S6.</p><p>(a) 2PPE spectra (hν = 3.35–3.75 eV, 370–330 nm, p-[001]) measured from AA-R110. Three features corresponding to feature 1, Auger electrons, and valence band coherent 2PPE (c2PPE) from TiO2 are labeled. Spectra are normalized to the Auger feature peak intensity. (b) Peak intensity of feature 1 between (hν = 3.35–3.75 eV, 370–330 nm, p-[001]), normalized to the Auger feature, for both FA-R110 and AA-R110. (c) Spin density contour of BGS for Tiint at the L1 site in the C-R110, FA-R110, and AA-R110 terminations. Arrows showing electron transfer to represent the relative attractive and repulsive properties of acid adsorbates relative to the clean surface. The red, light blue, brown, and pink spheres represent Ti, O, C, and H atoms, respectively. (d) Comparison of FA-R110 and AA-R110 2PPE (3.54 eV photons, 350 nm, s-[001]). The inset shows the difference spectrum between the two terminations. (e) Calculated oscillator strengths from BGS to the conduction band with L1 Tiint in the acetate-terminated system. Red [001], green [110], and blue [110] represent directions of the transition dipole moments.</p><p>Figure 3(b) compares the wavelength-dependent intensity of feature 1 in FA- and AA-R110 (p-[001]). This comparison was made by normalizing to the Auger feature, which is present in both terminations. FA-R110 has an increased intensity of feature 1 relative to AA-R110 in all wavelengths tested. Furthermore, in both terminations 2PPE with hν = 3.54 eV (350 nm) produces the most intense peak, as for the adsorbate-free surface. This demonstrates that there is no distinct adsorbate-induced splitting of the occupied and unoccupied t2g orbitals undergoing excitation.</p><p>There are a number of potential reasons for the differences in spectral intensity for the two adsorbates, with Tiint migration and photoelectron attenuation important factors. However, DFT results suggest an additional important element. Due to the electron-donating effects of the methyl substituent, acetate repels excess electrons from the adsorbate. This is in contrast to formate, which attracts them. This is evidenced in Figure 3(c), where the spin density contour of four distinctly located excess electrons in C-, FA-, and AA-R110 are shown. Further modifications by the adsorbates can also be seen in this model. Specifically, in C-R110 the occupied states contain only orbitals of t2g-like character. However, following adsorption of FA and AA, new orbital characters arise. Focusing on the excess electron localized at Tiint in FA- and AA-R110, a dz2-like orbital character can be identified. This change can be understood as an adsorbate-induced local crystal field. Specifically, the original octahedral crystal field is tilted into a trigonal prismatic field. In this new field, dz2 orbitals are lower in energy than the other 3d orbitals and subsequently appear in the spin density contour (Figure 3(c)) and PDOS (Figure 2(d)) (see also Figure S4). The density of those electrons in a trigonal prismatic field is governed by the electronegativity of the acid. In FA-R110, electrons are attracted away from Tiint, resulting in a higher proportion of surface localized t2g-like states compared to AA-R110. These surface states can undergo additional couplings between t2g and dz2, which result in the appearance of feature 2 in the 2PPE spectra of FA-R110 and its absence in AA-R110. This comparison is shown in Figure 3(d) (s-[001], 3.54 eV, 350 nm, see Figure S7 for further AA-R110 spectra). The absence of feature 2 in the 2PPE spectra of AA-R110 is also corroborated by the results of oscillator strength calculations in Figure 3 (e), where no clear peaks at the position of feature 2 are observed (compare Figures 2(e) and 3(e)). Furthermore, we assign feature 2 to states localized at the surface based on oscillator strength calculations, which show that feature 2 is present only in the formate termination with Tiint located at L1 and L2 (see Figure S4).</p><p>In summary, we have established that the facile formation of formate and acetate overlayers has dramatic, yet differing, implications for the behavior of polaronic states in rutile TiO2(110). Carboxylate adsorption leads to polaron redistribution toward the surface, driven by the migration of Tiint. This occurs more prominently in FA-R110, compared to AA-R110. Adsorbates subsequently couple with polaronic states to form unique crystal fields which alters the orbital character. The extent of this coupling is determined by the electrostatic properties of the carboxylate. For example, at the formate termination, polarons are attracted toward the adsorbate, increasing the oscillator strength of higher energy transitions. Specifically, polarons undergo photoexcitation via an intermediate state ∼3.83 eV above EF, characterized as a t2g → eg transition. It is also observed that the 2PPE spectra of both carboxylate-terminated TiO2(110) contain significant contributions from an Auger feature. Understanding how polarons interact with adsorbates is crucial if we are to describe the role of defects in TiO2 catalysis. This work provides an understanding of how carboxylates may enhance the activity of polarons by increasing their density at the surface, protecting them against oxidation (see Figure S8) and giving access to alternative photoexcitation channels.</p><p>Experimental details, computational details, and Figures S1–S8, showing further DFT calculations and PES spectra with extended discussion (PDF)</p><p>jz1c00678_si_001.pdf</p><!><p>⊥ Y.Z.: Central Laser Facility, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0QX, U.K.</p><p># J.O.: Departamento de Química, Universidad Técnica Particular de Loja, San Cayetano Alto, Loja 1101608, Ecuador.</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Evolving the [Myoglobin, Cytochrome b5] Complex from Dynamic Toward Simple Docking: Charging the Electron-Transfer Reactive Patch

We describe photo-initiated electron transfer (ET) from a suite of Zn-substituted myoglobin (1Mb) variants to cytochrome b5 (b5). An electrostatic interface redesign strategy has led to the introduction of positive charges in the vicinity of the heme edge through D/E \xe2\x86\x92 K charge-reversal mutation combinations at `hotspot\' residues (D44, D60, E85), augmented by the elimination of negative charges from Mb or b5 by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners\' total charges: \xe2\x88\x925 < \xe2\x88\x92qMbqb5 < 40. The binding affinity (Ka) increases a thousand-fold as \xe2\x88\x92qMbqb5 increases through this range, and exhibits a surprisingly simple, exponential dependence on \xe2\x88\x92qMbqb5. This is explained in terms of electrostatic interactions between a `charged reactive patch\' (crp) on each partner\'s surface, defined as a compact region around the heme edge that (i) contains the total protein charge of each variant, and (ii) encompasses a major fraction of the `reactive region\' (Rr) comprising surface atoms with large matrix elements for electron tunneling to the heme. As \xe2\x88\x92qMbqb5 increases, the complex undergoes a transition from fast to slow exchange dynamics on the triplet ET timescale, with a correlated progression in the rate constants for intracomplex (ket) and bimolecular (k2) ET. This progression is analyzed by integrating the crp and Rr descriptions of ET into the textbook steady-state treatment of reversible binding between partners that undergo intracomplex ET, and found to encompass the full range of behaviors predicted by the model. The generality of this approach is demonstrated by applying it to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome c2. Deviations from this model also are discussed.

evolving_the_[myoglobin,_cytochrome_b5]_complex_from_dynamic_toward_simple_docking:_charging_the_ele

Introduction<!>Bacterial Strains and Plasmids<!>Expression and Purification<!>Brownian Dynamics Simulations<!>Flash Photolysis Experiments<!>Electron Transfer Measurements<!>Analysis<!>BD Simulations<!>Generality of the Pre-equilibrium model of Eq 4<!>Summary and Conclusions

<p>Protein-protein binding is at the heart of much of biology, with affinity constants ranging over many orders of magnitude,(1, 2) and many diseases involving the disruption and/or elimination of vital interactions.(3) (4–6) Two limiting energy landscapes (Fig 1) have been used to describe the correlation between protein-protein binding and inter-protein reactivity): i) weakly-bound complexes can exhibit `dynamic docking' (DD), where reactive configurations are rare and geometrically distinct from more-abundant, thermodynamically favored unreactive configurations, and thus binding and reactivity are decoupled;(7–10) ii) tightly-bound complexes can exhibit `simple docking' (SD) where a dominant, strongly-binding configuration is also the most reactive.(7) We are exploring the principles governing reactive protein-protein binding by undertaking a systematic re-design of the interface of the [myoglobin (Mb), cytochrome b5 (b5)] electron transfer (ET) complex, with the aim of taking this weakly-bound complex and strengthening the binding of reactive configurations, so as to evolve the DD landscape on which it functions into an SD landscape and thereby enhance ET.(11–13)</p><p>A reverse process, the identification of `hotspot' residues that provide the most favorable interactions in the protein-protein interface of a tightly-bound, SD complex, is typically carried out through inspection of an x-ray structure, coupled with a traditional alanine replacement strategy.(14, 15) (16, 17) The studies of Okamura and coworkers with the [photosynthetic reaction center (RC), cytochrome c2 (c2)] ET complex provide a compelling example of the use of this crystallographic/mutational strategy to study the coupling between binding affinity and ET. By employing both charged(18, 19) and hydrophobic (20) interface mutants, these workers were able to identify residues essential to tight binding by monitoring mutationally-induced changes in ET, with the elimination of hotspot interactions lowering the binding constant by over three orders of magnitude relative to the wild-type complex. They further developed an empirical function to correlate the corresponding changes in binding affinity and ET reactivity generated by these interface mutations. (20)</p><p>To take a weakly-bound complex that reacts on a DD landscape, and identify potential `hotspot' surface residues where interactions can be most successfully introduced so as to evolve the complex towards a tight-binding complex with SD behavior, presents a challenge of a different order. Weakly-bound protein pairs such as the [Mb, b5] ET complex (K ~103 M−1)(21, 22) do not in general form crystals, precluding the use of X-ray structures to identify sites where modification would stabilize the protein-protein interface and enhance reactivity. Indeed, such complexes do not even have a well-defined `structure'.(23, 24) This has led us to develop an interface redesign strategy that employs Brownian Dynamics (BD) docking simulations to identify residues where charge reversal mutations will strengthen electrostatic interactions(25–27) in the protein-protein interface of ET-active conformations of such transiently-formed, DD-type complexes.(13) Application of this strategy to the [Mb, b5] partners identified residues D44, D60, and E85 of Mb as surface residue `hotspots' where the progressive introduction of D/E → K mutations in the vicinity of the Mb heme edge should create a positively charged `patch'(28) on the Mb surface with enhanced Coulombic attraction to the complementary pattern of negative surface charges on the b5 template, progressively stabilizing reactive configurations of the complex and `evolving' it towards a reactive SD behavior (Fig 1).</p><p>Guided by these predictions, we have now prepared all seven mutants in which positive charges have been added to the Mb surface by D/E → K charge-reversal mutations at one, two or all three of the hotspot positions on Mb, augmented with variants in which negative charge is eliminated from the Mb surface by neutralization of the Mb heme propionates. The binding affinity and ET reactivity for each member of this suite have been measured at two different pH values by examination of ET quenching of the Zn-deuteroporphyrin IX (ZnD) or ZnD-(dimethylester)-reconstituted Mb photo-donors by either the ferri-b5 electron acceptor or its diester heme reconstituted analog.</p><p>Whereas b5(wt) has a large negative total charge, qb5 e = −5.7 e, Mb(wt) is nearly charge-neutral, qMb e = −0.3 e (pH 7; Table SI_1). The charge variation through mutation and/or heme-neutralization yields a set of [Mb, b5] complexes that spans an unprecedentedly large range in the product of the total charges on the partners: −5 < −qMbqb5 < 40 (Table SI_1). As −qMbqb5 increases within the set, the binding constant (Ka) increases by over three orders of magnitude and the complexes shift from the fast-exchange (FE) dynamics regime to the slow-exchange (SE) regime on the triplet-state ET timescale. The second-order ET rate constant (k2) concomitantly increases by ~103; intra-complex protein-protein ET is observed in the SE regime and it too increases with −qMbqb5. The correlated increases in binding and reactivity are found to have remarkably simple dependences on the charge product, −qMbqb5. To describe these consequences of charge buildup, we have been led to extend the idea of a charged patch(28–31) by defining a charged reactive patch (crp) on each protein that satisfies two criteria. (i) It is the most compact area that surrounds a selected surface point/region and in which the surface charge of the protein equals its total charge. (ii) The selected region, here taken to be the heme edge, is chosen so that the crp encompasses a major fraction of the ET-active surface, the `reactive region (Rr)' comprising the surface atoms with large matrix elements for electron tunneling to the heme (Fig 2). Because the hot-spot mutation sites of Mb and the heme propionates of both partners fall within their WT crp, the crp for each of the ET partners remains compact and essentially invariant as its surface charge is changed. With the crp of each partner of every complex containing its total protein charge, while the remaining surface has no net charge, complex formation can be treated in terms of the interaction between the total charges, localized within the complementary, reactive regions on the protein surface — the crp. The correlated progression of k2 and Ka with increasing - qMbqb5, and the transition from fast to slow exchange dynamics, then arise naturally when the crp description of protein-protein binding is integrated into the textbook steady-state treatment of reversible pre-equilibrium binding between partners that exhibit intracomplex ET.(32–34)</p><p>This simple approach completely captures the global aspects of the correlated electrostatic control of binding and reactivity, while at the same time illuminating the `granularity' that results from alternative arrangements of charges on the Mb surface. The generality of this approach is evidenced by its ability to treat the correlated decreases in binding and ET rate constants between the reaction center (RC) and cytochrome c2 (c2), as produced by separate suites of hydrophobic and electrostatic mutations within the interface.</p><!><p>Escherichia coli strains used for plasmid propagation and protein expression were DH5a and BL21(DE3) respectively. Coding sequences for target proteins were cloned under control of the T7lac promoter by placing the initiation codon at the NcoI site of pET28a(+) (Novagen), which confers resistance to kanamycin. The plasmid for expressing horse heart myoglobin (Mb) was obtained from Professor A. Grant Mauk (University of British Columbia, Vancouver). The wild-type gene was then amplified via PCR from the constitutive expression vector pEMBL18 containing the template(35) with the forward (ACTAGCCATGGGTCTGTCTGATGGTGA) and reverse (ACTAGGGATCCTTAACCCTGGAAACCCAGTTC) primers. The PCR product was digested with NcoI and BamHI enzymes, and cloned into the pET28a(+) vector, similarly digested with the same enzymes. Protein mutants were created via site-directed mutagenesis using a Stratagene QuikChange II XL Site-Directed Mutagenesis Kit. The constructs for Mb wild-type and all mutants were verified by DNA sequencing (SeqWright).</p><!><p>Terrific broth medium (0.17mM KH2PO4, 0.72mM K2HPO4, 12g tryptone, 24g yeast extract/L) containing kanamycin (100ug/mL) was inoculated with an overnight culture of BL21(DE3) E. coli transformed with the desired Mb construct. The culture was grown at 37°C, 350rpm until it reached an OD600 ~ 2.4–2.7. Protein expression was induced with IPTG (final concentration of 1mM) and an increase in temperature to 42°C. The resulting protein formed inclusion bodies as described by Ribeiro, et al.(36) High cell densities (OD600 > 6) are desired, so to provide sufficient aeration of the expression culture the volume of culture was limited to < 25% of the flask volume and a high shaking speed (>350rpm) was maintained.</p><p>After 3h, the culture was pelleted by centrifugation at 20,000g for 15 minutes, re-suspended in 20ml/L 100mM Tris-HCl, 5mM EDTA, 1% Triton X-100 (pH 8.0) and lysed by sonication. This process was repeated 3 times with the same buffer, taking care to completely re-suspend the pellet after each centrifugation step. The pellet was then re-suspended in water and sonicated twice, as before, to remove excess Triton X-100. Finally, the pellet was re-suspended in 100mM Tris-HCl, 8M guanidine-Cl (pH 8.0) and sonicated to homogeneity.</p><p>An excess of the desired metalloporphyrin (Frontier Scientific) was added along with 5mM sodium dithionite, and then very slowly with stirring, the volume was doubled with 100mM Tris-HCl, pH 8.0. This solution was then dialyzed overnight in 14L of 10mM Tris-HCl (pH 7.2) with stirring. The next day, precipitated protein was removed by centrifugation, and the reconstituted Mb was further purified by ion-exchange chromatography, using a CM-52 column equilibrated with 10mM Tris-HCl (pH 7.2). Purity was assayed by measuring the A414,408/A280 ratio and verified by SDS-PAGE. The protein was then exchanged into 10mM KPi, pH 7.0, concentrated, pelleted in liquid nitrogen, and stored at −80°C. The protein yield was found to be mutation-dependent, and decreased with added mutations; wild-type was obtained at about 100mg/L, D44K/D60K/E85K at about 50mg/L. Concentrations were calculated using the Soret absorbance (Zn2+-DPIX, Zn2+-DPIX dme: ε414 nm = 361 M−1cm−1;(7) Fe3+-PPIX: ε408 nm = 188 M−1cm−1)(37).</p><p>Bovine Fe3+b5 was expressed in E.coli, isolated and purified according to previously described methods.(38, 39)</p><!><p>Mutants were designed as described previously using a Brownian dynamics docking protocol implemented in the Linux version of MacroDox.(13) Total charges were calculated for each of the Mb and b5 variants using the Tanford-Kirkwood method(40) with the default intrinsic pKa values, adjusted as implemented in MacroDox,(41) using the experimental parameters: i) pH7, μ = 18 mM, 293K; ii) pH6, μ = 5 mM, 293K. All Arg, Tyr, and Lys residues (including the mutation sites) were fully charged; all Asp and Glu residues were essentially deprotonated with the charge distributed between their carboxylate oxygen atoms. Consistent with the His pKa values measured by NMR for met-Mb(horse),(42, 43) the computed pKa for His 36 is larger than the default intrinsic pKa = 6.3, while those for His 48 are less than the intrinsic pKa. The computed pKa and corresponding charges on the Nε and Nδ atoms of the His residues are slightly smaller for the more highly charged mutants in comparison to their values in Mb(wt). The formal charges on the metal atoms in Mb and b5 were assigned as +2 and +3, respectively. We have suggested previously that the pKa of charged residues within the protein-protein interface may change upon binding,(7) but given the diversity of the geometries resulting from the BD simulations, this complication has not been addressed in the current work.</p><!><p>Cuvettes filled with 2 mL of buffer were stored open to the atmosphere overnight in a N2-filled glove box. Stock solutions of protein were thawed on a cold block in the glove box for several hours prior to the experiment. The final concentration of Mb in the sample was 5 μM. Protein stock concentrations were determined using a Hewlett-Packard UV-vis spectrophotometer: ε414 (ZnDMb) = 361 mM−1cm−1; (7) ε414 (Fe3+b5) = 117 mM−1cm−1.(44)</p><p>Samples were excited with a Nd:YAG Quanta-Ray INDI laser (Spectra-Physics) tuned to 532 nm. The output power was set so that it was approximately 100 mJ at the sample holder. Triplet quenching measurements were performed with an LKS.60 laser flash photolysis spectrometer (Applied Photophysics) fitted with a xenon lamp with pulsing capabilities. For slow triplet decays (<1000 s−1), a setup modified from Applied Photophysic's stopped-flow instrument was employed with a 9-stage photomultiplier tube for detection. Data for shorter timescales was taken with an Agilent Infiniium 600 MHz digitizer and 5-stage photomultiplier tube detector. The xenon lamp was pulsed to achieve the shortest timescales. The triplet decay timecourses were monitored at 475 nm. Typically, 10 shots were averaged on the slow timescale setup and at least 20 shots were averaged for the fast setup data collection. All kinetic experiments were performed at 20°C.</p><!><p>In the absence of b5, the photo-excited triplet states of all the ZnMbs decay exponentially, with a rate constant (kd) that ranges from 50 to 500 s−1. Upon addition of b5, photo-initiated ET quenches the triplet excited state, with the form of the resulting triplet decay trace being dependent on the strength of the binding interaction. The traces are exponential throughout a quenching titration when binding is weak and the complex is in fast exchange (FE) with its components; they are bi-exponential when the binding has been strengthened to the point that the complex is in slow exchange (SE) with unbound partners. The upper panel of Fig 3 shows triplet decay traces for a set of Mb surface charge mutants in solutions with excess b5 ([b5]/[Mb] = 2). The [Mb, b5] pairs with low values of the protein-protein charge product (−qMbqb5 ≤ 16.5), e.g., [ZnMb(wt), b5] and [ZnMb(D44K), b5] (Table S1), are all in the FE limit, and their triplet decay traces are well described with an exponential function. For these [Mb, b5] pairs, the quenching rate constant (kq) obtained by subtracting kd from the observed rate constant (kobs) increases linearly (Fig 3, lower) as a function of [Fe3+b5]. The slope of the resultant line gives the bimolecular quenching rate constant, k2. Complexes with high values of the charge product (−qMbqb > 16.5), e.g., [ZnMb(44/60), b5] and [ZnMb(44/60/85), b5], fall in the SE regime, with biphasic triplet decay traces. For these complexes, the rate constant for the faster decay phase (kf) is invariant with b5 concentration (Fig 3, lower) and is attributed to protein-protein ET within the pre-formed complex; the rate constant for the slower component (ks) varies linearly with [Fe3+b5] and like the single kinetic phase exhibited by the FE (DD)-type pairs, its slope (k2) is associated with second-order ET-quenching of the free ZnMb by Fe3+b5.</p><p>For the SE complexes, the fraction of the faster phase in the triplet decay typically is taken to correspond to the fraction of bound complex, and the measured variation in this fraction during a titration with an ET quencher is fit to a binding isotherm to determine the protein-protein binding constant, Ka. In contrast, Ka generally cannot be measured from a triplet quenching titration for complexes in the FE regime. However, we have shown that the progressive decrease in the early-time absorbance (A0) during a triplet-quenching titration results from quenching of the singlet state by b5.(13) On the singlet timescale, all the [Mb, b5] complexes are in SE and as a result, the variation in the absorbance decrease can be fit to a binding isotherm to calculate Ka, regardless of whether the complex is in FE or SE on the triplet timescale. This procedure has been applied to each of the complexes studied here.</p><p>The upper panel of Fig 4 is a semi-logarithmic plot of Ka for all the [Mb, b5] complexes, measured at both pH 6 and 7, as a function of the product of the total protein charges determined from Poisson-Boltzmann electrostatic computations, −qMbqb5. As −qMbqb5 increases from −5 to 40, the [Mb, b5] binding affinity increases by nearly three orders of magnitude. The exponential increase in Ka revealed in this plot shows that the driving force for complex formation, −ΔG0a = RTlnKa, increases linearly with the total-charge product for the [Mb, b5] ET pairs, −qMbqb5. This surprisingly simple dependence on total protein charges confirms that the progressive increase in Ka is electrostatically driven and that the mutations change only the electrostatic interactions between the partners.</p><p>The lower panel of Fig 4 is a semi-logarithmic plot of k2 versus −qMbqb5 for all complexes at both pH 6 and 7. At low values of −qMbqb5, where FE dynamics are obtained, k2 varies exponentially with −qMbqb5, increasing by over three orders of magnitude, from 8.0×105 M−1s−1 at −qMbqb5 = −5 to 1.2 × 109 M−1s−1 at −qMbqb5 ~ 16. This behavior was observed previously for Mb mutants with −qMbqb5 ≤ 12.(7) (45) As the charge product is increased beyond this value, the [Mb, b5] pairs show SE-type dynamic behavior (Fig 3) and the increase in k2 with −qMbqb5 slows markedly, resembling the weak charge dependence expected for the diffusion rate describing the association of charged partners.(32, 46, 47). The inset to the lower panel of Fig 4 displays the intracomplex ET rate constant, ket, measured for the complexes in the SE regime as a function of the charge product. Although ket ~ 106 over the accessible range of −qMbqb5, closer examination reveals that ket increases nearly an order of magnitude in this range, from ket ~ 2×105 s−1, toward a limiting rate constant in excess of 106 s−1.</p><p>Although k2 increases smoothly with increasing −qMbqb5, the dynamics of complex formation changes from FE to SE; complexes with −qMbqb5 ≤ 16.5 show FE dynamics; complexes with a larger value show SE dynamics. An interesting aspect of the presentation in Fig 4 is that an apparent pH dependence of k2 for the [Mb, b5] pairs (Table S1) is shown to be merely a consequence of the differing pH dependences of −qMbqb5 for each pair: the points for both pH values can be plotted together. Likewise, no special influences of heme neutralizations are seen beyond the resulting changes in −qMbqb5.</p><!><p>The interface redesign strategy employed here leads to the progressive generation of a patch in the vicinity of the ET-active Mb heme edge with a high local density of positive charge, complementary to the high negative charge of the b5 surface. Figure 4 shows that the consequence of this charge buildup is that Ka and k2 for the suite of [Mb, b5] complexes can be treated as functions of a single variable, the product of the total charges on the two partners, −qMbqb5. Although this confirms that binding and reactivity are both under electrostatic control, at first glance the simple logarithmic dependence of Ka on −qMbqb5, corresponding to a simple coulombic interaction between total protein charges, is a surprise. One might expect that at relatively low values of −qMbqb5, where there has been little localized charge buildup, the electrostatic interactions would involve charges over a wide area, and not be so simply parametrizable.(48)(49, 50)</p><p>The development of a model for the observed behavior begins with the recognition that for all complexes studied, the measurements of both binding affinity and ET probe only configurations of the complex in which b5 is bound at a site on the Mb surface that has a good ET pathway to the heme(51); such sites comprise a `reactive region (Rr)' on the Mb surface surrounding its exposed heme edge. Considering first the binding constant, Ka, this restriction arises because binding is assayed by measuring singlet ET quenching. As this quenching process must compete with the intersystem crossing rate (kisc ~ 2×108 s−1), it can only report on complexes with reactive configurations whose pathways for intracomplex ET can compete effectively, namely configurations in which b5 is bound at the Rr. Fig 5A shows a Pathways coupling map for the Mb surface,(52) color-coded according to the magnitude of the matrix element for electron tunneling from that site to the heme-iron. The heme edge, of course has the largest tunneling matrix element, with concentric `rings' of decreasing coupling as the distance from the heme increases. A more precise definition of the Rr could be developed, but for the present purposes, one may loosely imagine that the Rr for Mb comprises the surface atoms colored red and yellow (or yellowish-green) in Fig 5A.</p><p>A quantitative description of binding in terms of total protein charges is obtained by linking the idea of a Rr around the heme edge to total protein charge, through the definition of a `charged reactive patch (crp)'. Although one might have expected that a wide area of the Mb surface contributes to the energetics of binding at low to moderate values of −qMbqb5, we have examined the surface of Mb and found that a compact surface region that surrounds its heme edge contains a net surface charge equal to the total charge of the wild-type protein, leaving the rest of the protein as charge-neutral. The well-defined surface region identified in this way, shown in both panels of Fig 5, includes the Mb surface atoms that lie within ~10Å of any heme atom. It encompasses: (i) much of the Rr (Fig 5A), and thus an area whose effective ET pathways from the Mb surface support rapid electron tunneling between the heme of a bound b5 and the Mb heme; and (ii) all of the Mb mutation sites employed in this study (Fig 5B). Thus, the total charge on every Mb charge variant can be treated as being concentrated within the crp. The corresponding crp for b5 and its heme-neutralized charge variant, likewise defined to contain the total protein charge, is displayed in Fig SI_1. The definition of a crp naturally leads to a picture in which the ET partners react only when the Rr within their complementary crp are electrostatically drawn together.</p><p>This picture is supported by the finding that the crp of the two partners are relatively small and have effectively the same solvent-accessible surface area: 1410Å2 (Mb) and 1560Å2 (b5) at pH7. The crp on Mb includes only ~22% of the solvent-accessible Mb surface, which supports the physical reality behind the concept of a well-defined patch at which both binding and reactivity occur. Furthermore, BD simulations show that as the charge product is increased, there is a corresponding large increase in the likelihood that a reactive configuration (BD `hit') involves binding through a protein-protein interface that includes the complementary crp of Mb and b5 (Fig SI_2). We of course recognize that one could use any surface location to define a charged patch that encompasses the total protein charge. What makes the crp construct useful is that reactivity is associated only with the Rr, which mostly is contained within the crp (Fig 2,5).</p><p>A simple formulation of the reactive, electrostatic binding between the crp of the partner proteins follows naturally. As ET quenching arises from binding of b5 at surface sites of the Mb Rr that lie within its crp, and as the total charges for the Mb and b5 partners are defined to be localized within the relatively small surface areas of their crp, the electrostatic driving force for reactive complex formation must be dominated by the Coulombic interaction between the charges within the complementary crp of the partner proteins. The further simplification of expressing this driving force as the electrostatic interaction of the total protein charges leads to a form for the driving force that corresponds to the experimental findings: (1)−ΔGa0=RTln[Ka]=−[ΔGhyd0+V(qMbqb5)]=−[ΔGhyd0+qMbqb5f(∊o,∊i,ri,j)]=RTlnK0−qMbqb5f(∊o,∊i,ri,j). Here we have taken the electrostatic interaction energy, V(qMbqb5), to be proportional to the product of the complementary patch charges (qMbqb5), and a function f that could be formulated at differing levels of sophistication in terms of the distances between charged Mb and b5 patch residues (ri,j) and the protein and solvent dielectrics (εo, εi), but whose modeling is far beyond the scope of this report.(53) The additional contribution, ΔG0hyd, takes into account non-electrostatic interactions, as well as an entropy term that reflects the low fractional surface area at which reaction can occur. Definition of such a term represents an extension of the partitioning of the binding free energy, discussed earlier.(9) With this definition, the last line of Eq 1, rewrites −ΔG0a in terms of K0, the binding constant that would apply when Mb is completely uncharged (qMb = 0).</p><p>Eq 1 has been used to fit the dependence of Ka on −qMbqb5 for experiments at both pH 6 and 7. This fit yields K0 = 2 × 103 (−ΔG0a = 4.6 kcal/mol), f = 0.093 kcal/mol; as shown in Fig 4, upper panel, it gives an excellent description of the roughly thousand-fold increase in Ka with increasing −qMbqb5 over the entire range explored. Conversely, the experimentally observed linear dependence of −ΔG0a on −qMbqb5 supports the simplified description of electrostatic binding as governed by the total protein charge concentrated within the protein-protein interface formed by the complementary crp. We note, however, that because the actual magnitude of the parameter f is dependent on the spread in −qMbqb5, it might vary somewhat with the method for computing protein charges.(54) The success of this description (Eq 1) further supports the implicit assumption that hydrophobic interactions between the partners remain largely invariant as the charge in the Mb crp is increased.</p><p>We next consider k2, the bimolecular rate constant for ET. In the FE/DD regime, as we previously discussed,(7) k2 can be written as the sum of rate constants for all individual configurations weighted by their individual binding constants (Eq 2). Here, the non-reactive (NR) configurations are in the majority (NNR in number), but they do not contribute to reactivity as kNR ~ 0. Instead, the minority of reactive (R) conformations (nR in number; large intracomplex ET rate constant, kR) dominate the reactivity, leading to Eq 3, (2)k2=ΣKiki~nRkRKR+NNRkNRKNR (3)~nRkRKR As the binding constant measured by ET quenching, Ka, monitors only reactive sites, one may equate, Ka = KR. Taken together, Eqs 1, 3 then predict the exponential dependence of k2 on −qMbqb5 in the DD/FE regime that is observed (Fig 4). For the more tightly bound complexes that populate the SE regime, the interface redesign has enhanced binding to the ET-active R-configurations sufficiently so that one directly observes the first-order, intracomplex ET rate constant (Fig 4, inset). As this monitors only configurations in which b5 is bound to reactive sites within the crp, one can assign ket = kR = kf, which is taken to be invariant with −qMbqb5.</p><p>The second-order rate constant (Fig 4, lower) however continues to increase with increasing −qMbqb5 through the FE/SE transition and into the SE regime, but the rate of increase slows markedly. To understand the full −qMbqb5 dependence of k2, and in particular the transition from FE to SE, requires that the description of the dependence of the electrostatic binding energetics on −qMbqb5 be incorporated into a kinetic scheme. We find that this can be successfully carried out through use of the textbook steady-state treatment of intracomplex ET between partners involved in a binding pre-equilibrium. (4)D+A⇄koffkonDA→ketD+A− This scheme predicts that in the limit of tight binding, intracomplex, photo-initiated ET will be first order and its rate constant will be ket = kR. Most importantly, we find that it provides an excellent description of the variation of k2 with −qMbqb5 over the full range of behaviors from FE and weak binding through the transition to SE and tight binding, (Fig 4, upper). The scheme of Eq 4 leads to the classical steady-state expression for k2 as a function of the intracomplex ET rate constant (ket), the diffusion-limited association rate constant (kon), and the affinity constant (Ka = kon/koff). (5)K2=ketkon∕(ket+koff)=ketkonKa∕(kon+ketka) In the FE limit, defined by the rate ratio, ket/koff ⪡ 1, the scheme reduces to k2 = ketKa, as discussed above. In the slow-exchange limit, defined by ket/koff ⪢ 1, the rate constant for the second-order ET quenching of unbound, photo-excited ZnMb reduces to the diffusion rate constant, k2 → kon.</p><p>To apply this model to the experimental results, Ka = K0 exp(− qMbqb5f/RT), as described in Eq 1, and kon is given the charge dependence for diffusion of charged species.(32) (6)kon−kD0{−aqMbqb5∕[1−exp(aqMbqb5)]}≡kD0p(qMbqb5) Here kD0 is the diffusion-limited rate constant for the proteins that do not interact electrostatically (−qMbqb5 = 0), and p represents the contribution to diffusion from the electrostatic interactions between the partners, with a = f/RT.(32) As a simplifying first approximation, we further take ket to be identical for all [Mb, b5] protein pairs, ignoring the small fractional change observed experimentally (Fig 4, inset). Substitution of Eqs 1,6 into Eq 5 leads to the following form for the dependence of k2 on the product of the charges, qMbqb5, where c = kD0/ket, (7)k2=kD0p(qMbqb5)Ka∕[cp+Ka]</p><p>The fit of Eq 7 to the full dataset composed of k2 measured for the suite of complexes at both pH = 7 and 6 (Fig 4, solid line) gives a remarkably good description of its overall dependence on −qMbqb5, with equivalent scatter in the data at both pH values (Fig4, Fig SI_4). This fit is essentially identical that obtained with the pH 7 data alone (Fig 4, dashed line); the more limited range in −qMbqb5 available at pH = 6 (noted above) makes an independent fit of this data infeasible.</p><p>The parameters that result from the fit of the entire dataset to eq 7 are f = RTa = 0.22 kcal/mol, c = kd0/ket = 17.3, and kD0 = 2.6×108 M−1s−1. The nominal limiting intracomplex ET rate constant, ket = kD0/c = 1.5×107 s−1, is ~ 10-fold larger than the intracomplex ET rate constant determined for the SD-type complexes: ket ~ 106 s−1 (Fig 4, Table SI_1), suggesting that the rapid intracomplex ET reaction obtained in the quenching measurements (Fig 3) may be dynamically controlled, and may actually correspond to a rate constant for conformational interconversion. Motion(s) that might be involved include `diffusion' of b5 among the ensemble of forms bound to the crp, or the conversion between a weakly-bound `encounter complex' and the ensemble of bound forms (see next subsection). The parameter f ~ 0.2 kcal/mol obtained from the fit of k2 to Eq 7 is larger than the parameter f ~ 0.1 kcal/mol determined above from the dependence of Ka on −qMbqb5. Further work will be required to determine whether this difference merely reflects experimental limitations, or whether it reflects the fact that Ka is determined from singlet-quenching, while k2 is determined from triplet-quenching, and the two measurements sample different conformational ensembles.</p><p>According to Eq 5, the SE-regime comprises complexes with ket/koff ⪢ 1, the FE-regime, complexes with the reverse of this inequality. Surprisingly, the change between regimes occurs without any apparent transitional behavior at −qMbqb5 ~ 16.5, which corresponds to ket/koff ~ 250, based on the parameters a, kD0 and ket determined by the fit to Eq 7. To compare this finding with expectations based on the simple model of Eq 4, the parameters from the fit in Fig 4 were used to calculate values for kon and koff as a function of −qMbqb5 and these in turn were used as input parameters for computing kinetic decay traces through numerical solutions of the differential equations for the kinetic scheme of Eq 4. The calculated decay traces were then fit to exponential and bi-exponential decay functions. These fits show that for low values of −qMbqb5 the simulated traces are monophasic throughout a b5 titration, whereas for high values of −qMbqb5 they are biphasic. Of particular note, they further show that the transition from FE to SE kinetic behavior is sharp, and occurs at a ratio, ket/koff ~225, very similar to that found experimentally, but at a slightly lower value of −qMbqb5 ~10.5. This correspondence in the observed and calculated transitions supports the analysis based on the model of Eq 4, with the small differences likely attributable to experimental limitations on the ability to detect a second component in the experimental decay traces because of noise on those traces.</p><!><p>Fig 4 shows that the complex formation and ET reactivity between Mb and b5 are governed by electrostatic interactions between the total protein charges, viewed as localized in the crp, and that Eq 4 provides an excellent global characterization of the response of the kinetic behavior of this system to changes in the protein-protein charge product. To explore the structural basis for this relationship between k2 and −qMbqb5 we have performed paired Brownian Dynamics (BD) simulations of b5 docking to the series of Mb mutants created using the three hotspot charge residues. Here, as in previous studies,(12, 13) we employ the center-of-mass distance `hit' criterion (dCOM) as a measure of overall binding and a heme propionate – heme propionate distance criterion (dOO) as a measure of reactive binding. The variation of k2 with −qMbqb5 can be visualized by comparing the number of reactive OO hits to the number of COM hits (R), with an expectation that the two numbers approach unity as the energy landscape approaches the SD limit in which all hits are reactive.</p><p>Fig SI_2 shows the numbers of hits from the two types of simulations as a function of −qMbqb5. With the COM criterion, nearly half of the 104 trajectories result in hits, even for the [Mb(wt), b5] complex where −qMbqb5 ~ 0, and this number increases only modestly as a function of increasing −qMbqb5. However, the number of hits with the OO criterion is quite low when −qMbqb5 is small, increases rapidly as −qMbqb5 increases, and approaches the COM value as a limiting value at high −qMbqb5.</p><p>The global response of k2 to changes in −qMbqb5 is accompanied by noticeable variations (`granularity') at fixed values of −qMbqb5, which offers additional insights into the overall progression. For example, Fig 4 contains 12 reaction pairs with −qMbqb5 = 19.5 ± 2, among which k2 varies by roughly an order of magnitude, and similar behavior is observed for the five pairs with −qMbqb5 = 7.5± 2. BD simulations indicate that the variation in k2 at a given value of −qMbqb5 is not reflective of experimental uncertainties, but arises because the interactions within each pair involves an ensemble of conformations with b5 bound within the crp, and the nature of the ensemble depends on the number and placement of the surface charges.</p><p>The variations in the nature of these ensembles can be understood by comparing `hit profiles' from Brownian Dynamics simulations for protein pairs with similar values of −qMbqb5. The insets in Fig 6 show profiles of the reactive hits for the FE/DD-type [Mb(wt), b5] complex and three SE/SD-type [Mb(+4), b5] complexes with −qMbqb5 ~19.5 that involve different pairs of altered hotspot residues. For each of these [Mb(+4), b5] complexes, the reactive hits have coalesced from the dispersed array seen for the complex with Mb(wt)(13) into a `reactive' ensemble (`cluster') of hits in the crp, with the majority of the hits having the requisite short metal-metal distance needed for rapid ET. However, the histograms of the metal-metal distances for the hit ensembles of these three complexes are significantly different.</p><p>Viewed qualitatively, the distributions for the complexes with Mb(44/85) and Mb(60/85) are expanded in comparison to the distribution for the complex with Mb(44/60), extending towards the E85 hotspot mutation site. Looked at more quantitatively, the histograms for each of the three Mb(+4) mutants are well-described by a sum of two Gaussian functions with peak maxima near 16.5Å and 17.5Å (Table 1). While the breadths of the distributions for the shorter distance component (peak 1) are quite similar among the three pairs, the width at half-maximum (Δw1/2) for the longer-range component (peak 2) is much smaller for the [Mb(44/60), b5] pair than it is for the other two pairs. Thus, the differences in the hit profiles largely arise from differences in the less-reactive, longer-range component and in the relative populations of the short and long-range components. Overall, these computations confirm that increasing the charge of the crp serves to increase the attraction of the b5 partner to this Mb surface region, but variation in the location of the charges within the crp introduces `granularity' into the details of the ensemble of contributing conformations.</p><p>It is useful to note that this granularity exists within the context of a design strategy that successfully enhances binding/reactivity by making variants with increased total charge. By localizing all mutation sites within the crp, this design strategy generates a progression in charge product while minimizing granularity. With alternative design strategies, it is conceivable that the granularity in k2 at fixed −qMbqb5 could be made to encompass the full range of behaviors seen in the progression, thus illuminating the importance of the overall pattern of charged residues. Indeed, such an alternative strategy was elegantly implemented by Margoliash and coworkers,(55–57) (58) who mapped the binding interface of Cc with several of its redox partners through kinetic measurements of ET that employed a suite of site-selective, chemically-modified Cc proteins in which the dipole moment of Cc was varied while the charge was fixed.</p><!><p>It is widely thought that the association and docking of protein partners is a complicated process, with the partners first forming an `encounter complex' before proceeding to the most stable bound state.(23, 47, 59–62) Nonetheless, application of the analysis based on the pre-equilibrium binding/ET scheme of Eq 4 to the [RC, c2] complex suggests that this simple approach offers a general basis for discussions of protein-protein ET within suites of mutants formed by varying the properties of one or both of the partners. For the [Mb, b5] system, the linear relationship between the charge product −qMbqb5, and the driving force for binding (−ΔGa = RT log Ka, Eq 1), allows the abscissa of the semi-logarithmic plot of k2 to be assigned to either −ΔGa or −qMbqb5 (Fig 4, top and bottom axes). The use of a free energy axis then permits a comparison of the behavior of the [Mb, b5] system with ET between other suites of partners where the driving force for binding is varied without such a simple correlation with total-charge product.</p><p>The pre-eminent example of such a study involved measurements by Okamura and coworkers of ET between c2 and a suite of variants of the RC. Guided by the crystallographic structure of the [RC, c2] complex,(63) they initially prepared charged RC interface mutants (Δq = −1,1,2,4), to study long-range electrostatic effects on the formation, stabilization and reactivity of the [RC, c2] complex.(18, 19) As the crystal structure of the [RC, c2] complex suggested the presence of hydrophobic residues in the core of the binding interface, they subsequently prepared RC single-site variants at three hydrophobic hotspot positions.(20)</p><p>The inset to Fig 7 presents values of k2 for ET between c2 and the suite of RC variants prepared by mutating hydrophobic residues without changing the charge. The parent [RC, c2] complex is so tightly bound that despite the large decreases in binding affinity that were achieved, all the complexes from this suite remain in the SE regime. For this regime the pre-equilibrium model predicts that k2 = kon ~ kD. As kD should be independent of the binding free energy for complexes in which the RC differs only through hydrophobic mutations, k2 should be invariant in this regime, exactly as observed. In short, within this suite, the reactivity is kinetically decoupled from the large changes in binding.</p><p>The study with the charge-altering RC variation corresponds more closely to the present work with the [Mb, b5] complex. Fig 7 shows the variation of k2 as a function of RTlnKa = −ΔGa for the suite of RC with charge-altering mutations. As the binding is weakened by mutation, the tightly-bound complex shifts from the SE regime into the FE regime in a progression that reverses the progression from FE to SE generated by the charge reversal mutations in the weakly-bound [Mb, b5] complex. An independent fit of k2 vs −ΔGa for the [RC, c2] complex to Eq 7 is not feasible, as there are too few points in the FE regime, but the variation of k2 for the [RC, c2] complexes with –ΔGa, nonetheless, is well-described by offsetting the fitting curve for the [Mb, b5] pair. The offset to lower k2 reflects, in part, a smaller value of ket, as might be expected because the bacteriochlorophyll special pair of the RC is buried whereas the Mb heme edge is exposed.</p><p>Unlike the driving force for the [Mb, b5] system, –ΔGa for the [RC, c2] complexes does not monotonically follow the nominal mutation-induced changes in RC charge. This difference likely occurs because the `binding patch' of the RC (Fig SI_3) is quite large in comparison with the crp of Mb, and the charged residues on the RC reactive surface are dispersed within this much larger area. In addition, the crp of c2 is small compared to the RC binding surface, in contrast to the excellent match between the crp of Mb and b5. As a result, the binding interfaces between c2 and different modified RCs will involve residues in different electrostatic microenvironments, with different solvent exposure and different degrees of screening. As a consequence, charge-changing mutations at different sites will have different influences on the binding, preventing any simple correlation between the binding driving force and the product of total charges. Furthermore, the binding affinities for the RC hydrophobic variants span an equally large range in –ΔGa as do the complexes with the RC charge variants, yet the hydrophobic variations do not `push' the complex appreciably into the FE regime, whereas the charge variations do.</p><p>The large variability in the influence of individual contacts within the [RC, c2] interface contrasts with the considerably smaller variability/granularity for [Mb, b5] complexes, discussed above. This difference reflects the fact that the BD,designed Mb mutation sites are not dispersed over a large binding surface, as with the RC, but by design are closely clustered within the small Mb crp.</p><!><p>We have examined photo-initiated ET quenching within a set of [ZnMb, b5] complexes prepared from Mb variants identified by our interface electrostatic redesign strategy. The addition of positive charges to the reactive region of the Mb surface through D/E → K charge-reversal mutations at one, two or all three of the hotspot positions (D44, D60, E85), augmented with variants in which negative charge is eliminated from the Mb or b5 surface by neutralization of their heme propionates, has produced a remarkably large range in the product of the total charges of the two partners, −5 < −qMbqb5 < 40. As −qMbqb5 is increased within this range, the affinity constant for binding of b5 to Mb increases by 103 and the complex undergoes a transition from FE to SE kinetic behavior on the triplet ET timescale. The second-order triplet ET quenching rate constant (k2) is measurable throughout the entire range in −qMbqb5, increasing progressively as −qMbqb5 increases. The rate constant for intracomplex protein-protein ET from the 3ZnP excited state of the ZnMb (ket) can be directly measured only for complexes in the SE regime where it increases by roughly an order of magnitude (Fig 4, inset). Furthermore, intracomplex ET from the singlet excited state used here to measure the binding constants for the complex (Ka) becomes progressively more effective with increasing total-charge product. Indeed, the singlet ET process has been directly monitored on the ns/ps timescale for the [Mb, b5] complex with the highest −qMbqb5 value.</p><p>The binding affinity (Ka), which probes the population of ET-active configurations of the [Mb, b5] complex, shows a simple, exponential dependence on −qMbqb5 (Fig 4, Eq 1). To understand this behavior we introduced the idea of a charged reactive patch (crp) on the surface of each partner: the compact surface area around the heme edge that contains a charge equal to the total charge of the wild-type protein and encompassing much of the reactive surface region (Rr). All of the Mb mutation sites employed in this study fall within the crp (Fig 5B), so the total charge on every Mb charge variant can be treated as being concentrated within this surface region. A corresponding crp for b5 and its heme-neutralized charge variant likewise contains the total b5 charge and its good ET pathways (Fig SI_1). The crp definition naturally leads to a picture in which the ET partners react only when the Rr within the two complementary crp are electrostatically drawn together.</p><p>The progression in k2 with increasing −qMbqb5 and the transition from FE to SE kinetic behavior (Fig 4) can be understood by integrating the crp description of a protein-protein interface and the corresponding charge-product dependences of the binding free energy (Eq 1) and association rate (Eq 6) into the simplest steady-state, pre-equilibrium treatment of reversible binding between the ET partners (Eq 4). The suite of complexes studied here is found to encompass the full range of behaviors predicted by the model, and as shown in Fig 4, its simple approach captures the global aspects of the correlated electrostatic control of binding and reactivity extremely well. At the same time it reveals a `granularity' in the dependence of k2 on total charge product - the variations in k2 at fixed value of −qMbqb5. As illustrated in Fig 6, this variation results from a response of the populations of different bound configurations to variations in the electrostatic surface potentials resulting from the alternative arrangements of charges within the Mb crp.</p><p>The general applicability of this simple, textbook, pre-equilibrium model to suites of ET complexes prepared with surface mutants is supported by its ability to fully describe the behavior of ET for suites of [RC, c2] complexes. The analysis reveals a strong difference between the behavior of the hydrophobic RC mutants and the electrostatic RC mutants, yet the behavior of both subsets can be explained with the pre-equilibrium model. The model further illuminates the basis of the strong variation in the response of the binding affinity to mutations at different sites on the RC surface.</p><p>In summary, guided by the BD/mutation surface redesign strategy, we have made considerable progress in electrostatically strengthening the binding of the intrinsically, weakly-bound [Mb, b5] complex, thereby coupling reactivity to binding at the crp as the total-charge product, −qMbqb5, is increased. The example set by the tightly-bound [RC, c2] complex shows how much more remains to be achieved in strengthening the binding of the [Mb, b5] complex, further pushing it toward the SD limit where NMR structure determination or even crystallization of a tightly bound complex will offer a welcome comparison to the ensemble of structures predicted by the BD simulations. It is further anticipated that the strengthened binding will be accompanied by the direct observation of enhanced ultrafast ET on the singlet timescale. As a complement to these efforts, we are employing computational approaches to understand the dynamic and structural factors that govern protein-protein recognition and ET. In particular, the integration of inter-protein pathways and molecular dynamics with Brownian dynamics ensemble-generation provides a promising approach for further exploring the heterogeneity in the electrostatic interactions within the protein-protein interface that govern the progression in behaviors of the [Mb, b5] complex, and others, and the additional granularity that is not incorporated in the model presented here.</p>

PubMed Author Manuscript

FGF1 folding is critical for its nonclassical release

Fibroblast growth factor 1 (FGF1), a ubiquitously expressed proangiogenic protein that is involved in tissue repair, carcinogenesis and maintenance of vasculature stability, is released from the cells via a stress-dependent nonclassical secretory pathway. FGF1 secretion is the result of transmembrane translocation of this protein. It correlates with FGF1 ability to permeabilize membranes composed of acidic phospholipids. Similarly to several other nonclassically exported proteins, FGF1 exhibits \xce\xb2-barrel folding. To assess the role of FGF1 folding in its secretion, we applied targeted mutagenesis in combination with a complex of biophysical methods and molecular dynamics studies, followed by artificial membrane permeabilization and stress-induced release experiments. It has been demonstrated that a mutation of proline 135 located in the C-terminus of FGF1 results in: (i) partial unfolding of FGF1, (ii) decrease of FGF1 ability to permeabilize bilayers composed of phosphatidylserine, and (iii) drastic inhibition of stress-induced FGF1 export. Thus, FGF1 folding is critical for its nonclassical secretion.

fgf1_folding_is_critical_for_its_nonclassical_release

<!>DNA constructs<!>Recombinant proteins<!>Molecular dynamics simulation studies<!>Differential Scanning Calorimetry<!>Limited Trypsin Digestion<!>ANS Binding Studies<!>NMR<!>Liposome permeabilization<!>Cell culture<!>Adenovirus production and transduction<!>FGF1 export studies<!>Importance of Proline 135 located in the C-terminal portion of FGF1 for protein folding<!>Backbone conformation of wild-type and P135G FGF1 are similar<!>Mutation of Proline 135 attenuates the permeabilization of phosphatidylserine membrane by FGF1 and inhibits its stress-induced export<!>Discussion

<p>Proteins released through the classical secretion pathway, which involves the endoplasmic reticulum (ER) and Golgi, usually have in their primary structure a cleavable hydrophobic signal peptide that determines their translocation to the lumen of the ER (1). However, a large group of secreted proteins are devoid of signal peptides, and their export does not depend on the ER-Golgi (2, 3). Among these nonclassically secreted proteins are potent regulators of cell differentiation, motility, proliferation, viability, and senescence (4, 5). Although the phenomenon of nonclassical protein secretion has been known for the last two decades, its exact mechanisms remain elusive. The export of several secreted signal peptide-less proteins, such as HMGB1 (6), IL1β (7, 8) and tissue transglutaminase (9) is mediated by non-ER-Golgi vesicular cytoplasmic organelles, which fuse with the cell membrane and release their contents to the extracellular compartment. In contrast, other nonclassically secreted proteins, including potent pro-angiogenic regulators fibroblast growth factor (FGF) 1 (10, 11) and FGF2 (12) exhibit diffuse distribution in the cytosol and are exported by direct translocation through the cell membrane.</p><p>Hyperthermia, hypoxia and other stress conditions exist in damaged and inflamed tissues and stimulate the export of FGF1 in tumors (13–15). We have shown that the stress-stimulated export of FGF1 is preceded by its translocation to the cell membrane (10). The export of FGF1 through the cell membrane correlates with the transmembrane translocation of the acidic phospholipid (PL), phosphatidylserine (PS) (11). Sum frequency generation vibrational spectroscopy (SFS) of immobilized PL membranes (16) and studies of induced fluorochrome release from liposomes (17, 18) demonstrated that FGF1 induces the disorganization of acidic PL membranes. Although several basic amino acids of the C-terminal domain FGF1 are important for its export (18), structural determinants underlying the export of FGF1 remain insufficiently studied. The experiments with FGF-DHFR chimeras inducibly locked in folded conformation demonstrated that unfolding is not required for the export of either FGF2 (19) or FGF1 (20). The passage of a protein across the membrane should require the contact with the hydrophobic membrane core. Interestingly, nonclassically released proteins FGF1, FGF2, and IL1α are characterized by the β-barrel folding (4), which exposes hydrophobic residues and is found in many membrane proteins (21, 22). Most of the positively charged amino-acid residues of FGF1 are located in the C-terminal domain of this protein: in the β-sheets 10 and 11, and in the loops between β-sheets 10 and 11, and 11 and 12. We suggest that the β-barrel structure could be important for FGF1 export at the steps of membrane binding and/or transmembrane translocation. In this study, we applied a range of biophysical methods and the all-atom explicit-solvent molecular dynamics (MD) to further identify the importance of FGF1 folding for its export.</p><!><p>Wild type (WT) α form of human FGF1 cDNA (23) was cloned into pET20b expression vector (24). FGF1 mutants were generated using Quick Change – II site-directed mutagenesis. All plasmids were subjected to DNA sequencing to verify their correctness. For expression in mammalian cells, WT and P135G mutant of FGF1 were cloned in pAdlox vector, as described (25).</p><!><p>E. coli BL-21 pLysS cells were transformed with the respective plasmids. Cells were grown in LB broth and induced with 1mM IPTG for protein overexpression. Bacterial cells were harvested by centrifugation at 6000 rpm and subjected to cell lysis using ultra-sonication procedure with an output of 12 watts and an alternate on/off cycles for 10 seconds each in lysis buffer (10 mM sodium phosphate buffer; pH 7.2). Post-lysed cell suspension was centrifuged at 20000 rpm for 30 minutes to separate the clear cell lysate, which was loaded onto the pre-equilibrated heparin sepahrose column. Using a step gradient of sodium chloride concentration in the same buffer, WT and mutant proteins were eluted and further subjected to concentration and desalting using Millipore centrifugal ultra-concentrators. Concentration of the purified protein was estimated using spectrophotometry with A280 value.</p><!><p>Two solvated systems, one containing WT FGF1 and the other the P135G mutant, were constructed for all-atom explicit-solvent molecular dynamics simulations. FGF1 coordinates were from chain A in the 1.1 Å crystal structure of FGF1 (26). The N-terminal His tag was deleted and replaced with a single Met residue that corresponds to Met14 in the FGF1 sequence. Missing non-hydrogen atoms and the missing C-terminal residues Ser, Ser, Asp were constructed using force field geometries. The REDUCE algorithm (27) was used to add hydrogen atoms and to optimize the orientation of Asn and Gln sidechain amides and His sidechain imidazoles. Crystallographic water molecules within 5 Å of the protein were retained, and the protein was centered in a cube of bulk water molecules at experimental density and with edge length 20 Å longer than the FGF1 dimension along its longest principal axis. Bulk water molecules overlapping the protein were deleted, a random bulk water molecule was replaced with Cl− to neutralize the system, and additional random water molecules were replaced with Na+ and Cl− ions to yield 140 mM NaCl. Construction of the mutant followed the same protocol as for the WT FGF1 with the exception of computational introduction of the P135G mutation prior to solvation. Harmonic restraints were applied to FGF1 non-hydrogen atom positions, the system was energy minimized, and heated over 40 ps to 310 K. Harmonic restraints were then removed, and a 200 ns MD simulation at 310 K and 1 atm followed. Simulations employed periodic boundary conditions (28), Langevin thermostating with a friction coefficient of 0.1 ps−1 (29), Langevin barostating (30), a 10 Å spherical cutoff, an energy-switching function applied to Lennard-Jones interactions between 8 to 10 Å (31), an isotropic correction to the pressure to account for Lennard-Jones interactions beyond the cutoff (28) and particle-mesh Ewald to account for electrostatic interactions beyond the cutoff (32). System construction and MD trajectory analysis utilized the CHARMM program (33), and MD simulation utilized the NAMD program (34). All simulations used the CHARMM biomolecular force field (35–38) and the TIP3P water model (39). Each of the two systems was simulated five times, with a different initial random seeds for each trajectory.</p><!><p>Recombinant protein samples of WT FGF1 and mutants at a concentration of 1mg/ml in 10 mM sodium phosphate buffer containing 100 mM NaCl, pH 7.2 was used to determine the melting temperature (Tm) on a Nano III DSC instrument (Delaware, USA). Prior to loading, all samples were subjected to degassing at 25°C for 15 minutes and after loading, the cells were equilibrated for 10 minutes at the same temperature. DSC was performed in the presence or absence of 0.5 mM sucrose octasulfate (SOS). To obtain a stable baseline, buffer blanks experiments were conducted before running the protein scans. Data obtained was processed using the software provided by the manufacturer to derive the thermodynamic parameters like Tm, ΔH and ΔS.</p><!><p>Limited trypsin digestion of FGF1 WT and FGF1 mutants was performed in the absence or presence of SOS.with the addition of trypsin to protein at a ratio of 1 to 10 in 10mM sodium phosphate buffer containing 100 mM NaCl; pH 7.2. An initial sample of undigested protein was removed prior to the addition of the enzyme, and the remaining trypsin-containing sample was incubated in a water bath at 37°C. Samples were removed every 5 minutes for the first 20 minutes, and then every 10 minutes until the 60-minute mark was reached. The reaction was stopped with the addition of 100% trichloroacetic acid. Samples were resolved on a 15% SDS-PAGE gel and subjected to Coomassie blue staining. Using UN-SacnIT software (Silk Scientific Inc, Utah, USA), the bands corresponding to FGF1 were scanned and subjected to densitometry analysis to identify the percent digestion over time of incubation.</p><!><p>To determine the surface hydrophobicity of proteins, we used the sensitive and reliable 8-anilino-1-naphthalene sulfonic acid (ANS) binding assay, which requires very low concentrations of the protein sample. All spectra were collected on a Hitachi F-2500 spectrofluorometer with a slit width of 2.5 nm. Recombinant protein samples of WT FGF1 and mutants with a concentration of 50 μM in 10 mM sodium phosphate buffer containing 100 mM NaCl, pH 7.2 were loaded into a quartz cuvette, ANS dye was added at increasing concentrations. ANS bound protein sample was excited at 380 nm at 25°C. Appropriate blank corrections were made to account for the effect of dilution. Spectra obtained were subjected to smoothing function provided by the manufacturer before collecting the values at 520 nm. Relative fluorescence intensity at 520 nm (emission maxima) was plotted against concentration of ANS.</p><!><p>Bacterial expression host E. coli BL-21 (DE3) cells were grown in M9 minimal medium containing 15NH4Cl. To achieve maximal expression yield, medium was supplemented with necessary vitamins. Recombinant overexpressed proteins were subjected to uniform 15N labeling. Multi-dimensional NMR experiments were carried out at 25°C on a Bruker Avance-μ10mM sodium phosphate buffer pH 7.2 with 10% D2O added prior to acquisition of data. Spectra obtained were processed on Windows workstation using TopSpin v 2.5 and Sparky software.</p><!><p>Phospatidylserine (PS) (AvantI) emulsion containing carboxyfluorescein dye was passed through the 1μm pore extruder to generate liposomes of uniform size as described (18). Unbound dye was separated from the dye containing liposomes using a size-exclusion chromatography on Superdex 75 (16/60 mm) column. WT, P25G or P135G FGF1 were added to liposomes to the concentration of 500 nM. Triton X100 (positive control of liposome permeabilizaton) was added to the concentration of 0.1%. Unquenching of fluorescence due to the release of dye from liposomes was quantified on Hitachi F-2500 spectrofluorometer. The excitation wavelength was at 492 nm with a slit width of 5nm and emission was monitored at 517 nm with a slit width of 10 nm.</p><!><p>Murine NIH 3T3 cells (ATCC, Manassas, VA) were maintained in DMEM (HyClone, Logan, UT) supplemented with 10% bovine calf serum (HyClone).</p><!><p>Recombinant FGF1 WT and FGF1 P135G adenoviruses were produced, purified, and titered as described (25). Briefly, CRE8 cells were transfected with SfiI-digested pAdlox-derived constructs, and infected with the ψ5 virus. The lysates were prepared 2 days after infection. The virus was passed twice through CRE8 cells, and purified from the second passage using a cesium density gradient. The virus was quantified by optical density at 260 nm, and the bioactivity was determined by a plaque-forming unit assay. Adenoviral transduction was performed in serum-free DMEM with approximately 103 viral particles/cell in the presence of poly-D-Lysine hydrobromide (Sigma) (5X103 molecules/viral particle) for 2 h at 37°C. Then the adenovirus-containing medium was removed and replaced with serum-containing medium. The cells were plated for experiments 24–48 h after transduction.</p><!><p>NIH 3T3 cells were used to study FGF1 release 48 h after adenoviral transduction with FGF1 WT or P135G. The heat shock-induced FGF1 release assay was performed by incubation of the cells at 42°C for 110 min in serum-free DMEM containing 5 U/ml of heparin (Sigma), as previously described (13). Control cultures were incubated at 37°C for the same period. Conditioned media were collected, briefly centrifuged at 1,000 g to remove detached cells, and FGF1 was isolated for immunoblot analysis using heparin–Sepharose chromatography as described (13). Cell viability was assessed by measuring lactate dehydrogenase (LDH) activity in the medium using the Promega CytoTox kit (Promega, Madison, WI).</p><!><p>Analysis of the 3D structure of FGF1 based on NMR results from the Kumar laboratory identified a number of amino acid residues as potential targets of mutagenesis intended to disrupt the β-barrel structure of these proteins (Figure 1). Particularly, it suggests that residue P135 located in the β-turn region of FGF-1 between the β-sheets 11 and 12 could be important for maintenance of the β-trefoil architecture of this protein. In the attempt to assess the role of FGF1 3D structure in its nonclassical secretion, we attempted to design a point mutation interfering with FGF1 folding. Single proline/glycine mutants in the positions 25, 41, 93, 135, 148 and 150 were produced and their folding characteristics were studied using biophysical methods. Differential scanning calorimetry demonstrated that all the studied mutations induced a moderate (3–6°C) decrease of the melting temperature of FGF1 (Table 1). We also used the limited proteolytic digestion to assess the conformational changes induced by proline mutations. Time-dependent trypsin digestion of FGF1 was monitored by SDS-PAGE analysis. The degree of digestion was measured by densitometry on the basis of the intensity after Coomassie Blue staining of the 17kDa band on the polyacrylamide gel corresponding to undigested FGF1. Most studied mutations except P93G resulted in the increase of trypsin susceptibility of FGF1 (Figure 2A). Especially strong was the effect of P135G indicating that it induced a significant increase of the accessibility of normally hidden GHG1 domains to trypsin. In a parallel series of experiments, the effect of proline mutations on FGF1 folding was assessed by fluorometric determination of the binding of ANS, which is a hydrophobic fluorescent probe detecting the solvent-accessible non-polar surfaces in proteins (40). While most of proline mutations did not influence ANS binding, P135G mutation resulted in its significant increase (Figure 2B). Based on the results of trypsin digestion and ANS binding, we applied NMR to analyze the effect of P135G mutation on FGF1 structure. The overlay of the 1H-15N HSQC spectra of WT and P135G FGF1 forms have shown the perturbation of many residues including those located more than 5Å from the mutation site indicating that this mutation significantly disrupted protein folding (Figure 3).</p><!><p>Molecular dynamics simulation time evolution of the root mean squared deviation of atomic positions relative to the starting crystallographic conformation (RMSD) shows FGF1 to be a flexible protein for its small size. WT RMSD values for the Cα atoms reach as high as 8 Å, and this was also the case for P135G mutant (Figure 4A). Of note, sampling of conformations with large RMSD relative to the starting crystallographic conformation is often transient, with subsequent conformations from the same trajectory being closer to the crystal conformation. This suggests that the protein as simulated is not denaturing, but rather is highly flexible. This is confirmed by analysis of the root-mean-squared-fluctuation (RMSF) in Cα positions. RMSF data for both WT and P135G FGF1 showed that, while the protein was flexible, this flexibility was limited to the N-terminal and C-terminal residues (Figure 4B). Starting at the N-terminal Met14, flexibility was very high (RMSF > 10 Å) but rapidly declined such that by residue 25, the RMSF value was less than 1 Å. Similarly, until the C-terminal residues was reached, RMSF values stayed low, consistent with a stable, folded protein. Only after residue 150 did RMSF values increase substantially, reaching values greater than 5 Å for the terminal residue Asp154. The final snapshots after 200-ns of MD sampling showed the consistency in the conformations for residues 25 to 150 as compared to the crystal structure (Figure 5A). In all cases, for both WT and P135G, the crystallographic barrel structure of this small protein was preserved.</p><p>From the RMSF data, there appears to be no significant effect of the P135G mutation on the ensemble of conformations that constitutes the folded state of FGF1. Notably, RMSF values for residues adjacent to and including the P135G mutation were unchanged relative to the WT protein (Figure 4B). Visual inspection of the P135G loop in the final snapshots largely confirmed this finding. All five of the WT trajectory final conformations had a loop structure like that of the starting crystal structure. This also held true for four of the five for P135G (Figure 5B). In the case of the fifth, there was loss of the short span of helical secondary structure. This lone example suggests a local destabilizing effect of the point mutation. Taken in the context of the NMR data, it appears that P135G does not change the conformational properties of the folded state itself, but rather changes the thermodynamic balance between the folded state and the unfolded state, such that the folded state becomes relatively less stable.</p><!><p>We have previously shown that the mutations of Lysines 114,115, and 125,126 not only inhibit the stress-induced nonclassical export of FGF1, but also decrease the FGF1-induced destabilization of artificial membranes composed of acidic phospholipids (18). To assess the role of FGF1 folding in its ability to destabilize the membranes, we have chosen for the study of PS liposome permeabilization the P135G FGF1 mutant characterized by the strongest increase of protease sensitivity and ANS binding. We have used as controls WT FGF1 and the mutant P25G FGF1, which exhibited only a modest increase of protease sensitivity and ANS binding. While WT and P25G FGF1 caused a strong leakage of fluorochrome from PS liposomes, similar to that induced by Triton X100, the effect of P135G was significantly weaker (Figure 6A).</p><p>Next, the role of Proline 135 in FGF1 export was assessed. Preliminary studies demonstrated that P135G mutation did not interfere with FGF1 binding to heparin (Supplementary Figure 1), thus we used the standard heparin chromatography approach to assess the release of P135G FGF1 to the medium. NIH 3T3 cells were adenovirally transduced with WT or P135G FGF1 and subjected to 100 min incubation at 37°C or 42°C. Released FGF1 was isolated from the medium by adsorption to heparin-sepharose, resolved by SDS PAGE and detected by immunoblotting. We found that unlike WT FGF1, heat shock failed to induce the export of P135G FGF1 (Figure 6B)</p><!><p>Understanding the structural determinants critical for nonclassical export of FGF1 is important for elucidating the mechanisms of its transmembane translocation and developing approaches to regulation of the availability of this important biological regulator. Earlier studies (18, 41) demonstrated that the C-terminal portion of FGF1 enriched in basic amino acids is critical for its interaction with acidic PL present in the internal membrane leaflet. Here we report that the mutation of proline 135 localized in this domain disturbed the folding of FGF1 and blocked its stress-induced secretion. We have demonstrated that aminopterin-dependent stable folding of the dihydrofolate reductase (DHFR) moiety in FGF1-DHFR chimera does not prevent its stress-induced release (20). These data and earlier results of experiments with FGF2-DHFR (19) indicate that nonclassical FGF secretion does not require protein unfolding. The present study goes further by demonstrating the importance of FGF1 folding for its export. Although MD studies did not reveal significant conformation differences between folded WT and P135G FGF1, several biophysical and biochemical approaches including DSC, NMR, ANS binding and limited protease digestion clearly demonstrated a significant attenuation of FGF1 folding induced by P135G mutation. Rather than resulting in a permanent partial unfolding of FGF1, we suggest that this mutation changes the equilibrium between its folded and unfolded forms.</p><p>Nonclassical secretion of FGF1 and FGF2 involves their translocation through the plasma membrane and correlates with the ability of these proteins to destabilize bilayers containing acidic phospholipids (4, 42). Thus, mutations of basic amino acid residues in the C-terminal domain of FGF1 suppressed both its secretion and ability to destabilize artificial PS membranes (18). Similarly, as we have shown in the present study, a drastic inhibition of stress-induced FGF1 release by P135G mutation correlates with significantly attenuated permeabilization of the PS bilayer. The effect of P135G mutation on FGF1 secretion and membrane permeabilization may have different explanations. Thus, the specific folding of FGF1 could be critical for the exposure of C-terminal basic amino acids to the acidic phospholipids of the cell membrane and subsequent local membrane destabilization facilitating the translocation of this protein. On the other hand, the deterioration of the β-barrel structure by the P135 mutation may interfere with the formation of hydrophobic protein surface needed for FGF1 incorporation into the cell membrane. In this regard, it will be interesting to assess the effects of point mutations interfering with protein folding upon the nonclassical secretion of other β-barrel proteins such as FGF2 and IL1α (4, 42). The finding about the importance of FGF1 folding for its secretion is promising for the development of cell permeable small molecules specifically binding FGF1 and inducing its partial unfolding, which would result in the inhibition of nonclassical protein secretion.</p><p>FGF1 is known to translocate across the membrane in a complex involving S100A13, a Ca2+ binding protein, and the p40 form of synaptotagmin 1 (Syt1), a cytosolic product of alternative translation of mRNA normally coding for transmembrane protein synaptotagmin 1 (4, 43, 44). FGF1 shares an interface with S100A13 and Syt1 interacts with phosphatidylserine, disrupting the lipid bilayer allowing transfusion of the complex. By mapping the chemical shift perturbations of 1H-15N HSQC, Mohan and coworkers revealed that the FGF1 binding site for S100A13 is composed of 14 residues: K98-F101 and R112-A122 (44). As a whole, FGF1 is characterized as a non-covalent complex during exportation (44). P135 is not reported to be involved in the interfacial region of the FGF1-S100A13 interaction, and therefore will have no direct effect on the stability of the formation of the FGF1-S100A13-p40Syt1 complex or complex attachment to the lipid bilayer. Furthermore, changing a hydrophobic proline to a hydrophobic glycine does not disrupt the charge character of the protein, thus effects on the stability of the secondary structure better evidence attenuation of membrane interaction as reported in this study.</p>

PubMed Author Manuscript

Cation Ordering and Exsolution in Copper‐Containing Forms of the Flexible Zeolite Rho (Cu,M‐Rho; M=H, Na) and Their Consequences for CO2 Adsorption

AbstractThe flexibility of the zeolite Rho framework offers great potential for tunable molecular sieving. The fully copper‐exchanged form of Rho and mixed Cu,H‐ and Cu,Na‐forms have been prepared. EPR spectroscopy reveals that Cu2+ ions are present in the dehydrated forms and Rietveld refinement shows these prefer S6R sites, away from the d8r windows that control diffusion. Fully exchanged Cu‐Rho remains in an open form upon dehydration, the d8r windows remain nearly circular and the occupancy of window sites is low, so that it adsorbs CO2 rapidly at room temperature. Breakthrough tests with 10 % CO2/40 % CH4 mixtures show that Cu4.9‐Rho is able to produce pure methane, albeit with a relatively low capacity at this pCO2 due to the weak interaction of CO2 with Cu cations. This is in strong contrast to Na‐Rho, where cations in narrow elliptical window sites enable CO2 to be adsorbed with high selectivity and uptake but too slowly to enable the production of pure methane in similar breakthrough experiments. A series of Cu,Na‐Rho materials was prepared to improve uptake and selectivity compared to Cu‐Rho, and kinetics compared to Na‐Rho. Remarkably, Cu,Na‐Rho with >2 Cu cations per unit cell exhibited exsolution, due to the preference of Na cations for narrow S8R sites in distorted Rho and of Cu cations for S6R sites in the centric, open form of Rho. The exsolved Cu,Na‐Rho showed improved performance in CO2/CH4 breakthrough tests, producing pure CH4 with improved uptake and CO2/CH4 selectivity compared to that of Cu4.9‐Rho.

cation_ordering_and_exsolution_in_copper‐containing_forms_of_the_flexible_zeolite_rho_(cu,m‐rho;_m=h

<!>Introduction<!><!>Introduction<!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!>CO2 adsorption isotherms and kinetics<!><!>CO2 adsorption isotherms and kinetics<!><!>CO2 adsorption isotherms and kinetics<!><!>CO2 adsorption isotherms and kinetics<!><!>CO2 adsorption isotherms and kinetics<!><!>CO2 adsorption isotherms and kinetics<!>Conclusion<!>Experimental Section<!>Conflict of interest<!>

<p>M. M. Lozinska, S. Jamieson, M. C. Verbraeken, D. N. Miller, B. E. Bode, C. A. Murray, S. Brandani, P. A. Wright, Chem. Eur. J. 2021, 27, 13029.</p><!><p>Zeolites find widespread use as adsorbents in a range of commercially‐important gas separations involving small molecules, including air separation (where N2/O2 selectivity is required) and hydrogen purification (CO2/H2).[ 1 , 2 , 3 ] Furthermore, advanced materials and chemical engineering research continues to drive improved performance in these and similar applications, [4] and also in CO2 adsorption in natural gas and biogas upgrading (CO2/CH4)[ 5 , 6 ] and carbon capture from power plant and industrial emissions (CO2/N2 and CO2/CO,H2).[ 7 , 8 ]</p><p>The performance of zeolites in gas separation relates directly to their high chemical and thermal stability and also to their structural features: high internal surface area accessible via well‐defined pores and the presence of extra‐framework cations. These cations affect their adsorption properties in a number of ways. First, the direct cation‐adsorbate interaction enables molecules to be differentiated based on their dipole moment or polarizability – cationic zeolites can separate N2 from O2 due to its higher polarizability, for example.[ 1 , 2 ] Extra‐framework cations can also control the effective pore size, if they are located close to windows, as shown by the increasing pore size of K‐, Na‐ and Ca‐forms of zeolite Linde A (known as 3A, 4A, and 5A respectively). There is also strong evidence that cations in single eight‐membered ring (S8R) sites (8R refers to the size of the ring, which contains 8 tetrahedral Si or Al atoms and 8 O atoms) can exert trapdoor behaviour, where the cation must move to allow passage of adsorbates, leading to selectivity on the basis of the strength of cation – molecule interactions.[ 7 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ]</p><p>The extra‐framework cation composition can be modified by aqueous ion exchange. Many studies have investigated the adsorption behaviour as the cation type (and charge) is varied, typically examining the alkali and alkaline earth metal cations. For example, the adsorption of N2 and CO2 on alkali metal cation forms of the commercially‐important zeolites A and X has been compared.[ 16 , 17 , 18 , 19 ] Most such studies have concentrated on single cation forms of zeolites, which allows for straightforward rationalisation and computational modelling, but there are examples where mixed cation forms of zeolites have important advantages in industrial applications. For example, whereas fully Li‐exchanged forms of chabazite have excellent properties for N2/O2 separation, the high lithium content compromises their structural stability, due to the strong interactions of Li cations with O atoms of the framework, and mixed Li/Na forms of chabazite have improved stability while retaining high separation.[ 20 , 21 ] In these cases, site ordering of cations over different framework sites is observed, and therefore determination of the resulting distribution is essential to understand the properties. In Li,Na‐chabazite, for example, Li cations favour the smaller single six‐membered ring (S6R) sites whereas the Na cations prefer the larger 8R sites, due to their different cationic radii (Li+, 0.76 Å; Na+, 1.02 Å).[ 20 , 21 ]</p><p>Some zeolites possess flexible frameworks, which, when dehydrated, adapt to achieve optimal coordination with the extra‐framework cations. Zeolite Rho is the archetypal flexible zeolite, but other zeolites have been found to exhibit similar types of behaviour.[ 9 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ] Varying the cations can change the framework configuration of the 'activated' adsorbent and thereby the effective pore size, particularly for molecules that interact weakly with cations, such as O2 and N2. Additionally, as the framework distorts, the geometry of cation sites changes, so that cation coordination with framework O atoms, and therefore cation site preferences, are likely to change.</p><p>The CO2 adsorption properties of univalent cation forms of zeolite Rho have been studied extensively.[ 9 , 22 , 23 , 29 ] Rho is a promising zeolite for adsorption applications because of its large, three‐dimensionally connected pore volume, which comprises two identical, interpenetrated, but unconnected pore systems comprising lta cages connected by d8r windows, in which all of the internal space is accessible to small molecules (Figure 1a). When in the hydrated form, zeolite Rho adopts Im3‾m symmetry, with a=15.0352(2) Å, [22] but upon dehydration the framework can distort as the d8r unit twists, to give the acentric variant, space group I4‾3m (Figure 1b).</p><!><p>Two symmetries of zeolite Rho framework: (a) Im3‾m space group including three main cation sites: single eight‐membered ring (S8R), double eight‐membered ring (D8R) and single six‐membered ring (S6R) and (b) I4‾3m space group. Oxygen atoms=red spheres, T atoms (Si or Al)=grey spheres and cations=blue spheres.</p><!><p>The Rho framework offers three main cation sites: single eight‐membered ring (S8R) and double eight‐membered ring (D8R) sites in the d8rs connecting lta cages, and single six‐membered ring (S6R) sites in the lta cages (Figure 1a). In the proton form, zeolite H‐Rho(3.9), the structure retains Im3‾m symmetry, even when dehydrated, but in Na9.8‐Rho (9.8 cations per unit cell) the structure distorts to I4‾3m , with unit cell a parameters of 14.3771(2) Å. [22] This enables metal cations to achieve closer coordination with framework O atoms and therefore more favourable coulombic interaction (Figure 1b). In Na‐Rho, Na cations show a preference for the distorted S8R sites, with S6R sites being filled only when the d8r windows are occupied by at least one Na cation (in S8R sites). The cations in S8R sites block the windows and must move to allow sorbate uptake via trapdoor mechanism. This imparts a very high CO2/CH4 selectivity, even at high CH4 pressure, but the diffusion in Na‐Rho is very slow.[ 22 , 30 ]</p><p>In this study we aimed to improve transport properties of Na‐Rho by ion exchange with a divalent cation likely to prefer S6R sites. This should reduce the number of cations in total, due to charge balance considerations, and also decrease the likelihood that cations occupy sites in the d8r window. Cu2+ was chosen as a divalent cation likely to show a preference for S6R sites. Much is known of the siting of Cu2+ in high Si/Al ratio, small pore zeolites because of the catalytic application of such materials in the selective catalytic reduction of NO by NH3 (SCR) in diesel engine exhausts. The structural study of the Cu‐form of the high Si/Al form of zeolite A, performed as part of an examination of the remarkable SCR activity of this material, is particularly relevant in our case, because the Cu2+ is found to occupy S6R sites within the lta cage, which is also found in the zeolite Rho structure. [31] (Notably, the Cu‐form of high silica zeolite Rho (with Si/Al up to 12.5) shows promising SCR activity. [32] ) Furthermore, measurements of the O2, N2 and Ar uptake on Cu4.9‐Rho indicate very low O2/Ar and O2/N2 selectivity and very high O2 diffusion rates similar to those of H‐Rho, [4] which suggests Cu cations are primarily located away from d8r windows.</p><p>Here, we have prepared Cu,H‐ and Cu,Na‐Rho materials by aqueous ion exchange and deammoniation. Copper cations are found to prefer S6R sites strongly in the dehydrated forms of these materials. This favours open structures, and leads to exsolution phenomena in some Cu,Na‐Rho compositions. Determination of the kinetics and selectivity of CO2 adsorption via zero length column and breakthrough curve measurements indicate that a mixed cation Cu3.4Na3‐Rho has an improved combination of uptake, kinetics and CO2/CH4 selectivity over end‐member Cu‐ or Na‐Rho compositions.</p><!><p>Cu,H‐Rho samples were prepared with compositions Cu0.9H8.0‐Rho, Cu2.1H5.6‐Rho, Cu3.0H3.8‐Rho and Cu4.9‐Rho as described in the Experimental section and Supporting Information. The diffraction patterns were measured before and after calcination (see Figure S1 in Supporting Information) and no extra peaks corresponding to extra copper‐containing phases were observed. The gradual decrease in intensity of the {110} peak indicated greater exchange of NH4 + cations for Cu2+ cations in each consecutive sample.</p><p>To establish the presence of Cu2+ cations in Cu,H‐Rho samples, EPR spectroscopy was conducted on Cu0.9H8.0‐Rho, Cu2.1H5.6‐Rho and Cu4.9‐Rho samples (see Figure S2 in Supporting Information and Figure 2). The EPR spectrum for Cu0.9H8.0‐Rho sample showed the best resolution, due to less spin‐spin interactions of neighbouring Cu2+ cations in similar environments, therefore it was investigated in detail. The spectrum (Figure 2) showed well resolved hyperfine coupling to 63/65Cu giving four peaks at ca. 3000 G in the parallel region of the spectrum (A|| hyperfine splitting on g||) and a small peak with a deep trough at ca. 3300 G (g⊥) with no resolved hyperfine splitting. This was attributed to a planar coordination (g||=2.37 and A||=153×10−4 cm−1) of Cu2+ cations to three oxygen atoms in the S6R sites as also observed by Rietveld refinement, at a distance of 2.434(15) Å (Figure 3a and see Table S2 in Supporting Information). A previous EPR study on Cu‐Rho by Anderson and Kevan also suggested that copper strongly favours sites in the α‐cage rather than in the octagonal prism. [33] There was an additional broad peak visible at ca. 3150 G and this may indicate the presence of copper cations nearby.</p><!><p>EPR spectrum of dehydrated Cu0.9H8.0‐Rho sample measured at 295 K; the g|| region is highlighted.</p><p>Position of Cu2+ cations in S6R sites in (a) Cu0.9H8.0‐Rho, (b) Cu4.9‐Rho and (c) Cu1.0Na7.8‐Rho. Oxygen atoms=red spheres, T atoms (Si or Al)=light blue spheres and Cu cations=dark blue spheres.</p><!><p>Additionally, XPS analysis was conducted on Cu4.9‐Rho before and after heating to determine the oxidation state of copper (Figure S3 in Supporting Information). The XPS spectra for both samples showed a major peak at 934.9 eV identified as Cu2+ species. This is in agreement with previously reported XPS Cu 2p3/2 binding energy for mazzite (MAZ) zeolite, where Cu2+ species were found in s6r sites. [34] From the deconvolution an additional (unresolved) peak at 932.2 eV might be fitted and assigned to Cu0/Cu+1 species. [34] On this basis, while the presence of some Cu+ cannot be ruled out, at least 70 % of the copper close to the surface of the heated sample was 2+.</p><p>Remarkably, upon dehydration at 543 K the samples did not experience the contraction of their unit cells that is observed for zeolite Rho exchanged with other metal cations (e. g. univalent Li, Na, K, Cs or divalent Sr, Ca, Cd)[ 9 , 23 , 35 , 36 , 37 , 38 ] (see Figure S4 in Supporting Information). Structural refinements revealed that the frameworks of Cu,H‐Rho retained a large unit cell parameter, between 14.9947(1) Å and 14.9258(9) Å, although they were better refined in the acentric space group I4‾3m (Table 1 and see Table S1 and Figure S5 in Supporting Information).</p><!><p>Space group, unit cell parameter and cation site occupancies in dehydrated zeolite Rho as determined by Rietveld refinement.</p><p>Sample</p><p>Unit cell parameter [Å]</p><p>Space group</p><p>S6R site</p><p>S8R site</p><p>Frac</p><p>Atoms per unit cell</p><p>Frac</p><p>Atoms per unit cell</p><p>H9.8‐Rho[a]</p><p>15.0352(2)</p><p>Im3‾m</p><p>–</p><p>–</p><p>–</p><p>–</p><p>Cu0.9H8.0‐Rho</p><p>14.9947(1)</p><p>I4‾3m</p><p>0.1161(17)</p><p>0.92(8)</p><p>–</p><p>–</p><p>Cu2.1H5.6‐Rho</p><p>14.9743(5)</p><p>I4‾3m</p><p>0.2635(19)</p><p>2.11(1)</p><p>–</p><p>–</p><p>Cu3.0H3.8‐Rho</p><p>14.9352(1)</p><p>I4‾3m</p><p>0.3171(19)</p><p>2.53(7)</p><p>0.0108(5)</p><p>0.51(8)</p><p>Cu4.9‐Rho</p><p>14.9258(9)</p><p>I4‾3m</p><p>0.3592(12)</p><p>2.87(4)</p><p>0.0423(4)</p><p>2.03(1)</p><p>Na9.8‐Rho[a]</p><p>14.3771(2)</p><p>I4‾3m</p><p>0.372(11)</p><p>2.98(9)</p><p>0.539(7)</p><p>6.47(8)</p><p>Cu1.0Na7.8‐Rho</p><p>14.3449(6)</p><p>I4‾3m</p><p>0.1262(16) (Cu2+) 0.21077 (Na+)</p><p>1.01(7) 1.68</p><p>0.2474(15) (Na+)</p><p>5.94(6)</p><p>Cu3.4Na3.0‐Rho</p><p></p><p></p><p></p><p></p><p></p><p></p><p>acentric phase: Cu3.0Na3.9‐Rho</p><p>14.4052(9)</p><p>I4‾3m</p><p>0.373(13) (Cu2+)</p><p>2.98(5)</p><p>0.166(13) (Na+)</p><p>3.98(7)</p><p>centric phase: Cu4.9‐Rho</p><p>15.0324(2)</p><p>Im3‾m</p><p>0.289(19) (Cu2+)</p><p>4.62(7)</p><p>–</p><p>–</p><p>[a] The experimental data is taken from Ref. [22].</p><!><p>In Cu0.9H8.0‐ and Cu2.1H5.6‐Rho, the Cu cations occupied only S6R sites and while the unit cell is acentric, a remains close to that of the H‐form. In Cu3.0H3.8‐ and Cu4.9‐Rho additional scattering attributed to copper cations was found in the S8R sites (Table 1 and see Table S2, Figure S6 in Supporting Information) but again the effects on the unit cell are minor. Therefore in dehydrated zeolite Rho the Cu cations preferentially occupy S6R sites and even in fully exchanged Cu4.9‐Rho there is no strong distortion of the Rho framework.[ 36 , 37 , 38 ]</p><p>Refinement of Cu4.9‐Rho (Figure 4) showed that 2.9 of the Cu cations per unit cell occupy a central position in the plane of the 6r windows where they are coordinated by 3 framework oxygen atoms at a distance of 2.323(4) Å (Figure 3b and see Table S2 in Supporting Information), a similar distance to that previously observed for Cd2+ cations in S6R sites in zeolite Rho (2.49(2) Å). [37] Two copper cations per unit cell are present in the S8R sites, situated off‐centre in the ring, coordinated to two O atoms at 2.282(12) Å (see Table S2 in Supporting Information). This occupancy of window sites leaves 4 out of 6 per unit cell unoccupied, which is above the percolation limit. As a result, N2 adsorption at 77 K showed high uptake, reaching ca. 9 mmol g−1 at p/p0=0.9, characteristic of filling the open Rho structure. By contrast, N2 adsorption at 77 K on Na9.8‐Rho shows no uptake, due to the Na cations blocking the d8r windows, rendering the pore volume inaccessible (see Figure S7 in Supporting Information). This combination of undistorted d8r windows and relatively low cation occupancy in these windows explains the low O2/Ar selectivity and rapid O2 diffusion observed previously for Cu4.9‐Rho. [4]</p><!><p>(a) (left) Rietveld plot of synchrotron PXRD data (λ=0.8263980 Å, T=298 K) of dehydrated Cu4.9‐Rho (Observed: black, calculated: red, difference: blue, phase: pink and background: green) and (right) a capillary with Cu4.9‐Rho sample which remained blue upon dehydration. (b) Generalised model of the structure of zeolite Cu4.9‐Rho obtained from synchrotron data. The Cu2+ cations=blue spheres. Framework O atoms are omitted for clarity and T−T linkages are represented by grey rods.</p><!><p>The structural chemistry of copper in zeolite Rho was studied further in a series of Cu,Na‐Rho samples, prepared as described in the Experimental section and Supporting Information. In contrast to Cu cations, Na cations favourably occupy S8R sites in dehydrated Rho and the electrostatic attraction of Na+ cations to framework O atoms of the S8R sites leads to a strong distortion of the structure upon dehydration.[ 9 , 22 ] Consequently, it was of interest to examine how a combination of copper and sodium cations would affect the framework of zeolite Rho, and two mixed cation compositions were prepared (Cu1.0Na7.8‐Rho and Cu3.4Na3.0‐Rho) (Table 1).</p><p>In hydrated form, both samples contain a single phase (see Figure S8 in Supporting Information). Upon dehydration Cu1.0Na7.8‐Rho adopts I4‾3m symmetry with a unit cell of 14.345(1) Å, similar to fully exchanged zeolite Na9.8‐Rho, [22] and the same occupancy of S8R sites, ca. 66 % (Table 1 and see Table S2, Figure S9 and Figure S10 in Supporting Information). The d8r windows are very narrow since most of the Na cations occupy preferential S8R sites and this prevents the unit cell from expansion. The Cu cations occupy a slightly off‐centre position in the 6Rs where they are coordinated by three framework O atoms at a distance of 2.132(1) Å (Figure 3c and see Table S2 in Supporting Information).</p><p>Increasing the copper content to Cu3.4Na3.0‐Rho has a more marked effect, as two different zeolite Rho forms are seen to co‐exist in the dehydrated sample, with different cubic unit cell sizes (Figure 5). To eliminate the possibility of partial dehydration, the sample was kept under vacuum and heated at 623 K for 20 h, double the usual dehydration time, with the same result (see Figure S11 in Supporting Information).</p><!><p>Synchrotron XRD patterns of dehydrated (a) Na9.8‐Rho, (b) Cu1.0Na7.8‐Rho, (c) Cu3.4Na3.0‐Rho and (d) Cu4.9‐Rho with magnified views of 2θ range from 9° to 30°.</p><!><p>A sample of the heated Cu3.4Na3‐Rho was investigated by TEM and associated selected area EDS analysis which revealed an uneven distribution of copper and sodium cations (Figure 6). Mixed copper and sodium regions were observed with a range of Cu/Na ratios (Figure 6a and b): in these mixed cation regions the Cu cations tend to concentrate close to the edge of the crystals (Figure 6c). Additionally, copper‐only regions were observed (Figure 6d). This indicates that Cu cations migrate over hundreds of nanometres, and is consistent with the powder diffraction data that indicates crystalline domains of at least these dimensions are present in the exsolved mixture of phases.</p><!><p>TEM/EDS analysis of the Cu3.4Na3.0‐Rho crystals. Analysed regions are in yellow squares.</p><!><p>Two‐phase Rietveld refinement of synchrotron data identified that this Cu3.4Na3.0‐Rho consists of an acentric phase (I4‾3m symmetry, Cu3.0Na3.9‐Rho, a=14.4052(9) Å) and a centric phase (Im3‾m symmetry, Cu4.9‐Rho, a=15.0324(2) Å) (Table 1 and Figure 7). The centric phase accounts for 20 % of the overall composition (I4‾3m :Im3‾m =4 : 1) and the average unit cell composition was estimated as Cu3.4Na3.0‐Rho. The acentric phase was found to contain ca. 4 Na cations in S8R sites, and 3 Cu cations in the S6R sites (Table 1). The structure of the centric phase exhibits the unit cell size of fully open zeolite Rho. The 8r windows are too large for extra‐framework cations to coordinate with framework oxygen atoms, hence the 4.6 Cu cations were found in S6R sites. Nevertheless, since the phase fraction of acentric phase is 4× higher than centric phase it is very likely that some Cu cations are present in the acentric phase.</p><!><p>(a) (left) Rietveld plot of synchrotron PXRD data (λ=0.8263980 Å, T=298 K) of dehydrated Cu3.4Na3.0‐Rho (Observed: black, calculated: red, difference: blue, phase: pink (I4‾3m symmetry) and bright blue (Im3‾m symmetry), background: green) and (right) a capillary with the sample which remained blue upon dehydration. (b) Generalised model of the two coexisting structures (centric on the left and acentric on the right) of zeolite Cu3.4Na3.0‐Rho obtained from synchrotron data. The Cu2+ cations=blue spheres, the Na+ cations=yellow spheres. Framework O atoms are omitted for clarity and T−T linkages are represented by grey rods.</p><!><p>This PXRD and TEM/EDS analysis suggests that an exsolution process has occurred during dehydration. Exsolution commonly occurs upon cooling of solid solutions[ 39 , 40 ] but is rarely seen in zeolites. In zeolitic materials that have been reported, such as those of zeolite P, amicite and merlinoite, it is, as in this case, observed upon dehydration of a zeolite with a flexible framework.[ 25 , 41 , 42 ] During dehydration, cations must be able to diffuse through the structure on a micron length scale to achieve thermodynamically favourable locations. Since phase separation is associated with an entropy loss, this process must result in a reduction in enthalpy, as the zeolite distorts to coordinate cations more closely.</p><p>Upon dehydration of Cu3.4Na3.0‐Rho, the enthalpy can be reduced by concentrating Na cations within a distorted Rho where the Na cations can occupy narrow d8r windows and achieve better coordination. This is achieved by intrazeolitic cation exchange for copper cations. The copper cations that leave form Cu4.9‐Rho, in which they can be well coordinated in S6R sites without the framework needing to distort. To investigate the strength of the tendency of Na cations in copper‐rich samples to drive exsolution, an additional sample, Cu4.3Na1.0‐Rho, with a sodium content of 1 cation per unit cell was prepared. This also showed exsolution upon dehydration (see Figure S12 in Supporting Information). Furthermore, previously reported Na4.5H5.3‐Rho sample (a=14.3447(2) Å, see Figure S13 in Supporting Information) [22] did not show exsolution upon dehydration indicating a requirement of presence of Cu cations in zeolite Na‐Rho for the exsolution to occur. The exsolution occurs in Rho because of the flexibility of its framework, and its ability to adopt two very different conformations that are well adapted to the different cation types. Exsolution phenomena during dehydration are likely to be common within flexible zeolite structures for mixed cation zeolites of certain compositional ranges where the cations have very different ionic radii, charge or electronegativity.</p><!><p>The synthetic and structural investigation showed that it is possible to control the size of the d8r windows in Rho and their fractional cation occupancy through exchange of Cu cations into zeolite Na9.8‐Rho. The small window size in Na9.8‐Rho results in very high CO2/CH4 selectivity but prohibitively slow adsorption kinetics. By relocating cations away from the windows, and thereby enlarging their free diameter, we expect an improvement of the kinetics while retaining good selectivity. The CO2 adsorption isotherms, the kinetics of CO2 adsorption and the CO2/CH4 selectivity under dynamic conditions were therefore measured for selected Cu,Na‐Rho materials and compared with the Na9.8‐ and Cu4.9‐Rho end‐members.</p><p>CO2 adsorption isotherms (with desorption branches) were measured at 298 K on Na9.8‐Rho, Cu1.0Na7.8‐Rho, Cu3.4Na3.0‐Rho and Cu4.9‐Rho (Figure 8). As described previously, the sodium end‐member shows high uptakes of 3.1 mmol g−1 at 0.1 bar and 4.2 mmol g−1 at 0.9 bar as a result of the strong interaction of CO2 with the abundant Na cations. At very low pressures (<0.025 bar) the uptake increases sigmoidally during adsorption, characteristic of structural changes (the zeolite expands) which result in hysteresis on the desorption branch. Notably, the equilibration time for Na‐Rho is long, especially at low p CO2, which is explained by the cation gating effect of Na cations, requiring cation movement to allow uptake. [22]</p><!><p>CO2 isotherms at 298 K on Na9.8‐Rho (▪), Cu1.0Na7.8‐Rho (•), Cu3.4Na3.0‐Rho (▴) and Cu4.9‐Rho (▾). Adsorption, closed symbols; desorption, open symbols.</p><!><p>By contrast, the uptake on Cu4.9‐Rho is rapid, Type I and fully reversible. It achieves a lower uptake at 0.1 bar than Na9.8‐Rho (1.8 mmol g−1), but similar uptake at 0.9 bar (4.5 mmol g−1). Indeed, the isotherm characteristics are similar to those of H9.8‐Rho,5 which also possesses an open structure in the dehydrated form. The low uptake at lower pressures indicates weak electrostatic interactions of CO2 with the copper cations.</p><p>Introduction of one Cu2+ cation into Na9.8‐Rho has little effect on the CO2 adsorption properties so that it also shows hysteresis at low pressures caused by the cation gating effect. In this material there remains around one Na cation per d8r window, [22] so that cation migration is still required to permit uptake. By contrast, uptake is much faster for the adsorption of CO2 on the two‐phase Cu3.4Na3.0‐Rho sample and negligible hysteresis was observed. This two‐phase material, as a result from exsolution upon dehydration, exhibits Type I adsorption which indicates that both phases display this type of adsorption behaviour. Type I behaviour is expected for the minority Cu‐rich phase, as it is close to the Cu end‐member. For the Na‐rich phase, which has a unit cell size similar to that of the Na end‐member (a=14.4052(9) Å cf. 14.3771(2) Å), there are fewer cations in window sites than in the Na end‐member (4/6 windows occupied), which introduces sufficient permeation to strongly reduce the effect of cation gating and thus yield Type I adsorption behaviour. (This is also implied by the N2 adsorption at 77 K, which at 3.2 mmol g−1 (at 0.1 bar) is more than that expected for the minority Cu‐rich phase alone, see Figure S14 in Supporting Information.) At 0.1 bar the uptake of CO2 of the mixed phase is higher than for Cu4.9‐Rho, but well below that of the Na9.8‐Rho: at 1 bar the CO2 uptake is close to that of the Na9.8‐Rho, ca. 4.2 mmol g−1.</p><p>Isosteric heats of adsorption were determined for Cu3.4Na3.0‐Rho and Cu4.9‐Rho and compared with those reported previously for Na9.8‐Rho, [9] (see Figures S15 and S16 in Supporting Information). Na9.8‐Rho has a heat of adsorption of 38–42 kJ mol−1 over range of 1.5‐3.5 mmol g−1. The Cu3.4Na3.0‐Rho and Cu4.9‐Rho materials showed lower heats of adsorption, 30–38 kJ mol−1 over the same range of uptakes, which is attributed primarily to the presence of fewer cations.</p><p>The kinetics and CO2/CH4 selectivity were measured by a combination of ZLC and extended ZLC measurements. The ZLC measurements of the very slow uptake of CO2 on Na‐Rho have been reported previously but were repeated for consistency. [22] At 308 K, with a 4.0 mg sample and a 3 mL min−1 flow of 10 % CO2 in He, the equilibrium time is in excess of 1 h, and desorption from a sample loaded with 1.86 mmol g−1 is strongly kinetically limited and cannot be fitted in a straightforward way, because the structure undergoes structural changes. At low loadings, the last molecules to be desorbed show a D/R2 value of 5.2×10−5 s−1 (see Figures S17 and S18 in Supporting Information and Table 2).</p><!><p>Comparison of uptakes of CO2 and CH4, selectivity and kinetic diffusion parameters for Na9.8‐Rho, Cu4.9‐Rho and Cu3.4Na3.0‐Rho in flowing 10 % CO2/40 % CH4/He at 308 K.</p><p>Sample</p><p>Uptake of CH4 [mmol g−1]</p><p>Uptake of CO2 [mmol g−1]</p><p>α (CO2/CH4)</p><p>D/R2 [s−1]</p><p>Na9.8‐Rho</p><p>0.05</p><p>2.55</p><p>209</p><p>5.2×10−5</p><p>Cu4.9‐Rho</p><p>0.17</p><p>0.85</p><p>20.1</p><p>2.3×10−2</p><p>Cu3.4Na3.0‐Rho</p><p>0.15</p><p>1.27</p><p>33.7</p><p>2.5×10−4</p><!><p>Breakthrough curves were measured for 36.8 mg Na9.8‐Rho, in 40 % CH4 in He (2 mL min−1) and in a mixture of 10 % CO2/40 % CH4 in He (1 mL min−1), typical of some CO2‐rich natural gases and biogas (Figure 9). No CH4 is adsorbed in the first experiment. For the mixture, the poor CO2 adsorption kinetics result in rapid breakthrough of this component, so that under these conditions there is no period where pure CH4 is produced. Some small amounts of CH4 are taken up in the mixed gas experiment, because the structure opens up as CO2 is adsorbed. After saturation, desorption reveals a high CO2 uptake, as expected (2.55 mmol g−1) and very high selectivity (209), as evidenced by the very small amount of CH4 in the desorption curve.</p><!><p>Binary breakthrough experiment for Na9.8‐Rho in 10 % CO2/40 % CH4/He at 308 K. Blue symbols: CO2, pink symbols: CH4, black/red line: CO2/CH4 blank experiment. (a) Fast breakthrough of CO2 during adsorption indicates poor kinetics. (b) No CH4 is observed on desorption (overlap with blank experiment), indicating good selectivity for CO2.</p><!><p>The adsorption and desorption of CO2 on the Cu4.9‐Rho sample is, by comparison with Na9.8‐Rho, very fast. The rapid kinetics of Cu4.9‐Rho can be explained by its nearly circular windows, and the low concentration of blocking cations near them. ZLC measurements of desorption at 308 K from an equilibrated 10 % CO2/He‐loaded sample indicate desorption is nearly complete after 1–2 minutes, and the entire desorption process can be described using the standard ZLC model for linear isotherms with a D/R2 of 2.3×10−2 s−1, some three orders of magnitude higher than observed for Na‐Rho (see Figures S19 and S20 in Supporting Information and Table 2). The breakthrough curve of methane at 308 K (40 % CH4 in He) shows some uptake (0.20 mmol g−1), which is possible due to the circular and unblocked d8r windows (Figure 10). The rapid CO2 diffusion enables the production of pure CH4 in a mixed gas breakthrough experiment, but with lower CO2 capacity than Na9.8‐Rho because of the weaker interaction, showing an uptake of 0.85 mmol g−1 and a selectivity of 20. In the desorption profile a clear difference between the blank experiment can be seen for both components, showing that some CH4 is desorbing, which is indicative of a reduced selectivity compared to Na‐Rho.</p><!><p>Binary breakthrough experiment for Cu4.9‐Rho in 10 % CO2/40 % CH4/He at 308 K. Blue symbols: CO2, pink symbols: CH4, black/red line: CO2/CH4 blank experiment.</p><!><p>For the mixed Cu3.4Na3.0‐Rho sample, ZLC measurements at 308 K show rapid uptake at 1 % CO2, and the desorption is only kinetically limited at very low loadings with a D/R2 of 2.5×10−4 s−1 (see Figures S21 and S22 in Supporting Information and Table 2). This accounts for the lack of hysteresis in the adsorption isotherms and is due to the open Cu‐Rho phase and the ability for gases to permeate through the Cu,Na‐Rho phase. Breakthrough curves for methane show some CH4 can adsorb (0.21 mmol g−1), and the mixed gas breakthrough curve (Figure 11) shows an extended period where pure methane is produced compared to Cu‐Rho, due to the greater capacity of Cu3.4Na3.0‐Rho than Cu4.9‐Rho. The zeolite shows an adsorption capacity of 1.39 mmol g−1 and a selectivity of 33.7, a significant improvement over Cu4.9‐Rho. Given that some 20 % of the exsolved mixture is essentially Cu‐Rho, this indicates that the Cu3.4Na3.0‐Rho itself would possess a selectivity significantly above 35.</p><!><p>Binary breakthrough experiment for Cu3.4Na3.0‐Rho‐Rho in 10 % CO2/40 % CH4/He at 308 K. Blue symbols: CO2, pink symbols: CH4, black/red line: CO2/CH4 blank experiment.</p><!><p>The results show that the kinetics and selectivity of CO2 adsorption by zeolite Rho are strongly dependent on the extra‐framework composition. There is a trade‐off between the uptake rate, which is faster when fewer windows are blocked by cations that occupy S8R sites, and selectivity, which is enhanced when there are more cations in S8R sites and the windows are more elliptical and therefore narrower, giving shape selectivity for CO2 against the larger CH4. It should be noted that this will be modified at different CO2 concentrations, because the presence of CO2 is known to cause structural changes in zeolite Rho.</p><!><p>A series of Cu,H‐Rho samples have been prepared, up to the fully exchanged Cu4.9‐Rho, by Cu2+ ion exchange of the ammonium‐form, followed by deammoniation. In the dehydrated form, the Cu,H‐Rho samples only show a slight decrease in unit cell size, remaining close to the fully open framework '15 Å' form. Cu cations show a strong preference for the S6R sites, where they are located in trigonal coordination in the plane of the 6Rs, up to ca. 3 per unit cell, although some are also found in the S8R sites in samples with higher Cu content. The preferred occupancy of the S6R sites by Cu cations stabilises the open framework, which is unusual for dehydrated cationic forms other than H9.8‐Rho.</p><p>Cu,Na‐Rho samples were also prepared by ion exchange of Na9.8‐Rho, with the aim of improving the kinetics of CO2 adsorption of Na‐Rho and the CO2/CH4 separation of Cu‐Rho. Inclusion of one Cu cation per unit cell (Cu1.0Na7.8‐Rho) does not change the structural behaviour upon dehydration and like the Na9.8‐Rho gives a strongly distorted unit cell with full occupancy of d8r windows with Na cations. However, inclusion of 3.4 Cu cations per unit cell (Cu3.4Na3.0‐Rho) gives a solid that upon dehydration exhibits exsolution to give Cu4.9‐Rho and Cu3.0Na3.9‐Rho. This is observed by PXRD and by selected area EDS in TEM, which suggests long‐range diffusion of Cu and Na cations (of the order of 0.5 μm) and the generation of Cu‐rich Rho near the surface of particles. This exsolution behaviour is made possible by the flexibility of the structure, and its resulting ability to adapt to give sites of appropriate coordination geometry for cations depending on their size, charge and electronic structure. While the smaller cell (14.4052(9) Å) gives distorted S8R sites favourable for Na cations, the open (15.0324(2) Å) cell is favoured by Cu cations in the S6R sites.</p><p>The CO2 adsorption properties of the Cu‐, Na‐ and Cu,Na‐Rho materials are strongly dependent on composition, and there are three orders of magnitude variation in the measured diffusion time constants: the very slow adsorption of Na9.8‐Rho results from Na cations blocking elliptical windows, while the fast adsorption of Cu4.9‐Rho is possible due to its circular unblocked windows. Although Na9.8‐Rho shows very high selectivity for CO2/CH4 adsorption in dynamic breakthrough experiments, its slow kinetics for CO2 adsorption do not allow for clear separation of the two gas components. The open structure of Cu4.9‐Rho, on the other hand, allows very fast CO2 uptake and the production of pure CH4, but with reduced capacity at 0.1 bar CO2 and lower selectivity. The exsolved Cu3.4Na3.0‐Rho sample shows fast diffusion, in the Cu,Na‐Rho as well as the Cu‐Rho phases, because Na cations no longer block all the windows in the mixed cation form. Furthermore, it shows enhanced uptake of CO2 and produces more pure CH4 in the breakthrough tests, as well as being more selective for CO2 over CH4 overall.</p><p>These results confirm that the performance of mixed cation zeolite Rho in selective gas adsorption is highly sensitive to the composition, charge, size and electronic structure of its extra‐framework cations, and this is at least partly because of the flexibility of its framework. This offers many opportunities for the design of task‐specific Rho‐based adsorbents.</p><!><p>Zeolite Na,Cs‐Rho (RHO; (Na,Cs)9.8Al9.8Si38.2O96) was synthesised in the presence of the crown ether, 18‐crown‐6, using a previously reported procedure (see S1 in the Supporting Information). [43] The organic was removed by calcination at 823 K in flowing oxygen gas. The synthesised zeolite Na,Cs‐Rho was fully exchanged to the ammonium form with 3 M ammonium chloride solution at 333 K, eight times for 5 h. Subsequently the ammonium form was converted to sodium form by extended cation exchange treatments at 353 K using 10 wt % metal nitrate solutions. To prepare mixed cation Cu,NH4‐Rho and Cu,Na‐Rho samples the ion exchange with low concentration (0.05 M) copper nitrate solution at 333 K for 2–4 h was performed until desired compositions were achieved. A low concentration of copper nitrate solution was required to avoid precipitation of copper hydroxide on the zeolite surface. [44] The mixed Cu,H‐Rho samples were prepared via deammoniation of the Cu,NH4‐samples by heating under shallow bed conditions in dry flowing nitrogen at 823 K for 12 h. The compositions of mixed cation samples, determined by Rietveld refinement of diffraction data, are given in the forms CuxHy‐Rho or CuxNay‐Rho, where x and y are the numbers of Cu2+ and H+ or Na+ cations per unit cell, respectively.</p><p>The sample of Cu3.4Na3.0‐Rho for TEM/EDS analysis was crushed in a mortar and pestle, dispersed in ethanol and deposited on a holey carbon film supported on a copper grid. EDS measurements were carried out using a spherical aberration corrected (Cs‐corrected) FEI Titan Themis 200 transmission electron microscope equipped with a high brightness Schottky X‐FEG emitter and operated at 200 kV with a convergence angle of 20 mrad.</p><p>For EPR analysis, Cu1.0H7.8‐Rho, Cu2.1H5.6‐Rho and Cu4.9‐Rho samples were packed into 25 cm long, 0.4 cm diameter quartz EPR tubes (1 cm length of sample) and dehydrated on the glass line at 623 K at 5×10−5 mbar for 10 h before flame‐sealing. Measurement was performed in an ELEXSYS Super High Sensitivity Probehead (Bruker ER4122SHQE) using a Bruker EMX 10/12 spectrometer operating at 9 GHz with 100 kHz modulation frequency. The EPR spectra were recorded at 295 K using 2 mW microwave power, a 3000 G field sweep centred at 2700 G with 3000 points resolution, a time constant and conversion time of 40.96 ms each, and a modulation amplitude of 3 G.</p><p>For XPS analysis, Cu4.9‐Rho was measured before and after heating at 453 K under vacuum for 10 h, using a Scienta 300 spectrometer operating at or below 1×10−9 mbar. The X‐ray source is an SPECS monochromated Al Kα source (photon energy 1486.6 eV) operating at approx. 12 kV and 200 watts. The instrument maintains a pass energy set to 150 eV for all spectra. Survey scans were collected at a dwell time of 133 msec, step size 200 meV and 2 scans were added. Detailed scans were 2 to 5 scans depending on the S : N ratio, a dwell time of 533 msec and a step size of 20 meV. Commercially available CuO was measured as a standard. The FWHM of the Ag 3d5 peak at 368.4 eV is routinely below 0.55 eV with a similar value for Au 4f7 at 84 eV and experimental drift as a function of time is negligible over a period of 24 h.</p><p>The crystallinity of as‐prepared, cation‐exchanged and dehydrated samples was confirmed by laboratory powder X‐ray diffraction (PXRD) using a Stoe STAD I/P diffractometer with Cu Kα1 X‐radiation (1.54056 Å). To determine the structure of dehydrated zeolites, the powders were loaded into 0.7 mm quartz capillaries and dehydrated at 623 K at 5×10−5 mbar on a glass vacuum line for 10 h. The PXRD patterns of the dehydrated samples were obtained from these loaded and sealed capillaries. Additionally, synchrotron X‐ray powder diffraction at beamline I11 of the Diamond Light Source was performed on Cu,Na‐Rho and Cu4.9‐Rho samples.</p><p>The structures were determined by Rietveld refinement against the PXRD, using the GSAS suite of programs. [45] For the zeolite Rho, Im3‾m and I4‾3ms ymmetries, starting framework models were adapted from the literature with the unit cell modified to that derived from the diffraction patterns. [22] Samples with unit cell parameter a equal to and above 15.0 Å were refined in Im3‾m symmetry and those below 15.0 Å in I4‾3m symmetry. The background for all patterns was fitted by an 8‐term shifted Chebyshev function. The framework atomic positions were initially refined with geometric restraints on T−O (T=Si or Al; 1.64±0.02 Å) and O−O (2.65±0.02 Å) distances to maintain regular tetrahedral coordination. Three starting extra framework cation sites: single 6‐ring (S6R), single 8‐ring (S8R) and double 8‐ring (D8R) were estimated from literature models and their fractional occupancies and atomic coordinates refined. [22] No electron density in D8R sites was found for any refined samples. For the Cu,H‐Rho series and Cu4.9‐Rho sample, all electron density in S6R and S8R sites was attributed to Cu2+ cations. For mixed cation Cu,Na‐Rho samples, the Na+ and Cu2+ cations can simultaneously occupy S6R and S8R sites, therefore a combination of Rietveld refinement and compositional analysis was applied. For Cu1.0Na7.8‐Rho and Cu3.4Na3.0‐Rho samples, the Na+ cations are known to preferentially occupy the S8R site[ 9 , 22 ] in the dehydrated Rho structure therefore, they were firstly refined in that site and Cu2+ cations in the S6R site. Additionally, from the Fourier mapping analysis for Cu1.0Na7.8‐Rho extra scattering was observed in the S6R site, therefore Na+ cations were added at the position at which they occupy this site in Na9.8‐Rho and refined. The crystallographic data for all RHO structures is given in the Supporting Information and cif files.</p><p>CO2 and N2 adsorption isotherms were measured volumetrically at 298 K and 77 K, respectively, using a Micromeritics ASAP 2020 Gas Adsorption Analyzer connected to a Julabo F25 Chiller Unit. The samples were activated to 573 K at 5 K min−1 under vacuum and held at this temperature for 6 h before cooling and measurement. At each adsorption or desorption step the pressure was sampled every 7 s until no further change is observed, so that step times ranged from 10 to 100 min.</p><p>Additionally, high pressure CO2 adsorption isotherms from 0–10 bar at 283, 298 and 313 K, used to calculate the isosteric heats of adsorption, were measured gravimetrically on a Hiden Intelligent Gravimetric Analyzer (IGA). All samples were activated at 573 K for 6 h prior to measurements. The mass change for each adsorption/desorption step was followed, and a final reading was taken when it had reached 98 % of the asymptotic equilibrium value or after 90 min, whichever was shorter. The isosteric heats of adsorption for Cu4.9‐Rho and Cu3.4Na3.0‐Rho samples were determined using the Clausius‐Clapeyron equation at uptakes from 1.5 to 3.5 mmol g−1. The isotherms were first fitted by virial equations using Desmos software [46] and subsequently pressures giving specific uptakes were obtained from these fits. The heats of adsorption for Na9.8‐Rho sample were sourced from previously published data. [9]</p><p>The Zero‐Length Column (ZLC) experimental setup is described in detail in ref.. [47] In summary, small amounts (5–10 mg) of (Na,Cu)‐Rho sample were packed into a 1/8" stainless steel union (Swagelok®), fitted with two porous metal discs to keep the powder in place. The column and gas connections are placed either within an oven (Carbolite) with thermostatic control (Eurotherm) or inside a cooling jacket, connected to a thermostatic bath for temperature control (Julabo F‐25). The pure helium carrier and dosing gas mixtures (1–10 vol.% CO2 in helium) are supplied through mass flow controllers (Brooks Instrument) and a combination of four solenoid valves is used to direct either of the two gas streams to the ZLC. Both helium (BOC, CP grade, 99.999 % purity) and CO2 (BOC, 99.8 % purity) are additionally dried using columns packed with a combination of silica gel and zeolite 5 A molecular sieve. The gas leaving the ZLC is analysed by mass spectrometry (Dycor Residual Gas Analyzer, Ametek Process Instruments). Prior to ZLC measurements, the sample was activated overnight at 473 K under a flow of helium.</p><p>The ZLC method is in essence a chromatographic technique, whereby the desorption of a previously equilibrated adsorbent is monitored.[ 48 , 49 ] Equilibration occurs in the dilute mixture of adsorbate in inert carrier, whereas desorption takes place in the pure inert carrier. The small amount of sample allows for neglecting external mass and heat transfer resistances and the short length of the column allows for treating the system as a well‐mixed cell (CSTR), due to negligible axial concentration gradients. [50] Pressure drops are additionally assumed to be negligible and the system is treated as being isothermal.</p><p>Breakthrough experiments were used to assess the materials' potential for gas separation. In these experiments, a special "elongated" version of the zero‐length column (E‐ZLC) is used. The E‐ZLC consists of a Swagelok 1/8′′ bulkhead union with an internal diameter of 2.286 mm and a length of 25.9 mm. As a result the columns can hold up to five times the amount of sample that is normally used in a typical ZLC experiment, allowing a clear identification of the separation performances. Apart from the extended column, the experimental apparatus used for this study is identical to the ZLC setup described above. The experiments were carried out at 308 K at ambient pressure and at different flow rates, i. e. 1, and 2 mL min−1, in a gas mixture which is taken as representative for a CO2 containing natural gas, with composition 10 % CO2/40 % CH4 (BOC, 99.995 % purity)/50 % He. In order to minimize the pressure drop across the column, the samples were made as binderless pellet fragments of ca. 2 mm in size. The amounts of material used was 41.2 mg for Cu4.9‐Rho, 37.6 mg for Cu3.4Na3.0‐Rho and 36.8 mg for Na9.8‐Rho.</p><p>The selectivity is described by the following Equation (1), where qi is the equilibrium amount adsorbed for component i at its partial pressure,pi in the binary system. The equilibrium amounts adsorbed are determined by appropriate integration of the desorption curves of the breakthrough experiments. (1) αCO2/CH4=qCO2/pCO2qCH4/pCH4</p><p>To enable analysis of both breakthrough and ZLC results, blank runs were also carried out. [51] These consist of repeating the column experiments under the same conditions as described above, but without adsorbent. In this case the column is filled with 2 mm glass beads to give a pressure drop and void fraction close to that observed in the presence of the samples. This allows for measuring the dead volume and intrinsic kinetics of the system when no adsorption occurs.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supporting Information</p><p>Click here for additional data file.</p>

PubMed Open Access

Stereoselective synthesis of trans-fused iridoid lactones and their identification in the parasitoid wasp Alloxysta victrix, Part I: Dihydronepetalactones

Starting from the enantiomers of limonene, all eight stereoisomers of trans-fused dihydronepetalactones were synthesized. Key compounds were pure stereoisomers of 1-acetoxymethyl-2-methyl-5-(2-hydroxy-1-methylethyl)-1-cyclopentene. The stereogenic center of limonene was retained at position 4a of the target compounds and used to stereoselectively control the introduction of the other chiral centers during the synthesis. Basically, this approach could also be used for the synthesis of enantiomerically pure trans-fused iridomyrmecins. Using synthetic reference samples, the combination of enantioselective gas chromatography and mass spectrometry revealed that volatiles released by the endohyperparasitoid wasp Alloxysta victrix contain the enantiomerically pure trans-fused (4R,4aR,7R,7aS)-dihydronepetalactone as a minor component, showing an unusual (R)-configured stereogenic center at position 7.

stereoselective_synthesis_of_trans-fused_iridoid_lactones_and_their_identification_in_the_parasitoid

<!>Introduction<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!>Route to trans-fused dihydronepetalactones a and b starting from (S)-pulegone<!><!>Route to trans-fused dihydronepetalactones a and b starting from (S)-pulegone<!><!>Route to trans-fused dihydronepetalactones a and b starting from (S)-pulegone<!>Route to stereochemically pure trans-fused dihydronepetalactones from (R)-limonene<!><!>Route to stereochemically pure trans-fused dihydronepetalactones from (R)-limonene<!>Synthesis of the key intermediate 16<!><!>Synthesis of trans,trans-dihydronepetalactone b<!><!>Synthesis of trans,trans-dihydronepetalactone b<!><!>Synthesis of trans,trans-dihydronepetalactone b<!>Synthesis of cis,trans-fused dihydronepetalactone c<!><!>Synthesis of cis,trans-fused dihydronepetalactone c<!><!>Synthesis of cis,trans-fused dihydronepetalactone c<!>Synthesis of a mixture of trans-fused dihydronepetalactones c and d<!><!>Synthesis of a mixture of trans-fused dihydronepetalactones c and d<!>Formal synthesis of a mixture of trans-fused dihydronepetalactones a and b from (R)-limonene<!><!>Formal synthesis of a mixture of trans-fused dihydronepetalactones a and b from (R)-limonene<!>Conclusion<!>Identification of a trans-fused dihydronepetalactone in the parasitoid wasp Alloxysta victrix<!>

<p>This article is part of the Thematic Series "Biosynthesis and function of secondary metabolites". Part II [1] describes the synthesis of enantiomerically pure trans-fused iridomyrmecins by this approach.</p><!><p>The endohyperparasitoid wasp Alloxysta victrix is part of the tetratrophic system of oat plants (Avena sativa), grain aphids (Sitobion avenae), primary parasitoids (Aphidius uzbekistanicus) and hyperparasitoids (Alloxysta victrix). Chemical communication by volatile signals is considered to play a major role in interactions between these trophic levels, and some semiochemicals of the lower trophic levels such as oat plant and grain aphid have been identified [2–4]. However, there is nearly no information about the intra- and interspecific signaling pathways between primary parasitoids and hyperparasitoids. In order to gain further information about the chemical structures and the biological significance of corresponding signals, we examined the volatile components of pentane extracts from dissected heads as well as headspace volatiles of Alloxysta victrix by coupled gas chromatography/mass spectrometry (GC/MS). Figure 1 shows one major component and several trace components which could be identified as 6-methyl-5-hepten-2-one (1), neral (2), geranial (3), actinidine (4), and geranylacetone (5). Bioassays revealed the main compound 1 to be repellent to the aphid-parasitoid, Aphidius, by warning the primary parasitoid of the presence of the hyperparasitoid [5]. The prenyl-homologue of 1, geranylacetone (5), seems to be a component of the sex pheromone of Alloxysta victrix [6]. In addition, GC/MS of cephalic secretions of both sexes showed a minor component X, the mass spectrum of which suggested it to be a trans-fused dihydronepetalactone. Since no synthetic reference compounds were available, we had to synthesize all eight trans-fused dihydronepetalactones to unambiguously identify the natural product X. The realization of this task is the subject of the present paper.</p><!><p>Terpenoids 1–5 present in Alloxysta victrix and cis-fused bicyclic iridoids known from other insects (6–8).</p><!><p>Apart from a couple of known acyclic terpenoids (Figure 1), analysis by gas chromatography coupled with mass spectrometry (GC/MS) revealed the presence of an unknown minor component X in both sexes of Alloxysta victrix. Chemical ionization analysis (GC/CIMS) showed the molecular mass of the compound to be M+ = 168, while high resolution mass spectrometry (GC/HRMS) proved its atomic composition to be C10H16O2, suggesting an oxygenated monoterpene as the target structure. The fragmentation pattern, exhibited in the 70 eV EI-mass spectrum (Figure 2), showed some similarities to that of the known cis-fused dihydronepetalactone (6) [7], however, differences in relative abundances of fragment ions pointed to a trans-fused dihydronepetalactone as the target structure [8]. In the mass spectrum of the cis-fused compound m/z 67 and m/z 95 were of similarly low intensity (30%), while in that of the unknown natural product X the two fragments were highly abundant (80%). The most striking differences in the spectra were pronounced signals for the molecular ion M+ = 168 and M+ − 15 (70% and 40%, respectively) for the cis-fused dihydronepetalactone whilst in the spectrum of X the two signals were of only low abundance (Figure 2).</p><!><p>70 eV EI-mass spectrum of the iridoid X, a component of the volatile secretions of the parasitoid wasp Alloxysta victrix.</p><!><p>Dihydronepetalactones are derivatives of nepetalactone (7) which was first isolated by Mc Elvain in 1941 from the essential oil of catmint, Nepeta cataria (Figure 1) [9]. Relative configurations of cis-fused nepetalactones and some related derivatives have been investigated [10–11]. Nepetalactone and cis- as well as trans-fused dihydronepetalactones have been isolated from the leaves and galls of the plant Actinidia polygama [8]. In addition, dihydronepetalactones are components of the defensive secretions of some ant species [12], while nepetalactone and the corresponding lactol showing (1R)-configuration have been identified as pheromones of aphids [13–14]. (1R,4S,4aR,7S,7aR)-Dihydronepetalactol (8) was characterized as a semiochemical for lacewings [15].</p><p>The dihydronepetalactone skeleton shows four contiguous stereogenic centers, giving rise to eight trans-fused stereoisomers a–d and the corresponding enantiomers a'–d' (Figure 3).</p><!><p>Structures and gas chromatographic retention times of trans-fused dihydronepetalactones on a conventional FFAP column (FFAP) and on an enantioselective cyclodextrin column (cyclo). For experimental details see Supporting Information File 1. The racemates b/b' and d/d', which coeluted on FFAP, could be separated from each other on DB5 (data not shown).</p><!><p>Whilst several stereoselective syntheses of the relatively widespread and well known cis-fused nepetalactone and its dihydro derivatives have been carried out [16–19], only very few approaches specifically aiming at the synthesis of trans-fused iridoid lactones have been published. Starting from (S)-pulegone (9) or its enantiomer, Wolinsky [20–21] described a route to this group of iridoids that can be applied to synthesize pure stereoisomers of dihydronepetalactones as well as the structurally related iridomyrmecins, another class of iridoids. However, Wolinsky's method suffers from several major disadvantages such as high costs of (S)-pulegone and difficult separations of diastereomeric mixtures. Therefore, as an alternative, we present a novel stereoselective route to trans-fused dihydronepetalactones starting from pure, cheaply available enantiomers of limonene.</p><!><p>For comparison, the synthesis of a and b was carried out following Wolinsky's approach: (S)-Pulegone (9) was transformed to trans-pulegenic acid 10 via bromination, Favorskii rearrangement, and subsequent elimination (Scheme 1). Stereoselective addition of hydrochloric acid afforded the chloride 11, and subsequent elimination of hydrochloric acid gave a mixture of the methyl esters 12 and 13 (methyl trans-pulegenate) [20–22] which could be separated by chromatography on silica gel. Hydroboration and lactonization of 12 furnished a mixture of the C4-epimers a and b that once again needed to be separated by chromatography on silica gel [23].</p><!><p>Route from (S)-pulegone to the mixture of dihydronepetalactones a and b, consequently following Wolinsky's approach [23]. Reaction conditions: a) Br2, HOAc, 0 °C; b) KOH, H2O, reflux (15%, 2 steps); c) MeOH/HCl, rt, 96 h (82%); d) 2,6-lutidine, reflux, 72 h (75% of 12 + 13); e) BH3·SMe2, 0 °C, THF, NaOH, H2O2 (86% mixture of diastereomers); f) p-TsOH, toluene, reflux (17% of a + b).</p><!><p>Analytical data of the first eluting component a were in accordance with those reported in the literature [24]. The same sequence starting from (R)-pulegone yielded a mixture of diastereomers a' and b'. The relative configuration of a at C4 was assigned according to NOESY experiments. Decisive NO-effects were found between the protons 4-H and 7a-H as well as between 7a-H and 7-CH3 (Figure 4).</p><!><p>Configuration of the dihydronepetalactone a.</p><!><p>Basically, the sequence developed by Wolinsky could also provide access to the diastereomers c and d (and their enantiomers) if trans-pulegenic acid (10) would be replaced by cis-pulegenic acid. A mixture of the latter and its trans-isomer (60:40) can be obtained by using a different base in the Favorskii-rearrangement step [22], again requiring a difficult chromatographic separation. Furthermore, this multistep route has several major disadvantages: The formation of mixtures of epimers entails to separations at several stages which have proven to be problematic. Moreover, several reaction steps afford unsatisfactory yields [23]. In addition, one of the main disadvantages is the fact that (S)-pulegone (S-9) is a highly expensive starting material for the synthesis of four of the eight trans-fused dihydronepetalactones. That excludes this route for the synthesis of larger amounts.</p><!><p>Due to the shortcomings of the route described above, we designed an improved strategy towards trans-fused dihydronepetalactones. Starting from 1-formyl-2-methyl-5-(1-methylethenyl)-1-cyclopentene (15) as the key intermediate, the stereoselective synthesis of all eight stereoisomers could be achieved (Figure 5). The aldehyde 15 could be readily prepared from commercially available pure and cheap (R)-limonene (14) [25–27]. Non-selective hydroboration of the double bond in the side chain of 15 would yield a pair of diastereomers 16/16* which would have to be separated. However, we expected that the chiral center at C5 would cause stereocontrol by forcing the reaction to proceed through the sterically least hindered transition state. We envisioned that stereoselective hydrogenation of the endocyclic double bond of the key intermediate 16 (and its diastereomer 16*) in either a "syn"- or "anti"-fashion could yield two pairs of diastereomeric hydroxy carboxylic acids 17/17* or 18/18* after some simple functional group transformations. These hydroxy acids would then yield the desired trans-fused dihydronepetalactones a–d during a final lactonization step.</p><!><p>Route to stereochemically pure trans-fused dihydronepetalactones a–d from (R)-limonene.</p><!><p>Starting from cheap and pure (S)-limonene (14'), the corresponding trans-fused dihydronepetalactones a'–d' could be synthesized in the same way, showing our novel route to be a versatile alternative to Wolinsky's sequence [20–21]. In contrast to the latter, which fixed the stereogenic center of pulegone at position 7 of the final dihydronepetalactone, in our route the stereogenic center of limonene is retained at position 4a of the target compound and used for the stereoselective introduction of additional chiral centers.</p><!><p>The synthesis of the key intermediate 16 – which shows two differentiated primary alcohol functions – started from enantiomerically pure (R)-limonene (14, Scheme 2). Ozonolysis followed by reductive workup with dimethyl sulfide produced (3R)-3-(1-methylethenyl-6-oxoheptanal), which yielded the formyl cyclopentene 15 upon intramolecular aldol condensation [25–27]. Subsequently, the aldehyde 15 was reduced to the allylic alcohol 19 with LiAlH4 and converted into the acetate 20 [28]. Hydroboration of 20 using disiamylborane proceeded with high stereoselectivity affording 16 as a single stereoisomer [17,28]. Similar results of highly stereoselective hydroborations of structurally related chiral cyclopentene derivatives have been reported [20–21].</p><!><p>Synthesis of the key compound 16. Reaction conditions: a) O3, MeOH, −50 °C (86%); b) AcOH, piperidine, C6H6, reflux (85%); c) LiAlH4, Et2O (82%); d) Ac2O, pyridine, rt (91%); e) B2H6, 2-methyl-2-butene, 0 °C, THF, NaOH, H2O2 (66%).</p><!><p>To install a trans,trans-configuration between the substituents at C5-C1 and C1-C2 of the cyclopentane backbone – which would later reflect the trans,trans relationship between substituents at C7-C7a and at C7a-C4a of the dihydronepetalactones a and b – a formal "anti"-addition of hydrogen to the cyclopentene 16 had to be carried out (Scheme 3). Usually, both homogeneous and heterogeneous catalytic hydrogenation reactions proceed via "syn"-addition of hydrogen to olefinic double bonds. Only subsequent isomerization processes may lead to a formal "anti"-addition. To obtain a suitable precursor, which, due to enolization of the hydrogenation product, might allow this formal "anti"-addition of hydrogen, the key intermediate 16 was transformed to the aldehyde 23.</p><!><p>Synthesis of trans,trans-substituted dihydronepetalactone b. Reaction conditions: a) TBDMSCl, imidazole, DMF, 0 °C (99%); b) KOH, MeOH, 0 °C (87%); c) PDC, MS, CH2Cl2, 0 °C to rt (61%); d) ammonium formate, Pd/C, MeOH, reflux (48%); e) KMnO4, t-BuOH, pH 4.5, 0 °C (22%); f) TBAF, THF (100%); g) DCC, DMAP, CH2Cl2 (66%).</p><!><p>In the course of this short sequence, the free hydroxy group of 16 was protected as the TBDMS ether to yield 21 which afforded the mono-protected diol 22 after treatment with KOH in methanol. Subsequently, 22 was oxidized with pyridinium dichromate to give aldehyde 23.</p><p>We expected that catalytic hydrogenation of the trisubstituted cylopentene 23 with a heterogeneous catalyst would preferentially take place from the sterically less hindered side of the molecule. This would lead to an all-cis configured hydrogenation product which would endure considerable steric strain. Due to the CH-acidity at the α-position of the formyl group, epimerization of the all-trans product 24 under acidic or basic conditions could be expected. Lange et al. reported the catalytic hydrogenation of a structurally close analogue, (5R)-1-formyl-2-methyl-5-isopropylcyclopent-1-ene, over Pd/C (10%) to give a 9:1 mixture of the all-cis versus the all-trans product [29]. In our case, the application of Lange's method to the aldehyde 23 led to the formation of a 3:1 mixture of the all-cis versus the all-trans epimer. Subsequent treatment with sodium methoxide in MeOH at rt for 20 h completely shifted the equilibrium to the thermodynamically more stable all-trans product 24. Unfortunately, these results could not be reproduced on larger reaction scales (>5 mmol). After screening of a variety of other hydrogenation conditions, we found the hydrogenation of 23 with ammonium formate over palladium on carbon (10%) to be the method of choice [30]. Using this approach, the all-trans aldehyde 24 was almost exclusively formed. The presence of ammonium formate in the reaction mixture probably leads to an "in-situ" epimerization at C2 from the kinetically formed all-cis to the thermodynamically more stable all-trans product. Relative configurations of all substituents of compound 24 were confirmed by NOESY experiments (Figure 6). Strong NO-effects were found between the protons 5-CH3 and 1-H, 1-H and 1'H (the proton at C1 of the side chain), 5-H and 2-H as well as between protons of 1-CHO and 2-H which is in line with a trans,trans-configuration (using the nomenclature described above).</p><!><p>Configurations of compound 24 and the dihydronepetalactone b.</p><!><p>Starting from 24, the trans,trans-dihydronepetalactone b was synthesized in three subsequent steps (Scheme 3). First, oxidation of the aldehyde group with potassium permanganate in the presence of a phosphate buffer (pH 4.5) afforded the carboxylic acid 25 without epimerization at C1 [23,31]. Subsequent deprotection of the TBDMS ether with tetrabutylammonium fluoride (TBAF) yielded 17, and lactonization with N,N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in dichloromethane afforded the trans,trans-dihydronepetalactone b.</p><p>The relative configuration of the trans,trans-dihydronepetalactone b was confirmed by NOESY experiments. Decisive NO-effects could be observed between 4-CH3 and 7a-H as well as between 4-CH3 and 5-Ha, and furthermore, between 5-Ha and 7a-H as well as between 7a-H and 7-CH3 (Figure 6). The enantiomer b' was synthesized via the same route starting from (S)-limonene. Analytical data of compound b' were identical to those which were obtained of b when Wolinky's route was followed (see above).</p><!><p>For the synthesis of the cis,trans dihydronepetalactone c, a cis,trans-configuration between the substituents at C5-C1 and C1-C2 of the cyclopentane backbone needed to be established. With acetate 16 as the key intermediate, a stereoselective "syn"-addition of hydrogen from the same side as the (R)-configured side chain at C5 would provide the desired stereochemical outcome of the hydrogenation reaction (Scheme 4). We expected the free hydroxy group of 16 to coordinate to an appropriate homogenous hydrogenation catalyst, controlling the stereochemical course of the hydrogen transfer from the same side as the side chain at C5 through chelation. We chose Crabtree's iridium catalyst ([Ir(cod)PCy3(py)]PF6) which has been reported to furnish excellent facial selectivities during directed hydrogenations of cyclic olefins [32–34].</p><!><p>Synthesis of cis,trans-substituted dihydronepetalactone c. Reaction conditions: a) Crabtree's catalyst [Ir(cod)PCy3(py)]PF6 (11 mol %), 1 bar H2, CH2Cl2, rt (81%); b) TBDMSCl, imidazole, DMF, 0 °C (100%); c) KOH, MeOH, rt (93%); d) RuCl3·3H2O (2 mol %), NaIO4, CCl4, CH3CN, phosphate buffer (pH 7), rt (81%); e) HF, CH3CN, rt (100%); f) DCC, DMAP, CH2Cl2 (66%).</p><!><p>Hydrogenation of acetate 16 in the presence of 11 mol % of Crabtree's catalyst under 1 bar hydrogen pressure for 1.5 h yielded the desired product 26 as a single diastereomer. Alternative hydrogenation methods using optically active catalysts failed. In one case we investigated the hydrogenation of the endocylic double bond of the allylic alcohol 19 (Scheme 2) with one of Noyori's ruthenium BINAP catalysts ([Ru((S)-BINAP)](OAc)2) [35–36] but reduction occurred only at the side chain.</p><p>Relative configurations of all substituents of the acetate 26 were confirmed by NOESY experiments (Figure 7). Strong NO-effects were observed between the protons 5-CH3 and 1'-H (protons of the acetoxymethyl group at C1), 5-CH3 and 2-H, 1'-H and 2-H as well as 5-H and 1''-CH3 (protons of the methyl group at C1 of the side chain) which is in line with a cis,trans-configuration (using the nomenclature described above).</p><!><p>Configurations of compound 26 and the dihydronepetalactone c.</p><!><p>Starting from 26, the synthesis of the cis,trans-dihydronepetalactone c was completed in five subsequent steps (Scheme 4). First, the free hydroxy group of 26 was protected as the TBDMS ether to yield 27. Then, the acetate group was removed with methanolic KOH to afford the alcohol 28. Careful oxidation of the primary alcohol function [37–38] with ruthenium(III) chloride and sodium periodate in a biphasic mixture of carbon tetrachloride, acetonitrile, and phosphate buffer (pH 7) produced the carboxylic acid 29 without epimerization at C1. After removal of the TBDMS protecting group with HF in acetonitrile, the hydroxy acid 18 was lactonized in the presence of DCC and catalytic amounts of DMAP in dichloromethane at rt to afford cis,trans-dihydronepetalactone c. Its enantiomer c' was synthesized from enantiomerically pure (S)-limonene, following the same route. The relative configuration of c was confirmed by NOESY experiments (Figure 7). Decisive NO-effects could be observed between 4a-H and 7-CH3 as well as between 4-CH3 and 7a-H, and furthermore, between 4-CH3 and 5-Ha as well as between 5-Ha and 7a-H.</p><!><p>The stereogenic center at C1' of the acetate 26 keeps (R)-configuration which resulted from highly stereoselective hydroboration of the acetate 20 to yield the key intermediate 16 as shown above (Scheme 2). For the synthesis of the cis,trans-dihydronepetalactone d, this stereocenter needed to be isomerized to keep the (4S)-configuration in the final product (Scheme 5). To achieve the required inversion, the acetate 26 was oxidized to the aldehyde 30, which could be epimerized using p-toluenesulfonic acid in benzene under reflux conditions to provide a 2:3 mixture of the desired aldehydes 30 and its epimer 30*. Subsequent steps were carried out with the mixture of diastereomers. Reaction of 30/30* with sodium borohydride at −20 °C reduced the aldehyde function to yield a mixture of the diastereomers 26/26*. The following sequence, yielding a mixture of the dihydronepetalactones c and d was essentially the same as described above (Scheme 4).</p><!><p>Synthesis of a 2:3 mixture of dihydronepetalactones c and d. Reaction conditions: a) (COCl)2, DMSO, CH2Cl2, −70 °C to 0 °C (71%); b) p-TsOH, benzene, reflux (96%); c) NaBH4, MeOH, −20 °C; d) TBDMSCl, imidazole, DMF, 0 °C (78%) (over two steps); e) KOH, MeOH, rt (94%); f) RuCl3·3H2O (2 mol %), NaIO4, CCl4, CH3CN, phosphate buffer (pH 7), rt (69%); g) HF, CH3CN, rt (98%); h) DCC, DMAP, CH2Cl2 (62%).</p><!><p>Transformation of the free hydroxy group to the TBDMS ethers 27/27* was followed by cleavage of the acetate moiety with methanolic KOH to give a mixture of the alcohols 28/28*. Oxidation of the primary alcohol function with ruthenium(III) chloride and sodium periodate in a biphasic mixture of carbon tetrachloride, acetonitrile and phosphate buffer (pH 7) afforded the carboxylic acids 29/29* without epimerization at C1 [37–38]. After cleavage of the TBDMS ether with HF in acetonitrile, a mixture of dihydronepetalactones c and d was formed by lactonization of the hydroxy acids 18/18* with DCC and DMAP in dichloromethane at rt. The C4 epimeric dihydronepetalactones c and d could be separated by column chromatography over silica. Starting from the enantiomer of 26, a mixture of dihydronepetalactones c' and d' was synthesized by following the same reaction sequence.</p><!><p>As outlined above, six of the eight possible stereoisomers of trans-fused dihydronepetalatones were synthesized from the enantiomers of limonene following a new route. Compound a and its enantiomer a' were prepared according to the procedure described by Wolinsky [20–21]. However, our new approach also includes a formal synthesis of a and a'. A mixture of a and b will be easily obtained from the protected hydroxy aldehyde 24 by the straight forward procedure outlined in Scheme 6.</p><!><p>Formal synthesis of a mixture of dihydronepetalactones a and b from (R)-limonene.</p><!><p>Reduction of the aldehyde function of 24 and acetylation of the resulting primary alcohol followed by cleavage of the silyl group will furnish the primary alcohol 31, which upon oxidation will yield the corresponding aldehyde that can be epimerized to the diastereomers 32/32* as shown above. Subsequent reduction of 32/32*, silylation of the resulting primary alcohols and saponification will produce a mixture of the diastereoisomers 33/33*. Oxidation of the primary alcohol moiety, followed by cleavage of the silyl group will yield the epimeric hydroxy acids 34/34* which will form a mixture of the dihydronepetalactones a and b after lactonization. As shown above, this mixture can be separated upon column chromatography.</p><!><p>In summary, we synthesized all eight trans-fused stereoisomeric dihydronepetalactones. After having used the enantiomers of pulegone as educts in Wolinsky's route to (4S,4aS,7S,7aR)-dihydronepetalactone (a) and its enantiomer a' [23], we developed an improved and general way for the synthesis of all trans-fused dihydronepetalactones, starting from pure enantiomers of limonene. Our approach is also superior to that starting from optically active carvone that yields the starting material for the synthesis of trans-fused iridoid lactones only as a byproduct [15].</p><!><p>Upon gas chromatography using FFAP as a polar achiral stationary phase, the stereoisomers a and c could be well separated while b and d coeluted. However, the latter pair could be resolved on a less polar DB5-capillary, where b/b' eluted after d/d' (data not shown). As a result, the relative configuration of each of the trans-fused dihydronepetalactones could be unambiguously assigned by GC/MS.</p><p>With the exception of (4S,4aS,7R,7aR)-dihydronepetalactone (d) and its enantiomer d', the stereoisomers could well be distinguished by enantioselective gas chromatography using a 1:1-mixture of OV1701 and heptakis-(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin as an optically active stationary phase. Figure 3 shows the corresponding retention times of all eight stereoisomers that were obtained with the two used capillary column systems. Coupled GC/MS using FFAP as the stationary phase revealed the target natural iridoid lactone X to show the same mass spectrum and the same retention time as a/a', the first eluting pair of the synthetic dihydronepetalactones (Figure 3). Enantioselective gas chromatography on a cyclodextrin column showed X to coelute with a' which was well separated from its enantiomer by an α-value of a':a = 1.01 (Figure 3). Consequently, the structure of X was unambiguously assigned to be (4R,4aR,7R,7aS)-dihydronepetalactone. It should be noted that Meinwald et al. identified a/a' (absolute configuration not assigned) as a component of secretions of the abdominal defense glands of the rove beetle Creophilus maxillosus [39]. Interestingly, the structure of a' is relatively close to that of nepetalactone 7 and lactol 8, the sex pheromone of the grain aphid S. avenae [13] which keeps the second level in the investigated tetratrophic system. Grant et al., found the trans-fused (1R,4aS,7R,7aR)-1-methoxy-4,7-dimethyl-1,4a,5,6,7,7a)-hexahydrocyclopenta[c]pyran, called (1R)-1-methoxymyodesert-3-ene, among the volatiles of the Ellangowan poison bush, which they transformed to the corresponding lactone a' [24]. Apart from this compound and very few others, the stereogenic center carrying the methyl group in the five-membered ring of iridoid lactones including insect semiochemicals [13–15] generally shows (S)-configuration. Only recently, two isomeric iridoid lactones showing (7R)-configuration have been identified from the Drosophila parasitoid Leptopilina heterotoma [40]. Compound X has been identified in the mandibular gland secretions of other Alloxysta species, too, [41]. However, its biological significance is not yet clear and will need further investigations.</p><p>The differentiation of the oxygen containing functional groups in the trisubstituted cyclopentene 16, a key-compound in our synthetic approach, provides access to a large number of iridoids including nepetalactones but also iridomyrmecins and monocyclic compounds. Consequently, having reference compounds at hand, structures of hitherto unknown iridoids [42] may now be assigned. It may turn out that the chiral center carrying the methyl group in the five-membered ring of iridoids may much more often show (R)-configuration than it is known today.</p><!><p>Experimental details and characterization data for synthesized compounds.</p>

PubMed Open Access

Heteroaromatic and aniline derivatives of piperidines as potent ligands for vesicular acetylcholine transporter

To identify suitable lipophilic compounds having high potency and selectivity for vesicular acetylcholine transporter (VAChT), a heteroaromatic ring or a phenyl group was introduced into the carbonyl-containing scaffold for VAChT ligands. Twenty new compounds with ALog D values between 0.53-3.2 were synthesized, and their in vitro binding affinities were assayed. Six of them (19a, 19e, 19g, 19k and 24a-b) displayed high affinity for VAChT (Ki = 0.93 \xe2\x80\x93 18 nM for racemates) and moderate to high selectivity for VAChT over \xcf\x831 and \xcf\x832 receptors (Ki = 44 \xe2\x80\x93 4400-fold). These compounds have a methyl or a fluoro substitution that provides the position for incorporating PET radioisotopes C-11 or F-18. Compound (-)-[11C]24b (Ki = 0.78 for VAChT, 900-fold over \xcf\x83 receptors) was successfully synthesized and evaluated in vivo in rats and nonhuman primates. The data revealed that (-)-[11C]24b has highest binding in striatum and has favorable pharmacokinetics in the brain.

heteroaromatic_and_aniline_derivatives_of_piperidines_as_potent_ligands_for_vesicular_acetylcholine_

Introduction<!>Chemistry<!>In Vitro Binding Studies<!>In Vivo Evaluation in Rats<!>MicroPET Studies in Monkeys<!>Conclusion<!>General<!>Resolution of (4-aminophenyl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)metha-none (6) to obtain the minus isomer (-)-6 and the plus isomer (+)-6<!>tert-Butyl 4-(methoxy(methyl)carbamoyl)piperidine-1-carboxylate (11)<!>4-Bromo-1-methylpyridin-2(1H)-one (15h)<!>6-Bromo-1-methylpyridin-2(1H)-one (15i)<!>tert-Butyl 4-(1-methyl-1H-pyrrole-2-carbonyl)piperidine-1-carboxylate (16a)<!>tert-Butyl 4-(1-methyl-1H-pyrrole-4-carbonyl)piperidine-1-carboxylate (16b)<!>tert-Butyl 4-nicotinoylpiperidine-1-carboxylate (16c)<!>tert-Butyl 4-(6-methylnicotinoyl)piperidine-1-carboxylate (16d)<!>tert-Butyl 4-(6-methoxynicotinoyl)piperidine-1-carboxylate (16e)<!>tert-Butyl 4-(3-methoxypicolinoyl)piperidine-1-carboxylate (16f)<!>tert-Butyl 4-(3-fluoronicotinoyl)piperidine-1-carboxylate (16g)<!>tert-Butyl 4-(1-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl)piperidine-1-carboxylate (16h)<!>tert-Butyl 4-(1-methyl-6-oxo-1,6-dihydropyridine-2-carbonyl)piperidine-1-carboxylate (16i)<!>(1-Methyl-1H-pyrrol-2-yl)(piperidin-4-yl)methanone (17a)<!>(1-Methyl-1H-pyrrol-3-yl)(piperidin-4-yl)methanone (17b)<!>Piperidin-4-yl(pyridin-3-yl)methanone (17c)<!>(6-Methylpyridin-3-yl)(piperidin-4-yl) methanone (17d)<!>(6-Methoxypyridin-3-yl)(piperidin-4-yl)methanone (17e)<!>(3-Methoxypyridin-2-yl)(piperidin-4-yl)methanone (17f)<!>(5-Fluoropyridin-3-yl)(piperidin-4-yl)methanone (17g)<!>1-Methyl-5-(piperidine-4-carbonyl)pyridin-2(1H)-one (17h)<!>1-Methyl-6-(piperidine-4-carbonyl)pyridin-2(1H)-one (17i)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(1-methyl-1H-pyrrol-2-yl)methanone (19a)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(1-methyl-1H-pyrrol-3-yl)methanone (19b)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(pyridin-3-yl)methanone (19c)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-methoxypyridin-3-yl)methanone (19d)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-methylpyridin-3-yl) methanone (19e)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(3-methoxypyridin-2-yl)methanone (19f)<!>(5-Fluoropyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)methanone (19g)<!>4-(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidine-4-carbonyl)-1-methylpyridin-2(1H)-one (19h)<!>6-(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidine-4-carbonyl)-1-methylpyridin-2(1H)-one (19i)<!>(6-Fluoropyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)methanone (19j)<!>(2-Fluoropyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)methanone (19k)<!>1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-N-methoxy-N-methylpiperidine-4-carboxamide (20)<!>1-(3-((tert-Butyldimethylsilyl)oxy)-1,2,3,4-tetrahydronaphthalen-2-yl)-N-methoxy-N-methylpiperidine-4-carboxamide (21)<!>3-(4-(4-Acetamidobenzoyl)piperidin-1-yl)-1,2,3,4-tetrahydronaphthalen-2-yl acetate (22)<!>N-(4-(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidine-4-carbonyl)phenyl)-N-methylacetamide (23)<!>(4-(Dimethylamino)phenyl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)methanone (24a)<!>(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-(methylamino)phenyl) methanone (24b)<!>Resolution of (1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-(methylamino)phenyl) methanone (24b) to obtain the entantiomerically minus isomer, (-)-24b and plus isomer (+)-24b<!>(1--3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-hydroxypyridin-3-yl)methanone (25)<!>(6-(2-Fluoroethoxy)pyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)methanone (26a)<!>(3-(2-Fluoroethoxy)pyridin-2-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)methanone (26b)<!>2-Bromo-3-(2-fluoroethoxy)pyridine (28)<!>(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(1-methyl-1Hpyrrol-2-yl)methanone (30a)<!>(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(pyridin-3-yl)methanone (30c)<!>(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-methylpyridin-3-yl)methanone (30d)<!>(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-methoxypyridin-3-yl)methanone (30e)<!>(1-(5-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(1-methyl-1H-pyrrol-2-yl)methanone (31a)<!>tert-Butyl (4-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidine-4-carbonyl)phenyl)-carbamate, (-)-33<!>Radiochemistry<!>Radiosynthesis of (-)-[11C]24b<!>Log D Measurement<!>Vesicular acetylcholine transporter binding assays<!>Sigma receptor binding assays<!>Biodistribution in Rats<!>MicroPET Studies in Nonhuman Primate Brain<!>MicroPET Image Data Processing and Analysis

<p>Dementia is a clinical syndrome characteristic of loss or decline in memory and other cognitive abilities that result in functional impairment in the elderly. It is a global public health problem.1, 2 The prevalence in the USA has been recently estimated at 5.4 million individuals, including 5.2 million over the age of 65, and 200,000 individuals under the age of 65.1 Increasing lifespan of people will lead to increased number of patients with dementia. The severity of dementia in neurodegenerative diseases is linked to loss of cholinergic neurons and synapses in the central nervous system (CNS). The use of 11C-labeled Pittsburgh Compound-B ([11C]PIB) to assess β-amyloid plaques and diagnose Alzheimer's disease (AD) was a significant breakthrough.3 However, [11C]PIB cannot specifically assess the loss of cholinergic neurons and synapses in the brain, which correlates to severity of cognitive dysfunction in AD.4-9 For Parkinson's disease associated with dementia (PDD), the cholinergic deficit in cortical regions is more severe than for non-demented patients with Parkinson's Disease (PD).10 A PET tracer that can assess the loss of cholinergic synapses would provide a useful tool for assessing the severity of cognitive dysfunction and monitoring the efficacy of cholinergic therapies for dementia in neurodegenerative disorders.</p><p>Vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) are essential for a cholinergic neuron.11 VAChT is localized in cholinergic terminals, and it transports acetylcholine (ACh) from the cytoplasm into the synaptic vesicles. Anti-ChAT preferentially stains cell bodies, whereas anti-VAChT preferentially stains nerve terminals.4, 12 Anti-ChAT is more useful for monitoring the death of cholinergic cells, whereas anti-VAChT is more useful for monitoring changes in the density of cholinergic terminals. It is widely accepted that VAChT is a reliable biomarker to study cholinergic function in the brain. Currently, (-)-5-[123I]iodobenzovesamicol ([123I]IBVM) is the only radiotracer used for imaging VAChT levels in living human brain using single-photon emission computed tomography (SPECT). The relative distribution for specific binding of [123I]IBVM in human brain corresponds well with postmortem results reported for ChAT.13, 14 When [123I]IBVM was used to assess cholinergic deficiency in patients with dementia, it was found that PDD and AD patients have globally reduced cortical binding.15 In addition, a significant decrease in [123I]IBVM binding (47-62%) in cingulate cortex and parahippocampalamygdaloid in AD subjects compared to control patients has been observed.16 However, the slow binding kinetics of [123I]IBVM requires scanning for approximately 6 hours post-injection, which can be stressful for patients. Positron emission tomography (PET) imaging will be able to carry out scans with higher sensitivity and spatial resolution (3-5 mm) compared to SPECT (10 mm).17, 18 The demand to provide higher accuracy in clinical imaging of VAChT levels in humans makes the identification of a PET tracer for VAChT very important. To date, this goal has not been achieved because of the lack of suitable PET radiotracers.</p><p>In efforts to develop a PET tracer for VAChT, investigators have put tremendous effort into optimizing the structure of vesamicol analogues with the goal of identifying highly potent and selective ligands.4, 19-27 Among various radioligands developed, only a small number have been evaluated in vivo in non-human primates and humans.19, 27-29 Despite promising in vitro, ex vivo and initial in vivo studies, most of the ligands are unsuitable for clinical use due to poor selectivity over σ receptors in brain, low extraction from the blood, slow brain kinetics or fast metabolism. Among the physicochemical properties of ligands, lipophilicity is a one of the key properties that plays a pivotal role in absorption, distribution, metabolism, and elimination of ligands.30 For central nervous system drugs, it was found that the blood-brain-barrier (BBB) penetration is optimal with the ALog D values in the range of 1.5 – 3.0, with a mean value of 2.5.31-33 Although other properties of compounds affect the BBB penetration, those ligands with moderate lipophilicity often exhibit highest brain uptake.30 Highly lipophilic radiotracers are usually cleared slowly from the brain.</p><p>Our group has reported a new class of VAChT inhibitors containing a carbonyl group attached to the 4 position of the piperidine ring and discussed the structure-activity relationship (SAR) of this new class of compounds.4, 19, 34-36 Among them (as shown in Figure 1), compounds 5, 7 and 8 displayed high potencies and good selectivity for VAChT in vitro.4, 19 In particular, a fluorine-18 labeled version of (-)-2-hydroxy-3-(4-(4-fluorobenzoyl)piperidino)tetralin, (-)-[18F]7 was successfully radiosynthesized and used to conduct in vivo evaluation in rats and monkeys; the initial results were very promising.19 The possibility that (-)-[18F]7 can serve as a clinical PET tracer for quantifying the level of VAChT in vivo is under investigation. The current manuscript focuses on 1) optimizing the structures of this new class of VAChT ligand to identify highly potent ligands having lipophilicity suitable to efficiently cross the BBB. 2) Separate the enantiomers of the optimal compound, and radiosynthesize with carbon-11 PET isotope. 3) In vivo evaluate the new C-11 radiotracer in rodent and non-human primate. The strategies to achieve optimization include: (1) replacing the thiophenyl group in (1-((2S,3S)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(thiophen-2-yl)methanone 9 with N-methyl pyrrole4 (the methyl group provides a position for conveniently labeling with carbon-11 isotope), (2) replacing the 4-fluorophenyl group in 74 with pyridine, substituted pyridines and pyrroles and optimizing the substitution so that radiolabeling with carbon-11 or fluorine-18 can be achieved, and (3) converting the primary amino group of compound 6 to monomethyl amino or dimethyl amino, which will provide access for radiolabeling with carbon-11. This investigation was inspired by (1) the observation that a series of carbonyl group containing analogues have high affinity for VAChT and low affinity for σ receptors;4, 19, 34 (2) our in vivo validation of (-)-[18F]719 and its analogue (-)-[11C]8,35 which showed high binding in the striatum, the VAChT enriched regions in the brain; and (3) the high demand for a clinically suitable PET probe for investigating the correlation between loss of cognitive function and loss of cholinergic synapses, which will help improve the early diagnosis of dementia and monitor the therapeutic efficacy of treating Alzheimer's and other neurodegenerative diseases.</p><!><p>The synthesis of a new series of vesicular acetylcholine transporter inhibitors was accomplished according to Schemes 1-5. t-Boc-protected piperidine-4-carboxylic acid (10) was treated with 1,1'-carbonyldiimidazole (CDI), and N,O-dimethylhydroxylamine to afford Weinreb amide 11, which served as a versatile intermediate for synthesizing piperidines bearing a substituted heteroaromatic carbonyl group at the 4-position. Bromo substituted heteroaromatic compounds were lithiated with n-butyllithium (n–BuLi) and reacted with Weinreb amide to afford intermediates 16a-i. When synthesizing N-methyl pyrrole derivative 16a, via treatment of N-methylpyrrole with n-BuLi36 followed by compound 11, it was observed that the yield was much higher if the reaction vessel was pre-cooled to -78 °C while adding n-BuLi. To synthesize N-methyl pyridine derivatives 16h and 16i, commercially available 4 or 6-bromine substituted pyridin-2-amines 12h and 12i were first converted to 2-hydroxypyridines 13h and 13i or 2-pyridones 14h and 14i with sodium nitrite in sulfuric acid37, 38 followed by methylation using iodomethane in acetone to form bromo substituted N-methyl-2-pyridones 15h and 15i as major products.39 These pyridones reacted with 11 to afford the corresponding carbonyl group containing intermediates 16h and 16i. Trifluoroacetic acid (TFA) was used to remove the t-Boc group in 16a-i to afford the key intermediates 17a-i. Treatment of 17a-i with epoxide 1a,2,7,7a-tetrahydronaphtho[2,3-b]oxirene (18)4 afforded the target compounds 19a-i as shown in Scheme 1. To confirm whether the methyl group was on the N- or O-atom of 19h and 19i, the X-ray crystal structure of the oxalate salt of 19i was obtained as shown in Figure 2. The X-ray crystal structure revealed that the methyl group is on the nitrogen.</p><p>Although direct condensation of substituted piperidines and epoxide 18 was a common procedure for synthesizing vesicular acetylcholine transporter inhibitors,19, 40 it proved challenging when synthesizing 19j and 19k due to the low solubility of substituted heteroaromatic piperidines in commonly used solvents such as methanol, ethanol, dichloromethane, DMF and DMSO. Thus, an alternative approach was followed in which the t-Boc group in compound 11 was removed first, and the resulting piperidine was treated with 18 to afford compound 20. The free hydroxyl group of compound 20 was protected with the tert-butyldimethylsilyl group by reacting with tert-butyldimethylsilyl chloride to afford compound 21. Commercially available 5-bromo-2-fluoropyridine (17j) or 3-bromo-2-fluoropyridine (17k) was treated with n-BuLi in THF followed by compound 21, and further removal of TBDMS group afforded target compounds 19j and 19k as shown in Scheme 2.</p><p>Compounds 24a and 24b were synthesized as depicted in Scheme 3. Compound 6 was synthesized following the reported procedure.19 Direct dimethylation of the aromatic amine in compound 6 with iodomethane in the presence of NaH afforded compound 24a very easily. However, the synthesis of the N-monomethylaniline analogue 24b was more complicated. The free hydroxyl group and the primary aniline group of 6 were protected by treating with acetic anhydride to form diacetylated compound 22.41 N-methylation of aromatic amide 22 gave intermediate 23, which upon hydrolysis afforded the target compound 24b.</p><p>Synthesis of fluoroethoxy analogues 26a and 26b followed two different approaches as shown in Scheme 4. The first approach was to demethylate 19e in the presence of boron tribromide (BBr3) or trimethylsilyl iodide (TMSI) to generate the corresponding alcohol 25, which was reacted with 1-bromo-2-fluoroethane to afford the target O-alkylated compound 26a. However, use of a similar approach to synthesize compound 26b was not successful with either BBr3 or TMSI to remove the methyl group in 19f. To overcome this challenge, an alternative approach was used. Commercially available 2-bromopyridin-3-ol (27) was reacted with 1-bromo-2-fluoroethane via O-alkylation to afford 2-bromo-3-(2-fluoroethoxy)pyridine (28). This compound was lithiated and treated with compounds 11, followed by 18 to afford target compound 26b following the procedure described for synthesis of compounds 19j and 19k.</p><p>To make aminobenzovesamicol (ABV) analogues containing substituted heteroaromatic carbonyl groups, namely 30a-d and 31a, the corresponding piperidine precursors 17a-d containing substituted heteroaromatic carbonyl groups were reacted with 2,2,2-trifluoro-N-(1a,2,7,7atetrahydronaphtho[ 2,3-b]oxiren-3-yl)acetamide (29), which was synthesized by following the literature protocol.4 The trifluoroacetyl protection group was then removed via hydrolysis in the presence of sodium hydroxide to afford the regioisomers 30a-d and 31a as shown in Scheme 5.</p><p>In vitro binding studies revealed that 24b was highly potent. Therefore, the (-)-24b and (+)-24b were obtained by separating the enantiomers on HPLC using chiralcel OD column. The precursor (-)-33 for the radiolabeling of (-)-[11C]24b was synthesized as shown in Scheme 6. Briefly, the enantiomeric separation of 6 was performed on chiral HPLC using Chiralcel OD column to give (+)-6 and (-)-6. The (-)-6 isomer was treated with Boc anhydride in the presence of triethylamine to give the tri-Boc protected intermediate (-)-32. 4-Dimethylaminopyridine (DMAP) was used in stoichiometric amount in this reaction. The tri-Boc protected compound upon treatment with potassium carbonate in methanol under reflux selectively deprotected one Boc group on the aniline nitrogen to give the precursor (-)-33, which was used for radiosynthesis of (-)-[11C]24b.</p><p>The radiosynthesis of (-)-[11C]24b was successfully accomplished in two steps from the precursor as shown in Scheme 7. The precursor was first reacted with [11C]CH3I to obtained the intermediate [11C]methylated Boc protected compound. This intermediate was then treated with trifluoroacetic acid in situ to obtain the Boc deprotected product (-)-[11C]24b in high radiochemical yield (40-50%). The new PET radiotracer obtained was subjected to biodistribution evaluations in Sprague–Dawley rats and microPET imaging studies in nonhuman primates.</p><!><p>VAChT binding was assayed using highly expressed human VAChT with gently homogenized and partially clarified PC12123.7 cells by displacement of bound 5 nM (-)-[3H]vesamicol. Apparent equilibrium dissociation constants (Ki, nM) are reported in Table 1. The σ1 and σ2 binding affinities were assayed in rat brain and in guinea pig membranes, respectively. Apparent equilibrium dissociation constants (Ki, nM) are reported in Table 1. The ligand selectivity is defined in terms of an index that is the Ki for σ1 or σ2 receptors divided by Ki for VAChT (Ki σ1/Ki VAChT or Ki σ2/Ki VAChT). A larger number represents good selectivity for binding to VAChT.</p><p>In the previous studies, we had reported that a new class of carbonyl group containing analogues displayed high potency and high selectivity for VAChT;4 particularly, compound (1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(thiophen-2-yl)methanone (9), which replaces the phenyl group in the structure of compound 5 with a 2-thiophene moiety, displays comparable affinity for VAChT, with a Ki value of 5.00 ± 1.20 nM. In the current study, to explore the structure-activity relationship, we first replaced the thiophen-2-yl group in compound 9 with 1-methyl-1H-pyrrol-2-yl or 1-methyl-1H-pyrrol-3-yl group to obtain compounds 19a and 19b. Both of these two compounds displayed moderately potent binding toward VAChT with the Ki values of 18.4 ± 2.5 and 26.6 ± 3.8 nM for compounds 19a and 19b, respectively (Table 1) for binding to VAChT. Compared to 9, compounds 19a and 19b displayed about 4-5-fold decrease in affinity for VAChT. However, the selectivity of 19a and 19b for VAChT versus σ1 receptor reached at least 71-fold and 114-fold respectively. More importantly, these modified structures (with a methyl group on the N-atom of the pyrrole) provide a site for labeling with carbon-11 via [11C]methyl iodide.</p><p>When the benzoyl group was replaced with pyridin-3-carbonyl, 6-methyl-pyridin-3-carbonyl, 6-methoxy-pyridin-3-carbonyl, or 6-fluoro-pyridin-3-carbonyl, compounds 19c, 19d, 19e, and 19j were obtained. The pyridine-3-carbonyl analogue 19c (Ki = 24.1 ± 2.8 nM) displayed 5-fold lower affinity for VAChT compared to its benzoyl counterpart 5 (Ki = 4.30 ± 1.00 nM). When the hydrogen on the 6-position of the pyridine-3-carbonyl group is replaced with a methyl group, methoxy group, or fluoride group, the order of the binding potency for VAChT is -OCH3> -F ≈ -CH3, with Ki values of 8.36 ± 0.68, 26.1 ± 2.4, and 27.9 ± 8.0 nM for compounds 19e, 19j, and 19d, respectively. This demonstrates that an electron donating group, -OCH3, at the 6-position of pyridin-3-carbonyl results in increased affinity for VAChT. When further replacing the methoxy in 19e (Ki value of 8.36 ± 0.68 nM) with fluoroethoxy to afford compound 26a (Ki value of 38.0 ± 3.8 nM), the affinity for VAChT decreased 4.5-fold.</p><p>When the substitution pattern in the pyridine ring of 19e is changed from the 6-methoxy-pyridin-3-carbonyl to that of 3-methoxy-pyridin-2-carbonyl (19f), binding affinity (Ki value) for VAChT dropped 15-fold from 8.36 ± 0.68 nM to 121 ± 29 nM. Replacing the methoxy group in 19f with a fluoroethoxy group to obtain compound 26b resulted in slight improvement in VAChT binding (Ki value of 76.4 ± 8.3 nM). The affinities of compounds 19f and 26b are lower than those of the structural isomers 19e and 26a.</p><p>The only difference among compounds 19g, 19j and 19k was the position of fluoro substitution in the pyridin-3-carbonyl group. The order of binding affinity toward VAChT is 19g ≈ 19k >19j, in which the fluorine atom is at 5-, 2- and 6- positions of the pyridine-3-carbonyl ring and the dissociation constants for VAChT are 10.1 ± 1.5, 12.7 ± 1.1 and 26.1 ± 2.4 nM, respectively. Compounds 19h and 19i are N-methyl-2-pyridone derivatives. The only difference between them is the position of the bridging carbonyl group located at the 4-position in 19h and at the 6-position in 19i. The binding affinities for VAChT are 66.2 ± 10.2 nM for 19h and 182 ± 65 nM for 19i.</p><p>Compound (4-aminophenyl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)methanone (6) has higher potency for VAChT (Ki = 1.68 ± 0.14 nM) and has suitable lipophilicity (ALog D = 1.56) for crossing the BBB. Introducing methyl group(s) on the aniline nitrogen by N-methylation is a conventional way to convert compound 6 into carbon-11 radiolabeled tracer using [11C]methyl iodide. Monomethylation and dimethylation afforded 24a (Ki = 0.93 ± 0.09 nM) and 24b (Ki = 3.03 ± 0.48 nM). Their VAChT binding affinities are comparable to that of compound 6 (Ki = 1.68 ± 0.14 nM). The ALog D values of 24a and 24b are 3.24 and 2.61 respectively, which suggests they have suitable lipophilicity for the BBB penetration. We experimentally measured the Log D value of 24b and found to be 2.60.</p><p>To test whether an amino group in the 5'-position or 8'-position of the 3'-hydroxy-1',2',3',4'-tetrahydronaphthalen-2'-yl group affects affinity for VAChT, compounds 30a-d and 31a were synthesized and their binding affinities were determined. Compound 30a (Ki = 38.7 ± 7.0 nM) displayed 60-fold higher VAChT binding affinity than that of 31a (Ki = 2310 ± 390 nM). This result is consistent with the reported results in which the 8'-amino analogues displayed much higher potency than that of 5'-amino analogues.4 When aniline compounds 30a, 30b, 30c and 30d are compared with the non-aniline counterparts, 19a, 19c, 19d and 19e, there is no any significant change in binding affinities towards VAChT. This suggests that introducing the 5'-amino group into heteroaromatic carbonyl containing analogues may not be beneficial to VAChT binding affinity. Nevertheless, a large difference in binding affinity between the regioisomeric pairs 30a and 31a was observed.4</p><p>These substituted heteroaromatic carbonyl containing VAChT analogues have moderate to high VAChT binding affinities. Consequently, we screened their sigma receptor binding affinity. The in vitro data suggest that these new analogues had very low affinities for the sigma receptors. Except for compound 24a with Ki = 40.9 ± 8.2 nM, all other new analogues exhibit greater than 300 nM for σ1 receptor, as shown in Table 1. Among these new analogues, 6 compounds 19a, 19e, 19g, 19k, 24a and 24b have Ki values less than 20 nM toward VAChT. Compounds 19a and 19g have >70-fold selectivity ratios for VAChT vs either sigma receptor type. Importantly, compounds 19e, 19k and 24b not only display high affinity toward VAChT with Ki values of 8.36 ± 0.68, 12.7 ± 1.1 and 3.03 ± 0.48 nM respectively, but they also display greater than 100-fold selectivity ratios for VAChT versus sigma receptors. Compound 24a has the highest potency for VAChT (0.93 ± 0.09 nM) and is highly selective for VAChT versus σ1 (44-fold) and σ2 (4400-fold) receptors. Thus, compound 24a can be a good blocking agent for validating the selective binding of other VAChT radioligands.</p><p>To further test the feasibility of the lead compounds reported in this manuscript to sever as PET tracer for imaging VAChT in vivo in living animal, racemic compound 24b was resolved by HPLC using chiralcel OD column to obtain the (+)-24b and (-)-24b. The in vitro data revealed that (-)-24b (Ki-VAChT = 0.78 nM) is the more potent isomer than the (+)-24b (Ki-VAChT = 19.0 nM). This is consistent to that ligand binding to VAChT is stereoselective.</p><!><p>To check the in vivo distribution of (-)-[11C]24b and its washout kinetics from brain regions of interest and peripheral tissues, ex-vivo biodistribution was conducted in male Sprague-Dawley rats (185 – 205 gram). Rats were euthanized at 5 and 30 min post i.v. injection of (-)-[11C]24b. To test the ability of (-)-[11C]24b to cross the blood brain barrier (BBB), rats were pretreated with Cyclosporine A (CycA) 30 min prior to injection of the radiotracer dose and then euthanized at 30 min post injection of the radiotracer. The distribution data obtained in this study were shown in Figure 3 and 4. For the normal rats, the uptake of radioactivity at 5 min was 0.34, 0.49, 1.77, 0.22, 0.18, 0.89, 1.19, 1.47, 3.45, 0.57 % injected dose per gram (%I.D./g) in blood, heart, lung, muscle, fat, pancreas, spleen, kidney, liver and brain respectively post intravenous (i.v.) injection and the liver has the highest uptake; the radioactivity decreased in all the tissues that were collected in the studies from 5 to 30 min as shown and the liver retained the highest uptake as 2.44 at 30 min. For the rats pretreated with CycA, except the uptake (%I.D./g) in kidney displayed slightly increase (0.77 in control rats, 1.21 in rats pretreated with CycA), the other peripheral tissues didn't show significant change (Figure 3). For the brain regions of interest, the uptake (%I.D./g) at 30 min in cerebellum, striatum, brain stem, cortex, thalamus, hippocampus, and brain were 0.157, 0.593, 0.245, 0.262, 0.293, 0.265 and 0.272 respectively for the control rats; for the rats pretreated with CycA, the uptakes (%I.D.) were 0.404, 1.020, 0.577, 0.637, 0.600, 0.562 and 0.596 respectively for corresponding brain regions of interest. The uptake in brain was observed to increase 2.2-fold when the rats were pretreated with CycA. P-glycoprotein (P-gp) is a 170-kDa protein that is able to binding with compounds with diversify structures and it has widespread tissue distribution belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) transporters42, 43 and it is expressed in the capillary endothelial cells which comprise the blood-brain barrier.42 CycA, is a modulator/inhibitor of the adenosine triphosphate (ATP)-binding cassette (ABC) transporters including P-gp. It was frequently used to evaluate the ABC transporter-mediated efflux of PET radiotracers and it suggested that ABC transporter-mediated efflux of PET radiotracers has species difference.44 If a radioligand is a substrate of P-gp, rats pretreated with CycA will results in significantly increase of the uptake in the brain of animals, for example, radioligands, [11C]verapramil,45, 46 [11C]GR21823147 and benzamide D3 radioligands.48 For radioligand, (-)-[11C]24b, using CycA pretreated the rats displayed only 2.2-fold increase in the brain uptake of the rats. Considering the brain uptake (%I.D./g) of radioactivity reached 0.57 at 5 min. we concluded that the P-gp modulating the brain uptake of (-)-[11C]24b is very weak and (-)-[11C]24b is able to penetrate the BBB and displayed sufficient uptake in the brain of the rats.</p><!><p>To further confirm that (-)-[11C]24b is able to bind with VAChT in the brain in vivo, microPET studies of (-)-[11C]24b in the brain of nonhuman primate, male cynomolgus monkey were performed (n = 3). The microPET studies demonstrated that (-)-[11C]24b is able to enter the brain and has highest accumulation in striatum, the VAChT enriched area in the regions of interest in the brain (Figure 5). (-)-[11C]24b is able to give sufficient contrast for the striatum versus other non-target regions. The tissue-time activity curves post injection of (-)-[11C]24b revealed that the radioactivity accumulation was the highest at 10 min post-injection. The ratio of radioactivity accumulation in the target (striatum) and non-target (cerebellum) reaches > 2.5 fold after 70 min post injection (Figure 5). This data suggests that (-)-[11C]24b is a promising PET tracer to quantify VAChT in vivo.</p><!><p>In the present study, we successfully synthesized a series of new VAChT inhibitors by replacing the benzoyl group in 5 with a heteroaromatic carbonyl group or replacing the 4-aminobenzoyl group with the 4-methylaminobenzoyl group or the 4-N,N-dimethylaminobenzoyl group. The six compounds 19a, 19e, 19g, 19k, 24a and 24b displayed high VAChT binding affinities (Ki values ranging from 0.93 nM to 18.4 nM) and good selectivity for VAChT over sigma receptors. In particular, compounds 19e, 19k and 24b are very potent for VAChT with Ki values of 8.36 ± 0.68, 12.7 ± 1.1 and 3.03 ± 0.48 nM, respectively; and binding selectivity ratios equal to or greater than 100-fold for VAChT over sigma receptors. The racemate 24b was Successfully resolved on HPLC to obtain the minus and plus isomers. In vivo validation of (-)-[11C]24b in rodents and nonhuman primates demonstrated that (-)-[11C]24b is able to penetrate the BBB and has highest accumulation in the striatum, the VAChT enriched area in the brain. The time tissue-activity of (-)-[11C]24b in the brain of cynomolgus monkey revealed that it has favorable washout pharmacokinetics in the brain. Further imaging studies of (-)-[11C]24b in nonhuman primate are warranted to test the feasibility of (-)-[11C]24b to be a promising candidate for assessing the level of VAChT in the brain.</p><!><p>All reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. All anhydrous reactions were carried out in oven-dried glassware under an inert nitrogen atmosphere unless otherwise stated. When the reactions involved extraction with methylene chloride (CH2Cl2), chloroform (CHCl3), or ethyl acetate (EtOAc), the organic solutions were dried with anhydrous Na2SO4 and concentrated on a rotary evaporator under reduced pressure. Melting points were determined on the MEL-TEMP 3.0 apparatus and left uncorrected. 1HNMR spectra of majority of the compounds were recorded at 300 MHz on a Varian Mercury-VX spectrometer with CDCl3 as solvents and tetramethylsilane (TMS) was used as the internal standard. Varian 400 MHz NMR was used for some compounds and was reported in the experimental section where appropriate. Elemental analyses (C, H, N) were determined by Atlantic Microlab, Inc. and were found to be within 0.4% of theoretical values. Chiralcel OD column was used for normal phase HPLC to resolve enantiomers. Phenomenex Luna C18 column was used for reverse phase HPLC conditions to purify the radioactive product on HPLC, and Phenomenex Prodigy was used for QC analysis of radiotracer.</p><!><p>Approximately 105.0 mg of racemate 6 was resolved on HPLC using a Chiralcel OD column (250 mm × 10 mm) under isocratic conditions (34% isopropanol in hexane) at a flow rate of 4.0 mL/min and UV wave length at 254 nm to give 41.0 mg of (+)-6 (Rt = 20.8 min) and 48.0 mg of (-)-6 (Rt = 33 min). The specific rotation was determined on an automatic polarimeter (Autopol 111, Rudolph Research, Flanders, NJ). The optical rotation was [α]D = -50° for (-)-6 and [α]D = +63° for (+)-6 at the concentration of 1.0 mg/mL in dichloromethane at 20 °C.</p><!><p>To a solution of 10 (2.5 g, 10.9 mmol) in CH2Cl2 (30 mL) at room temperature, CDI (1.77 g, 11.0 mmol) was added. The mixture was stirred at room temperature for 1 h and N, O-dimethylhydroxyamine hydrochloride (1.3 g, 13.3 mmol) followed by Et3N were added. The reaction mixture was stirred overnight and was washed with aqueous Na2CO3, water, dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 11 as a white solid (2.70 g, 90%). mp: 54 °C. 1H NMR (CDCl3): δ 1.46 (s, 9H), 1.64-1.72 (m, 4H), 2.78 (s br, 3H), 3.18 (s, 3H), 3.71 (s, 3H), 4.13 (s br, 2H).</p><!><p>A solution of 4-bromopyridin-2-amine 12h (0.60 g, 3.5 mmol) in a mixture of 2 N H2SO4 (20 mL) and 2 N NaNO2 (10 mL) was stirred at 0-5 °C for 2 h. The reaction mixture was extracted with CH2Cl2. Crude product was used in the next step without further purification. To the above crude product, potassium carbonate (0.50 g, 3.6 mmol) and methyl iodide (0.53 g, 3.7 mmol) were added and heated at 80 °C in acetone (100 mL) in a sealed tube for 4 h. The reaction mixture was cooled, and potassium carbonate was filtered off. Acetone was evaporated and a small amount of water was added to the residue. This solution was extracted with CH2Cl2 and the product was purified with silica gel column chromatography to afford 15h as a yellow solid (355 mg, 57%). 1H NMR (CDCl3): δ 3.49 (s, 3H), 6.31 (d, J = 6.0 Hz, 1H), 6.82 (s, 1H), 7.14 (d, J = 6.0 Hz, 1H).</p><!><p>Compound 15i as a yellow solid was synthesized starting with compound 12i as described in procedure A. mp: 105 °C. Yield, 54 %. 1H NMR (CDCl3,): δ 3.73 (s, 3H), 6.46-6.52 (m, 2H), 7.10-7.16 (m, 1H).</p><!><p>To a schlenk flask containing N-methylpyrrole (0.49 g, 6.08 mmol), 20 mL of THF was added while stirring at room temperature until a clear solution was formed. The solution was cooled down to -78 °C for 15 min and 12.0 mL (6.08 mmol) of n-BuLi was added. The solution was allowed to warm up to 0 °C for 20 min, resulting in the formation of the yellow lithium intermediate. The resulting mixture was added via cannula into a solution of 11 (0.83 g, 3.04 mmol) in THF (20 mL) at the same temperature and the reaction mixture was stirred at -78 °C for 4 h, then quenched with saturated aqueous NH4Cl and allowed to warm to room temperature. The mixture was washed with brine and dried over Na2SO4, filtered, and evaporated under reduced pressure to give the crude product. The crude compound was purified by silica gel chromatography to obtain the desired compound 16a (0.61 g, 34%). 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.66-1.80 (m, 4H), 2.83 (s, 2H), 3.17 (m, 1H), 3.94 (s, 3H), 4.18 (s br, 2H), 6.15 (dd, J = 4.2, 2.4 Hz, 1H), 6.84 (pseudo t, J = 1.8 Hz, 1H), 6.99 (dd, J = 4.2, 1.5 Hz, 1H).</p><!><p>To a solution of 3-bromo-1-methyl-1H-pyrrole (2.19 g, 13.8 mmol) in anhydrous THF at -78 °C, n-BuLi (1.6 M, 10 mL) was added. The solution was stirred for 45 minutes at -78 °C. The resulting mixture was cannulated into a solution of 11 (2.5 g, 9.2 mmol) in THF (80 mL) at the same temperature and the reaction mixture was stirred at -78 °C for 4 h, then quenched with sat. aqueous NH4Cl and allowed to warm to room temperature. The mixture was washed with brine and dried over Na2SO4, filtered, and evaporated under reduced pressure to give the crude product. The crude compound was purified by silica gel chromatography to obtain the desired compound 16b (1.07 g, 40%). 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.66-1.77 (m, 4H), 2.83 (s, 2H), 3.15-3.18 (m, 1H), 3.70 (s, 3H), 4.13 (s br, 2H), 6.57-6.61 (m, 2H), 7.26-7.29 (m, 1H).</p><!><p>To a solution of 3-bromopyridine (1.13 g, 7.2 mmol) in anhydrous THF (45 mL) at -40 °C under argon atmosphere, lithium dibutyl(isopropyl)magnesate (5.2 mL, 0.7 M, 3.6 mmol) was added. Stirring was continued at the same temperature for 1 h. The resulting mixture was cannulated into a solution of 11 (1.5 g, 5.5 mmol) in THF (50 mL) at -78 °C. The solution was maintained at -78 °C for 1 h, and quenched with saturated aqueous NH4Cl and allowed to warm to room temperature. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, dried over Na2SO4, filtered, and evaporated under reduced pressure to give the crude product. The crude compound was purified by silica gel chromatography to obtain the desired compound 16c (0.49 g, 31%). 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.63-1.89 (m, 4H), 2.92 (m, 2H), 3.38 (m, 1H), 4.20 (m, 2H), 7.44 (ddd, J = 8.1, 5.1, 0.9 Hz, 1H), 8.22 (dt, J = 8.1, 2.1 Hz, 1H), 8.79 (dd, J = 5.1, 1.8 Hz, 1H), 9.16 (d, J = 1.8 Hz, 1H).</p><!><p>Compound 16d was prepared from 5-bromo-2-methylpyridine following the procedure described above for the preparation of 16c. Yield, 42%. 1H NMR (CDCl3): δ 1.49 (s, 9H), 1.68-1.87 (m, 4H), 2.63 (s, 3H), 2.90 (m, 2H), 3.35 (m, 1H), 4.18 (s br, 2H), 7.28 (d, J = 8.4 Hz, 1H), 8.11 (dd, J = 8.4, 2.1 Hz, 1H), 9.04 (d, J = 2.1 Hz, 1H).</p><!><p>Compound 16e was prepared from 5-bromo-2-methoxypyridine following the procedure described above for the preparation of 16b. Yield, 61%. 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.62-1.86 (m, 4H), 2.88 (m, 2H), 3.31 (m, 1H), 4.01 (s, 3H), 4.19 (s br, 2H), 6.81 (d, J = 8.7 Hz, 1H), 8.13 (dd, J = 8.7, 2.4 Hz, 1H), 8.79 (d, J = 2.4 Hz, 1H).</p><!><p>Compound 16f was prepared from 2-bromo-3-methoxypyridine following the procedure described above for the preparation of 16b. Yield, 36%. 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.66-1.87 (m, 4H), 2.89 (s, 2H), 3.67-3.81 (m, 1H), 3.90 (s, 3H), 4.07 (s br, 2H), 7.35-7.45 (m, 2H), 8.23-8.25 (m, 1H).</p><!><p>Compound 16g was prepared from 3-bromo-5-fluoropyridine following the procedure described above for the preparation of 16b. Yield, 41%. 1H NMR (CDCl3): δ 1.44 (s, 9H), 1.56-1.61 (m, 2H), 1.86-1.90 (m, 2H), 2.87 (s, 2H), 3.17-3.24 (m, 1H), 4.06 (s br, 2H), 7.54-7.58 (m, 1H), 8.53-8.60 (m, 2H).</p><!><p>Compound 16h was prepared from compound 15h following the procedure described above for the preparation of 16b. Yield, 38%. 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.56-1.87 (m, 4H), 2.82 (s, 2H), 3.05-3.07 (m, 1H), 3.50 (s, 3H), 4.17 (s br, 2H), 6.49 (d, J = 6.0 Hz, 1H), 6.73 (d, J = 6.0 Hz, 1H), 7.36 (s, 1H).</p><!><p>Compound 16i was prepared from compound 15i following the procedure described above for the preparation of 16b. Yield, 35%. 1H NMR (CDCl3): δ 1.45 (s, 9H), 1.58-1.87 (m, 4H), 2.81 (s, 2H), 3.05-3.07 (m, 1H), 3.47 (s, 3H), 4.14 (s br, 2H), 6.44-6.47 (m, 1H), 6.69-6.72 (m, 1H), 7.30-7.36 (m, 1H).</p><!><p>To a solution of 16a (0.67 g, 2.3 mmol) in CH2Cl2 (30 mL), TFA (2 mL) was added. The reaction mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure and the residue was neutralized with 1 N NaOH solution and extracted with CH2Cl2 (2 × 15 mL). The organic layer was washed with brine, dried and concentrated to give crude product. The crude product was purified by silica gel column chromatography to afforded 17a (0.32 g, 72%). 1H NMR (CDCl3): δ 1.76-1.87 (m, 4H), 2.81 (m, 2H), 3.17-3.23 (m, 4H), 3.94 (s, 3H), 6.14 (m, 1H), 6.83 (m, 1H), 6.98 (m, 1H).</p><!><p>Compound 17b was prepared from compound 16b as described in procedure B. Yield, 80 %. 1H NMR (CDCl3): δ 1.76-1.80 (m, 4H), 2.65-2.73 (m, 2H), 3.01-3.17 (m, 4H), 3.68 (s, 3H), 6.57-6.59 (m, 2H), 7.25 (s, 1H).</p><!><p>Compound 17c was prepared from compound 16c as described in procedure B. Yield, 85%. 1H NMR (CDCl3): δ 1.62-1.77 (m, 2H), 1.86-1.89 (m, 3H), 2.79 (td, J = 12.6, 2.7 Hz, 2H), 3.21 (dt, J = 12.9, 3.0 Hz, 2H), 3.37 (tt, J = 11.4, 3.9 Hz, 1H), 7.43 (ddd, J = 8.1, 4.8, 0.9 Hz, 1H), 8.22 (ddd, J = 4.8, 2.1, 1.5 Hz, 1H), 8.78 (dd, J = 4.5, 1.5 Hz, 1H), 9.16 (dd, J = 2.4, 0.9 Hz,1H).</p><!><p>Compound 17d was prepared from compound 16d as described in procedure B. Yield, 70%. 1H NMR (CDCl3): δ 1.62-1.87 (m, 5H), 2.63 (s, 3H), 2.77 (m, 2H), 3.16-3.22 (m, 2H), 3.33 (m, 1H), 7.27 (d, J = 8.4 Hz, 1H), 8.12 (dd, J = 8.1, 2.4 Hz, 1H), 9.04 (d, J = 2.4 Hz, 1H).</p><!><p>Compound 17e was prepared from compound 16e as described in procedure B. Yield, 77%. 1H NMR (CDCl3): δ 1.65-1.89 (m, 4H), 2.40 (s, 1H), 2.78 (td, J = 12.0, 2.7 Hz, 2H), 3.21 (dt, J = 9.0, 3.6 Hz, 2H), 3.31 (m, 1H), 4.00 (s, 3H), 6.79 (d, J = 8.4 Hz, 1H), 8.13 (dd, J = 8.4, 2.4 Hz, 1H), 8.78 (d, J = 2.4 Hz, 1H).</p><!><p>Compound 17f was prepared from compound 16f as described in procedure B. Yield, 78%. 1H NMR (CDCl3): δ 1.51-1.81 (m, 4H), 2.69 (s, 2H), 3.08-3.25 (m, 3H), 3.49-3.58 (m, 1H), 3.83 (s, 3H), 7.27-7.36 (m, 2H), 8.16-8.18 (m, 1H).</p><!><p>Compound 17g was prepared from compound 16g as described in procedure B. Yield, 79%. 1H NMR (CDCl3): δ 1.56-1.61 (m, 2H), 1.86-1.90 (m, 2H), 2.87 (m, 2H), 3.17-3.21 (m, 4H), 7.56 (s, 1H), 8.53-8.60 (m, 2H).</p><!><p>Compound 17h was prepared from compound 16h as described in procedure B. Yield, 79%. 1H NMR (CDCl3): δ 1.57-1.88 (m, 4H), 2.83 (s, 2H), 3.03-3.09 (m, 4H), 3.50 (s, 3H), 6.50 (d, J = 6.0 Hz, 1H), 6.72 (d, J = 6.0 Hz, 1H), 7.38 (s, 1H).</p><!><p>Compound 17i was prepared from compound 16i as described in procedure B. Yield, 75%. 1H NMR (CDCl3): δ 1.58-1.87 (m, 4H), 2.82 (s, 2H), 3.02-3.10 (m, 4H), 3.47 (s, 3H), 6.42-6.48 (m, 1H), 6.67-6.72 (m, 1H), 7.31-7.36 (m, 1H).</p><!><p>A mixture of 17a (0.27 g, 1.4 mmol), 18 (0.10 g, 0.68 mmol) and Et3N (0.3 mL, 2.2 mmol) in ethanol (5 mL) was stirred at 75 °C for 36 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with EtOAc (3 × 15 mL). The residue was purified by silica gel column chromatography to give 19a as a white solid (0.11 g, 47%). 1H NMR (CDCl3, free base): δ 1.81-2.03 (m, 4H), 2.37 (m, 1H), 2.76-3.13 (m, 8H), 3.30 (m, 1H), 3.88 (m, 1H), 3.95 (s, 3H), 4.45 (s br, 1H), 6.14 (m, 1H), 6.83 (m, 1H), 6.98 (m, 1H), 7.09-7.16 (m, 4H). Free base was converted to the corresponding oxalate salt by adding oxalic acid in ethyl acetate to 19a in CH2Cl2. mp: 212 °C (decomposed). Anal. (C21H26N2O2 • H2C2O4) C, H, N.</p><!><p>Compound 19b was prepared from compound 17b as described in procedure C. Yield, 50%. 1H NMR (CDCl3, free base): δ 1.83-2.00 (m, 4H), 2.38 (m, 1H), 2.76-3.13 (m, 8H), 3.29-3.35 (m, 1H), 3.71 (s, 3H), 4.32 (s br, 1H), 6.61 (m, 2H), 7.12-7.16 (m, 4H), 7.30-7.31 (m, 1H). The free base was converted to the oxalate salt. mp: 212.6 °C (decomposed). Anal. (C21H26N2O2• H2C2O4•0.25H2O) C, H, N.</p><!><p>Compound 19c was prepared from compound 17c as described in procedure C. Yield, 40%. 1H NMR (CDCl3, free base): δ 1.77-1.98 (m, 4H), 2.43 (m, 1H), 2.75-3.02 (m, 7H), 3.20-3.38 (m, 2H), 3.88 (m, 1H), 7.08-7.15 (m, 4H), 7.42-7.46 (m, 1H), 8.21-8.25 (m, 1H), 8.77-8.79 (m, 1H), 9.16 (s, 1H). The free base was converted to the oxalate salt. mp: 214 °C (decomposed). Anal. (C21H24N2O2 • H2C2O4) C, H, N.</p><!><p>Compound 19d was prepared from compound 17d as described in procedure C. Yield, 44%. 1H NMR (CDCl3, free base): δ 1.80-2.02 (m, 4H), 2.43 (m, 1H), 2.62 (s, 3H), 2.78-3.06 (m, 7H), 3.20-3.38 (m, 2H), 3.89 (m, 1H), 6.83 (d, J = 8.7 Hz, 1H), 7.11-7.18 (m, 4H), 8.17 (dd, J = 8.7, 2.4 Hz, 1H), 8.82 (d, J = 2.4 Hz, 1H). The free base was converted to the oxalate salt. mp: 223 °C (decomposed). Anal. (C22H26N2O2 • 2H2C2O4) C, H, N.</p><!><p>Compound 19e was prepared from compound 17e as described in procedure C. Yield, 42%. 1H NMR (CDCl3, free base): δ 1.74-1.96 (m, 4H), 2.41 (m, 1H), 2.75-3.01 (m, 7H), 3.26-3.33 (m, 2H), 3.86 (m, 1H), 4.04 (s, 3H), 4.17 (s br, 1H), 7.06-7.14 (m, 4H), 7.27 (d, J = 8.1 Hz, 1H), 8.11 (dd, J = 8.1, 2.1 Hz, 1H), 9.03 (d, J = 2.1 Hz, 1H). The free base was converted to the oxalate salt. mp: 240 °C (decomposed). Anal. (C22H26N2O3 • H2C2O4) C, H, N.</p><!><p>Compound 19f was prepared from compound 17f as described in procedure C. Yield, 36%. 1H NMR (CDCl3, free base): δ 1.72-1.96 (m, 4H), 2.33-2.41 (m, 1H), 2.72-2.96 (m, 7H), 3.26-3.33 (m, 1H), 3.55-3.63 (m, 1H), 3.81-3.90 (m, 3H), 4.30 (s br, 1H), 7.06-7.14 (m, 4H), 7.26-7.39 (m, 2H), 8.23-8.25 (m, 1H). The free base was converted to the oxalate salt. mp: 146.6 °C (decomposed). Anal. (C22H26N2O3• H2C2O4•0.25H2O) C, H, N.</p><!><p>Compound 19g was prepared from compound 17g as described in procedure C. Yield, 44%. 1H NMR (CDCl3, free base): δ 1.70-2.04 (m, 4H), 2.34-2.43 (m, 1H), 2.75-3.34 (m, 8H), 3.82-3.90 (m, 1H), 4.13 (s br, 1H), 7.06-7.11 (m, 4H), 7.57-7.61 (m, 1H), 8.56-8.62 (m, 2H). The free base was converted to the oxalate salt. mp: 203.7 °C (decomposed). Anal. (C21H23FN2O2• H2C2O4) C, H, N.</p><!><p>Compound 19h was prepared from compound 17h as described in procedure C. Yield, 37%. 1H NMR (CDCl3, free base): δ 1.72-1.99 (m, 4H), 2.33-2.39 (m, 1H), 2.75-3.01 (m, 6H), 3.27-3.34 (m, 2H), 3.50 (s, 3H), 3.82-3.91 (m, 1H), 4.08 (s br, 1H), 6.45 (d, J = 6.0 Hz, 1H), 6.72 (d, J = 6.0 Hz, 1H), 7.12-7.13 (m, 4H), 7.35 (s, 1H). The free base was converted to the oxalate salt. mp: 93.8 °C (decomposed). Anal. (C22H26N2O3•H2C2O4•1.5H2O) C, H, N.</p><!><p>Compound 19i was prepared from compound 17i as described in procedure C. Yield, 39%. 1H NMR (CDCl3, free base): δ 1.72-2.04 (m, 4H), 2.32-2.39 (m, 1H), 2.81-3.01 (m, 6H), 3.27-3.35 (m, 2H), 3.50 (s, 3H), 3.82-3.89 (m, 1H), 4.06 (s br, 1H), 6.43-6.46 (m, 1H), 6.70-6.73 (m, 1H), 7.06-7.14 (m, 4H), 7.31-7.37 (m, 1H). The free base was converted to the oxalate salt. mp: 122.4 °C (decomposed). Anal. (C22H26N2O3•H2C2O4•1.5H2O) C, H, N.</p><!><p>To a solution of 17j (2.42 g, 13.8 mmol) in anhydrous THF at -78 °C, n-BuLi (1.6 M, 10 mL) was added. The solution was stirred for 45 minutes at -78 °C. The resulting mixture was cannulated into a solution of 21 (3.97 g, 9.2 mmol) in THF (80 mL) at the same temperature and the reaction mixture was stirred at -78 °C for 4 h, then quenched with sat. aqueous NH4Cl and allowed to warm to room temperature. The mixture was washed with brine and dried over Na2SO4, filtered, and evaporated under reduced pressure to give the crude product. The crude compound was purified by chromatography on silica gel to obtain the TBDMS-protected intermediate (1-(3-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-fluoropyridin-3-yl) methanone (1.55 g, 36%). 1H NMR (CDCl3): δ 0.12 (s, 6H), 0.91 (s, 9H), 1.65-1.77 (m, 4H), 2.46-3.05 (m, 9H), 4.13 (s br, 2H), 7.04-7.12 (m, 4H), 8.23-8.37 (m, 2H), 8.78 (s, 1H).</p><p>A mixture of the intermediate (1-(3-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-fluoropyridin-3-yl) methanone (1.55 g, 3.3 mmol) and concentrated HCl (5 mL) in THF were stirred for 4 h at room temperature until TLC indicated that deprotection was complete, and then it was carefully neutralized with 1 N NaOH and extracted with CH2Cl2. The crude product was purified by column chromatography to afford 19j as white solid (0.92 g, 79%). 1H NMR (CDCl3, free base): δ 1.84-2.03 (m, 4H), 2.38-2.46 (m, 1H), 2.75-3.02 (m, 6H), 3.21-3.34 (m, 2H), 3.82-3.89 (m, 1H), 4.30 (s br, 1H), 7.03-7.11 (m, 4H), 8.33-8.39 (m, 2H), 8.79 (s, 1H). Free base was converted to the corresponding oxalate salt by adding oxalic acid in ethyl acetate to 19j in CH2Cl2. mp: 204.3 °C (decomposed). Anal. (C21H23FN2O2• H2C2O4•0.5H2O) C, H, N.</p><!><p>Compound 19k was prepared from compound 17k as describe in procedure D to give TBDMS-protected intermediate (1-(3-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(2-fluoro-pyridin-3-yl) methanone (1.68 g, 39%). 1H NMR (CDCl3): δ 0.11 (s, 6H), 0.90 (s, 9H), 1.63-1.78 (m, 4H), 2.45-3.08 (m, 9H), 4.13 (s br, 2H), 7.04-7.11 (m, 4H), 7.31-7.39 (m, 1H), 8.23-8.30 (m, 1H), 8.38-8.40 (m, 1H). Removal of TBDMS with HCl gave 19k (0.87 g, 75%). 1H NMR (CDCl3, free base): δ 1.84-2.03 (m, 4H), 2.38-2.46 (m, 1H), 2.75-2.99 (m, 6H), 3.25-3.33 (m, 2H), 3.80-3.90 (m, 1H), 4.31 (s br, 1H), 7.03-7.13 (m, 4H), 7.30-7.41 (m, 1H), 8.22-8.31 (m, 1H), 8.38-8.40 (m, 1H). The free base was converted to the oxalate salt. mp: 205.8 °C (decomposed). Anal. (C21H23FN2O2• H2C2O4•H2O) C, H, N.</p><!><p>TFA (2 mL) was added into a solution of 11 (0.38 g, 1.4 mmol) in CH2Cl2 (30 mL). The reaction mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure and the residue was neutralized with 1 N NaOH solution and extracted with CH2Cl2 (2 × 15 mL). The organic layer was washed with brine, dried and concentrated to give the crude compound, which was used in the next step without further purification. Crude compound, 18 (0.10 g, 0.68 mmol) and Et3N (0.3 mL, 2.2 mmol) in ethanol (5 mL) were stirred at 75 °C for 36 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with EtOAc (3 × 15 mL). The organic layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 20 as a white solid (86.4 mg, 40%). 1H NMR (CDCl3): δ 1.81-1.93 (m, 4H), 2.28-2.31 (m, 1H), 2.75-2.97 (m, 8H), 3.27-3.20 (s, 3H), 3.27-3.34 (m, 1H), 3.72 (s, 3H), 3.85-3.90 (m, 1H), 4.27 (s br, 1H), 7.09-7.13 (m, 4H).</p><!><p>To a solution of 20 (0.4 g, 1.25 mmol) and imidazole (0.4 g, 5.88 mmol) in CH2Cl2 (50 mL), TBDMSCl (0.38 g, 2.5 mmol) was added. The reaction mixture was stirred overnight at room temperature until TLC indicated that the reaction was complete. The reaction mixture was washed with brine and 1 N NaH2PO4 (30 mL × 3). The organic layer was dried and concentrated to a residue. The crude product was purified by silica gel column chromatography to afford 21 as a white solid (0.38 g, 70 %). 1H NMR (CDCl3,): δ 0.12 (s, 6H), 0.91 (s, 9H), 1.65-1.77 (m, 4H), 2.46-3.05 (m, 9H), 3.17 (s, 3H), 3.69 (s, 3H), 4.13 (s br, 2H), 7.04-7.12 (m, 4H).</p><!><p>Acetic anhydride (1.89 mL, 18.5 mmol) was added dropwise over 15 min to a magnetically stirred solution of 6 (0.54 g, 1.54 mmol) and Et3N (3.12 g, 30.8 mmol) in dry</p><p>CH2Cl2 (10 mL) overnight. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated under reduced pressure. Water was added to wash the resulting solid and the residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:2) to give 22 (0.55 g, 82%). 1H NMR (CDCl3): δ 1.24-1.28 (m, 2H), 1.70-1.84 (m, 2H), 2.12 (s, 3H), 2.22 (s, 3H), 2.44-2.60 (m, 2H), 2.84-3.05 (m, 6H), 3.16-3.23 (m, 2H), 5.27-5.34 (m, 1H), 7.05-7.16 (m, 4H), 7.49 (s br, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H).</p><!><p>Sodium hydride (96 mg, 2.4 mmol) was added to 3-(4-(4-acetamidobenzoyl)piperidin-1-yl)-1,2,3,4-tetrahydronaphthalen-2-yl acetate 22 (0.87 g, 2 mmol) in anhydrous THF, and iodomethane (0.34 g, 2.4 mmol) was added dropwise to the mixture, which was maintained below 5 °C for 0.5 h and then stirred at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was partitioned between saturated aqueous NH4Cl and ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers dried and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate: hexane = 3:2) to give 23 (0.63 g, 78%). 1H NMR (CDCl3): δ 1.77-1.90 (m, 4H), 1.96 (s, 3H), 2.44 (t, J = 10.3 Hz, 1H), 2.76-3.03 (m, 7H), 3.27-3.35 (m, 2H), 3.31 (s, 3H), 3.84-3.89 (m, 1H), 4.21 (s br, 1H), 7.09-7.28 (m, 4H), 7.32 (d, J = 8.1 Hz, 2H), 8.01 (d, J = 7.8 Hz, 2H).</p><!><p>Sodium hydride (96 mg, 2.4 mmol) was added to 6 (0.87 g, 2 mmol) in anhydrous THF, and iodomethane (4.8 mmol) was added dropwise to the mixture, which was stirred at room temperature for 0.5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was partitioned between saturated aqueous NH4Cl and ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried and contentrated. The crude product was purified by silica gel column chromatography to give 24a as a white solid (0.63 g, 83%). 1H NMR (CDCl3, free base): δ 1.81-1.98 (m, 4H), 2.36-2.44 (m, 1H), 2.74-3.06 (m, 13H), 3.23-3.33 (m, 2H), 3.83-3.91 (m, 1H), 4.46 (s br, 1H), 6.66 (d, J = 9 Hz, 2H), 7.09-7.15 (m, 4H), 7.88 (d, J = 9 Hz, 2H). Free base was converted to the corresponding oxalate salt by adding oxalic acid in ethyl acetate to 24a in CH2Cl2. mp: 228.9 °C (decomposed). Anal. (C24H30N2O2 • H2C2O4) C, H, N.</p><!><p>Concentrated HCl (12 M, 0.25 mL) was added to a stirred solution of 23 (0.4 g, 1.0 mmol) in ethylene glycol (0.75 mL). The reaction mixture was heated to reflux for 3 h and the reaction was monitored by TLC. When the reaction was complete, the mixture was partitioned between water and ethyl acetate. The organic layer was separated and the aqueous layer was extracted. The combined organic layers dried and contentrated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 1/4) to give 24b (0.3 g, 82%). 1H NMR (CDCl3): δ 1.85-1.92 (m, 4H), 2.40-2.44 (m, 1H), 2.77-2.99 (m, 8H), 3.27-3.34 (m, 2H), 3.81-3.89 (m, 2H), 4.19-4.23 (m, 1H), 4.46 (s br, 1H), 6.59 (d, J = 8.1 Hz, 2H), 7.09-7.15 (m, 4H), 7.86 (d, J = 8.1 Hz, 2H). Free base was converted to the corresponding oxalate salt by adding oxalic acid in ethyl acetate to 24b in CH2Cl2. mp: 215.7 °C (decomposed). Anal. (C23H28N2O2• H2C2O4) C, H, N.</p><!><p>Approximately 200 mg of (±)-24b was separated on chiral HPLC using a Chiralcel OD column (250 mm × 10mm). The mobile phase used was 35% isopropanol in hexane at a flow rate of 4.0 mL/min to give 83.3 mg of (+)-24b (Rt = 15 min) and 94.9 mg of (-)-24b (Rt = 30 min). The specific rotation was determined on an automatic polarimeter (Autopol 111, Rudolph Research, Flanders, NJ). The optical rotation of (-)-24b was [α]D = -30.5° at the concentration of 1.8 mg/mL in dichloromethane and that of (+)-24b was [α]D = +21.8 ° at the concentration of 1.1 mg/ mL in dichloromethane at 20 °C. The (-)-24b and (+)-24b were converted to oxalates by treating one equivalent of (-)-24b or (+)-24b with one equivalent of oxalic acid. The salt obtained was used for the in vitro studies. mp of (+)-24b: 199.8 °C (decomposed), mp of (-)-24b: 200.2 °C (decomposed).</p><!><p>Into an oven-dried argon-purged 50 mL round bottom flask was placed 19e (73.3 mg, 0.2 mmol) and 3.0 mL chloroform was added followed by TMSI (57 μL, 0.4 mmol). The reaction mixture was heated to 55 °C for 1 h until TLC (EtOAc/hexane, 1/1) indicated the disappearance of starting material. The reaction mixture was cooled to room temperature and 0.6 mL methanol was added. The reaction mixture was concentrated. Ethyl acetate was added followed by aqueous sodium bicarbonate solution. The ethyl acetate layer was separated and the aqueous layer was extracted with ethyl acetate. Combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give compound 25 as a white solid (69 mg, 97%). 1H NMR (CDCl3): δ 1.40-2.00 (m, 3H), 2.20-2.25 (m, 1H), 2.60-3.10 (m, 8H), 3.30 (dd, J = 6.2, 13.9 Hz, 1H), 3.80-4.00 (m, 1H), 4.20-4.25 (m, 1H), 6.60 (d, J = 9.6 Hz, 1H), 7.00-7.20 (m, 4 H), 8.05 (dd, J = 2.7, 9.6 Hz, 1H), 8.16 (s, 1H).</p><!><p>To the mixture of 25 (69 mg, 0.195 mmol) and 1-bromo-2-fluoroethane (30 μL, 0.4 mmol) in DMF (2 mL) was added potassium carbonate (60 mg, 0.43 mmol). The resultant reaction mixture was stirred at the room temperature for 72 h. Ethyl acetate was added to the reaction mixture and washed with aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel chromatography to give colorless sticky solid (40 mg, 51%). 1H NMR (CDCl3): δ 1.75 -1.99 (m, 1H), 2.20 - 2.50 (m, 1H), 2.70-3.10 (m, 11H), 3.30 (dd, J = 16.0, 6.0 Hz, 1H), 3.80-3.95 (m, 1H), 4.27 (t, J = 4.5 Hz, 1H), 4.36 (t, J = 4.5 Hz, 1H), 4.67 (t, J = 4.5 Hz, 1H), 4.83 (t, J = 4.5 Hz, 1H), 6.61 (d, J = 9.6 Hz, 1H), 7.00-7.20 (m, 4H), 7.88 (dd, J = 2.7, 9.6 Hz, 1H), 8.15 (s, 1H). Free base was converted to the corresponding oxalate salt by adding oxalic acid in ethyl acetate to 26a in CH2Cl2. mp: 84 °C (decomposed). Anal. (C24H27FN2O3• H2C2O4•H2O) C, H, N.</p><!><p>To a solution of 28 (1.60 g, 7.3 mmol) in THF was added n-BuLi (1.6 M, 7 mL) at -78 °C under N2 atmosphere. The solution was stirred at -78 °C for 1 h and a solution of tert-butyl-4-(methoxy(methyl)carbamoyl)piperidine-1-carboxylate 11 (1.0 g, 3.6 mmol) in THF was added. The solution was maintained at -78 °C for 4 h, and then quenched with saturated aqueous NH4Cl that was allowed to warm to room temperature with stirring. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined extracts were washed with water, dried over Na2SO4, filtered, concentrated, and the residue chromatographed on a silica gel column with ethyl acetate : hexane (1:4, v/v) to give the t-Boc protected compound tert-butyl-4-(3-(2-fluoroethoxy)picolinoyl)piperidine-1-carboxylate (0.51 g, 40%), 1H NMR (CDCl3): δ 1.47 (s, 9H), 1.62-1.70 (m, 4H), 2.83 (s, 2H), 3.64-3.73 (m, 1H), 4.17 (s br, 2H), 4.25-4.36 (m, 2H), 4.68-4.87 (m, 2H), 7.23-7.26 (m, 1H), 7.38-7.40 (m, 1H), 8.25-8.30 (m, 1H).</p><p>To a solution of the above compound (0.49 g, 1.4 mmol) in CH2Cl2 (30 mL), TFA (2 mL) was added. The reaction mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure, and the residue was neutralized with 1 N NaOH solution and extracted with CH2Cl2 (2 × 15 mL). The organic layer was washed with brine, dried and concentrated to give crude 4-(3-(2-fluoroethoxy)picolinoyl)piperidine. The crude product was used in the next step without further purification.</p><p>A mixture of the above compound, 18 (0.10 g, 0.68 mmol) and Et3N (0.3 mL, 2.2 mmol) in ethanol (5 mL) was stirred at 75 °C for 36 h. After cooling to room temperature, the mixture was poured into water, extracted with EtOAc (3 × 15 mL). The organic layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 26b (0.12 g, 43%). 1H NMR (CDCl3, free base): δ 1.73-1.98 (m, 4H), 2.32-2.40 (m, 1H), 2.71-2.96 (m, 8H), 3.31-3.33 (m, 1H), 3.49-3.56 (m, 1H), 3.80-3.89 (m, 1H), 4.24-4.36 (m, 2H), 4.69-4.87 (m, 2H), 7.09-7.25 (m, 4H), 7.34-7.38 (m, 2H), 8.27-8.29 (m, 1H). The corresponding oxalate salt was obtained by adding oxalic acid in ethyl acetate the solution of 26b in CH2Cl2 and then recrystallized. For the oxalate salt, mp: 119.9 °C (decomposed). Anal. (C23H27FN2O3•H2C2O4•H2O) C, H, N.</p><!><p>A mixture of 2-bromopyridin-3-ol 27 (0.17 g, 1.0 mmol), 1-bromo-2-fluoroethane (0.25 g, 2.0 mmol) and K2CO3 (0.55 g, 4.0 mmol) in acetonitrile were stirred at reflux for one day until TLC indicated that the reaction was complete. The reaction mixture was filtered, dried and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to afford 28 (0.16 g, 68%). 1H NMR (CDCl3): δ 4.25-4.34 (m, 2H), 4.73-4.89 (m, 2H), 7.20-7.22 (m, 2H), 8.01 (s, 1H).</p><!><p>A mixture of 29 (0.20 g, 0.78 mmol), 17a (0.44 g, 2.3 mmol) and Et3N (0.5 mL) in ethanol (10 mL) was stirred at 60 °C for 48 h. To the mixture, 1 N NaOH (3 mL) was added and the stirring was continued overnight. The solvent was removed, the residue was extracted with EtOAc (3 × 30 mL) and the organic layer was washed with aqueous Na2CO3 solution, dried and concentrated. The crude product was purified by column chromatography to give 30a (0.11 g, 37%). 1H NMR (CDCl3, free base): δ 1.85-1.96 (m, 4H), 2.43-2.53 (m, 2H), 2.67-2.89 (m 5H), 2.96-3.28 (m, 3H), 3.60 (s br, 2H), 3.85 (m, 1H), 3.95 (s, 3H), 6.14 (m, 1H), 6.57 (m, 2H), 6.83 (s br, 1H), 6.96-7.02 (m, 2H). Free base was converted to the corresponding oxalate salt by adding oxalic acid in ethyl acetate to 30a in CH2Cl2. mp: 131 °C (decomposed). Anal. (C21H27N3O2 • 2H2C2O4 • 2H2O) C, H, N.</p><!><p>Compound 30c was prepared from compound 17c as described in procedure E. Yield, 61%. 1H NMR (CDCl3, free base): δ 1.80-2.05 (m, 4H), 2.36-2.53 (m, 2H), 2.68-2.92 (m, 5H), 3.01-3.35 (m, 4H), 3.61 (s br, 2H), 3.87 (m, 1H), 6.54-6.60 (m, 2H), 6.99 (td, J = 7.5, 3.3 Hz, 1H), 7.45 (dd, J = 8.1, 4.8 Hz, 1H), 8.24 (dt, J = 8.1, 1.8 Hz, 1H), 8.80 (dd, J = 4.8, 1.8 Hz, 1H), 9.17 (d, J = 2.1 Hz, 1H). The free base was converted to the oxalate salt. mp: 109 °C (decomposed). Anal. (C21H25N3O2 • 2H2C2O4 • 0.5H2O) C, H, N.</p><!><p>Compound 30d was prepared from compound 17d as described in procedure E. Yield, 44%. 1H NMR (CDCl3, free base): δ 1.79-1.99 (m, 5H), 2.35-2.53 (m, 2H), 2.64 (s, 3H), 2.67-3.28 (m, 8H), 3.62 (s br, 2H), 3.87 (m, 1H), 6.57 (m, 2H), 6.99 (m, 1H), 7.29 (d, J = 8.1 Hz, 1H), 8.13 (dd, J = 8.1, 2.1 Hz, 1H), 9.04 (d, J = 2.1 Hz, 1H). The free base was converted to the oxalate salt. mp: 98 °C (decomposed). Anal. (C24H29N3O4 • H2C2O4) C, H, N.</p><!><p>Compound 30e was prepared from compound 17e as describe in procedure E. Yield, 43%. 1H NMR (CDCl3, free base): δ 1.65 (s br, 1H), 1.80-2.02 (m, 4H), 2.42-2.53 (m, 2H), 2.67-3.03 (m, 6H), 3.21-3.28 (m, 2H), 3.61 (s br, 2H), 3.86 (m, 1H), 4.02 (s, 3H), 6.57 (m, 2H), 6.82 (d, J = 8.7 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 8.15 (dd, J = 8.7, 2.4 Hz, 1H), 8.80 (d, J = 2.4 Hz, 1H). The free base was converted to the oxalate salt. mp: 81.4 °C (decomposed). Anal. (C22H27N3O3 • 2H2C2O4 • 1.5H2O) C, H, N.</p><!><p>Compound 31a was prepared from compound 17a as describe in procedure E. Yield, 20%. 1H NMR (CDCl3, free base): δ 1.85-1.96 (m, 4H), 2.36-2.45 (m, 2H), 2.76-2.89 (m 5H), 2.96-3.16 (m, 3H), 3.62 (s br, 2H), 3.92 (m, 1H), 3.96 (s, 3H), 6.15 (m, 1H), 6.56 (m, 2H), 6.83 (s br, 1H), 6.96- 7.02 (m, 2H). The free base was converted to the oxalate salt. mp: 114 °C (decomposed). Anal. (C21H27N3O2 • 2H2C2O4 • 1.5H2O) C, H, N.</p><!><p>To a solution of (-)-6 (153.6 mg, 0.438 mmol) in THF was added Di-tert-butyl dicarbonate (Boc2O, 287.0 mg, 1.314 mmol), Et3N (0.5 mL) and DMAP (5.4 mg, 0.044 mmol). The reaction mixture was stirred at room temperature while monitoring by TLC. After 1.5 hrs the starting material completely disappeared, at which point the reaction mixture was concentrated on Rotary evaporator and partitioned the residue with brine and ethyl acetate. Aqueous phase was washed with ethyl acetate. The organic phase was dried over sodium sulfate, concentrated and purified on silica gel column (8:1 to 6:1 hexanes: ethyl acetate) to give the tri-Boc protected intermediate (-)-32 which was dissolved in methanol and excess K2CO3 was added and refluxed overnight. The product was partitioned between brine and dichloromethane. Aqueous phase was washed twice with dichloromethane. The organic phase was dried over sodium sulfate and concentrated to give (-)-33 as a white solid in 28% yield. 1H NMR (400 MHz, CDCl3): δ 1.55 (s, 9H), 1.78-1.95 (m, 4H), 2.42 (t, J = 12.0 Hz, 1H), 2.78-3.05 (m, 7H), 3.25-3.35 (m, 2H), 3.83-3.90 (m, 1H), 6.73 (s, 1H), 7.08-7.07 (m, 4H), 7.46 (d, J = 11.6 Hz, 2H), 7.92 (d, J = 12.4 Hz, 2H).</p><!><p>[11C]CH3I was produced at our institution from [11C]CO2 using a GE PETtrace MeI Microlab. Up to 1.4 Ci of [11C]CO2 was produced from Washington University's JSW BC-16/8 cyclotron by irradiating a gas target of 0.5% O2 in N2 for 15-30 min with a 40 μA beam of 16 MeV protons. The GE PETtrace MeI microlab coverts the [11C]CO2 to [11C]CH4 using a nickel catalyst (Shimalite-Ni, Shimadzu, Japan P.N.221-27719) in the presence of hydrogen gas at 360 °C; it was further converted to [11C]CH3I by reaction with iodine that was held in a column in the gas phase at 690 °C. Several hundred millicuries of [11C]CH3I were delivered as a gas at approximately 12 min after end of bombardment (EOB), to the hot cell where the radiosynthesis was accomplished.</p><!><p>Approximately 1.2 - 1.5 mg of (-) enantiomer of the precursor was placed in a reaction vessel and 0.2 mL of DMF was added followed by 3.0 μL of 5N aqueous sodium hydroxide. The mixture was thoroughly mixed on a vortex. [11C]CH3I was bubbled into the reaction vessel. The reaction mixture was heated at 85 °C for 5 min. The reaction vessel was removed from heating, and 200 μL trifluoroacetic acid was added. The reaction vessel was mixed well and heated again at 85 °C for 5 min, and then quenched by adding 1.4 mL of HPLC mobile phase (29% acetonitrile in 0.1M ammonium formate buffer pH 4.5) and 200 μL 5N aqueous NaOH to neutralize the reaction mixture. The solution was loaded on to reverese phase C-18 column (Phenomenex Luna C18, 250mm × 9.6 mm, 10 μm with UV wave length at 254 nm); using the above HPLC mobile phase, at a flow rate of 4.0 ml/min, the radioactive product was collected between 17.0 and 19.0 min, and diluted with 50 mL of sterile water. The aqueous solution was passed through C18 Sep-Pak Plus (to trap the product) by applying nitrogen pressure. The collection bottle was rinsed by adding 10 mL water and passed through Sep-Pak plus. The product was collected in a vial by eluting C18 Sep-Pak plus with 0.6 mL of EtOH and 5.4 mL of saline to formulate the injection dose. The injection dose sample was authenticated using analytical HPLC system by co-injecting with cold standard compound (-)-24b. The HPLC system for quality control is: Column: Phenomenex Prodigy C18 analytical column, 250 × 4.6 mm; Mobile phase: 37% acetonitrile in 0.1 M ammonium formate buffer pH=4.5; Flow rate: 1.2 mL/min; the retention time for (-)-[11C]-24b was 4.9 min. The entire process was completed in approximately 1 hr. The radiochemical yield was 40-50% (decay corrected to EOB) with the radiochemical purity of > 99% and the specific activity was > 2000 Ci/mmol (decay corrected to end of synthesis).</p><!><p>Partition coefficient was measured by mixing the (-)-[11C]24b sample with 3 mL each of 1-octanol and buffer that is 0.1 M phosphate and pH equals 7.4 in a test tube. The mixture in the test tube were vortexed for 20 s followed by centrifugation for 1 min at room temperature. 2 mL of organic layer was transferred to a second test tube, 1 mL 1-octanol and 3 mL PBS buffer was added. The resulting mixture was vortexed for 20 s followed by centrifugation for 1 min at room temperature. 1 mL of organic and aqueous layer were taken separately for measurement. The radioactivity content values (count per minute) of two samples (1 mL each) from the 1-octanol and buffer layers were counted in a gamma-counter. The partition coefficient Log D7.4 was determined by calculating as the decimal logarithm the ratio of cpm/mL of 1-octanol to that of buffer. The measurements were repeated three times. Value of partition coefficient is 2.60.</p><!><p>In vitro binding assays to VAChT were conducted with human VAChT permanently expressed in PC12 cells at about 50 pmol/mg of crude extract. No significant amounts of σ1 or σ2 receptors were present. The radioligand used was 5 nM (-)-[3H]vesamicol, and the assay was conducted at final concentrations of 10-11 M to 10-5 M novel compounds.4, 19 Unlabeled (-)-vesamicol was used as an external standard, for which Ki = 15 nM, and the mixture was allowed to equilibrate at 23 °C for 20 hours. Duplicate data were averaged and fitted by regression of a rectangular hyperbola to estimate the Ki value of the novel compounds.</p><!><p>The compounds were dissolved in DMF, DMSO or ethanol, and diluted in 50 mM Tris-HCl buffer containing 150 mM NaCl and 100 mM EDTA at pH 7.4 prior to performing the σ1 and σ2 receptor binding assays. The detailed procedures for performing the binding assays have been described.19, 49 For the σ1 receptor assays, guinea pig brain membrane homogenates (~300 μg protein) were the receptor resource and ~5 nM (+)- [3H] pentazocine (34.9 Ci/mmol, Perkin-Elmer, Boston, MA) was the radioligand. The incubation was performed in 96-well plates for 90 min at room temperature. Nonspecific binding was determined from samples that contained 10 μM of nonradioactive haloperidol. After 90 min, the reaction was quenched by addition of 150 μL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). The harvested samples were filtered rapidly through a 96-well fiberglass filter plate (Millipore, Billerica, MA) that had been presoaked with 100 μL of 50 mM Tris-HCl buffer at pH 8.0 for 60 min. Each filter was washed 3 × 200 μL of ice-cold wash buffer, and the filter counted in a Wallac 1450 MicroBeta liquid scintillation counter (Perkin-Elmer, Boston, MA). The σ2 receptor binding assays were determined using rat liver membrane homogenates (~300 μg protein) and ~5 nM [3H]DTG (58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the presence of 1 μM of (+)-pentazocine to block σ1 sites. The incubation time was 120 min at room temperature. Nonspecific binding was determined from samples that contained 10 μM of nonradioactive haloperidol. All other procedures were same as those described for the σ1 receptor binding assay above.</p><p>The IC50 value was determined using nonlinear regression analysis. Competitive curves were best fit with a one-site model and gave pseudo-Hill coefficients of 0.6-1.0. Ki values were calculated using the method of Cheng and Prusoff50 and are presented as the mean (± 1 SEM). For these calculations, we used a Kd value of 7.89 nM for [3H](+)-pentazocine binding to σ1 receptor in guinea pig brain and a Kd value of 30.7 nM for [3H]DTG binding to σ2 receptor in rat liver.</p><!><p>All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals established by Washington University's Animal Studies Committee. For the biodistribution studies, ~350 μCi of (-)-[11C]24b in about 175 μL of 10% ethanol/saline solution (v/v) was injected via the tail vein into mature male Sprague–Dawley rats (185 - 205 g) under anesthesia (2.5% isoflurane in oxygen at a flow rate of 1 mL/min). A group of at least four rats were used for each time point. For the control group, at 5 and 30 min post injection, the rats were anesthetized and euthanized. For the CycA pretreated group, CycA (Sandimmune diluted 1:1 with saline) at a dose of 25 mg/kg were administrated by intravenously (i.v.) 30 min prior to radioligand injection; at 30 min post injection of the radioligand, (-)-[11C]24b, the rats were euthanized. The whole brain was quickly harvested and various organs dissected comprising cerebellum, brain stem, cortex, striatum, thalamus, hippocampus and the brain. The remainder of the brain was also collected in order to determine total brain uptake. Simultaneously, samples of blood, heart, lung, liver, spleen, pancreas, kidney, muscle, fat, and tail were dissected. All the tissue samples were collected in the tared tubes and counted in an automated gamma counter (Beckman Gamma 8000 well counter) along with the standard solution of (-)-[11C]24b prepared by diluting the injectate. The counted tissues were then weighed, and the %ID/g was calculated.</p><!><p>A microPET Focus 220 scanner (Concorde/CTI/Siemens Microsystems, Knoxville, TN) was used for the imaging studies of (-)-[11C]24b in male cynomolgus monkey (4–6 kg). The animals were fasted for 12 h before the study. The animals were initially anesthetized using an intramuscular injection with ketamine (10 mg/kg) and glycopyrulate (0.13 mg/kg), and intubated with an endotracheal tube under anesthesia (maintained at 0.75–2.0% isoflurane in oxygen) throughout the PET scanning procedure. After intubation, a percutaneous venous catheter was placed for radiotracer injection. A 10 min transmission scan was performed to check the positioning; once confirmed, a 45 min transmission scan was obtained for attenuation correction. After that, the animal was administrated 5–7 mCi of (-)-[11C]-24b via the venous catheter, Subsequently, a 100 min dynamic PET scan (3 × 1 min, 4 × 2 min, 3 × 3 min, and 20 × 5 min) was acquired. During the whole procedure, core temperature was kept constant at 37 °C with a heated water blanket. In each microPET scanning session, the head was positioned supine in the adjustable head holder with the brain in the center of the field of view. For each subject, at least three independent PET studies were performed (n = 3).</p><!><p>PET image reconstructed resolution was < 2.0 mm full width half maximum for all 3 dimensions at the center of the field of view. Emission scans were corrected using individual attenuation and model-based scatter correction and reconstructed using filtered back projection as described previously.51 The first baseline PET image for each animal acted as the target image with the MPRAGE MRI scan and subsequent PETs coregistered to it using automated image registration program AIR.52, 53 All MPRAGE-based volume of interest (VOI) analyses were done by investigators blinded to the clinical status of the monkeys.4 For quantitative analyses, three-dimensional regions of interest (ROI) (cerebellum, frontal, occipital, striatum, temporal, white matter, midbrain and hippocampus) were transformed to the baseline PET space and then overlaid on all reconstructed PET images to obtain time–activity curves. Activity measures were standardized to body weight and dose of radioactivity injected to yield standardized uptake value (SUV).</p>

PubMed Author Manuscript

Impaired beta-oxidation increases vulnerability to influenza A infection

Influenza A virus (IAV) infection casts a significant burden on society. It has particularly high morbidity and mortality rates in patients suffering from metabolic disorders. The aim of this study was to relate metabolic changes with IAV susceptibility using well-characterized inbred mouse models. We compared the highly susceptible DBA/2J (D2) mouse strain for which IAV infection is lethal with the C57BL/6J (B6) strain, which exhibits a moderate course of disease and survives IAV infection. Previous studies showed that D2 has higher insulin and glucose levels and is predisposed to develop diet-induced type 2 diabetes. Using high-resolution liquid chromatography–coupled MS, the plasma metabolomes of individual animals were repeatedly measured up to 30 days postinfection. The biggest metabolic difference between these strains in healthy and infected states was in the levels of malonylcarnitine, which was consistently increased 5-fold in D2. Other interstrain and intrastrain differences in healthy and infected animals were observed for acylcarnitines, glucose, branched-chain amino acids, and oxidized fatty acids. By mapping metabolic changes to canonical pathways, we found that mitochondrial beta-oxidation is likely disturbed in D2 animals. In noninfected D2 mice, this leads to increased glycerolipid production and reduced acylcarnitine production, whereas in infected D2 animals, peroxisomal beta-oxidation becomes strongly increased. From these studies, we conclude that metabolic changes caused by a distortion of mitochondrial and peroxisomal metabolism might impact the innate immune response in D2, leading to high viral titers and mortality.

impaired_beta-oxidation_increases_vulnerability_to_influenza_a_infection

<!>Results<!><!>Analysis of metabolomics data<!><!>Acylcarnitines<!>Fatty acids<!>Oxidized FAs<!>Glycerolipids<!>Carbohydrates<!>Amino acids and derivatives<!>Nucleobases<!>NAM<!>Phenols<!><!>Omitted metabolites<!>Discussion<!>Mitochondrial beta-oxidation is decreased in noninfected D2 mice<!><!>Mitochondrial beta-oxidation is decreased in noninfected D2 mice<!><!>Glycerolipid synthesis is increased in noninfected D2 mice<!>Interstrain difference in NAM and purine pathways for noninfected animals<!>Similarities in metabolic effects during IVA infection in both strains and recovery in B6<!>Interstrain differences in energy metabolism after infection<!>Interstrain differences in immune response<!>Conclusion and future perspectives<!>Chemicals<!>Mouse husbandry and infections<!>Sample preparation<!>Metabolite analysis<!>Data treatment and analysis<!><!>Data treatment and analysis<!>Data availability<!>Supporting information<!>Conflict of interest<!>Supporting information

<p>Edited by Qi-Qun Tang</p><p>Every year, the influenza epidemic costs the lives of hundreds of thousands of people worldwide. The severity of disease is determined by many environmental and intrinsic factors. Besides age, diabetes, obesity, and metabolic syndrome are major risk factors for severe outcome (1, 2). However, the complexity of factors that impact susceptibility to influenza makes it difficult to assess the underlying mechanism in humans and necessitate studies in well-controlled experimental models. In this work, we compared two well-characterized mouse strains with different metabolic phenotypes and different susceptibilities to influenza A virus (IAV) infections. We aimed to find metabolic pathways correlated with adverse outcome of IAV infection.</p><p>This study makes use of the C57BL/6J (B6) and DBA/2J (D2) mouse strains. B6 mice recover readily from infection with a low virulent IAV strain, whereas such an infection is lethal in D2 mice. Genetic mapping has identified several genomic regions that are associated with the increased susceptibility of D2 mice to infections. However, the detailed mechanisms are still elusive (3, 4, 5).</p><p>Furthermore, both mouse strains were previously studied in different metabolic contexts, including diabetes, energy metabolism (6, 7, 8, 9, 10, 11, 12, 13), and hepatic steatosis (14). These studies revealed that under normal and nonchallenge conditions, levels of insulin, triacylglycerides, and fat mass are about twice as high in D2 compared with B6. In addition, D2 is more predisposed to the development of diet-induced type 2 diabetes. Therefore, these interstrain metabolic differences might further explain the difference in susceptibility to IAV infections.</p><p>By repeated sampling of individual animals early after infection and up to 30 days postinfection (dpi), we compared the plasma metabolomes of healthy and infected B6 and D2 animals. We used an untargeted and high-resolution liquid chromatography–coupled mass spectrometric (hrLCMS) approach to detect and quantify plasma metabolites. After multistep data analysis, changes in several metabolite families were found between strains and within strains upon infection. Observed differentially expressed metabolites were mapped to canonical pathways. Taking in account prior knowledge and the newly observed interstrain metabolic differences in healthy and infected animals, we propose a hypothesis for the high susceptibility of D2 mice to IAV infection. This may shed light on the cause for severe disease in human patients and aid in development of novel therapeutic approaches in patients with metabolic comorbidities.</p><!><p>Metabolic responses were measured by an untargeted hrLCMS approach. Infection with IAV invoked physiological and metabolic changes in both mouse strains. Some effects were similar between strains but many differed. Moreover, animals in the control arm of the experiment (mock infection and 0 dpi) also showed metabolic interstrain differences. The results with respect to hrLCMS data analysis, metabolic effects, and physiological behavior are described later.</p><!><p>Changes in body weights after IAV infection for D2 and B6 mouse strains. CI, 95% confidence interval for the LOESS fit; IAV, influenza A virus.</p><!><p>To track the plasma metabolomes of the animals, raw hrLCMS data were cleaned up, adjusted, and modeled. After automatic peak extraction, 10,350 features in positive electrospray ionization mode and 1461 features in negative electrospray ionization mode were collected over all the samples. Noise reduction and other cleaning steps reduced these numbers by about 63%, leaving the total number of features at 4318. After subsequent data adjustment, 1641 features showed a p value of less than 0.001 in a linear mixed-effect (LME) model. Subsequent post hoc analysis resulted in a selection of 706 features that showed differences between either mock and infected groups or between strains. This feature set was then manually integrated. The reintegrated signals were again subjected to adjustments and statistical filtering. Finally, 106 selected features were identified, and 52 more features were added by pathway enrichment, resulting in a final set containing 158 identified metabolites (Fig. S1). Most of these metabolites were related to specific canonical metabolic pathways, that is, beta-oxidation, phospholipid, nucleotide, and nicotinamide (NAM) metabolism. The final set was used to form a hypothesis explaining the increased vulnerability of D2 mice to IAV infection. The entire set, including chemical details, is included in Table S1.</p><!><p>Responses in lipid metabolism in healthy and infected animals for D2 and B6 strains. Heatmaps of percent change from baseline (Δ%) for the lipid-associated metabolites in serum (A) and correlations between Δ% for these metabolites within each lipid class for both strains at 3 dpi (B). The colors of the heatmap tiles define the direction of the effects, whereas their sizes define the credibility of this direction. For clarity, color scales for positive effects (red) are cut off at 100% so that extreme values do not dominate the scale. The top row shows the differences between strains (interstrain) for baselines (BL, i.e., pooled 0 dpi and mock 3 and 5 dpi results). Since the reference strain was taken to be B6, a positive Δ% means an increase in D2 levels (e.g., MCar). The second row shows the Δ% for D2 between 0 dpi (reference) and infected 3 dpi. Rows 3 to 7 show the Δ% for B6 baseline and infected day 3 to day 30. The relative magnitudes for the raw signals with respect to the median within these finer classifications are shown in the bottom rows. The magnitude gray scale starts at 0 and is cut off at 1 where 1 would be the metabolite with the median value per class. Note that these magnitudes are only informative for metabolites that have similar ionization efficiencies; hence, no magnitude is displayed for the "polar" group. Interstrain correlations for Δ% per lipid class at 3 dpi (log scales) are displayed in panel B. The diagonal gray line indicates the identity line (i.e., equal effects in both strains). The ellipses indicate the 50% posterior percentile intervals per metabolite. Red dots indicate metabolites with opposite effects between strains. The panel "Glycerolipids" in (B) include all detected DAGs, (L)PIs, (L)PCs, and LPEs. DAGs, diacylglycerols; LCFAs, long-chain fatty acids; (L)PCs, (lyso)phosphatidylcholines; LPE, lysophosphatidylethanolamines; (L)PIs, (lyso)phosphatidylinositols; MCFAs, medium-chain fatty acids; OxFAs, oxidized fatty acids; VLCFAs, very long–chain fatty acids.</p><p>Heatmaps of percent change from baseline (Δ%) for metabolites in serum. The colors of the heatmap tiles define the direction of the effects, whereas their sizes define the credibility of this direction. For clarity, color scales for positive effects (red) are cut off at 100% so that extreme values do not dominate the scale. Interstrain correlations for Δ% at 3 dpi (log scales) are displayed in panel B. For abbreviations of metabolites, see Table S1. CHs, carbohydrates and related; NAM, nicotinamide.</p><!><p>The most important differences between and within strains were observed for acylcarnitines (ACars; Fig. 2A). Levels in healthy animals for most ACar species were approximately −50% lower in D2 compared with B6. Conversely, malonylcarnitine (MCar) was +505% higher in D2.</p><p>During infection at 3 dpi, B6 ACar levels with six or more acyl-carbons stayed more or less stable, whereas D2 levels showed a strong increase, ranging from +56% for Car(6:0) to +670% for Car(18:1/O) (Fig. 2, A and B). Note from Figure 2B that there is a positive correlation between relative effect sizes in both strains with a positive offset for D2. This means that ACars that are upregulated in B6 are stronger upregulated in D2 (e.g., Car(18:1/O)); ACars that were downregulated in B6 are moderately upregulated in D2 (e.g., Car(18:2), red points, opposite effects); and ACars that were strongly downregulated in B6 were moderately downregulated in D2 (e.g., Car(4:0)).</p><p>During 5 dpi to 8 dpi, the medium-chain and long-chain ACar levels in B6 decreased between −38% and −83%. During recovery, a divergence of ACar levels with respect to oxidation state and chain length was observed in B6 mice. Most oxidized short-chain and medium-chain ACars were increased, whereas the remaining species stayed equal or showed a decrease, compared with healthy baseline.</p><!><p>In healthy animals, the most abundant long-chain fatty acids (LCFAs) and very long–chain fatty acids (VLCFAs), that is, FA(16:0), FA(18:0), FA(18:1), FA(20:4), and FA(22:6), showed no important differences between strains (Fig. 2A).</p><p>After infection, most unsaturated medium-chain fatty acids (MCFAs) and LCFAs showed no change or an increase in both strains. Increases were slightly higher in D2, equivalent to ACars (Fig. 2, A and B). Exceptions were FA(18:1) and FA(16:1), which showed opposite interstrain effects, namely an increase of about +20% in B6 and a decrease of about −30% in D2 (red points in the panel of MCFAs and LCFAs; Fig. 2B). VLCFAs showed decreases upon infection in both strains. Overall, these decreases were stronger in D2 compared with B6.</p><p>FAs showed a strong decease in B6 at 5 and 8 dpi, similar to ACars. Most saturated and monounsaturated MCFAs and LCFAs (e.g., FA(12:0) and FA(16:1)) showed a strong upregulation (from +36% up to +153%) during recovery. Conversely, all the polyunsaturated VLCFAs showed a decrease, ranging from −31% to −65%, after the virus was eliminated at 30 dpi.</p><!><p>In healthy animals, oxidized MCFAs were all decreased in D2 compared with B6, ranging from −60% for FA(12:1/O) to −18% for FA(10:1/O). Oxidized LCFAs showed no interstrain differences in healthy animals. Upon infection, saturated oxidized species showed opposite effects between species (red points, OxFAs panel; Fig. 2B). In B6, the saturated and oxidized FA levels were higher (up to +70%), whereas in D2, these metabolites showed a decrease (down to −65%). Moreover, all oxidized LCFAs were strongly decreased in D2 at 3 dpi. During disease progression in B6, levels of most oxidized FAs dropped between 5 dpi and 8 dpi. Analogous to MCFAs and LCFAs, all OxFA species showed a strong increase in B6 upon recovery (up to +132%).</p><!><p>In healthy animals, the biggest interstrain differences for lipids were seen for diacylglycerols (DAGs) and phosphatidylinositols, which were around +80% higher in D2 compared with B6 (Fig. 2A). Other detected glycerolipids did not show important differences between strains. Variations for (lyso)phosphatidylcholines (LPCs, PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylinositols (LPIs) were approximately between +20% and −20%. The same was observed for polar, lipid, and carnitine-associated metabolites like glycerophosphorylcholine, carnitine, and γ-butyrobetaine. In general, the most abundant members of the glycerolipid families showed the lowest effects.</p><p>In general, all lipid species, including DAGs, PCs, LPCs, LPEs, and LPIs, became downregulated upon infection at 3 dpi (Fig. 2A). These downregulations were stronger for D2 (Fig. 2B). The biggest drops were observed in D2, especially for the DAGs. Plasma levels of DAGs in D2 decreased by −44% to −80% compared with healthy D2 animals, whereas in B6, these drops lay between −27% and −62%. Moreover, strong drops in polar lipid and carnitine-associated metabolites were seen in infected animals of both strains; for example, glycerophosphorylcholine, glycerophosphorylethanolamine, crotonobetaine, and trimethylamine N-oxide (TMAO). During the recovery period in B6, betaine, crotonobetaine, and TMAO were upregulated between 8 and 30 dpi.</p><!><p>Baseline glucose and succinate levels in healthy animals were more than −20% lower in D2 compared with B6 (Fig. 3) and were not clearly affected by infection in either strain. Succinate was about −50% lower during infection in both species at 3 dpi. In B6, succinate stayed decreased at 5 dpi to recover slightly under baseline after 18 dpi.</p><!><p>Interstrain differences in baseline plasma levels in healthy animals were detected for amino acids and their derivatives (Fig. 3). Decreases in D2 compared with B6 were found for the essential AA lysine (Lys; −38%) and branched chain AAs valine (Val; −55%) and isoleucine/leucine (Ile/Leu; −21%). Glutamine (Gln) levels were −23% lower in D2. The most important interstrain difference for AA derivatives was observed for acetylornithine and was −71% lower in D2.</p><p>Upon infection, Val levels at 3 dpi dropped by −88% in D2, whereas virtually, no change was observed in B6. Other differences between infected strains were seen for Ile/Leu and phenylalanine (Phe). These species were about +40% upregulated in D2, whereas a decrease of about −15% was observed in B6. A similar but less pronounced effect was observed for Gln (+9% in D2 versus −9% in B6). Furthermore, in infected animals, Lys, arginine, and tyrosine were decreased by about −50% in B6, whereas no change was observed in D2. Effects for AA derivates tracked in equal directions between strains, with the bigger effect sizes observed in D2. Upregulations were seen for sulfur-containing derivatives, isethionate and taurine, whereas most other derivatives showed downregulations. Especially, 3-indolepropionate was downregulated by about −80% in both species. Differential effects were seen for acetylated histidine and Phe and 3-methyldioxyindole, which were strongly upregulated in D2 (between +50% and +100%) and downregulated in B6 (all around −35%).</p><p>For B6, the amino acid profile on 5 dpi was the same as on 3 dpi. Levels of most AAs tended to normal between 8 dpi and 18 dpi, yet stayed somewhat below initial levels. However, some increase with respect to the healthy B6 baseline was seen for proline, Phe, serine, and Val. At 30 dpi, all detected species except Val were lower than they were initially.</p><!><p>In healthy animals, the pyrimidine nucleobases thymidine, uridine, and cytidine were all lower in D2 (respectively −76%, −35%, and −19%) with respect to B6 (Fig. 3). The purine-derived metabolite allantoin was also lower in D2 by −32%. After infection, at 3 dpi, the changes in both strains moved in the same direction except for cytosine, which was +36% higher in D2 and −16% lower in B6. In both strains, the biggest observed changes were the decreases of the pyrimidine base 5-methylcytidine (by about −40%), and all detected purine-related metabolites, that is, allantoin, urate, methylguanosine, and 5-hydroxyisourate (5HIU) (between −21% and −50%). Deoxycytidine was increased by about +35% in both strains. Moreover, compared with 0 dpi, this metabolite generated a substantial spike (+88%) in B6 at 8 dpi and returned to normal at 18 dpi. After recovery, most detected nucleobase-related metabolites were still lower after 8 dpi in B6.</p><!><p>Interstrain differences in healthy animals were detected for metabolites in the NAM pathway (Fig. 3). N1-methyl-n-pyridone-5-carboxamide (nPY, n = 2 or 4) was −83% lower in D2. Conversely, levels of N1-methylnicotinamide (MNA) were +23% elevated in D2.</p><p>During infection, different responses between strains were seen for nPY. Levels for this metabolite went up in D2 by +36% and down in B6 by −30% at 3 dpi. Other detected metabolites of the NAM pathway were downregulated in both strains during infection with the strongest effect seen for trigonelline, which was decreased in both strains by about −80%. In B6, except for trigonelline, all NAM-related metabolites stayed downregulated after recovery. The strongest downregulation during the recovery phase in B6 was seen for nPY (−86%). Trigonelline saw an increase of around +57% at 8 and 18 dpi to return to normal at 30 dpi.</p><!><p>Sulfated phenolic compounds showed interstrain differences in baseline concentrations, ranging from −49% decrease (dihydroxybenzoic acid sulfate) to +59% increase (ferrulic acid sulfate) in D2 with respect to B6 (Fig. 3). Moreover, hippuric acid was +71% decreased in D2. During infection, in both species, the plasma levels of all phenolsulfates dropped by around −80% at 3 dpi with stronger negative effects observed in D2 In B6, these levels stayed reduced by the same amount at 5 dpi. In the recovery phase, some of the phenols returned to normal, whereas most of them exceeded the initial levels up to +100%. Only vanillic acid sulfate kept on being reduced.</p><!><p>Correlations between Δ% for nutritional markers for both strains at 3 dpi. The diagonal gray line indicates the identity line (i.e., equal effects in both strains). The ellipses indicate the 50% posterior percentile intervals per metabolite.</p><!><p>To keep the article concise, some metabolite classes were omitted from the Results section. Omission was based on whether metabolites were used in the mechanistic discussion. Metabolite classes that were omitted were a miscellaneous group including vitamins. These omitted markers are included in the supporting information (Tables S1 and S2, and Figs. S2 and S3).</p><!><p>Influenza can be a life-threatening disease in immunocompromised individuals, which is clearly observed in patients suffering from metabolic disorders. Thus, new insights into pathophysiological metabolic mechanisms involved in IAV infection could help to ameliorate symptoms in these patients through tailored therapeutic or dietary interventions. Therefore, in this work, plasma from mouse models of high and moderate influenza disease was used to study differential metabolic responses to IAV infection. These responses were subsequently used to infer a mechanism as to why the D2 strain is highly vulnerable to IAV, whereas B6 is not.</p><p>However, some limitations are associated to plasma metabolic studies given that an important characteristic of metabolism is the compartmentalization of metabolic processes on organ, cell, or organelle level. Hence, information on the origin or destination of detected plasma metabolites is obscured since all organs and cell types contribute to or extract from the plasma metabolome. What is more, the plasma metabolome contains mainly metabolites that can, either actively or passively, traverse cell membranes depending on, for example, chemical nature or concentration. This means that in plasma, not all members of a certain metabolic pathway can be detected. This incompleteness problem is aggravated by the fact that the chemical diversity of metabolites is vast, that is, they vary in molecular weight, polarity, stability, or concentration. This is a challenge from an analytical chemical perspective. Despite hrLCMS being one of the more sensitive and selective methods available, it is simply not possible for a single technique to detect or to identify the entire metabolite set.</p><p>Nevertheless, a big corpus of information is available on most metabolic pathways, including their members, interactions, and locations. Moreover, genomic, transcriptomic, proteomic, and metabolomic information is available for the B6 and D2 strains. As such, it is known that both B6 and D2 mice have a predisposition for diet-induced type II diabetes, but D2 has the worst outcome. It is the D2 strain that shows a diabetic phenotype from the outset, including insulin resistance and lipid accumulation (6, 12). We used this prior information, together with the observed changes in the plasma metabolome from this untargeted metabolomics study, to deduce a hypothesis for the heightened vulnerability of D2 toward the IAV.</p><!><p>High baseline plasma levels of MCar in D2 suggest decreased mitochondrial beta-oxidation, which is offset by other energy-generating processes including increased tricarboxylic acid (TCA) cycle activity and peroxisomal beta-oxidation.</p><p>The main observed metabolic differences between strains at baseline were the vastly increased plasma levels of MCar in D2. MCar is a proxy for the production of malonyl-coenzyme A (MCoA), and differences in plasma levels of MCar reflect intracellular concentrations of MCoA (15, 16). MCoA plays a crucial role in lipid metabolism. It is a strong inhibitor of carnitine palmitoyltransferase I (CPT1) and an essential substrate for the fatty acid synthase complex and elongation of very long–chain fatty acid enzymes.</p><!><p>Transcription levels of various genes from previously published data (20).Dots represent the measured log2-transformed signals for both strains where B6 is represented in blue and D2 in orange. The vertical bars represent the 95% confidence intervals around the mean for a normal distribution.</p><p>Proposed changes in energy metabolism between strains in healthy and infected state. Proposed changes in energy metabolism between strains in a healthy state (A) and for D2 in infected state (B), which could explain the heightened vulnerability of D2 toward influenza A virus (IAV). Percentage changes (%Δ) within percentile intervals for relevant metabolic classes are added for comparison (C). Metabolites for which 80% of the posterior density was either above or below 0 are indicated with filled dots. Comparisons of healthy D2 and B6 energy metabolomes suggest that mitochondrial beta-oxidation is inhibited in D2 by malonyl-CoA (MCoA). Compensatory processes (anaplerosis) are increased in D2 to make up for decreased oxidative phosphorylation. This is likely driven by increased insulin levels in D2. During infection, D2 virus titers are extremely high compared with B6 (43), possibly leading to increased TLR4 signaling and fatty acid (FA) recruitment from adipose tissue. The surplus of FA cannot be efficiently utilized by D2 because of the continuous blockade of mitochondrial beta-oxidation by MCoA. Fatty acids in D2 are routed to peroxisomal beta-oxidation, thereby increasing the production of reactive oxygen species and ACars. Moreover, amino acids like valine and serine are used to fuel the TCA cycle in D2 during infection, thereby depleting energy deposits more than in B6. Thus, possible driving factors for the increased vulnerability of D2 are impaired mitochondrial beta-oxidation, depletion of energy reserves, and increased production of reactive oxygen species. Metabolites depicted in gray were not directly observed. Enzymes in green are upregulated by insulin. ABCD, ATP-binding cassette D; ACar, acylcarnitine; ACC, acetyl-CoA carboxylase; ACoA, acyl-CoA; AcCoA, acetyl coenzyme A; AP, anaplerotic precursor; β-Ox, beta-oxidation; CACT, carnitine-acylcarnitine translocase; Cit, citrate; CPT1/2, carnitine palmitoyl transferase ½; DAG, diacylglycerol; FATP1, fatty acid transporter protein 1; GLUT4, glucose transporter 4; MCar, malonylcarnitine; OA, oxaloacetate; OxFA, oxidized fatty acids (ml: medium/long, lvl: long/very long); PAMPs, pathogen-associated patterns; Pyr, pyruvate; TAG, triacylglycerol; TCA, tricarboxylic acid cycle.</p><!><p>In the context of beta-oxidation, it should also be mentioned that the D2 strain expresses an aryl-hydrocarbon receptor (Ahr) allele with a 10 to 100 times lower ligand affinity compared with B6. It was previously shown that stimulation of this receptor increased the transcription of Cpt1b, coding for the mitochondrial form of CPT1 in liver (22). On the other hand, under high-fat diet, liver-specific knockout of Ahr caused increased expression of lipogenesis-related genes coding for proteins ACC1 (Acaca) and glycerol-3-phosphate acyltransferase 1 (Gpam) (23). Moreover, indirect inhibition of AHR in CD4+ T cells led to the decrease of CPT1 protein expression and activity, whereas ACC2 was upregulated and its phosphorylated inactive form downregulated (24). Therefore, the D2 deficiency in AHR response might work in concert with increased insulin levels causing increased levels of MCar and decreased levels of ACars.</p><p>Decreased mitochondrial beta-oxidation and a subsequent deficit in ATP production might be offset by increased glucose utilization driven by increased D2 insulin levels. Evidence for this is found in decreased baseline plasma glucose levels in D2 as observed in this study and others (6, 7, 12). Furthermore, decreased beta-oxidation could in part be compensated by increased catabolism of amino acids (25). Evidence that this is the case in D2 can be found in lower plasma levels of the (branched-chain) amino acids Val, Ile/Leu, and Glu, which can be used as alternative energy sources. Increased mitochondrial utilization of glucose and amino acids would lead to increased TCA cycle activity and increased levels of TCA intermediates, including citrate. Excess citrate is transported from the mitochondria to the cytosol where it serves as a source of cytosolic acetyl coenzyme A (AcCoA) for subsequent production of MCoA and hence FAs (26).</p><!><p>Proposed changes in miscellaneous pathways showing interstrain differences in healthy and infected states. Pyrimidine metabolism (A), purine metabolism (B), and nicotinamide metabolism (NAM) (C). Percentage changes (%Δ) within percentile intervals for relevant metabolic classes are added for comparison (D). Pyrimidine metabolism flux toward anaplerotic substrates might be increased in healthy D2 animals. For purine metabolism, increased renal excretion could lead to a higher clearance of allantoin. Upon infection, 5-hydroxyisourate (5HIU) levels in D2 might be increased because of lower oxygen availability in peroxisomes. As for NAM metabolism, low baseline levels of N1-methyl-n-pyridone-5-carboxamide (nPY, n = 2 or 4) is likely because of impaired function of aldehyde oxidase homologs 1 (mAOX3) enzyme in D2 and an increased renal excretion.</p><!><p>Baseline metabolic signatures observed in D2, including high MCar and DAG levels, together with previously observed gene and protein expression profiles suggest that glycerolipid synthesis is upregulated in D2.</p><p>As suggested, increased levels of MCoA could lead to increased FA synthesis. Strain-specific increases in D2 healthy plasma levels of saturated FAs and VLCFAs were observed, which might suggest increased FA synthesis. However, FA levels are determined by a myriad of processes including chain elongation and shortening, desaturation, diffusion, and lipase activity. Moreover, with our analytical methods, the desaturation and oxidation positions, which play crucial roles in inferring their origins, could not be determined.</p><p>The best evidence of increased FA synthesis in D2 is found in increased baseline levels of DAGs and higher amounts of adipose tissue with respect to B6. Both DAGs and triacylglycerols (TAGs) are produced in hepatocytes and enterocytes from FAs and glycerol-3-phosophate. DAGs, together with TAGs and various other phospholipids, are transported to adipose and muscle tissue in low-density lipoprotein-containing vesicles. Although our analytical method did not allow for the detection of TAGs, lipoprotein-associated DAGs are correlated with the TAGs and reflect TAG synthesis (27). Excess TAGs are stored in adipose tissue as energy reserve. Assuming that dietary FA uptake is equal between noninfected strains, the 2-fold increase of adipose tissue mass in D2 with respect to B6 as observed in various studies (7, 10, 11) can be explained by increased FA production in D2 livers (Fig. 6, A and B).</p><p>Furthermore, the low responsiveness of the D2 AHR might play an additional role in the observed changes in lipid metabolism. The expression of many enzymes involved in TAG synthesis is modulated by AHR. As mentioned, glycerol-3-phosphate-O-acyltransferase is upregulated in Ahr-deficient mice under a high-fat diet (23). In addition, diacylglycerol O-acyltransferases and acylglycerol-3-phosphate-O-acyltransferases are downregulated after stimulation of AHR (28). Therefore, reduced AHR signaling in D2 might increase the metabolic flux through the DAG synthesis pathway, thereby increasing the formation of the associated lipids. This, together with increased FA synthesis, would further explain the increased levels of both DAGs and phosphatidylinositols as was observed in D2.</p><p>An indirect effect of increased FA synthesis in D2 might be the downregulation of a peroxisomal transporter protein. Transcriptomics results show that in D2 after 1 dpi, transcription levels of the ATP-binding cassette D3 gene (Abcd3) in lung tissue are decreased with respect to B6 (Fig. 5, (20)). Moreover, the same study that found higher PP2A transcription also found that RNA levels of Abcd3 in healthy lung tissue were about 40 times lower in D2 (19) although this effect did not show up in data from Ref. (20). ABCD3 is one of the three known peroxisomal transporters of medium-chain and long-chain ACoA esters and is essential for peroxisomal beta-oxidation (29). The physiological reason for the observed downregulation might be that peroxisomal beta-oxidation produces higher amounts of reactive oxygen species than mitochondrial beta-oxidation. Hence, a decrease of peroxisomal ACoA transport in the presence of elevated FA levels would reduce production of reactive intermediates and subsequent cell damage.</p><!><p>Finally, a basal interstrain difference was observed for nucleotide metabolism. Changes in purine metabolism are likely driven by changes in renal clearance, whereas changes in pyrimidine metabolism can be explained by difference in aldehyde oxidase function.</p><p>Since allantoin is the irreversible end product of purine catabolism, decreases in D2 with respect to B6 at baseline are either caused by decreased levels of upstream precursors or increased excretion. The direct precursors urate and 5-hydroxyisourate neither show interstrain differences nor does the purine nucleoside xanthosine. Therefore, an explanation for low D2 allantoin levels might be found in the higher renal clearance of allantoin (Fig. 7, A and C). This is supported by the fact that under control conditions, D2 mice have about 20% to 50% higher glomerular filtration rate as B6 (30).</p><p>Similarly, interstrain differences were found for nPY and MNA. Both metabolites are the end products of the NAM metabolic pathway (Fig. 7, A and D). The strong decrease in D2 for nPY can be explained by increased renal excretion in D2. However, D2 has a selective deficit in expression of Aox3 and Aox4 coding for aldehyde oxidase 3 and 4 (31, 32). Especially AOX3 is of interest here since it oxidizes MNA to form nPY (33), thus driving the nPY levels in D2 even further down. Transcriptomics data from Wilk et al. (20) clearly show that transcription of both Aox3 and Aox4 genes is reduced in D2 compared with B6 in both healthy and infected animals (Fig. 5).</p><!><p>Similarities in metabolic responses between strains during infection were seen for phospholipids, carnitine-related metabolites, amino acid derivatives, and phenolic compounds. These changes seem to be driven by nutritional changes during infection and recovery.</p><p>Most detected phospholipid species, including PC, LPCs, LPEs, and LPIs, show the same trends (mainly a decrease) between species upon infection. This overall reduction in phospholipid levels is observed in infections featuring strong inflammation, for example, community-acquired pneumonia and correlates with levels of C-reactive protein (34). Furthermore, polar metabolites associated with lipid degradation and carnitine metabolism are similarly decreased in infected animals of both strains. Carnitine-associated metabolites are probably downregulated because of lower food intake and gut metabolism. Crotonobetaine, γ-butyrobetaine, and TMA, the direct precursor of TMAO, are produced in the gut from carnitine (35) as well as the AA derivative 3-indolepropoinate. The strong decreases in sulfated phenolic compounds and their degradation product hippurate are also likely driven by reduced food intake. A similar explanation can be given for the decreases of trigonelline and NAM. Looking at Figure 2B (glycerolipid panel) and Figure 4, it seems that glycerolipids and nutritional markers are more decreased in D2, suggesting lower food intake in this strain.</p><p>Purine metabolites were downregulated at 3 dpi, which could also be caused by lower nutritional intake or lower de novo synthesis. Interesting is the increase of deoxycytidine in both strains, an effect previously observed in lung tissue of B6 under similar conditions (36). Moreover, an 88% spike for this metabolite was seen in B6 at 8 dpi. It was shown that an enzyme involved in the phosphorylation of deoxycytidine, deoxycytidine kinase, is necessary in the development of T and B lymphocytes (37). Therefore, deoxycytidine might play a role in immune response in both strains.</p><p>Since all D2 animals died between 5 and 8 dpi, metabolic disease progression could only be described for B6. In general, all detected metabolite classes were maximally decreased between 5 dpi and 8 dpi. Drops in the levels of ACars, FAs, and other lipids are probably because of the exhaustion of lipid reserves. However, upon recovery (from 18 dpi on), most metabolites did not return to their preinfection levels. MCFAs, LCFAs, and their oxidized forms were in general higher at recovery, whereas VLCFAs stayed down with respect to 0 dpi. Differential effects were also observed for ACars. These changes might be caused by altered metabolism because of renewed buildup of energy reserves or to age-related changes in energy expenditure. Increased levels of diet-dependent markers (e.g., trigonelline, crotonobetaine, TMAO, and phenols) indicated a higher food intake after recovery. Increased food intake just prior to recovery might also be the reason for the increase of most AAs, the tyrosine derivative dihydroxyindole and taurine at 8 dpi. Increased AA metabolism would lead to activation of the urea cycle and hence increased acetylornithine and citrulline as observed in B6.</p><!><p>We argue that most metabolic and interstrain differences during infection are due to lower mitochondrial beta-oxidation and subsequent infection-induced increase of peroxisomal beta-oxidation in D2.</p><p>After IAV infection, both strains showed significant weight loss, which was about 5% higher in D2 compared with B6 (Fig. 1). Weight loss could be explained by a decrease in nutritional intake and increased energy expenditure needed for the activation of the immune response (38) and higher breathing effort. To source the extra energy needed, high-energy substrates are released from adipose tissue in the form of FAs, from myocytes as AAs and from autophagic processes. In IAV infection, these catabolic processes are mediated by Toll-like receptors (TLRs), in particular TLR4, after interaction with pathogen-associated molecular patterns (PAMPs) (39). TLR4 is known to trigger lipase activity (40) and insulin resistance (41) in adipocytes and breakdown of myofiber proteins in muscle (42). This leads to increased availability of plasma glucose, production of FAs, and release of amino acids. Since virus titers were about 10 to 100 times higher in D2 (43, 44), it is likely that circulating PAMPs were also higher. This would lead to higher TLR activity and subsequent loss of adipose and muscle tissues, causing higher weight loss in D2 and increased release of FAs and AAs. Moreover, food intake is likely more reduced in D2 compared with B6 during IAV infection, which might lead to activation of pathways for releasing stored energy deposits.</p><p>In muscle cells, FAs serve as a direct energy source, mainly through mitochondrial beta-oxidation. In the liver, FAs are transformed to ketogenic bodies and glucose by mitochondrial processes to serve as energy sources elsewhere. However, high levels of MCar in D2 at 3 dpi suggest that MCoA levels are still highly upregulated in this strain during infection. Therefore, in D2 during IAV infection, mitochondrial oxidation of FAs would still be reduced by continuing inhibition of CPT1. This would lead to a reduction in ACar via this pathway. However, a strong increase in circulating ACars was observed in D2 plasma. Thus, an alternative pathway is likely responsible for the increased production of ACars.</p><p>Interestingly, highly increased ACar levels were detected in patients with influenza-associated encephalopathy (45). This was caused by a thermolabile variant of CPT2 that was rendered nonfunctional upon fever. Through the CPT2 defect, ACars in the mitochondrial matrix were not converted to their CoA esters, which led to impaired mitochondrial beta-oxidation with severe consequences. A thermolabile variant of CPT2 would increase plasma ACar levels, but it would not explain the interstrain decrease in ACar levels in healthy D2 animals.</p><p>Another candidate pathway leading to increased ACar levels upon infection in D2 is peroxisome metabolism. These organelles are also capable of producing ACars via carnitine O-octanoyltransferase and can compensate for a defective mitochondrial carnitine shuttle (46). Peroxisomes actively take up long- to very long–chain polyunsaturated ACoA via the aforementioned ABCD transporters. Moreover, passive transport of nonesterified FAs over the peroxisomal membrane is possible (47). Once in the peroxisomes, the acyl chains are shortened under the formation of AcCoA but not until completion. The shortened ACoA esters are then converted into ACars. The resulting ACars are excreted back into the cytosol where they can serve as fuel for mitochondrial beta-oxidation. In the liver, AcCoA leaves the peroxisome as acetate after hydrolyzation by ACOT12 to add to the cytosolic pool (48). Acetate in the cytosol is either excreted or converted to AcCoA and thus MCoA/MCar (Fig. 6, A and C). Thus, excessive peroxisomal beta-oxidation could lead to increased cytosolic levels of carnitine esters.</p><p>Compatible with increased peroxisomal beta-oxidation, it was found that the transcription of mediator complex subunit 1 (Med1, Pparbp) was upregulated in lungs of D2 (19) and at the same time identified as a quantitative trait locus for influenza virus resistance (5). This mediator enhances peroxisome proliferation and FA oxidation via peroxisome proliferator–activated receptor alpha signaling (49).</p><p>Because of the spatial separation of peroxisomes and mitochondria, peroxisomal ACar formation is less efficiently coupled to mitochondria as is the CPT1/CACT/CPT2 shuttle. Therefore, we hypothesize that in D2 during infection, peroxisomal ACars will leak away to the extracellular space instead of entering the mitochondria. This would explain the strongly increased plasma ACar levels in D2. Higher release of FAs from adipose tissue, because of increased TLR4 signaling, could augment this process in D2. On the contrary, mitochondrial beta-oxidation in B6 seems to increase in activity during IAV infection as increased OxFA levels suggest.</p><p>Since D2 cannot efficiently utilize FAs, Val might be used to compensate for energy shortages. This AA exhibited a −90% drop in D2 at 3 dpi, whereas B6 levels were stable. Serine and glutamate might be other AAs used in both strains to replenish the TCA cycle. This is supported by transcriptomics data, which clearly show that the transcription of the gene coding for the branched-chain amino acid transporter 1 (Bcat1) is higher in D2 compared with B6 at 5 dpi (Fig. 4). Conversely, Ile/Leu was increased in D2 and apparently cannot be efficiently used in anaplerotic reactions during infection.</p><p>Moreover, increased peroxisomal hepatic beta-oxidation in D2 might affect purine metabolism. The catalysis of urate to 5HIU by urate oxidase is a peroxisomal process using oxygen. In both species, urate and 5HIU levels were downregulated. However, relative to B6, 5HIU D2 levels were lower, whereas its precursor urate was higher. Since D2 oxygen levels are probably decreased with respect to B6 because of impaired lung function and increased peroxisomal beta-oxidation, less oxygen is available for urate catalysis. The downstream metabolite allantoin was reduced in both species, an effect previously observed in IAV-infected B6 lung tissue (50).</p><p>The proposed switch from mitochondrial to peroxisomal FA metabolism might in part explain the heighted vulnerability of D2 animals for IAV infection. Since peroxisomal beta-oxidation produces high amounts of ROS (51), oxidative stress and subsequent cell damage would increase in D2, at least in metabolic tissues like liver and muscles (52).</p><p>In lung tissue, another mechanism explaining a poor outcome in D2 might be involved. Higher virus titers in D2 very early (1 dpi) after infection (43) are either related to an impaired initial cellular host defense, an increased replication cycle, or both. While we discuss possible differences in immune response later, a distortion of peroxisomal beta-oxidation in healthy lungs of D2 animals might explain this increase in virus replication. Deficient FA oxidation in alveolar epithelial cells is specifically associated with pathogen-induced acute lung injury (53). Furthermore, it was found that in lung epithelial cells, IAV decreases peroxisomal beta-oxidation and increases peroxisomal ether lipid (plasmalogen) synthesis for viral envelope fabrication. Conversely, induction of peroxisomal beta-oxidation reduced viral replication in a lung-epithelial cell line (54). Moreover, IAV led to a downregulation of carnitine O-octanoyltransferase in airway epithelium (55). Since peroxisomal beta-oxidation of FAs is shut down by IAV in lung epithelial cells, these metabolites can now be used in the synthesis of ether lipids in these organelles. The increased levels of plasma FAs might be a cause of the high initial viral titers observed in D2 since they provide a source for the increased synthesis of ether lipids for viral assembly and thus.</p><p>In addition, ACars are pneumotoxic in high concentrations. Accumulation at the epithelial air–fluid interface reduces the effect of pulmonary surfactant, thereby reducing pulmonary compliance. This might lead to increased susceptibility to infections and hypoxia (56). Increased D2 plasma levels of ACars therefore might compromise lung function further.</p><!><p>Both mitochondria and peroxisomes play an important role in innate antiviral signaling via retinoic acid–inducible gene I–like receptor signaling (57, 58). It might be that the compromised peroxisomal–mitochondrial axis in D2 would affect these signaling pathways. It is known that mitochondria are specifically affected by the pathogenic influenza A subtype used in this study through the viral protein PB1-F2. This viral protein translocates to the mitochondrial inner membrane, thereby impairing innate immunity (59). Therefore, we hypothesize that initially compromised mitochondrial and peroxisomal functions, together with specific targeting of these organelles by IAV in lung epithelial cells, work in concert to increase viral replication and spreading in the lung. This would further aggravate the severe disease outcome in D2.</p><p>High lethality in D2 compared with B6 was previously observed for tuberculosis, which was contributed to reduced levels of CD103+ dendritic cells (DCs) in D2 (60). Specifically, this DC subtype is not affected by IAV and therefore able to induce virus-specific CD8+ T cells (61). Therefore, reduced levels of these DCs in D2 lungs would also impair an antiviral response. In addition, it was found that a ketogenic diet increased γδ T cells in lung tissue and protected against IAV infection. However, as a prerequisite for this protective effect, there should occur a metabolic switch favoring FA oxidation. Increasing the plasma levels of the ketogenic substrate betahydroxybutyric acid alone did not increase expansion of protective T cells (62). Therefore, a reduced T-cell response because of a decreased mitochondrial beta-oxidation might weaken the bridge toward an adaptive immune response in D2.</p><p>Finally, it is known that D2 is deficient in complement component 5 (C5, hemolytic complement), a protein involved in both innate and adaptive immune responses. Upon activation of the complement system via PAMPs, C5 is cleaved into C5a and C5b. C5b is part of the pore forming membrane attack complex, which causes targeted cells to lyse. C5a interacts with C5a receptors and is involved in chemotaxis, immune cell activation, and reactive oxygen species production. It was found that after crossing B6 with the C5-deficient A/J mouse strain, C5 was associated with higher IAV susceptibility in females than in males (63). However, this sex-dependent effect was not observed for D2 since both genders were equally susceptible (43).</p><p>In IAV infection, C5 is implicated in CD8+ T-cell response, and blocking C5a receptor results in the impairment of this response (64). However, in primary infection, this response has an onset at 5 to 7 dpi, which makes it less relevant to our observations. This is corroborated by a study in C5-deficient and sufficient B10.D2 mice where the beneficial effect of C5 on mortality only showed after 7 dpi (65). Moreover, a chimeric D2 model, which had its bone marrow replaced with that of B6, excluded the involvement of C5 in IAV susceptibility (5).</p><p>Conversely, C5aR−/− mice on a BALBc background recovered better than the WT mice, and blocking of the C5aR1 receptor had little to no effect on mortality during H1N1 infection in mice (66). Moreover, excessive C5 activation was shown to cause lung injury and thus can have an adverse effect. Targeting C5 with a specific inhibitor reduced excessive inflammatory reactions associated with severe IAV infections. Also, inhibition of C5 had no effect on viral load (67). Therefore, although important in later stages of IAV infection, C5 has a limited or even adverse effect at the onset of infection and thus would be less relevant in explaining the observed phenotype in D2.</p><!><p>By using an untargeted high-resolution LC–MS approach, we identified metabolic markers that differentiate the susceptible (D2) and resistant (B6) inbred mouse strains at baseline and during H1N1 influenza A viral infection. By combining the observed metabolic responses with prior metabolic, genetic, and transcriptional information, we derived a hypothesis for the increased susceptibility of the D2 strain. In brief, high insulin levels in the insulin-resistant prediabetic D2 strain leads to increased production of MCoA. MCoA inhibits mitochondrial beta-oxidation in healthy and infected D2 animals. In healthy D2 animals, we suggest that this leads to increased FA synthesis and adipose lipid storage. In infected animals, it leads to increased peroxisomal beta-oxidation resulting in increased levels of ROS. Increased FA mobilization through increased TLR4 signaling in D2 aggravates this process. Furthermore, since both mitochondria and peroxisomes are implicated in the antiviral innate immune response, the increased metabolic stress on these organelles by IAV leads to high uncontrolled virus replication and spreading in D2 mice.</p><p>It is well known that metabolic disorders like diabetes, insulin resistance, and obesity are risk factors for severe outcomes of influenza A infection. For example, it has been shown that in bona fide diabetic mouse models, hyperglycemia in the lung increases the severity of IAV infection by damaging the pulmonary epithelial–endothelial barrier and possibly compromises host defense (68, 69). However, in D2, although showing high insulin levels and some insulin resistance, plasma glucose levels are relatively low. Therefore, the D2 strain could be used as an alternative prediabetic in vivo model. This model could be used to get a better understanding in the role of the mitochondrial–peroxisomal axis in host defense. Furthermore, the model could be used to study infection progression under pharmacological therapies. As such, it would be interesting to test the effects of ACC inhibitors or insulin secretion blockers on disease outcome in D2.</p><p>Moreover, investigating dietary measures to ameliorate symptoms could be of interest. In this respect, one could think of administration of glucose, amino acids, or short-chain FAs in combination with TLR4 inhibitors. Furthermore, it would be interesting to investigate if some of the observed changes in D2 metabolic profiles during IAV infection are also observed in diabetic or obese mouse models or mouse models with severe outcomes for IAV or other viral infections. In this respect, it would be important in a follow-up study to investigate the difference in metabolomic changes during weight loss in both strains that were either induced by dietary restriction or by IAV infection. At the same time, it would then be important to follow gene transcription in metabolic tissues, such as liver, muscle, and adipose tissue rather than lung as used in this report.</p><p>From a translational perspective, it would be interesting to determine if correlations between plasma levels of MCar, ACars, oxidized FAs, and amino acids and disease outcome can also be found in human patients. As such, plasma levels of these metabolites could serve as predictor for high risk or biomarkers for the progression toward severe disease outcome. We expect that our metabolite study represents a stepping stone to protect an ever-growing population with insulin resistance, diabetes, and related syndromes from severe consequences of IAV infection.</p><!><p>HPLC-grade acetonitrile (ACN), water, and acetic acid were purchased from Fisher Scientific. Ammonium acetate was purchased from Sigma–Aldrich. Chemical standards were purchased from different suppliers. Supplier information can be found in Table S1.</p><!><p>All animal experiments were approved by an external committee according to the German regulations on animal welfare (permit numbers: 33.9.42502-04-051/09 and 3392 42502-04-13/1234). The protocol and all methods used in these experiments have been reviewed and approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (permit numbers: 33.42502-108/06; 33.19-42502-04-18/2922; 33.9.42502-04-051/09; and 33.19–42502-04-13/1234) after consultation with its ethics committee. All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with the ARRIVE guidelines (70).</p><p>C57BL/6J and DBA/2J mice were obtained from Janvier. All mice were maintained under specific pathogen-free conditions and according to the German animal welfare law (BGBl. I S. 1206, 1313 and BGBl. I S. 1934) at the animal facility of the Helmholtz Centre for Infection Research. Breeding and maintenance colonies were kept in individually ventilated cages (Sealsafe Plus GM500; Tecniplast) at a temperature of 22 ± 1 °C, humidity of 55 ± 5%, 75 air exchanges per hour in the cages, and a 14/10-h light/dark cycle with the lights on at 6:00 AM. The maximum caging density was five mice from the same litter and sex starting from weaning. As bedding, aspen wood bedding (Tapvei) was provided. Mice were fed a standardized mouse diet (V1534-300; Ssniff) and provided drinking water ad libitum. All materials, including individually ventilated cages, lids, feeders, bottles, bedding, and water were autoclaved before use. Health status was controlled quarterly by exhaust air dust PCR testing (Environmental Mouse Complete PRIA; Charles River Laboratories) and biannually by serum samples of stock animals (Federation of European Laboratory Animal Science Associations Annual Serology; Charles River Laboratories). Mice were negative for at least all Federation of European Laboratory Animal Science Associations–relevant murine infectious agents.</p><p>Mice were allocated to groups by chance; a specific randomization protocol was not used. Mice exhibiting abnormal behavior, posture, or injuries were excluded from the experiments. These criteria were established a priori. Treatment was not blinded because infected and noninfected groups had to be kept in separated cages to avoid crossinfection.</p><p>The mouse-adapted virus strain influenza A/Puerto Rico/8/34 (H1N1;PR8;PR8_Mun2_INFG_0312) was produced and titrated as described previously (4, 71). Female 10-week-old DBA/2J (D2) or C57BL/6J (B6) mice were anesthetized by intraperitoneal injection with ketamine/xylazine (85% isotonic NaCl solution, 10% ketamine, and 5% xylazine) with doses adjusted to the individual body weight. Mice were then intranasally infected with 20 μl virus solution (2 × 103 focus-forming unit PR8) or mock infected with PBS. Body weight changes were followed for each day after infection. In addition to mice that were found dead, animals with a body weight loss of more than 30% of the starting body weight were euthanized and recorded as dead. For each individual, animal eye blood (75 μl blood to 25 μl heparin) was taken by final bleeding on 0, 1, 3, 5, 8, 18, and 30 dpi for metabolite analysis. Per strain and challenge, five animals were used.</p><!><p>Heparin-blood samples were centrifuged 10 min/4 °C/1300g, and the supernatant was stored at −70 °C. All collected samples were processed and analyzed at the same time. Stored samples were thawed, and 150 μl ACN/water (50% v/v) was added to 50 μl of supernatant. Samples were shaken at 4 °C at 1400 rpm in a thermomixer and then centrifuged for 15 min at 4 °C and 13,000 rpm. Finally, 175 μl of the supernatant was transferred into a new tube and left 20 min at −70 °C, then placed in a speed vac to evaporate fluid to dryness in approximately 2 h. Pellets were shipped from Germany to Spain on dry ice and resuspended in 100 μl ACN/water (60%/40% v/v) prior to analysis.</p><!><p>Samples were analyzed on an ultraperformance liquid chromatographic system (Acquity; Waters, Inc) coupled to a time-of-flight mass spectrometer (SYNAPT G2; Waters, Inc). A 2.1 × 100 mm, 1.7 μm BEH HILIC (hydrophilic interaction) column (Waters, Inc), thermostated at 40 °C, was used for sample separation. Mobile phase solvent A (aqueous phase) contained 98.5% water, 1% ACN, 0.5% acetic acid, and 5 mM ammonium acetate, whereas solvent B (organic phase) contained 1% water, 98.5% ACN, 0.5% acetic acid, and 5 mM ammonium acetate.</p><p>Separation of the analytes was performed with the following gradient: from 5% A to 15% A in 2 min in a linear gradient, from 15% A to 99.9% A in 4.5 min in a curved gradient (#9; defined by Waters MassLynx software), constant at 99.9% A for 1.5 min, back to 5% A in 0.2 min, and constant at 5% A for 1.8 min. The flow rate was 0.5 ml. All samples were injected randomly with an injection volume of 2 μl. After every five injections, a quality control sample was injected.</p><p>The MS was operated in positive and negative ESI mode as described previously (72). The sampling cone voltage was set to 25 V, extraction cone voltage to 5 V, and capillary voltage to 500 V for both polarities. Source temperature was set to 120 °C and capillary temperature to 450 °C. The flow of the cone and desolvation gas (both nitrogen) were set to 5 and 800 l/h, respectively. Scan time was set to 0.2 scans/s covering a range from 50 to 1200 Da. A 2 ng/ml Leu-enkephalin solution was infused at 10 μl/min and used for a lock mass, which was measured each 15 s for 0.5 s, and peaks were automatically corrected for deviations. The MS was tuned to a resolution of 20.000 full-width half-maximum at the m/z of the lock mass (556.1227) with an intensity of 1 × 105 counts.</p><!><p>For clarity, we will start this section with some definitions that will be used throughout the article. We make a difference between features and metabolites. An LCMS feature is defined as a unique mass-to-charge/retention time pair, that is, a unique code based on raw data. An LCMS feature is not chemically identified yet and can be endogenous or exogenous of nature. If the LCMS feature is exogenous, it is referred to as noise, if it is endogenous it is referred to simply as feature. Features are metabolites or related to metabolites, that is, LCMS adducts or fragments of metabolites. Features are raised to metabolite status and assigned proper names after the identification process.</p><!><p>Data processing pipeline. (A) and contrasts used in the post hoc test to select relevant markers with a linear mixed-effects (LME) model (B). MFC, median fold change; MM, multilevel model; QC, quality control.</p><!><p>LME models of features with p values smaller than 0.001 were selected and subsequently subjected to post hoc analysis on relevant contrasts (Fig. 8B). Next, features that showed interstrain or intrastrain differences in the post hoc test (at least one adjusted p value < 0.05) were selected. If possible, these features were identified by accurate mass, isotope patterns, and in-source fragmentation ions (75) with the aid of HMDB and METLIN metabolite databases. Because of possible autointegration errors, selected features were manually reintegrated (QuanLynx software; Waters, Inc) and again subjected to data adjustment and statistical filtering (post hoc–adjusted p value < 0.01). The resulting metabolite set was enriched in an iterative process by mapping the previously identified members to canonical pathways (Kyoto Encyclopedia of Genes and Genomes) and looking for their nearest neighbors in the raw or autointegrated data. For initially selected acyl-containing metabolites like ACars, FAs, or phospholipids, data were mined for members of the same family with different acyl chains and desaturations. The raw data were subsequently mined in order to find, reintegrate, and include these neighbors. Where possible, after pathway enrichment, the putatively identified features were confirmed with chemical standards.</p><p>Metabolites of the final set were modeled with a Bayesian multilevel model in the probabilistic modeling language Stan. Posterior distributions of the percent change (%Δ) between relevant (pooled) sample groups were obtained with samples from the Markov-chain Monte Carlo process. The healthy interstrain %Δ is calculated by calculating the relative difference between strains using the pooled posterior samples of 0 dpi, mock-infected 3 dpi, and mock-infected 5 dpi, taking B6 as baseline. As such, for healthy interstrain comparisons, negative %Δ values represent lower levels of a particular metabolite in D2 with respect to B6. For the intrastrain %Δ, for a particular strain, the sample groups in the infected arm were individually compared with 0 dpi. Therefore, negative values for intrastrain %Δ reflect a decrease in metabolite levels during infection compared with 0 dpi.</p><p>The %Δ distributions are represented as the 50% percentile in the 50% (25% to 75%) and 90% (5% to 95%) percentile intervals. The 50% percentile of the %Δ distributions represents the most likely value for %Δ based on data, prior probability, and likelihood. Note that Bayesian models do not return p values, but the posterior distributions contain (unlike classical confidence intervals) the most likely value of the calculated metric. The posterior distributions were used to infer metabolic differences between strains in healthy and infected states.</p><!><p>Data that were used in this study are available at the following github repository: https://github.com/smvanliempd/IAVmetabolism.</p><!><p>This article contains supporting information.</p><!><p>The authors declare that they have no conflicts of interest with the contents of this article.</p><!><p>Figure S1 Figure S2 Figure S3 Table S1 Table S2</p>

PubMed Open Access

Interfacial cavity filling to optimize CD4-mimetic miniprotein interactions with the HIV-1 surface protein

Ligand affinities can be optimized by interfacial cavity filling. A hollow (Phe43 cavity) between HIV-1 surface protein (gp120) and cluster of differentiation 4 (CD4) receptor, extends beyond residue phenylalanine 43 of CD4 and cannot be fully accessed by natural amino acids. To increase HIV-1 gp120 affinity for a family of CD4-mimetic miniproteins (miniCD4s), we targeted the gp120 Phe43 cavity with eleven non-natural phenylalanine derivatives, introduced into a miniCD4 named M48 (1). The best derivative named M48U12 (13) binds HIV-1 YU2 gp120 with 8 pM affinity, and shows potent HIV-1 neutralization. It contained a methylcyclohexyl derivative of 4-aminophenylalanine and its co-crystal structure with gp120 revealed the cyclohexane ring buried within the gp120 hydrophobic core but able to assume multiple orientations in the binding pocket, and an aniline nitrogen potentially providing a focus for further improvement. Altogether, the results provide a framework for filling the interfacial Phe43 cavity to enhance miniCD4 affinity.

interfacial_cavity_filling_to_optimize_cd4-mimetic_miniprotein_interactions_with_the_hiv-1_surface_p

Introduction<!>Peptide Synthesis<!>Competitive Binding Assays on a M33 (16) Series<!>Chemistry<!>Competitive Binding Assays on a M48 (1) Series<!>Antiviral Activity and Cytotoxicity<!>Surface Plasmon Resonance (SPR) Analysis<!>Co-crystal Structure of 13 in Complex with HIV-1 gp120<!>Discussion<!>Conclusion<!>Synthesis Procedures<!>N,O-Bis-allyloxycarbonyl-L-tyrosine (2\xe2\x80\xb2)<!>N,O-Bis-allyloxycarbonyl-L-tyrosine immobilized on Wang resin (3\xe2\x80\xb2)<!>N-allyloxycarbonyl-L-tyrosine immobilized on Wang resin (4\xe2\x80\xb2)<!>N-allyloxycarbonyl-O-(c-alkyl or c-aryl-alkyl chain)-L-tyrosine immobilized on Wang resin (5\xe2\x80\xb2 to 13\xe2\x80\xb2)<!>O-(c-alkyl or c-aryl-alkyl chain)-L-tyrosine immobilized on Wang resin (14\xe2\x80\xb2 to 22\xe2\x80\xb2)<!>N-(9-fluorenylmethoxycarbonyl)-O-(c-alkyl or c-aryl-alkyl chain)-L-tyrosine immobilized on Wang resin (23\xe2\x80\xb2 to 31\xe2\x80\xb2)<!>N-(9-fluorenylmethoxycarbonyl)-O-(c-alkyl or c-aryl-alkyl chain)-L-tyrosine (32\xe2\x80\xb2 to 40\xe2\x80\xb2)<!>N-(9-fluorenylmethoxycarbonyl)-O-(cyclohexylmethyl)-L-tyrosine (32\xe2\x80\xb2)<!>4-benzyloxy-1-butanol (44\xe2\x80\xb2)<!>N-Cbz-(S)-(+)-2-pyrrolidinemethanol (48\xe2\x80\xb2)<!>2-((2-((tert-butyldimethylsilyl)oxy)ethyl)thio)ethanol (50\xe2\x80\xb2)<!>Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(2-((2-((tert-butyldimethylsilyl)oxy)ethyl)-thio)ethoxy)phenyl)propanoate (51\xe2\x80\xb2)<!>2-amino-3-(4-(2-((2-((tert-butyldimethylsilyl)oxy)ethyl)thio)ethoxy)phenyl)propanoic acid (52\xe2\x80\xb2)<!>2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(2-((2-((tert-butyldimethylsilyl)-oxy)ethyl)thio)ethoxy)phenyl)propanoic acid (53\xe2\x80\xb2)<!>2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(2-((2-((tert-butyldimethylsilyl)-oxy)ethylsulfinyl)ethoxy)phenyl)propanoic acid (54\xe2\x80\xb2)<!>N-(9-fluorenylmethoxycarbonyl)-p-(N-cyclohexylmethylamino)-L-phenylalanine (56\xe2\x80\xb2)<!>N-(9-fluorenylmethoxycarbonyl)-p-(N-cyclohexylmethyl-N\xe2\x80\x99-tert-butyloxycarbonyl-amino)-L-phenylalanine (57\xe2\x80\xb2)<!>N-(9-fluorenylmethoxycarbonyl)-p-(isopropyl)-L-phenylalanine (59\xe2\x80\xb2)<!>Peptides Synthesis, Refolding and Purification<!>Competitive Binding Assays<!>Antiviral Activity Assays<!>Cellular Toxicity Assays<!>SPR Assays<!>Complex Preparation, Crystallization, Data Collection, Structure Determination, Refinement, and Analyses

<p>Cell entry of the human immunodeficiency virus type 1 (HIV-1) is a multi-step process initiated by the binding of the HIV-1 surface protein (gp120) envelope glycoprotein on the viral spike to the cluster of differentiation 4 (CD4) receptor on appropriate target cells. This association stabilizes a conformation of gp120 that enables it to bind to a chemokine co-receptor, CCR5 or CXCR4.1-3 Co-receptor binding in turn activates the fusogenic properties of the noncovalently associated transmembrane component (gp41) of the viral spike, and subsequent spikeconformational rearrangements and virus-host membrane fusion lead to entry of the HIV-1 genetic material into the cell.4 Each of these steps represents a potential target for therapeutic intervention.5-8</p><p>For over a decade, we have been developing peptide CD4 mimics, called miniCD4s, which are based on the transplantation of the gp120-binding surface of CD4 into the context of various scorpion-toxin scaffolds.9 These disulfide rich peptides belong to the knottin family, which has been widely utilized as scaffolds for developing stable peptide ligands through loop grafting of bioactive peptides.10 and references therein The 31-residue scyllatoxin, from the scorpion Leiurus quinquestriatus hebraeus, was selected based on the similarity of the gp120-binding portion of CD4 with the scyllatoxin structure, which consists of a β-hairpin linked to a short helix. The scyllatoxin β-hairpin between residues 18-29scyllatoxin superimposes with a root-mean-square deviation (RMSD) of 1.1 Å with CD4 residues 36-47CD4, which are involved in binding gp1209 (to aid in clarity, residues are identified by a subscript of the parent macromolecule). Moreover, the scyllatoxin fold, stabilized by three disulphide bridges (Figure S1),11 is highly permissive to mutation,12 stable to acidic pH and high temperatures,12 relatively resistant towards proteolytic degradation,13 and poorly immunogenic.14 Optimization of the scyllatoxin peptide structure led to miniCD4s of only 27 residues and affinities for gp120 comparable to that of CD4 itself.15 These miniproteins stabilize the CD4-bound conformation of gp120 as observed by X-ray crystallography15,16 as well as by immunogenic elicitation: thus, cross-linking of a miniCD4 to HIV-1 gp120 or HIV-1 gp140 (gp120 coupled to the extracellular part of gp41) enhances its binding to the CCR5 receptor, and injection of this cross-linked complex into rabbits significantly increases the production of CD4-induced antibodies that recognize the site of CCR5 binding on gp120.17,18 MiniCD4s are also able to neutralize HIV entry in vitro15,16,19 and in vivo, as shown recently in a macaque vaginal simian-human immunodeficiency virus (SHIV) challenge.20</p><p>An interfacial cavity, called the Phe43 cavity, extends from the tip of the gp120-interactive β-hairpin of CD4 into the heart of HIV-1 gp120. This mostly hydrophobic cavity is capped by the phenyl side chain of residue 43CD4. The miniCD4, named M48 (1),15 recognizes the entrance of the Phe43 cavity in a manner similar to that of CD4.15 To enhance the interaction between gp120 and miniCD4, we synthesized peptide CD4 mimics, in which the side chain of residue 23miniCD4 – the miniCD4 residue positioned at the mouth of the Phe43 cavity– was replaced by a number of unnatural phenylalanine derivatives. The antiviral activity of four such peptides (2-4 and 14) was determined previously,15,19 with 2 being the most potent. In a related study, we determine the structural basis for 2 recognition of HIV-1 gp120 and show that optimal filling of the Phe43 cavity is a mechanism for enhancing affinity and neutralization potency of a CD4-binding site ligand.21 To provide a fuller understanding of how interactions with the Phe43 cavity might contribute to HIV-1 gp120 affinity, we synthesized miniCD4 derivatives of 1, each with a unique 23miniCD4 extension. This paper describes the synthesis of eleven novel unnatural amino acids, which were used to replace residue 23M48. Their incorporation, along with three commercially available unnatural amino acids, into 1, allowed us to compare the relative affinity for 15 miniCD4 (including 1) ligands to HIV-1, and we did so with two gp120s, one belonging to clade B and the other to clade C. The antiviral activity of the six tightest binding derivatives of 1 was assessed against two clade B lab strains, two clade C primary isolates and two transmitter/founder (T/F) viruses, one belonging to clade B and the other to clade C. The tightest binder and most potent neutralizer M48U12 (13) was analyzed for its gp120-binding affinity by surface plasmon resonance (SPR) and crystallized in complex with HIV-1 gp120 from the clade B YU2 isolate. The co-crystal structure at 1.9 Å-resolution provides atomic-level details for 13, which displays both extraordinary affinity and potent neutralization. Together, these results show how appropriately filling of an interfacial cavity through a combination of rational design, chemical synthesis, and functional/structural feedback allows for potent near-pan neutralization of HIV-1 by miniCD4 mimetics.</p><!><p>MiniCD4s were obtained by standard solid phase peptide synthesis using Fmoc chemistry and by introducing various commercial or homemade amino acid derivatives at position 23miniCD4. After deprotection, the miniCD4s were folded by adding the crude peptide mixtures to Tris buffer (pH 8) containing the glutathione redox couple. Addition of two molar guanidinium hydrochloride in the folding buffer was required to increase the solubility of the various linear peptides. MiniCD4s were purified by reverse phase HPLC. Two series of miniCD4s were synthesized. The first one was obtained on the M3313 (16) family (TpaNLHFCQLRCKSLGLLGKCAGSXCACV-NH2, where Tpa stands for thiopropionyl, and X for Phe, Trp or unnatural amino acid) with commercially available amino acids. The second was built with three commercially available and eleven homemade unnatural amino acids on the more recently developed M48 (1) family (TpaNLHFCQLRCKSLGLLGRCA-DPro-TXCACV-NH2, where X stands for Phe or unnatural amino acid). We thus obtained a panel of twenty-four miniCD4s, some of which have been partially described previously.13</p><!><p>Relative apparent gp120-binding affinities of the various miniCD4s, modified at position 23, were determined by competitive ELISA, using soluble CD4 (sCD4) as competitor. The miniCD4s were tested on clade B gp120SF162 and on clade C gp120CN54. A preliminary study done on the M33 (16) family, which contains commercially available, natural or unnatural amino acids at position 23, showed systematically higher potency with various miniCD4s for clade B virus compared to the clade C virus (Table S1; IC50CN54/IC50SF162 > 10). Most of the miniCD4s showed inhibition constants in the nanomolar range for clade B gp120SF162, but apart from the biphenyl- (16) or phenylalanine (17) derivatives, they all have micromolar IC50s for clade C gp120CN54. While the biphenyl derivative marginally improves the apparent affinity over the phenyl derivative, neither tryptophan nor the other six unnatural amino acids tested showed an improvement in affinity (see ratio A and B in Table S1). The modifications, whether extending the linker between the alpha carbon and the phenyl moiety (18 and 19) or using bicyclic residues (20 and 21), or introducing a methoxy (23), a hydrophobic methyl (22) or tert-butyl (24) group at the para position of the phenylalanine, all led to a loss in gp120-binding affinity of the miniCD4s. While both tryptophan and 2-naphtylalanine were deleterious, the latter was less deleterious, since 20 showed low nanomolar affinity for the SF162 surface protein. The most deleterious effect was observed on 24 and 18, where the tert-butyl or the phenyl moiety, respectively, probably clash with the narrowest part of the pocket (this narrowing can be seen in Figure 1). Lengthening the linker between the alpha carbon and the aromatic ring, as was done for 19, enhanced the affinity compared to 18 suggesting that the aromatic ring probably lies in the widest and deepest part of the cavity. The presence of a p-methyl group (22) on the phenylalanine resulted in a small loss of affinity for SF162 gp120, which could be reversed by replacing the p-methyl by a p-phenyl moiety (16), which again probably increases hydrophobic contacts with the deepest part of the pocket,16 while the indole entity of 21 appears to create a steric hindrance at the inlet of the cavity.</p><p>While these results provided useful structure activity relationship of ligand occupation of the gp120 Phe43 cavity, no improvements in affinity of 16 could be achieved using commercially available amino acids. We, therefore, decided to synthesize novel, unnatural amino acids to introduce into the more recently developed M48 (1) sequence.15</p><!><p>Novel unnatural amino acids suitable for Fmoc-SPPS were obtained using a synthetic route described earlier,22 producing a series of ethers derived from the tyrosine amino acid, using the Mitsunobu reaction on solid phase synthesis (Scheme 1). The tyrosine (1′) phenol and amino groups were first protected by an allyloxycarbonyl group. The obtained compound (2′) was immobilized onto a solid support (Wang resin) via its carboxylic moiety. Loading was estimated by cleavage and weight analysis of small samples of resin. 1H NMR of crude aliquots confirmed the reaction progress. An average 87% loading was obtained for the bis-allyloxycarbonyl protected compound (3′), which was close to the earlier 91% obtained by Morley.23 Piperidine treatment led to selective removal of the carbonate group in mild conditions thereby, avoiding detachment of the template from the solid support and deprotection of the allylcarbamate. With a 95% reaction yield, estimated by weighing the resin, an average loading of 1.03 mmol/g was obtained for the resin harboring the monoprotected allyloxycarbonyl derivative (4′). This selective removal of the phenolic protecting group generated an intermediate suitable for the synthesis of an ether library by using the Mitsunobu reaction. Commercially available alcohols were used to prepare resins 5′-12′. Monoprotected diols 44′-46′, used for the synthesis of resins 10′-12′, were prepared by one protecting step from commercially available compounds (Scheme 2). To obtain resin 13′, the N-Cbz-L-proline (47′) carboxylic acid had first to be transformed into an alcohol function. This was done by formation of a mixed anhydride with isobutyl chloroformate, followed by a reduction with sodium borohydride, yielding N-Cbz-(S)-(+)-2-pyrrolidinemethanol 48′ (Scheme 3). The Mitsunobu reaction was carried out in a 1/1 mixture of DCM/THF with PPh3, diisopropylazodicarboxylate (DIAD) and the various alcohols, in a double coupling strategy, as proton NMR showed that some phenol remained after the first coupling. Loading yield of the resins 5′-13′ obtained after the Mitsunobu reaction, as well as the next two steps yields were not determined. Instead, a global yield for these three steps was calculated at the end of the synthesis. The next step was the allyloxycarbonyl moiety removal on the nitrogen atom to reveal the primary amine, by activation with Pd(PPh3)4 in mild conditions where other functional groups remained inert. Then, the resulting amino-resins 14′-22′ were protected by a Fmoc moiety to give Fmoc-amino-acid resins 23′-31′. The last step, i.e. cleavage from the solid support, was carried out under acidic conditions in a mixture of TFA/DCM. After extraction and filtration, the final Fmoc-protected amino acid compounds 32′ to 40′ were obtained with overall yields ranging between 26% and 63% for the last three steps.</p><p>Two Fmoc-protected amino acids 54′ and 57′ were obtained by solution synthesis. For 54′ (Scheme 4), one of the two alcohol functions of 2,2′-thiodiethanol (49′) was first protected with a tert-butyldimethylsilyl moiety. The derivative that was obtained (50′) was coupled to Cbz-Tyr(OH)-OBn using the Mitsunobu reaction. The carboxylic and amine functions were then deprotected by catalytic reduction on Pd black, just before a standard Fmoc protection of the amine moiety. Finally, the sulfoxide (54′) was obtained in a 33% overall yield by oxidation with a tert-butyl hydroperoxide and thiourea dioxide mixture. The last unnatural amino acid that we developed was a methylcyclohexyl derivative of Fmoc-p-aminophenylalanine (57′). It is an analog of 32′, with a nitrogen atom leading to a phenylalkyl amine replacing the oxygen atom of the phenyl ether moiety. It was synthesized by reductive amination on Fmoc-p-amino-L-phenylalanine (55′) using sodium triacetoxyborohydride in CH3CN/AcOH with cyclohexanecarboxaldehyde (Scheme 5). The secondary amine that was obtained was then protected with the tert-butyloxycarbonyl moiety compatible with Fmoc peptide chemistry. The overall yield of 57′ was 73%. Fmoc-protected amino acid 59′ was obtained by reaction of the commercially available amino acid 58′ with Fmoc-chloroformate (Scheme 6).</p><!><p>Competitive ELISA assay showed the global superiority of the M48 (1) series over the M33 (16) series (Table 1 compared to Table S1). Eight derivatives shared subnanomolar and five low nanomolar apparent affinities for the clade B SF162 strain. Moreover, two derivatives had subnanomolar and five low nanomolar affinities for the clade C strain. Four derivatives (1, 11, 12, 14) were made with commercially available amino acids, and as was observed for the M33 (16) series, no improvement in binding affinity to gp120, over that of 1, could be achieved for 11, 12 or 14 (previously called M4715). Moreover, the results obtained with 12 confirmed the data obtained for 24, as these p-tert-butylphenylalanine derivatives share the worst IC50 values for both gp120s. However, the affinity loss, on changing the phenylalanine residue for a p-tert-butylphenylalanine, is much higher in the M48 (1) family versus the M33 (16) one (26-fold higher for gp120SF162 and about 13-fold higher for gp120CN54). Interestingly, replacing the p-tert-butyl group by a slightly less bulky isopropyl one (11) enhanced the apparent affinity on SF162 by a factor of seven, while no difference was observed on the C strain gp120, suggesting differences in the Phe43 cavity shape between these two gp120 proteins. Notably, this phenomenon is also present for 2, 4, 11, 13 and 15, with their B/A ratio (impact of the miniCD4 amino acid modification on position 23 on the affinity for gp120 on clade C compared to clade B; see Table 1) deviating significantly from 1. While for the M33 (16) series, the biphenyl moiety marginally increased the apparent binding affinity to gp120 compared to the phenyl group (Table S1; A = 0.9, B = 0.6), the effect was opposite in the M48 (1) family (Table 1; A = 2.6, B = 5.3), with the biphenyl substitution resulting in loss in gp120 binding affinity. X-ray crystallographic studies have already been published for the complexes between the clade B YU2 gp120 and 16, 17 (earlier called F23),16 1 (earlier called [Phe23M47]) and 14 (earlier called M47).15 These data are consistent with the overall small differences between these representative miniCD4s observed in our binding assays, especially on the clade B strain, as the maximum root-mean-square deviation of gp120 is within 0.5 Å for the biphenyl group and 0.3 Å for the phenyl moiety, when comparing the two families (Figure 1). Of the miniCD4s built with the eleven novel unnatural amino acids synthesized in our lab, six showed enhanced affinity for gp120SF162, compared to 1 and four also showed an affinity increase for the clade C gp120CN54. Interestingly, extending the linker between the phenyl ether and the cyclohexyl moiety one carbon atom at a time, going from 2 to 5 and then to 6, demonstrated that shorter the linker, better the affinity for the HIV-1 surface protein. A small loss of affinity was observed at each step, and the effect, once again, was greater on the clade C strain. Exchanging the cyclohexyl moiety in 5 for a phenyl group gave opposite results on the two strains, with an increase in affinity for gp120SF162 but slightly decreased affinity for gp120CN54. On the other hand, exchanging the cyclohexyl moiety for a less bulky cyclopentyl group (3) resulted in enhanced affinity for both strains. The difference between 2 and 10 is the replacement of the cyclohexyl group for a pyrrolidine, which again engendered a higher loss in affinity for clade C CN54 than for clade B SF162 gp120 surface proteins. Filling the Phe43 cavity with linear flexible ethers gave miniCD4s with high affinity for gp120, notably 8 and 9, while a shorter linker between the alcohol function and the phenyl group, as for 7, led to a small loss of affinity. Oxidation of the thioether in 9 to the racemic sulfoxide in 15 was dramatically deleterious for the interaction between the miniCD4 and gp120CN54 and dropped the affinity by a factor of over 300-fold, whereas, affinity for the SF162 strain was reduced by a factor of 29. This large difference in affinities between the two gp120 proteins due to this small chemical modification in the miniCD4 was unexpected (Table 1; B/A = 28). The two best miniCD4s identified in this study were 13 and the previously published miniprotein 2,19 both methylcyclohexyl derivatives of either an aniline (13) or a phenol (2) entity. The NHCH2 or OCH2 linkers appear to have the right length to place the cyclohexyl moiety in the largest part of the Phe43 cavity. The non-planar shape of the cyclohexane ring possibly allows better hydrophobic contacts with the deepest part of the pocket than the planar phenyl group in 14. Interestingly, these two miniCD4s are the only ones that share almost the same subnanomolar affinities for both gp120 (see Table 1, IC50CN54/IC50SF162 ≈ 1) suggesting that the methylcyclohexyl moiety contacts the regions of the cavity that are conserved between the two gp120s.</p><!><p>In addition to competitive binding assays, the antiviral activity of those CD4-mimetics with highest binding affinities was determined against two subtype B and two subtype C viruses. In an earlier paper19 we have described the antiviral activity of 2, 3 and 4 in pseudoviruses using GHOST target cells expressing CD4 and either CCR5 or CXCR4 HIV co-receptor. Nevertheless, we tested these molecules again in this study using a TZM-bl and replication-competent HIV and showed that the results are similar in both assays (Table 2), particularly for Bal, IIIB and VI829 viruses. Minor differences were however observed for VI1358 as the EC50s are about one order of magnitude smaller. Nanomolar EC50s were found for all six M48 miniCD4s tested on lab strain clade B viruses (Table 2). Overall, highest activity was associated with miniproteins 2, 3 and 13 against subtype B HIV-1. Of note, all tested M48 miniCD4s were more active against the CXCR4-tropic strain IIIB, with low nanomolar or picomolar EC50s. While 4, 8 and 9 lost potency against subtype C viruses, 2, 3 and 13 could still inhibit the entry of the HIV-1 subtype C virus. These miniCD4s were also tested against two transmitted/founder viruses (T/F, subtype B and C) (Table 2). Interestingly, while 4, 8 and 9 were highly active against the subtype B lab strains, their antiviral activity was dramatically reduced against the T/F subtype B strain. Although the EC50 of 2, 3 and 13 against the T/F virus was higher than the EC50 values against other viruses, submicromolar activity was retained. The most notable difference between 2 and 13 was against the T/F clade C strain, where 13 with an EC50 value of 32 nM was 8.5-fold more active than 2.</p><p>Cytotoxicity of the same series of miniCD4s was evaluated using the water-soluble tetrazolium-1 (WST-1) cell proliferation assay on TZM-bl cells. With minimum CC50 values of 15 μM, no significant cytotoxicity was associated with the six CD4 miniproteins tested (Table 2).</p><!><p>Results from the competitive binding and virological assays described above establish knottin 13 as the most potent miniCD4 synthesized to date, surpassing the in vitro biological properties of the closely related miniprotein 2, which has demonstrated efficacy as a microbicide in macaque challenge models.20 To determine the affinity of interaction of 13 with HIV-1 gp120, we performed SPR analysis with three Tier 2 HIV-1 viruses namely, clade B isolates YU2 and TRO.11, and clade C isolate ZM135. We compared 13 affinity for HIV-1 gp120 with that of the closely related miniCD4 2 (Table 3 and Figure S2). In a recently published manuscript21 we have reported an affinity for peptide 2 of 15.4 ± 0.8 pM for the clade B YU2 isolate. In this study we performed the SPR assays with extended dissociation time (30 min) to obtain more accurate estimates of affinity of these tight binding complexes. We obtained an affinity of 18.1 ± 0.1 pM for 2 binding to YU2 gp120, comparable to the value obtained in the related study.21 CD4-mimetic 13 showed an affinity of 8.4 ± 0.2 pM for YU2 gp120 with the increase in affinity primarily a result of an enhanced association rate. For TRO.11, the other clade B isolate tested, a ∼14-fold enhancement in affinity for 13 relative to 2, derived from both an increased on-rate as well as a reduced off-rate of binding, the latter suggesting the formation of a more stable complex with 13. At 6.20 × 106 M-1.s-1, the on-rate of 13 binding to TRO.11 gp120 is ∼3-fold enhanced compared to that of 2, and at 1.10 × 10-4 s-1, its off-rate is ∼5-fold slower than 2. For the clade C ZM135 isolate, 16 ± 1 pM binding affinity of 13 to gp120 is a ∼3-fold enhancement over 2, with the enhancement coming primarily as a result of a 3-fold increase in the on-rate of binding.</p><p>The SPR results show that, for all three HIV-1 strains tested, peptide 13 binds gp120 with greater affinity than 2, although the degree of enhancement was isolate dependent. Such isolate dependence is consistent with observations of isolate-specific differences observed with the relative binding affinities obtained by CD4 competition ELISA. With all three isolates tested, an increase in rate of association of 13 with gp120 was observed, and with the clade B TRO.11 gp120 we also observed a ∼5-fold reduction in the off-rate.</p><!><p>To obtain an atomic-level understanding of the extraordinary affinity and neutralization breadth of 13, we crystallized miniCD4 13 with an extended core version of gp120 from the HIV-1 clade B YU2 strain.24 Crystals diffracted to 1.9 Å and the structure was solved by molecular replacement and refined to an Rfree/Rwork of 22.4%/16.8% (Table S4).</p><p>Overall, and as expected, the conformation of gp120 bound to 13 resembled the conformation of gp120 bound to CD4 or other previously structurally characterized miniCD4s15,16 with an RMSD variation ranging from 0.3-0.8 Å. Miniprotein 13 bound to the CD4 binding site of gp120 (Figure 2A), with the non-natural amino acid at position 23 inserting its side chain into the gp120 Phe43 cavity (Figure 2B) at the intersection of three gp120 domains: the inner domain, the outer domain, and the bridging sheet. In a related study, we structurally characterized gp120 binding of the closely related miniCD4 2, which contains a methylcyclohexyl derivative of tyrosine at position 23.21 Miniprotein 13 contains a methylcyclohexyl derivative of 4-aminophenylalanine at position 23. Superposing the gp120 molecules in the 2-gp120 and 13-gp120 complexes revealed close overall similarity in the environment around the Phe43 cavity in the two complexes, including the near-identical placement of the water molecules in the conserved gp120 solvent channel (Figure S3). The orientation of the Phe43 insert side chain in 2 and 13 revealed however subtle differences in the two complexes.</p><p>The overall orientation of the side chain in the Phe43 cavity is unambiguously defined by the electron density in the miniprotein-13-bound cavity (Figure 3A-C and Figure S3), with the anilino group at position 23 appropriately positioned for interacting with the conserved gp120 solvent channel by hydrogen bonding with the proximal water molecule (Figure 3D and 3E). While the overall definition of the insert in the Phe43 cavity is clear for 13, its cyclohexane ring displayed asymmetric electron density, indicative of local ligand disorder in the bound state (Figures 3A-C, Figure S3 and Figure S4). Interestingly, the electron density indicates mobility or disorder at similar positions as was observed in different crystal forms of gp120-bound 2 (Figure S3), suggesting a precise and conserved recognition mode in the Phe43 cavity that allows these Phe43 cavity inserts to retain conformational mobility. The mono-substituted cyclohexane ring was modeled in the chair conformation with the sole substitution that connects the ring to the rest of the miniprotein placed in an equatorial orientation, thereby defining the cyclohexane conformation. The electron density in the cavity appeared to be well modeled by rotations about the flexible linker that connects the cyclohexane ring to the phenyl ring of 13 (Figure 3F).</p><p>The cyclohexane ring was packed against a hydrophobic patch in the Phe43 cavity comprised of atoms from residues Val255, Trp112, Phe382 and Ile424. The observation of residual ligand entropy in the gp120 Phe43 cavity was reminiscent of synthetic host-guest systems, where an enthalpic-entropic balance was found to be critical to achieve optimal binding.25 For complexes in confined apolar cavities, it has been surmised that optimal stability is attained when the guest (or ligand) occupies 55 ± 9% of the cavity volume.26 This rule, first observed in synthetic host-guest systems, was later found to also apply to enzymatic systems.27,28 To determine whether this rule also applies to miniCD4 13 binding to the predominantly apolar gp120 Phe43 cavity, we quantified the packing efficiency of its insert in the Phe43 cavity by calculating the packing coefficient, which is the fraction of the pocket volume occupied by the ligand. We also calculated shape complementarity, which related to the goodness of fit between protein and ligand surfaces. We observed that the insert moiety occupies 80% of the available volume in the Phe43 cavity (Figure 2B and Figure S5). The cyclohexane ring, on the other hand, only fills 66% of the volume of the cavity to which it binds. The observed packing coefficient for the cyclohexane ring of 66% is just above the conjectured optimum of 55 ± 9%. Taken together these calculations show that, while the overall occupation of the Phe43 cavity by the insert of 13 is enthalpy driven and suggestive of tight interactions of the ligand with the protein, local regions of looser packing allow retention of entropy in the bound state. In addition, results from packing coefficient calculations appear to be consistent with shape complementarity analysis: the cyclohexane ring shows lower shape complementarity compared to the overall insert moiety, where tight packing results in high shape complementarity.</p><!><p>Although crystal structures of full-length gp120 or trimeric HIV envelope protein gp160 are not yet available, the three-dimensional structures of HIV gp120 core variants have been determined in the CD4-bound conformation29-33 as well as in the unliganded state.24 CD4 binding stabilizes a cavity of roughly 150 Å3, which penetrates about 14 Å into the gp120 hydrophobic core at the intersection of three gp120 domains. The Phe43CD4 side chain caps the entrance of this pocket, hence the Phe43 cavity name. However, this pocket is induced not only by the CD4 receptor, but also by CD4-binding-site antibodies34 and by synthetic entry inhibitors24,35 including miniaturized CD4-mimetics,15,16 called miniCD4s. To increase the affinity of the miniCD4s for the gp120 surface protein and to improve their ability to neutralize HIV-1, we targeted the gp120 Phe43 cavity with various unnatural amino acid side chains. While the effect of modifying position 23 on the miniCD4 was often not identical on the two HIV-1 isolates tested, SF162 and CN54 (see ratio B/A in Tables 1 and S1), the overall trends were similar. For most derivatives, the variation of affinity compared to knottins 17, in the M33 series, or 1, in the M48 series, is more pronounced on the CN54 strain than on SF162. In both strains, all the cavity-lining residues are identical, except for Arg426 (HXBc2 numbering) from strain SF162 and Met426 from strain CN54 (Figure S6). With the non-conserved residue 426, the Phe43 cavity was lined by its invariant main chain component, with the non-conserved side chain facing away from the cavity. The differences in recognition of SF162 and CN54 thus likely relates to isolate-specific differences in Phe43 cavity shape, due to either the variation in residue 426 or the effect of residue variation outside the Phe43 cavity.</p><p>Since commercially available amino acids proved to be of limited utility for improving the activity of the miniCD4s, we developed unnatural residues designed to fill the gp120 Phe43 cavity. Of the miniCD4s synthesized and tested thus far, knottin 13 showed the highest affinity for gp120s tested as well as the most potent antiviral activity on laboratory-adapted strains, primary isolates and transmitter/founder viruses.</p><p>A cavity-filling analysis was previously performed on the CD4 mutant, called D1D2F43C,36 in which various chemical moieties were attached to the free cysteine at residue 43CD4 (analogous to residue 23miniCD4). One of the best derivatives of D1D2F43C contained a cyclohexyl group at a distance from residue 23miniCD4 Cα similar to that of 2 and 13. Introduction of the cyclohexyl group to D1D2F43C increased affinity ∼30-fold, to a level similar to the unmodified D1D2 (sCD4) case.</p><p>In the current study, introduction of a cyclohexyl moiety enhanced the relative affinity of 13 by a factor six for gp120CN54 and two for gp120SF162 compared to 1, resulting in a CD4-binding-site ligand with subnanomolar apparent affinity, and about one order of magnitude better IC50 of sCD4 on the SF162 receptor (4 nM, personal communication). SPR analyses on gp120 from three different strains from clades B and C show an increased affinity of 13 for gp120 compared to 2. The SPR results from this study and from a related study,21 together show that the affinity of 13 for YU2 gp120 surpasses that of 1, 2, 14 and sCD4. The in vitro antiviral activity of 13 improved more than 100-fold versus 1, and between 5 to 25-fold versus sCD4,19 on the laboratory adapted and primary isolates tested.</p><p>The crystal structure of 13 bound to YU2 gp120 shows that the introduction of the aniline nitrogen results in a hydrogen bond interaction with the gp120 solvent channel. Conjugation of this nitrogen may provide a means to improve affinity even further, by utilizing interaction with a conserved gp120 solvent channel, which runs adjacent and roughly perpendicular to the Phe43 cavity. Our results further suggest that binding of the insert moiety of 13 to the Phe43 cavity is primarily enthalpy driven while at the same time torsional movements about the flexible linker bonds allow different orientations of the cyclohexane ring to be accommodated in the wider, innermost recess of the cavity, thereby preserving conformational entropy of the ligand in its bound state. It remains to be tested whether modifications of the cyclohexane ring via structureguided substitution will result in better ring packing and higher affinity. While peptides 8 and 9 showed IC50 values similar to miniCD4 13 in the gp120SF162 (clade B) assay, their antiviral activities were not comparable to that of 13 even for clade B isolates. The differences in their pharmacological activity may result from the differences in their cavity-filling properties, described by X-ray crystallographic analyses in this study and in a related one.21 All these inserts displayed flexibility and hydrophobic interactions, but the inserts of 221 and 13 (this study) showed better shape complementarity with the Phe43 cavity than the insert of 821 and probably also of peptide 9. Taken together our results show that a subtle change in the chemistry of Phe43 cavity insert can result in improved chemical complementarity and substantially improved biological activity.</p><!><p>The miniCD4s built on the scyllatoxin scaffold are stable to acidic pH and high temperatures, relatively resistant towards proteolytic degradation, poorly immunogenic and are not cytotoxic. They stabilize the CD4-bound conformation of gp120 and one miniCD4 representative (2) was also shown to neutralize HIV entry in vitro and in vivo. Cost is a key consideration for the potential use of such compounds as microbicides especially in developing countries. One way to achieve low cost is to enhance the potency of the miniCD4s, thereby reducing the amount of drug that needs to be administered. In this paper, we showed that the synthesis of novel unnatural phenylalanine derivatives allowed us to design the most effective miniCD4 ever developed. It is able to block the viral activity of T/F viruses in the nanomolar range not only for clade B but also for clade C, being therefore more potent than the previously described miniprotein 2. From the perspective of synthesis of future miniCD4 variants, 13 has a distinct advantage over 2. Replacement of the phenol moiety by an aniline group, i.e. the presence of a secondary amine in place of ether, allows further modification in direction of the gp120 solvent channel. Such modifications may lead to development of miniCD4s with even greater potency and therefore lower cost in a potential use as a microbicide. It will also allow exploration of a very highly conserved but relatively poorly understood feature in the HIV-1 envelope structure.</p><!><p>All reagents and solvents used in the synthesis and purification steps were purchased from Sigma-Aldrich, Fluka, Novabiochem, SDS or Lancaster and were of the highest purity available. THF was distilled from sodium/benzophenone immediately prior to use. Wang resin (p-benzyl-oxybenzyl alcohol, polymer bound resin), used for amino acids synthesis, was purchased from Aldrich (100-200 mesh, loading level 1.82 mmol OH/g). Fmoc-PAL-PEG-PS, used for solid-phase peptide synthesis, was purchased from Applied Biosystems (100-200 mesh, loading level 0.2 mmol/g). Standard Fmoc-protected amino acids were obtained from Novabiochem. Unnatural amino acid Fmoc-L-4-tert-butyl-Phe was purchased from PepTech Corp., Fmoc-L-Phe(4-i-Pr)-OH from Carbone Scientific, Fmoc-L-2-naphtylalanine-OH from Senn Chemicals, Fmoc-L-styrylalanine from PolyPeptide, Fmoc-L-Phe(4-Ph)-OH and Fmoc-homoPhe-OH from Bachem, Fmoc-L-Tyr(Me)-OH and Fmoc-L-Phe(4-Me)-OH from Advanced ChemTech. Flash chromatography was carried out with Merck silica gel Si 60 (40-63 μm) as stationary phase. Melting points of the crystallized compounds were determined on a Kofler melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on Bruker AVANCE 250 and 400 NMR spectrometers. The molecular weights of the amino acids and peptides were determined by electrospray mass spectrometry (ES/MS), performed on a Quattro micro mass spectrometer (Micromass, Altricham, U.K.). The purity of Fmoc-amino acids, denoted in parentheses, was evaluated by analytical-HPLC, at 214 nm, on a thermo HPLC apparatus (32′-40′, 56′, 57′ and 59′) or a Waters 600E HPLC (54′) with a Chromolith SpeedROD RP-18e column (VWR, 50 × 4.6 mm, 2 μm) with a linear gradient from 10% to 90% solvent B over 6 min (0.1% TFA in water (solvent A), 90% CH3CN with 0.09% TFA in water (solvent B) at 3 mL/min constant flow rate (these conditions will be noted HPLC-A). The purity of the various tested peptides (see tables S2 and S3 in the supporting information) was checked on a Waters 600E HPLC, at 214 nm, by reverse phase chromatography at a constant 1 mL/min flow rate, on an analytical Discovery BIO Wide Pore C18, 5 μM (15 cm × 4.6 mm) column with a gradient going from 10% to 50% B in 30 min. Even if we could not always reach a purity ≥95% for the newly synthesized Fmoc-amino acids, all the tested compounds, i.e. the various miniCD4s, have a purity >95%. Retention times (Rt) are reported in minutes.</p><!><p>Tyrosine (1′) (5.44 g, 30 mmol) was dissolved in 15 mL of 4 N sodium hydroxide (2 equiv). The solution was cooled in an ice-bath and treated with 7 mL (2.2 equiv) of allyl chloroformate and additional 15 mL of 4 N sodium hydroxide, each added in five equal portions, with vigorous shaking for a few minutes after each addition. After the last addition, the mixture was shaken vigorously for 1.5 h, letting it warming up to rt. The water layer was extracted twice with ether, and then acidified to pH 4-5 with concentrated hydrochloric acid. After cooling overnight, crystals were filtered, washed with cold water and dissolved in DCM. The organic layer was washed with H2O and brine, dried over Na2SO4 and evaporated to yield 9.35 g (89%) of 2′ as a white solid: Rf (DCM/MeOH/AcOH: 90/8/2) = 0.75; mp 106-107 °C (105-106 °C);37 1H NMR (CDCl3) δ 3.14 (dt, J = 13.9, 5.2 Hz, 2H), 4.56 (d, J = 5.4 Hz, 2H), 4.73 (d, J = 5.8 Hz, 2H), 4.72-4.75 (m, 1H), 5.21 (d, J = 11.1 Hz, 1H), 5.34 (d, J = 10.4 Hz, 1H), 5.35 (d, J = 18.8 Hz, 1H), 5.43 (d, J = 18.6 Hz, 1H), 5.92-6.26 (m, 2H), 6.36 (br s, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.6 Hz, 2H); 13C NMR (CDCl3) δ 37.1, 55.1, 66, 69.2, 118, 119.6, 121.1, 130.4, 131, 132.4, 134.1, 150, 153.6, 156, 175.6.</p><!><p>Wang resin (8.5 g; 1.82 mmol OH/g) was swelled in NMP (50 mL) for 15 min in a reactor with external shaking. The resin was filtered and resuspended in NMP (50 mL). 7.27 mL of diisopropylcarbodiimide (3 equiv) and 189 mg of DMAP (0.1 equiv) were added and the mixture was stirred for 15 min. Compound 2′ (16.2 g, 3 equiv) in solution in NMP (50 mL) was added and shaking was continued for 48 h at rt. The resin was filtered, rinsed with DCM (3 × 100 mL) and finally dried in vacuo to afford 12.95 g of a 0.99 mmol/g (87% theoretical) resin-bound N,O-bis-allyloxycarbonyl-L-tyrosine 3′.</p><p>Estimated resin loading by cleavage. A small weighed sample of resin (between 50 and 100 mg) was cleaved by 5 mL of TFA/DCM: 3/7 during 1 h. The mixture was filtered, the resin rinsed with DCM (2 × 3 mL) and the filtrate evaporated. The oil was mixed with a few mL of water, frozen and lyophilized. The obtained crude compound was weighed and analyzed by 1H NMR. Loading was estimated by comparison with the theoretical obtained quantity.</p><!><p>12 g (11.92 mmol) of bis-protected tyrosine resin 3′ (0.99 mmol/g) were treated with a solution (150 mL) of 20% piperidine in DMF (v/v) for 24 h at rt. The resin was filtered, rinsed with DCM (3 × 100 mL) and finally dried in vacuo to afford 11.05 g of a 1.03 mmol/g (95% theoretical) resin-bound N-allyloxycarbonyl-L-tyrosine 4′.</p><!><p>General procedure (G1). PPh3 and the corresponding alcohol (4 to 8 equiv depending on the nature of the alcohol, see below) were added to resin 4′ (4.5 mmol, 4.37 g), suspended in an anhydrous 22 mL 1:1 (v/v) DCM/THF mixture. After 15 min, DIAD (4 to 8 equiv depending on the nature of the alcohol, see below), in solution in a few mL of DCM, was added portionwise, with shaking for a few minutes after each addition. The reaction continued for 24 h, at rt. The resin was filtered and rinsed with DCM (3 × 100 mL). This procedure was repeated a second time. After 24 hours shaking, the resin was filtered and rinsed with DMF (2 × 100 mL), DCM (1 × 100 mL), MeOH (1 × 100 mL) and DCM (2 × 100 mL). A small weighed sample of resin was cleaved in the same conditions as described above. Crude compound was used for 1H NMR analysis to check the disappearance of the phenol moiety. Loading was not estimated.</p><p>N-allyloxycarbonyl-O-(cyclohexylmethyl)-L-tyrosine immobilized on Wang resin (5′) was synthesized according to the procedure G1 using 3.9 mL of cyclohexylmethanol (7 equiv, 31.5 mmol) with 7 equiv of PPh3 and DIAD.</p><p>For 6′ to 13′: see supporting information</p><!><p>General procedure (G2). Under argon atmosphere, 2.6 g of tetrakistriphenylphosphine palladium (0.5 equiv, 2.25 mmol) were added to the resin 5′-13′ (loadings were estimated at 4.5 mmol) in suspension in 150 mL of THF/DMSO/0.5 M HCl/morpholine: 20/20/10/1 ratio. The reactor was protected against light, as Pd(Ph3)4 is light sensitive, and stirred for 24 h. The resin was filtered and rinsed with DMF (2 × 100 mL), a 0.1 M solution of diethyldithiocarbamic acid in DMF (5 × 100 mL), DMF (2 × 100 mL) and DCM (3 × 100 mL). A 100% yield was estimated whatever the 5′-13′ resins used.</p><p>Washings with 0.1 M solution of diethyldithiocarbamic acid in DMF were essential in order to remove all traces of catalyst from the resin.</p><p>O-(cyclohexylmethyl)-L-tyrosine immobilized on Wang resin (14′) was synthesized according to the procedure G2 using resin 5′.</p><p>For 15′ to 22′: see supporting information</p><!><p>General procedure (G3). To the resin 14′-22′ (loadings not calculated, estimated at 4.5 mmol), in suspension in DCM (90 mL), were added 3.14 mL of diisopropylethylamine (4 equiv, 18 mmol). The mixture was stirred for 15 min. 3.5 g of Fmoc-Cl (3 equiv, 13.5 mmol), in solution in 30 mL of DCM, were added to this suspension and the reaction continued for 24 h at rt. The resin was filtered, rinsed with DCM (3 × 100 mL) and dried in vacuo.</p><p>N-(9-fluorenylmethoxycarbonyl)-O-(cyclohexylmethyl)-L-tyrosine immobilized on Wang resin (23′) was synthesized according to the procedure G3 using resin 14′.</p><p>For 24′ to 31′: see supporting information</p><!><p>General procedure for resin cleavage (G4). The resin 23′-31′ were treated with 100 mL of TFA/DCM: 3/7 for 1 h at rt. The mixture was filtered and the obtained organic layer was washed with H2O (2 × 50 mL), dried over MgSO4 and evaporated. The crude residue was applied to silica gel flash chromatography with DCM and DCM/MeOH: 97/3 (flash chromatography condition 1: FC1) or DCM and DCM/AcOH: 99/1 (flash chromatography condition 2: FC2) as eluents to give compound 32′ to 40′ with yields comprised between 26 and 63%.</p><!><p>Procedure G4 was applied on 23′ and the crude residue was purified according to FC2 to yield 1.32 g of 32′ (59%) as a white solid: Rf (DCM/AcOH: 99/1) = 0.47; mp 136-137 °C; 1H NMR (CDCl3) δ 0.96-1.10 and 1.14-1.37 (2m, 6H), 1.63-1.91 (m, 5H), 3.11 (ddt, J = 14.0, 16.6, 5.8 Hz, 2H), 3.70 (d, J = 6.2 Hz, 2H), 4.21 (t, J = 7.1 Hz, 1H), 4.37 (dd, J = 9.8, 6.6 Hz, 1H), 4.45 (dd, J = 10.5, 7.1 Hz, 1H), 4.67 (dt, J = 6.0, 5.9 Hz, 1H), 5.17 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 8.1 Hz, 2H), 7.04 (d, J = 8.1 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.55 (t, J = 6.2 Hz, 2H), 7.76 (d, J = 7.3 Hz, 2H); 13C NMR (CDCl3) δ 25.79, 26.50, 30.30, 36.38, 37.66, 47.10, 54.57, 66.99, 73.43, 114.63, 119.98, 125.03, 127.05, 127.72, 130.32, 141.26, 143.76, 155.72, 158.51, 175.78; ES/MS for C31H33NO5 (negative ionization): Mol.wt calcd: 499.2, found: 499.1; Rt (HPLC-A): 5.11 (94%).</p><p>For compounds 33′ to 40′: see supporting information</p><!><p>To sodium hydride (4.45 g, 111.2 mmol, 60% in mineral oil) in suspension in dry THF (200 mL), under argon, cooled to 0 °C, were added slowly 50 g of 1,4-butanediol (41′) (5 equiv, 555 mmol). The reaction was stirred for 1 h. Benzyl bromide (13 mL, 111.2 mmol), in 200 mL of dry THF, was added dropwise and the reaction mixture was stirred 5 more hours. After addition of 300 mL H2O, the solution was extracted twice with Et2O. The organic layer was washed with water and brine, dried over MgSO4 and the solvent was evaporated under reduced pressure. After purification by column chromatography on silica gel (eluted with DCM and DCM/MeOH: 97/3), 13.2 g (66%) of 44′ were obtained as a slightly yellow oil: Rf (DCM/MeOH: 97/3) = 0.39; 1H NMR (CDCl3) δ 1.57-1.71 (m, 4H), 3.48 (t, J = 6.1 Hz, 2H), 3.53 (s, 1H), 3.56 (t, J = 6.3 Hz, 2H), 4.49 (s, 2H), 7.24-7.30 (m, 1H), 7.31-7.36 (m, 4H); 13C NMR (CDCl3) δ 26.33, 29.58, 62.10, 70.24, 72.86, 127.67, 128.35, 138.19.</p><p>For compounds 45′ and 46′: see supporting information</p><!><p>To 9 g of (-) Cbz-L-proline (47′) (36.1 mmol) in DME (55 mL) was added 4-methylmorpholine (1.1 equiv, 39.7 mmol). The reaction mixture was cooled to -15 °C and isobutylchloroformate (1.3 equiv, 46.9 mmol) was added slowly. After 20 min of stirring at this temperature, the white precipitate was quickly filtered off and washed with DME. To the obtained solution, cooled again to -15 °C, was added sodium borohydride (1.5 equiv, 54.2 mmol), in 23 mL of water, and the reaction mixture stirred during 1 h, letting it warming up to rt. After addition of 65 mL of water and evaporation of DME under reduced pressure, the solution was extracted twice with AcOEt. The organic layer was washed with 1 N aqueous KHSO4 solution, 1 N aqueous NaHCO3 solution and finally with brine. It was dried over MgSO4 and the solvent was evaporated under reduced pressure. 7.4 g (87%) of 48′ were obtained as a colorless thick oil: Rf (DCM/MeOH: 95/5) = 0.55; 1H NMR (CDCl3) δ 1.52-1.93 (m, 4H), 3.32 (t, J = 6.8 Hz, 1H), 3.42 (t, J = 6.8 Hz, 1H), 3.55 (d, J = 5.4 Hz, 2H), 3.86-3.96 (m, 1H), 5.05 (s, 2H), 7.20-7.29 (m, 5H); 13C NMR (CDCl3) δ 24.53, 29.01, 47.82, 61.10, 67.00, 67.72, 128.43, 128.97, 129.04, 137.06, 157.48;</p><!><p>5.8 mL (57.6 mmol) of 2,2′-thiodiethanol (49′) were dissolved in dry THF containing 3.2 g (63.4 mmol) of NaH (60% in oil), and stirred for 1.5 h, under inert atmosphere. The solution was cooled to 0 °C and 8.6 g (57.6 mmol) of tert-butyldimethylsilyl chloride were added slowly. The mixture was stirred overnight at rt. 10% K2CO3 was then added, THF was evaporated and diethyl ether was added and extracted with water and brine. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (eluted with hexane/AcOEt: 8/2) afforded 8.1 g of 50′, as an almost colorless oil (59%). 1H NMR (CDCl3) δ 0.86 (s, 6H), 0.91 (s, 9H), 2.68 (t, J = 6.5 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H), 2.88 (br s, 1H), 3.74 (t, J = 6.0 Hz, 2H), 3.79 (t, J = 6.75 Hz, 2H); 13C NMR (CDCl3) δ -5.3, 18.3, 25.9, 34.2, 35.8, 60.9, 63.4.</p><!><p>4.1 g (17.4 mmol) of 50′ were dissolved in toluene and cooled to 0 °C. 8.8 g (1.2 equiv) of Z-Tyr-OBn dissolved in 10 mL of dry DMF were added together with 5.7 g (1.2 equiv) of PPh3. After dissolution, DEAD (1.2 equiv) was added dropwise. The reaction was then stirred overnight at rt. Sodium phosphate buffer (0.5 M, pH 7) was added to the mixture, which was then extracted with AcOEt. The organic phase was washed with a saturated solution of NH4Cl, water and brine, dried over Na2SO4 and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (eluted with hexane/AcOEt: 9/1 to 85/15) afforded 10.45 g of 51′ as a colorless oil (96%). 1H NMR (CDCl3) δ 0.08 (s, 6H), 0.9 (s, 9H), 2.74 (t, J = 6.75 Hz, 2H), 2.92 (t, J = 6.75 Hz, 2H), 3.03 (d, J = 5.75 Hz, 2H), 3.81 (t, J = 7.0 Hz, 2H), 4.06 (t, J = 7.0 Hz, 2H), 4.65 (m, 1H), 5.11 (m, 4H), 5.27 (d, J = 8.25 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 7.30 (m, 10H); 13C NMR (CDCl3) δ -5.2, 18.3, 25.9, 31.4, 34.9, 37.2, 54.9, 63.4, 66.9, 67.2, 67.7, 114.6, 127.7, 128.1, 28.2, 128.4, 128.5, 130.3, 135.1, 136.2, 155.6, 157.6, 171.4.</p><!><p>10.45 g (16.8 mmol) of 51′ were dissolved in 20 mL of MeOH and reduced overnight with palladium black under 3.4 bars of H2. The slurry was filtrated. The catalyst was washed five times with MeOH and the filtrate was evaporated yielding 6.62 g of compound 52′ (quantitative). ES/MS for C19H33NO2SSi (positive ionization): Mol.wt calcd: 399.1, found: 399.1</p><!><p>Compound 52′, dissolved in 50 mL DMF, was added to 5.9 g (17.5 mmol) of Fmoc N-hydroxysuccinimide ester in a mixture of 750 mL of CH3CN, 370 mL H2O and 5.3 g of NaHCO3 and stirred overnight at rt. The solution was concentrated and extracted with AcOEt. The organic layer was washed with 10% citric acid, a saturated solution of NH4Cl, water and brine, dried over Na2SO4 and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (eluted with DCM/AcOH: 99.5/0.5 to DCM/MeOH/AcOH: 95/5/0.5) afforded 8 g of 53′ as a colorless oil (78%). 1H NMR (CDCl3) δ 0.09 (s, 6H), 0.9 (s, 9H), 2.74 (t, J = 6.75 Hz, 2H), 2.92 (t, J = 6.75 Hz, 2H), 3.09 (m, 2H), 3.81 (t, J = 6.75 Hz, 2H), 4.08 (t, J = 6.75 Hz, 2H), 4.19 (t, J = 6.85 Hz, 1H), 4.41 (m, 2H), 4.65 (m, 1H), 5.28 (t, J = 10.5 Hz, 1H), 6.80 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 7.16-7.43 (m, 4H), 7.53-7.58 (m, 2H), 7.76 (d, J = 7.5 Hz, 2H), 8.94 (br s, 1H); 13C NMR (CDCl3) δ -5.2, 18.4, 26.0, 31.5, 34.9, 36.9, 47.2, 54.8, 63.5, 67.1, 67.8, 114.8, 120.1, 125.2, 27.2, 127.8, 128.2, 130.5, 141.4, 143.8, 155.9, 157.8, 176.2. ES/MS for C34H43NO6SSi (positive ionization): Mol.wt calcd: 622.2, found: 622.2</p><!><p>1.24 g (2 mmol) of 53′ were dissolved in 6 mL of dry DCM and treated with 534 mL (1.5 equiv) of tert-butyl hydroperoxide (5.5 M in decane) and 10 mg (6% in mol) of thiourea dioxide for 3 days. The mixture was diluted with DCM and washed with water, brine, and dried over anhydrous Na2SO4. Purification by column chromatography on silica gel (eluted with DCM/MeOH/AcOH: 95/5/0.1) afforded 1.89 g of 54′ as a white foam (74%): mp not measurable, behaves like a wax; 1H NMR (CDCl3) δ 0.1 (s, 6H), 0.9 (s, 9H), 3.06-3.24 (m, 6H), 4.00-4.62 (m, 8H), 5.55 (d, J = 7.75 Hz, 1H), 6.78 (d, J = 8.25 Hz, 2H), 7.05 (d, J = 8.25 Hz, 2H), 7.25-7.41 (m, 4H), 7.54-7.59 (m, 2H), 7.75 (d, J = 7.5 Hz, 2H), 11.19 (br s, 1H); 13C NMR (CDCl3) δ -5.5, 18.2, 25.8, 36.9, 47.1, 51.43, 54.7, 55.2, 56.1, 60.3, 66.8, 114.5, 119.9, 125.1, 127.0, 127.7, 128.0, 130.6, 141.2, 143.8, 155.7, 156.9, 173.7. ES/MS for C34H43NO7SSi (positive ionization): Mol.wt calcd: 637.3, found: 637.3; Rt (HPLC-A on a Waters 600E HPLC): 5.23 (94%).</p><!><p>Cyclohexanecarboxaldehyde (900 mL, 1.5 equiv, 7.45 mmol) was added to a suspension of Fmoc-p-amino-L-phenylalanine (55′) (2 g, 4.98 mmol) in 50 mL CH3CN/AcOH (1% v/v). The reaction mixture was stirred for 2 h at rt. Solid NaBH(OAc)3 (2 equiv, 9.94 mmol) was then added portionwise and stirring was continued overnight, at rt. The mixture was poured into 250 mL of a 0.1 N aqueous HCl solution and the solution was extracted twice with AcOEt. The organic layer was washed twice with brine, dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. After purification by column chromatography on silica gel (eluted with DCM/AcOH: 100/0.5 to DCM/MeOH/AcOH: 99/1/0.5), 1.9 g (77%) of 56′ were obtained as a white solid: Rf (DCM/MeOH/AcOH: 99/1/0.5) = 0.18; mp 95 °C; 1H NMR (CDCl3) δ 0.86-0.98 (m, 2H), 1.02-1.34 (m, 5H), 1.42-1.83 (m, 6H), 2.88 (d, J = 6.6 Hz, 2H), 3.07 (d, J = 5.4 z, 2H), 4.21 (t, J = 6.9 Hz, 1H), 4.35 (dd, J = 10.3, 6.8 Hz, 1H), 4.43 (dd, J = 10.3, 7.3 Hz,1H), 4.63 (dt, J = 7.9, 5.3 Hz, 1H), 5.28 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 8.4 Hz, 2H), 6.97 (d, J =8.3 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.57 (d, J = 7.1 Hz, 2H), 7.76 (d, J = 7.4 Hz, 2H); 13C NMR (acetone-d 6) δ 26.4, 27.0, 37.0, 37.8, 47.6, 50.7, 56.8, 66.6, 112.5,120.5, 125.1, 126.1, 127.7, 128.2, 130.4, 141.6, 144.7, 148.7, 156.6, 174.3; ES/MS forC31H34N2O4 (positive ionization): Mol.wt calcd: 498.3, found: 498.3; Rt (HPLC-A): 3.25 (98.5%).</p><!><p>To 1.07 g (2.15 mmol) of 56′, dissolved in 45 mL of THF, were added 750 mL of diisopropylethylamine (2 equiv) and 1.64 g (3.5 equiv) of di-tert-butyldicarbonate. The reaction mixture was stirred at 45-50 °C for 3 days. The mixture was concentrated in vacuo. After addition of 100 mL AcOEt, the organic layer was washed with 10% citric acid, brine and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. After purification by column chromatography on silica gel (eluted with DCM/MeOH/AcOH: 97/3/1.5), 1.22 g (94.8%) of 57′ were obtained as a pale yellow foam: Rf (DCM/MeOH/AcOH: 97/3/1.5) = 0.45; mp not measurable, behaves like a wax; 1H NMR (CDCl3) δ 0.85-0.90 (m, 2H), 1.04-1.14 (m, 3H), 1.45 (s, 9H), 1.53-1.70 (m, 6H), 3.15 (d, J = 4.5 Hz, 2H), 3.44 (d, J = 7.2 Hz, 2H), 4.19 (t, J = 7.0 Hz, 1H), 4.27-4.44 (m, 3H), 4.71 (m, 1H), 5.64 (br s, 1H), 6.98-7.19 (m, 4H), 7.29 (dt, J = 7.2, 1.2 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.57 (t, J = 6.5 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H); 13C NMR (CDCl3) δ 25.8, 26.4, 28.3, 30.6, 36.7, 37.1,47.1, 54. 3, 56.1, 67.1, 80.36, 119.9, 125.2, 127.1, 127.4, 127.7, 130.0, 133.8, 141.3, 143.7,155.3, 173.6; ES/MS for C36H42N2O6 (positive ionization): Mol.wt calcd: 598.3, found: 598.4; Rt(HPLC-A): 5.29 (96%).</p><!><p>350 mg (1.69 mmol) of p-(isopropyl)-L-phenylalanine (58′) were dissolved in 6.8 mL (4 equiv, 6.76 mmol) of 1 N Na2CO3 aqueous solution and 5 mL of dioxane. Half of the solvent volume was removed under reduced pressure. Then, the residual was diluted in 7.5 mL of water (pH was controlled at 9-10) and 5 mL of dioxane, and cooled with an ice bath. 547 mg (1.25 equiv, 2.11 mmol) of Fmoc-chloroformate, dissolved in 5 mL of dioxane, were added slowly. The reaction mixture was stirred 2 h at 0 °C and overnight at rt (additional 2 mL of 1 N Na2CO3 aqueous solution were added in order to keep pH around 9). After addition of 40 mL H2O, the solution was acidified with a 2 N HCl aqueous solution until the product precipitated. The aqueous layer was quickly extracted twice with Et2O. The organic layer was washed twice with H2O to eliminate all traces of HCl, dried over Na2SO4 and the solvent was evaporated under reduced pressure. The crude residue was applied to silica gel flash chromatography (eluted with DCM and DCM/MeOH: 97/3) to yield 0.34 g of 59′ (46%) as a white powder: Rf (DCM/MeOH: 95/5) = 0.33; mp 113 °C; 1H NMR (CDCl3) δ 1.21 and 1.24 (2s, 6H), 2.88 (sept, J = 6.9 Hz, 1H), 3.15 (ddt, J = 14.5, 10.6, 5.1 Hz, 2H), 4.21 (t, J = 6.8 Hz, 1H), 4.36 (dd, J = 10.3, 7.2 Hz, 1H), 4.45 (dd, J = 10.6, 7.1 Hz, 1H), 4.67-4.72 (m, 1H), 5.21 (d, J = 8.1 Hz, 1H), 7.07 (d, J = 8.1 Hz, 2H), 7.16 (d, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.54-7.55 (m, 2H), 7.77 (d, J = 7.4 Hz, 2H); 13C NMR (CDCl3) δ 23.92, 23.95, 33.70, 37.22, 47.11, 54.50, 67.08, 119.98, 119.99, 126.76, 127.05, 127.73, 129.28, 132.63, 141.29, 143.67, 155.77, 175.58; ES/MS for C27H27NO4 (negative ionization): Mol.wt calcd: 429.1, found: 429.1; Rt (HPLC-A): 4.25 (100%).</p><!><p>The sequences of the various synthesized peptides were TpaNLHFCQLRCKSLGLLGKCAGSXCACV-NH2 for the M33 (16) series and TpaNLHFCQLRCKSLGLLGRCA-DPro-TXCACV-NH2 for the M48 (1) series. Tpa stands for thiopropionyl and × for phenylalanine or a phenylalanine derivative. Peptides were synthesized on an ABI-433A (Applied Biosystems) automated peptide synthesizer, using the stepwise solid-phase method and standard Fmoc chemistry. Synthesis was performed on a 0.1 mmol scale with 10 equiv Fmoc-protected amino acids, 20% piperidine in NMP for Fmoc-deprotection, DCC and Cl-HOBt for activation, and acetic anhydride for capping. For the M48 (1) series, N-terminal thiopropionyl group (Tpa) was introduced in its disulphide form. The miniprotein was cleaved from the resin with simultaneous removal of side-chain protecting groups by treatment with reagent K′ [TFA/H2O/phenol/thioanisole/1,2-ethanedithiol/triisopropylsilane: 81.5/5/5/5/2.5/1 (all v/v)], or a mixture of TFA/H2O/triisopropylsilane: 9.5/0.25/0.25, for 2.5 h at rt. The resin was then filtered off and the fully deprotected peptide was precipitated in methyl-tert-butyl ether at 4 °C. After centrifugation and washing with methyl-tert-butyl ether, peptides were dissolved in acetic acid (20% v/v) and freeze-dried. Refolding was done on the crude reduced peptide dissolved at 0.1 mg/mL in Tris/HCl buffer (0.1 M, pH 8.0), containing 2 M guanidinium chloride, 5.0 mM glutathione and 0.5 mM oxidized glutathione. Glutathione was added 15 min before oxidized glutathione in order to deprotect the Tpa moiety, when present, before refolding. The refolded peptides were purified by reverse phase chromatography on a Discovery BIO Wide Pore C18, 5 μM (25 cm × 10 mm), using H2O/5%CH3CN/0.1% TFA as solvent A and CH3CN/10%H2O/0.09%TFA as solvent B. A classical gradient was 10% to 50% B in 90 min. Peptides were lyophilized and dissolved in water. Their concentration was determined by total hydrolysis under highly acidic conditions. Molecular characteristics and purity of the various miniCD4s are described in the supporting information (Tables S2 and S3).</p><!><p>Competition binding assays in ELISA were performed in 96-well plates (Maxisorb; Nunc). Competition assay on gp120SF162: 50 ng/well antibody D7324 (Aalto Bio Reagents, Dublin, Ireland) was coated overnight at 4 °C. Wells were then saturated with phosphate-buffered saline containing 3% bovine serum albumin buffer and washed three times, and 15 ng/well gp120SF162 were added, followed by addition of 1.25 ng of sCD4 (Progenics) and different concentrations of soluble competitors. After one night at 4 °C, we successively added anti-CD4 monoclonal antibody L120.3 (Centralized Facility for AIDS Reagents, NIBSC, UK), goat anti-mouse peroxidase-conjugated antibody (Jackson ImmunoResearch, West Grove, PA, USA), and the 3,3′,5,5′-tetramethylbenzidine substrate (Sigma). After acidification, optical density was measured at 450 nm and expressed as the mean of experiments performed in duplicate. The conditions used for competition assay on gp120CN54 were the same as for gp120SF162, except that (i) D7324 was replaced by 500 ng of concanavalin A (Sigma-Aldrich), (ii) 5 ng/well of gp120CN54 were added and (iii) 1 ng of sCD4 was used.</p><!><p>The antiviral activity of peptides 2-4, 8, 9 and 13 was determined by pre-incubating 104 TZM-bl cells/well in a 96-well plate for 30 min at 37 °C and 5% CO2 with or without a serial dilution of compound. Next, 200 TCID50 of Bal (subtype B, CCR5), IIIB (subtype B, CXCR4), VI829 (subtype C, CCR5), VI1358 (subtype C, CCR5), pREJO.c/2864 (transmitted/founder, subtype B, CCR5) or p246F10 (transmitted/founder, subtype C, CCR5) viruses were added to each well, and cultures were incubated for 48 h before luciferase activity was quantified. Each compound was tested in triplicate in a single experiment. Antiviral activity was expressed as the percentage of viral inhibition compared to the untreated control and plotted against the compound concentration. Next, non-linear regression analysis was used to calculate the 50% effective concentration (EC50).</p><!><p>Cytotoxicity was determined using the water soluble tetrazolium-1 (WST-1) cell proliferation assay, which is based on the cleavage of the tetrazolium salt WST-1 to a formazan dye by cellular dehydrogenases. Because this bioreduction is dependent on the glycolytic production of NAD(P)H in viable cells, the amount of formazan dye formed is correlated directly to the number of viable cells in a culture. Quantification is done by measuring absorbance at 450 nm in a multiwell plate reader. 104 cells were plated per well in a 96-well plate and a serial dilution of compound was added. 48 h later, cell proliferation reagent was added and cell viability was measured compared to untreated control cultures. Cell viability was plotted against the compound concentration and non-linear regression analysis was performed to evaluate the 50% cytotoxic concentration (CC50).</p><!><p>Experiments were carried out on a Biacore 3000 or Biacore T200 instrument (GE Healthcare). gp120 was covalently coupled to a CM5 chip at 1000-2000 RU, and a blank surface with no antigen was created under identical coupling conditions for use as a reference. MiniCD4s 2 and 13 were serially diluted 2-fold, into 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% polysorbate 20 (HBS-EP) and injected over the immobilized gp120 and reference cells at 50 μL/min. The data were processed with SCRUBBER-2 and double referenced by subtraction of the blank surface and a blank injection (no analyte). Binding curves were globally fit to a 1:1 binding model.</p><!><p>HIV-1 clade B YU2 gp120 coree was expressed, purified and deglycosylated as previously described.24 The 13-gp120 complex was formed by mixing deglycosylated YU2 gp120 and the CD4-mimetic miniprotein (1:2 molar ratio) at room temperature and purified by size exclusion chromatography (Hiload 26/60 Superdex S200 prep grade, GE Healthcare) with buffer containing 0.35 M NaCl, 2.5 mM Tris pH 7.5, 0.02% NaN3. Fractions with gp120-miniprotein complexes were concentrated to ∼10 mg/ml, flash frozen with liquid nitrogen before storing at -80 °C and used for crystallization experiments.</p><p>Vapor-diffusion hanging drops were set up by mixing 0.5 μL of protein with an equal volume of reservoir solution composed of 9.0% PEG 4000, 14.0% isopropanol, 100 mM sodium citrate, pH 5.6.38 Droplets were allowed to equilibrate at 20 °C, and diffraction-quality crystals were obtained in 1-5 days.</p><p>Diffraction data were collected under cryogenic conditions using 15% (2R,3R)-butanediol as cryoprotectant. X-ray diffraction data were collected at ID-22 beamline (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory, with 1.0000 Å radiation, processed and reduced with HKL2000.39</p><p>The crystal structure of the 13-gp120 complex was solved by molecular replacement using Phaser40 in the CCP4 Program Suite.41 The structure of YU2 gp120 bound to CD4-mimetic miniprotein 221 was used as search model. Refinement was carried out with PHENIX.42 Starting with torsion-angle simulated annealing with slow cooling, iterative manual model building was carried out on COOT43 with maps generated from combinations of standard positional, individual B-factor, TLS refinement algorithms and non-crystallographic symmetry (NCS) restraints. Ordered solvents were added during each macro cycle. Throughout the refinement processes, a cross validation (Rfree) test set consisting of 5% of the data was used and hydrogens were included as riding model. Structure validations were performed periodically during the model building/refinement process with MolProbity44 and pdb-care.45 X-ray crystallographic data and refinement statistics are summarized in Table S4. For reporting resolution, the data were scaled to a highest resolution of 1.89 Å. For refinement, all data collected to a highest resolution of 1.79 Å were used.</p><p>Superpositions were performed within the CCP4 program suite. Figures were made using Pymol. LIGPLOT46 was used to generate the 2-dimensional representation of 13 environment within the gp120 Phe43 cavity. Pocket-Finder (http://www.modelling.leeds.ac.uk/pocketfinder/), the web interface based on the pocket detection algorithm LIGSITE47 was used to define the volume of the Phe43 cavity and the region of the Phe43 cavity that binds to the cyclohexane ring of 13. The volume of ligands was calculated within the ChemBio3D Ultra 12.0 module within ChemBioOffice 2010 (http://www.cambridgesoft.com/Ensemble_for_Biology/ChemBio3D).</p>

PubMed Author Manuscript

Angioplasticity in asthma

Plasticity of the lung vasculature is intrinsically more complex than other organs due to the presence of two blood supply systems under different arterial pressures, the pulmonary and bronchial arterial systems. The bronchial and pulmonary circulations may both contribute to vascular remodelling in lungs after injury or inflammation. Vascular remodelling in the airway is a long recognized component in asthma. Growing numbers of reports suggest that a pro-angiogenic milieu is not a consequence of, but rather dictates the chronic inflammation of asthma. The fairly recent discovery of EPCs (endothelial progenitor cells) has enabled us to study the bone-marrow-derived cells that regulate lung vascular plasticity in asthma. This mini review provides a concise synopsis of our present knowledge about vascular plasticity in adult lungs, summarizes our current view of angioplasticity in asthma and highlights yet unresolved areas of potential interest.

angioplasticity_in_asthma

The human lung circulation<!>Neovascularization, or new blood vessel formation<!>Plasticity of the lung vasculature<!>Pro-angiogenic microenvironment and vascular remodelling in asthma<!>Inflammation promotes angiogenesis<!>Airway angiogenesis in the genesis of airway inflammation<!>Future perspectives and conclusions<!>Neovascularization in asthma

<p>The human lung circulation consists of two blood supply systems: a bronchial vasculature that is the systemic arterial blood supply providing oxygenated blood and nutrients to the lung tissue and a pulmonary vasculature carrying deoxygenated blood from the right ventricle to the lungs for oxygenation. Although more than 95% of the blood flow through the lungs is from the pulmonary circulation, experimental systems clearly show that the pulmonary circulation and bronchial circulation are both crucial for proper lung function. For example, remodelling of the small pulmonary arteries results in pulmonary hypertension, while remodelling of the bronchial vasculature is associated with chronic airway inflammatory diseases. The pulmonary artery branches run in parallel to bronchi and bronchioles and terminate in a diffuse capillary network surrounding the alveoli for gas exchange, to ultimately converge with pulmonary venules and pulmonary veins. The bronchial circulation arises directly from the aorta as bronchial arteries networking into two capillary plexus, one on each side of the airway smooth-muscle layer, the subepithelial plexus and adventitial plexus. These capillary networks form bronchial veins, which then also drain into the common pulmonary veins and return to the left atrium [1].</p><!><p>Neovascularization, or new blood vessel formation, can occur by three major processes, namely vasculogenesis, angiogenesis and arteriogenesis. Vasculogenesis signifies de novo blood vessel formation during which primitive precursors migrate to vascularization sites, differentiate into endothelial cells and structurally become the newly formed blood vessel. Budding of new vessels from pre-existing ones is referred to as angiogenesis, while the increase in the luminal diameter of an artery to form a collateral is termed arteriogenesis [2,3].</p><p>New studies are revealing multiple functions and types of vascular stem and progenitor cells involved in neovascularization. Different biological assays and cell surface markers are used to define subsets of progenitors [4]. The EPC (en-dothelial progenitor cell) is one of the important participants in postnatal new blood vessel formation [5], and contributes to physiological and pathological neovascularization. EPCs are bone-marrow-derived cells and influence neovascularization through paracrine effects [6]. EPCs are enumerated by flow cytometric analysis using a combination of haemopoietic stem cells markers and endothelial cell antigens, such as CD34, CD133 and VEGF (vascular endothelial growth factor) receptor-2. Whether these bone-marrow-derived cells differentiate into true endothelial cells is a subject of debate [7,8]. However, culture of EPCs in vitro gives rise to CFU-ECs (colony-forming units of endothelial-like cells) or CFU-Hill colonies. CFU-ECs are heterogeneous colonies comprising proliferating progenitors [6] in the presence of T-cells [9]. The exact cellular composition of these colonies is currently unknown. Immunophenotypic analysis indicates low to intermediate expression of the progenitor cell markers CD34 and CD133. Cells are also positive for endothelial cell-like features such as uptake of acetylated low-density lipoprotein, lectin binding and expression of CD31, von Willebrand factor and vascular–endothelial cadherin. However, CFU-ECs also express the pan-haemopoietic marker CD45, myeloid marker CD33, CD11b and α-smooth-muscle actin [10,11], thus they are not considered to be true endothelial cells. In contrast with the debate on the endothelial nature of the CFU-ECs derived from EPCs, a newly described endothelial progenitor, termed the ECFC (endothelial colony-forming cell), is considered to be a true endothelial stem cell. Little is known about ECFCs. These cells are rare in the circulation, most probably derived from the vascular wall, but can be expanded numerous times when needed as the building blocks of blood vessels (Figure 1). In this review, we provide a concise overview of our current knowledge about vascular remodelling in postnatal lungs, focusing on the EPCs in the process, and then present a current overview of angioplasticity in asthma highlighting the yet unresolved areas of potential interest.</p><!><p>Early studies (reviewed in [1,12]) indicate that the lung vasculature has considerable adaptation capacity. Leonardo da Vinci is generally recognized as the first one to report neovascularization in lung pathology [1]. Pulmonary artery obstruction in patients with chronic thromboembolic disease results in rapid (within 2–3 days) substantial expansion of the bronchial circulation [13]. Studies in adult dogs reveal that unilateral ligation of the pulmonary artery is associated with pre-capillary anastomoses between the pulmonary and bronchial circulation in order to supply the ischaemic lung regions [14]. It is the bronchial arteries that proliferate to anastomose with the pulmonary vasculature after obstruction of the pulmonary artery, and result in ~ 10-fold increase in the systemic blood flow through the lungs [15]. Whether this bronchial-to-pulmonary blood flow improves gas exchange has been suggested but is unlikely given the fact that the bronchial blood is already oxygenated [1]. On the other hand, angiogenesis induced either by hypoxia in rats [16] or pneumonectomy in adult animal models and in humans [17] results in alveolar proliferation, which indicates the concept of vascularization-directed alveolar growth. In support of this paradigm, inhibition of endothelial cell migration in neonatal rats causes impaired postnatal lung development, i.e. decreased alveolarization [18]. Targeted induction of apoptosis of lung endothelial cells in mice leads to a loss of alveoli numbers and leads to an emphysema-like pheno-type [19]. There is intriguing support for vascularization-dependent alveolarization in humans. In women, angiogenesis that occurs in the uterus over the menstrual cycle is closely associated with increases in pulmonary gas transfer, which occurs due to cyclic expansion and contraction of the pulmonary vascular capillary bed [20].</p><!><p>Asthma is a chronic inflammatory disease pathologically defined by airway wall remodelling. Vascular remodelling of the airways including angiogenesis, vasodilation and microvascular leakage is one of most prominent and uniform findings in asthma. Angiogenesis is present in the earlyphases of adult asthma [21]. Compared with control subjects, the total number of vessels and vascular area in the airways are increased in adult asthmatics [22–24]. Analysis of the distribution of blood vessel sizes indicates an increase in the numbers of smaller vessels (<25 μm) [25]. The increased numbers and sizes of vessels and vascular leakage contribute to thickening of the airway wall resulting in narrowing of the lumen [26].</p><p>Under homoeostatic circumstances, vascular quiescence is maintained by an equilibrium between endogenous angiogenic activators and inhibitors. In pathological angiogenesis, overproduction of angiogenic factors and/or down-regulation of inhibitors disturb this balance favouring pro-angiogenic factors and resulting in increased vascularization. Several pro-angiogenic mediators are expressed or up-regulated in asthmatic lungs (reviewed in [27]), but VEGF (VEGF165) has been the most studied. Asthmatics have increased VEGF concentration in bronchoalveolar lavage fluid, and the levels correlate to blood vessel numbers in the asthmatic airway mucosa [10,28]. VEGF levels are also associated with other features of asthma such as basement membrane thickening [28], increased vascular leakage contributing to tissue oedema and airway hyperresponsiveness [29]. Compared with healthy controls, patients with asthma have increased amounts of VEGF and decreased amounts of the anti-angiogenic factor endostatin in sputum [28, 30, 31]. VEGF levels in sputum are inversely associated with airflow parameters in children [32]. Furthermore, the sputum of children during an acute asthmatic attack contains elevated levels of VEGF, which are correlated to asthma severity [32]. Pro-angiogenic activity of bronchoalveolar lavage fluid from asthmatics is confirmed by quantitative in vitro angiogenic assays [33]. Animal models of allergen-induced airway hyperresponsiveness also have increased VEGF in bronchoalveolar lavage [29,34] and vascular remodelling [34]. In industrial chemical-induced asthma in mice, administration of the VEGF receptor tyrosine kinase inhibitors (SU5614 and SU1498) reduces the development of airway hyperresponsiveness and inflammation [29]. Definitive evidence of a causal role for VEGF165 and angiogenesis in asthma comes from elegant studies by Elias and co-workers [35,36] in which VEGF is overexpressed in airway epithelium of mice. These transgenic animals spontaneously develop airway inflammation, angiogenesis and an asthma-like phenotype [35]. Subsequent mechanistic studies in these mice show that nitric oxide synthesis is essential for the VEGF-mediated angiogenesis [36]. Altogether, the studies support a role for VEGF and angiogenesis in asthma.</p><p>Immunohistochemical analyses show that many cell types intrinsic to the asthmatic lung are capable of producing VEGF, including mast cells, eosinophils, macrophages and CD34+ cells in airway biopsies [28,37,38]. In addition, airway epithelium produces VEGF during rhinovirus-associated asthma exacerbations [39], or during dust mite allergen exposure [34]. Other structural cells such as fibroblasts [40] and airway smooth-muscle cells can probably also contribute to the high levels of VEGF in asthma [33].</p><!><p>Angiogenesis and chronic inflammation are mutually supporting [41]. Inflammatory cells in asthma have the capacity to produce pro-angiogenic factors and induce angiogenesis. Eosinophils produce and release VEGF and other pro-angiogenic factors, including bFGF (basic fibroblast growth factor), IL-8 (interleukin-8), nerve growth factor, GM-CSF (granulocyte/macrophage colony-stimulating factor) and eotaxin [38,42]. Eosinophils directly induce endothelial cell proliferation in vitro, and neovascularization in rat aortic ring assays or in the chick embryo chorioallantoic membrane assay [43]. Mast cells produce high levels of VEGF and numerous other pro-angiogenic factors, such as FGF-2 (fibroblast growth factor 2), TGFβ (transforming growth factor-β), TNFα (tumour necrosis factor α) and IL-8, and are the critical cells that promote angiogenesis in tumours [42,44]. Pro-inflammatory mediators such as TNFα [45] and nitric oxide [36] that are found at high levels in asthmatic lungs also promote angiogenesis, in part by induction of VEGF expression. Altogether, the inflammatory cells and mediators in asthma almost certainly combine to accelerate the angiogenic remodelling events in asthma. On the other hand, human and murine studies indicate an early onset of vascular remodelling in asthma, prior to inflammation onset [10].</p><!><p>Analysis of bronchial biopsies indicates an increased number of blood vessels and eosinophils in children with asthma, but also in atopic children without asthma when compared with healthy non-atopic individuals [46]. An increased vascular network is detected in the early phases of chronic adult asthma [21]; however, temporal analysis of the onset of angiogenesis and asthma is not possible in observational studies of humans. At the time of diagnosis, the airway inflammation and vascular remodelling are both well established. The ovalbumin mouse model of asthma has been used to study the sequential occurrence of these events [10]. In this model, mice are sensitized with intraperitoneal ovalbumin in adjuvant, followed weeks later by daily ovalbumin inhalation challenge over 8 days. A switch to a pro-angiogenic milieu takes place within the first 48 h after the first allergen inhalation challenge, well in advance of an influx of eosinophils, which reaches a peak on days 4–6 of allergen challenge. Rather, EPC mobilization from bone marrow and recruitment into the lung occurs within a few short hours after the first allergen challenge, which is followed quickly by vascular remodelling and neovascularization. Similar to previous studies in vascular injury and remodelling, EPCs apparently initiate the switch to a pro-angiogenic environment [10]. These findings raise the possibility that the angiogenic process is one of the driving forces that initiate and/or perpetuate asthmatic airway inflammation. Based upon the current paradigm of angiogenesis, EPCs release paracrine signals upon arrival at vascular sites, which alter the local microenvironment and promote neovascularization. Endothelial cells of newly formed blood vessels accelerate and amplify inflammation by facilitating the adhesion of circulating inflammatory cells and migration into the tissues. In the case of asthmatic inflammation, the EPC-mediated angiogenic switch is dependent on Th1/Th2 lymphocytes as indicated by allergen-specific adoptive transfer of Th1- and Th2-cells [10]. Transfer of either Th1 or Th2 cells into naive mice induces EPC mobilization and recruitment into the lungs, but new vessels do not form. Neovascularization occurs only when both Th1 and Th2 cells are transferred together. Thus memory T-cells and their specific immunogenic products are required for the onset of angiogenic remodelling in asthma. In support of similar events in the genesis of human asthma, patients with asthma have greater than normal numbers of circulating EPCs, which exhibit increased proliferation potential and angiogenic activity [10].</p><!><p>Bronchial vascular remodelling is a universal finding in asthma. The mouse model of asthma identifies that the onset of neovascularization is before the recruitment of inflammatory cells into the lungs. In fact, several lines of evidence suggest that angiogenesis is an active component in the pathogenesis of inflammation in asthma. This raises several questions. Does bronchial angiogenesis promote airway inflammation? Does airway inflammation trigger angiogenesis? Or are inflammation and angiogenesis co-dependent? There is support for all three possibilities in the current literature (Figure 1). Do both vascular systems supplying the lung participate in the angiogenic remodelling? Or is the bronchial circulation primarily involved? Study of small rodents such as the mouse may be a limitation in answering this question as the murine lung vasculature and blood supply varies from the human. Only one report provides some evidence for the existence of both a pulmonary and bronchial circulation in mice [47]. Vascular corrosion casting of mouse lungs via the descending aorta followed by scanning electron microscopy analysis reveals the presence of a bronchial circulation in mice. However, interconnections between the two systems occur at the distal bronchial arteries and alveolar capillaries, and airway inflammation leads to an increase in bronchial perfusion of alveolar capillaries [47]. Nevertheless, observations of murine lungs provide useful information on the general plasticity of the pulmonary vascular bed.</p><p>The angiogenic switch has been shown to be an early event in asthma, but what are the triggers that disrupt the angiogenic equilibrium? VEGF alone is necessary but not sufficient to induce neovascularization [3]. What are the other (perhaps lung-specific) angiogenic factors involved? The functional role of EPCs in the angiogenic switch, their interactions with other cell types and how this may affect recruitment of inflammatory cells remain to be resolved. The angiogenic switch is dependent on both Th1 and Th2 cells in the murine asthma model, but it is yet unclear how T-cells contribute to the pro-angiogenic microenvironment. Insight into the immune mechanisms regulating lung vascular homoeostasis will be important for the design of more effective asthma therapies that may be based on anti-angiogenesis strategies to inhibit neovascularization and inflammation in the airway.</p><!><p>The pathophysiology of asthma is related to allergy, and/or infections with respiratory viruses. Inhalational exposures with allergens, or virus infections, trigger asthma exacerbations. In the case of an allergen, antigen-presenting cells capture the allergen and trigger immune responses skewed towards Th2 cells. Th2 cells induce allergen-specific IgE production by B-cells, which bind to the surface of mast cells. Mast cells degranulate upon their subsequent encounter with allergen(s). Mast cell and T-cell derived products induce a cascade reaction involving structural vascular cells and airway epithelial cells that result in the recruitment of eosinophils, typical effector cells in asthma. Damage to structural lung cells (epithelial cells, endothelial cells and smooth-muscle cells) is caused by eosinophil-derived products, which leads to airway hyperresponsiveness and airway remodelling. VEGF (and other yet unknown angiogenic triggers) may be released by activation of epithelial and mast cells and initiates the mobilization of EPCs from the bone marrow into the peripheral circulation. Homing signals recruit EPCs into the lungs. EPCs switch the local environment from vascular quiescence to a pro-angiogenic state. Mediators released by EPCs either directly or indirectly via interactions with vascular cells in the newly formed blood vessels facilitate the recruitment and activation of pro-inflammatory cells such as eosinophils. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2009. All Rights Reserved.</p>

PubMed Author Manuscript

An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity

Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. As these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH which is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail, and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by Co(II)-HDAC8 compared to peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-fold, 2.8-fold, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.

an_enzyme-coupled_assay_measuring_acetate_production_for_profiling_histone_deacetylase_specificity

Introduction<!>Reagents<!>Acetyl-CoA synthetase preparation<!>HDAC8 expression and purification<!>Fluor de Lys assay<!>Acetate assay kit<!>Optimized stopped coupled acetate assay<!>Optimized continuous coupled acetate assay<!>Fluorescamine assay<!>Assay<!>Optimization of Assay for Measuring HDAC Activity<!>Stopped Assay<!>Continuous Assay<!>Reactivity of HDAC8 with peptides<!>Discussion

<p>Histone (or acetyl-lysine) deacetylases (HDACs) are a family of 18 enzymes that catalyze the deacetylation of acetylated lysine side chains.[1-2] Acetylation is a post-translational modification identified on over 3,100 lysines within the mammalian proteome [3] that alter the activities and properties of modified proteins.[4] As many of these proteins are essential to cellular processes,[5-6] aberrant acetylation and deacetylation may contribute to disease states.[7] Attesting to the role of HDACs in diseases are two HDAC inhibitors (Vorinostat and Romidepsin) that have been approved by the FDA for the treatment of T-cell Lymphoma,[8] though the mechanism of action for these drugs is not well understood. One complicating factor to understanding the biological function and regulation of protein deacetylation is the lack of identified HDAC isozyme-substrate pairs. Determining the substrate specificity of HDACs would provide insight into cellular homeostasis and development of isozyme-specific inhibitors.</p><p>There are four classes of HDAC enzymes. Classes I, II, and IV use an active site divalent metal ion cofactor to catalyze deacetylation, yielding lysine and acetate as products.[9] Class III HDACs use NAD+ as a cofactor and produce 2'-O'acetylribose, nicotinamide, and the deacetylated protein.[10] Current in vitro assays for the measurement of HDAC activity use environmentally sensitive fluorophores,[9, 11-17] HPLC methods,[18] free amine reactive reagents,[11] radiolabeled acetate,[19-20] and mass spectrometry.[21-23] While these assays are useful, each has associated limitations. In particular, many of these techniques can only be used to measure deacetylation of short peptide substrates rather than the biologically-relevant acetylated proteins. Furthermore, in the most frequently used assay, the Fluor de Lys assay, the methylcoumarin substituent alters substrate recognition.[23] For assays that can be adapted to measure deacetylation of proteins, many cannot be adapted to high throughput formats, are hard to quantify, or require specialized equipment.</p><p>Here we optimize an enzyme-coupled assay for the measurement of low micromolar acetate concentrations that can be used to evaluate the activity of class I, II, and IV HDACs. This assay couples the formation of acetate to the production of NADH that is monitored via an absorbance or fluorescence signal. This assay quantitatively measures deacetylation independent of the substrate size or structure, does not require specialized equipment, and can be adapted to real-time and high throughput formats. Using this assay, we demonstrate that cobalt(II)-bound histone deacetylase 8 (Co(II)-HDAC8) catalyzes deacetylation of a peptide sequence from the H4 histone tail containing a C-terminal methylcoumarin fluorophore with a kcat/KM value that is 50-fold, 2.8-fold, and 2.3-fold greater than the kcat/KM value for deacetylation of the same peptide containing a C-terminal carboxylate, amide, or tryptophan, respectively. The loss of catalytic efficiency for deacetylation of the C-terminal carboxylate peptide is a result of a 2.3-fold increase in the value of KM and a 22-fold decrease in the kcat value. These data demonstrate that interactions between HDAC8 and the C-terminal moiety are important for substrate recognition and efficient chemistry.</p><!><p>ATP, Coenzyme A, NAD+, L-malic acid, citrate synthase (CS), and malate dehydrogenase (MDH) were purchased from Sigma. The acetic acid detection kit was purchased from R-biopharm. Fluor de Lys peptide and the developing reagent were purchased from Enzo Life Sciences. The unlabeled peptides (Ac-KGGAKac-COO−, Ac-KGGAKac-NH2, and Ac-KGGAKacW-NH2) were purchased from Peptide2.0 (>85 % purity). Cobalt and magnesium were purchased as ICP standards from GFS Chemicals and the acetic acid standard was purchased from the Ricca Chemical Company. Chelex 100 resin was purchased from Bio-Rad. HDAC3/NCOR1 was purchased from Enzo Life Sciences. Ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma-Aldrich at >99 % purity. All other materials were purchased from Fisher and were of a purity >95 % unless otherwise noted.</p><!><p>The chitin tagged acetyl-CoA synthetase (ACS) plasmid (Acs/pTYB1)[24] was a generous gift from Professor Andrew Gulick (Hauptman-Woodward Institute). To increase the yield of protein, the gene for ACS was subcloned from the chitin-tagged ACS plasmid into a pET vector containing a His6x affinity tag. The ACS gene from Acs/pTYB1was amplified using the polymerase chain reaction to add XhoI and XbaI restriction sites. The amplified DNA segment was digested using XhoI and XbaI and ligated into a pHD4 vector[25] containing a T7 RNA polymerase promoter and a C-terminal TEV (Tobacco Etch Virus) protease sequence followed by a His6x motif to form the pHD4-ACS-TEV-His6x expression vector. The plasmid sequence was confirmed by sequencing at the University of Michigan DNA Sequencing Core.</p><p>The pHD4-ACS-TEV-His6x vector was transformed into BL21(DE3) cells. An overnight culture (12.5 mL/L) was used to inoculate autoinduction TB medium (12 g/L tryptone, 24 g/L yeast extract, 4.6 g/L KH2PO4, 20.6 g/L K2HPO4, 4 g/L lactose, 1 g/L glucose, 10 mL/L glycerol) that was supplemented with 100 μg/mL ampicillin and grown at 30°C for 20 hours prior to harvest. The cells were pelleted by centrifugation (9,000 × g, 10 min) and then resuspended and lysed in low imidazole buffer (30 mM HEPES, 150 mM NaCl, 20 mM imidazole, 1 mM TCEP, pH 8) using an M110L microfluidizer (Microfluidics). The lysate was cleared by centrifugation (39,000 × g, 45 min) and the supernatant was loaded onto a 12 mL GE Chelating Sepharose column charged with NiCl2. The ACS was eluted with a gradient of low (20 mM) to high (200 mM) imidazole buffer. Fractions containing ACS were identified using SDS-PAGE chromatography and were concentrated to <1 mL using 30,000 MWCO Amicon Ultra-15 centrifugal units and loaded onto a GE HiPrep 16/60 Sephacryl S200 HR size exclusion column equilibrated with size exclusion buffer (30 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 8). The fractions containing ACS were collected, combined with his-tagged TEV(S219V) protease (0.5 mg per liter of culture) purified in our lab using the method of Tropea et al. [26] and dialyzed against >500 fold excess of low imidazole buffer at 4°C overnight. The dialyzed ACS was run over a second Ni2+-charged Sepharose column, and the flow-through containing ACS was collected and concentrated to ~2 mM. The protein was then flash frozen in liquid nitrogen and stored at −80°C. Frozen ACS can be used for at least 12 months with little to no effect on the assay.</p><!><p>HDAC8 was expressed and purified as previously described[9] with the exception that a 20 mL DEAE Sepharose column was used after the second Chelating Sepharose column to remove excess metal from HDAC8. This column utilized a gradient from low to high salt buffer (50 mM HEPES, 10 μM ZnSO4, 1 mM TCEP, 50 mM NaCl, 5 mM KCl, pH 7.8 and 50 mM HEPES, 10 μM ZnSO4, 1 mM TCEP, 1 M NaCl, 5 mM KCl, pH 7.8, respectively).</p><!><p>All assays were performed in metal free tubes using metal free tips before being quenched into 96 well black plates (Corning plate# 3638). The Fluor de Lys assay was performed as previously described.[9] Briefly, HDAC8 was reconstituted with stoichiometric cobalt(II) at a final concentration of 10 μM and incubated on ice for 1 hour. Fluor de Lys HDAC8 deacetylase substrate (0 to 100 μM) was resuspended in HDAC8 assay buffer (50 mM HEPES, 137 mM NaCl, 2.7 mM KCl, pH 8) and incubated at 30°C for 5 minutes. Reactions were initiated by adding 0.5 μM Co(II)-HDAC8 and the reaction was quenched by a10-fold dilution into 0.05x Enzo developer II and 1.2 μM Trichostatin A (TSA) in HDAC8 assay buffer at 0, 30, and 60 seconds. Samples were incubated at room temperature for 15 minutes and the fluorescence was measured using a Polarstar Galaxy fluorometer (ex. = 340 nm; em. = 450 nm and 380 nm). The initial rate of deacetylation was determined from the time-dependent increase in the fluorescence ratio (450 nm/380 nm) and the concentration of product was calculated using a standard curve.</p><!><p>Acetate standard curves were made by diluting the Ricca acetic acid standard with HDAC8 assay buffer (above). The acetic acid detection kit (R-biopharm) was used according to the instructions except that the reaction volume and coupled solution volume were decreased 10-fold and no additional water was added to dilute the reaction. Solutions 1, 2, 3 and 4 in the kit were preincubated at room temperature for 20 minutes before being mixed with acetate. The reaction was incubated at room temperature for 40 minutes and then NADH fluorescence (ex. = 340 nm, em. = 460 nm) was measured using a Polarstar Galaxy fluorometer in a 96 well plate.</p><!><p>To remove contaminating metals from peptide substrates, ~6 % (v/v) hydrated Chelex 100 was added to the Ac-KGGAKac-NH2 and Ac-KGGAKacW-NH2 peptides and incubated at room temperature for three hours. The Ac-KGGAKacW-NH2 peptide concentration was determined from the absorbance measurement using an ND-1000 Spectrophotometer (Nanodrop) with a calculated extinction coefficient of 5500 M−1 cM−1.[27] Additionally, the concentration of peptides containing a free amine (lysine) was measured using the fluorescamine assay described below. Peptide substrates without a fluorophore (0 – 1600 μM) were preincubated in HDAC8 assay buffer (above) at 30°C for 10 min. The reactions were initiated by adding 0.5 μM (final concentration) Co(II)-HDAC8 or HDAC3/NCOR1, and quenched by addition of 0.37 % (v/v, final concentration) HCl after 0, 30, 60, and 90 minutes of incubation. The reactions were flash frozen within 20 minutes of quenching and stored at −80°C. Upon thawing, the reactions were neutralized by addition of 0.6% (w/v, final concentration) NaHCO3. The coupler mixture (50 mM HEPES, 400 μM ATP, 10 μM NAD+, 30 μM CoA, 0.07 U/μL CS, 0.04 U/μL MDH, 50 μM ACS, 100 mM NaCl, 3 mM KCl, 50 mM MgCl, 2.5 mM L-malic acid, pH 8) were incubated for 20 minutes at room temperature and added to each quenched reaction (at a ratio of 10 μL coupler mix/65 μL reaction) in a 96 well black plate. The reactions were incubated at room temperature for 40 minutes and the NADH fluorescence (ex. = 340 nm, em. = 460 nm) was measured. The steady state kinetic parameters for the Ac-KGGAKac-COO− peptide were determined from fitting the Michaelis-Menten equation to the concentration dependence of HDAC-catalyzed deacetylation. Substrate inhibition is observed for the peptides Ac-KGGAKac-NH2 and Ac-KGGAKacW-NH2, therefore the kinetic parameters for these substrates were determined by fitting Equation 1 to the dependence of the initial velocities on peptide concentration. Equation 1 was derived from rearrangement of the Briggs-Haldane to report the value of kcat/KM and the standard error directly from the output.</p><!><p>The 96 well plates were soaked (>3 hours) in 100 mM divalent metal-free EDTA to strip the plate of contaminating metal. The continuous assay buffer (50 mM HEPES, 400 μM ATP, 10 μM NAD+, 30 μM CoA, 0.07 U/μL CS, 0.04 U/μL MDH, 50 μM ACS, 127 mM NaCl, 2.7 mM KCl, 2.5 mM L-malic acid, pH 8) was incubated with Chelex resin for 1 hour at room temperature. The mixture was clarified by centrifugation at 16,800 × g for 2 minutes and the supernatant was collected. Then 6 mM magnesium was added to the buffer and the mixture was incubated for 20 minutes to allow NAD+/malate and NADH/OAA to equilibrate. The peptide (100 μM final concentration Ac-KGGAKac-NH2) in HDAC8 assay buffer was added to this mixture at a ratio of 2:1, respectively. The reaction was initiated with the addition of Co(II)-HDAC8 (0.5 – 1 μM final concentration) and deacetylation was measured from the time-dependent increase in NADH fluorescence (ex. = 340 nm, em. = 460 nm).</p><!><p>The peptide substrate Ac-KGGAKac-COO− (0 - 1600 μM) was preincubated in HDAC8 assay buffer (above) at 30°C for 10 minutes. The reactions were initiated by adding 0.5 μM HDAC8, and quenched by addition of 1 μM TSA after 0, 30, 60, and 90 minutes of incubation. The reactions were used immediately or flash frozen and stored at −20°C. Upon thawing, solutions were filtered through Pall 10K mwco NanosepMF Centrifugal devices to remove HDAC8. The flow-through (80 μL) was mixed with 50 μL of 1 M boric acid (pH 9) and the mixture was added to a Corning 96 well black plate. 33 μL of 4.3 mM fluorescamine (dissolved in acetone) was then added to the sample, incubated at room temperature for 10 minutes, and fluorescence (ex. = 340 nm, em. = 460 nm) was measured.[28] A standard curve was created using N-α-acetyl lysine methyl ester (0 – 5 μM). To measure the peptide concentration, peptides were diluted into 1 M borate, pH 9, and 0.56 mM fluorescamine was added. The mixture was incubated at room temperature for 10 min and the fluorescence (ex. = 340 nm, em. = 460 nm) was measured. The peptide concentration was determined from the standard curve.</p><!><p>An acetate detection assay was initially described in the Official Collection of Assays according to § 35 of German food law [29] and a kit containing the assay components is distributed by the R-biopharm company (Figure 1). This assay system couples enzymatic reactions that produce one molecule of citrate, CoA, and NADH per molecule of acetate. The NADH concentration is monitored using fluorescence and/or absorbance, allowing determination of the acetate concentration from a spectroscopic signal. This assay is optimized to measure millimolar concentrations of acetate with a detection limit of ~100 μM (Figure 2 and S1), which is not sufficiently sensitive to measure the steady state kinetic parameters of many enzymes, including HDACs.</p><!><p>The KMvalues for HDAC-catalyzed hydrolysis of acetyl-lysine residues in peptides are typically in the low to mid micromolar range. Therefore, to measure the initial rate (≤10 %) of the reaction, the detection limit should be in the low micromolar range. To optimize the detection limit for the acetate-coupled assay, the signal to noise ratio was improved by: (1) using highly purified recombinant ACS, which decreased the background signal; (2) lowering the concentration of L-malic acid and NAD+ to decrease the background signal due to the equilibrium formation of OAA and NADH; and (3) increasing the ratio of the sample to the coupling solution volume to improve the signal intensity. With these alterations, a linear standard curve from 0 to 50 μM acetate with a limit of detection of ~1 μM (coefficient of variance 2.2; Figure 2) was produced using this assay. This assay can be altered to measure larger concentrations of acetate (>50 μM) by adding higher concentrations of the limiting reagents CoA and NAD+. This standard curve indicates that the optimized coupled assay is sensitive enough to measure the steady state kinetic parameters for HDACs and other enzymes.</p><p>Based on the assay design, each molecule of acetate should yield one molecule of NADH. To test this, the fluorescence change from the addition of acetate to the coupled assay was compared with the fluorescence of a comparable concentration of NADH (Figure 3). The slopes of the standard curves for NADH and acetate were 770 ± 61 and 790 ± 50 fluorescence units per μM, respectively. The equivalence of these slopes indicates that there is a one to one relationship between the concentration of acetate and the signal created by the production of NADH, allowing calculation of the acetate concentration from the fluorescence change.</p><!><p>We first optimized the acetate assay in a stopped format to measure HDAC8 activity. After reacting HDAC8 with the substrate of interest, the reaction was quenched by the addition of HCl and flash frozen in liquid nitrogen. Upon thawing, the pH was neutralized by addition of NaHCO3. Using the Fluor de Lys assay to measure activity, the HCl solution quenches HDAC8 activity immediately (<10 sec), and HDAC8 activity is not restored upon neutralization (data not shown). The acetate concentration in this sample is then measured using the coupled assay. Under these optimized conditions, formation of NADH from the addition of acetate occurs within minutes. Upon mixing the coupling enzymes with the assay substrates, NAD+ and L-malic acid equilibrate to form NADH and oxaloacetate. This equilibration is complete in 20 minutes (Figure 4), forming ~4 μM NADH, consistent with the equilibrium constant for the reaction catalyzed by malate dehydrogenase.[30] The reaction of up to 20 μM acetate is complete within 30 minutes and the signal remains stable for over an hour. The limiting step in this assay is the formation of citrate and CoA catalyzed by citrate synthase. Therefore, the rate of acetate production can be increased by the addition of higher concentrations of CS, if needed.</p><p>To demonstrate the effectiveness of the acetate assay in measuring deacetylation, we compared the rate of HDAC8-catalyzed deacetylation determined using the coupled assay with the fluorescamine assay. Fluorescamine is a reagent that increases in fluorescence intensity upon reaction with primary amines[31] and therefore a fluorescent signal is coupled to the formation of lysine generated by HDAC-catalyzed deacetylation. We measured the reactivity of Co(II)-HDAC8 with an unlabeled peptide (Ac-KGGAKac-COO−) mimicking the H4 histone K16 acetylation site (H4 K16ac). HDAC8-catalyzed deacetylation of this peptide (200 μM peptide, 0.5 μM HDAC8) measured by the coupled acetate assay and the fluorescamine assay yielded comparable rates within experimental error of 0.0021 ± 0.0003 μM s−1 and 0.0027 ± 0.0008 μM s−1, respectively. Therefore, both assays measure the deacetylation rate and are viable for measuring HDAC8 activity, though the fluorescamine assay is less accurate for substrates containing multiple lysine side chains due to a higher signal to noise ratio.</p><p>To demonstrate that this optimized stopped assay can serve as a general acetate assay, we measured deacetylation of 100 μM Ac-KGGAKac-NH2 catalyzed by another HDAC isozyme, 0.5 μM HDAC3/NCOR1 [Figure S3]. The initial rate for this reaction was 0.015 ± 0.0016 μM s−1, comparable to the value measured for HDAC8 (see below).</p><!><p>We next evaluated whether the coupled acetate assay could be carried out as a continuous, real-time assay to measure HDAC8 activity. Since HDAC8 is sensitive to inhibition by metals,[9] and monovalent cations [15], the acetate coupling solutions were reformulated with concentrations of NaCl and KCl typically used to assay HDAC8 activity (Biomol unpublished)[15] and treated with Chelex resin prior to addition of magnesium. The concentration of magnesium was decreased to 2 mM to minimize inhibition of HDAC8 activity (~2-fold inhibition under these conditions) [Figure S2]. To counteract the loss in activity of the coupling enzymes due to the lower concentration of magnesium, the concentration of these enzymes was increased by 2.3-fold to yield a final rate for the coupling reactions of 0.046 μM s−1. These assay conditions were used to measure HDAC8-catalyzed deacetylation of 100 μM Ac-KGGAKac-NH2 peptide yielding rates of 0.018 ± 0.00013 μM s−1 and 0.028 ± 0.00024 μM s−1 at 0.5 μM and 1 μM HDAC8, respectively [Figure 5]. The linear dependence on the HDAC8 concentration demonstrates that the assay rate is not limited by the coupling reactions. Furthermore, the HDAC8 activity measured using the stopped assay (0.5 μM HDAC8 and 100 μM Ac-KGGAKac-NH2) is 0.033 ± 0.0034 μM s−1 which is within the two-fold of the stopped assay measured rate, and represents the difference expected due to magnesium inhibition of Co(II)-HDAC8.</p><p>Additionally, we measured the HDAC8-catalyzed deacetylation of H3/H4 tetramer acetylated using acetic anhydride, where ~100% of the lysines were acetylated as indicated by mass spectrometry [data not shown]. The initial rate for deacetylation of ~0.076 μM acetylated H3/H4 tetramer catalyzed by 0.5 μM Co-(II)-HDAC8 is 0.0021 ± 0.0001 μM s−1 [Figure S4], demonstrating that this assay can measure deacetylation of both peptide and protein substrates.</p><!><p>Using a mass spectrometric assay, Gurard-Levin et al. [23] previously demonstrated that HDAC8 catalyzes deacetylation of a p53 peptide mimic containing a C-terminal methylcoumarin fluorophore significantly faster than a comparable peptide with a C-terminal cysteine followed by an amide terminus. To further analyze the recognition of the peptide C-terminus by HDAC8, we measured Co(II)-HDAC8-catalyzed deacetylation using the stopped coupled assay of an H4 peptide mimic containing varied C-termini, including: a carboxylate (Ac-KGGAKac-COO−); an amide (Ac-KGGAKac-NH2); and a tryptophan capped by an amide (Ac-KGGAKacW-NH2) (Figure 6 and Table 1). The steady state kinetic parameters for Ac-KGGAKac-COO− were determined from fitting the Michaelis Menten equation to the concentration dependence of activity, yielding values of kcat/KM, kcat and KM of 56 M−1 s−1, 0.041 s−1 and 730 μM, respectively (Figure 6). For the Ac-KGGAKac-NH2 and Ac-KGGAKacW-NH2 peptides, modest to substantial inhibition was observed at higher peptide concentrations. This inhibition is not due to metal contamination of the peptide[9] as pre-incubation of the peptide with Chelex 100 resin had no effect on the observed activity (Figure 6). Therefore, these data indicate that the activity is inhibited by high substrate concentrations. As many HDAC isozymes form multi-protein complexes,[32-33] the peptides may inhibit HDAC8 activity by binding to non-active site protein-protein interaction sites. For these peptides the kinetic parameters were determined by fitting an equation including terms for substrate inhibition (Equation 1) to the data; the values of kcat/KM for deacetylation of Ac-KGGAKacW-NH2 and Ac-KGGAKac-NH2 are 1200 ± 250 M−1s−1 and 980 ± 47 M−1s−1 parameters are coupled, independent values for these parameters could not be accurately determined. Substrate inhibition by the Ac-KGGAKac-NH2 peptide appears to be cooperative with a Hill coefficient (n) that is larger than 1. In general, substrate inhibition significantly complicates the analysis of the reactivity of HDAC8 with peptide libraries measured at a single peptide concentration.</p><p>These data demonstrate that the structure of the C-terminal moiety significantly affects the reactivity of Co(II)-HDAC8 with peptide substrates. Furthermore, a comparison of these data to the kcat/KM value of 2800 M−1 s−1 [25] for HDAC8-catalyzed deacetylation of the Ac-KGGAKac-methylcoumarin peptide demonstrates that the methylcoumarin fluorophore enhances (up to 50-fold) the catalytic efficiency of HDAC8-catalyzed deacetylation (Table 1).</p><!><p>The current HDAC assays have limitations that do not allow in-depth and/or high throughput analysis of HDAC substrate specificity. The first kinetic measurements of HDAC activity employed radioactively labeled peptides and proteins and detected the formation of radiolabeled acetate.[19-20] While this assay is effective and sensitive, radioactive peptide substrates are expensive to produce, and many radiolabeled protein substrates are acetylated non-specifically, preventing the determination of detailed kinetics at specific sites. Recently, the Fluor de Lys assay (Biomol) has become popular for measuring HDAC kinetics.[9, 11-17] This assay uses a methylcoumarin fluorophore-conjugated peptide, which upon deacetylation becomes a substrate for the serine protease trypsin, cleaving the fluorophore and altering the fluorescence spectrum of the methylcoumarin. The rate of deacetylation is measured by the change in the fluorescence signal.[12] However, the methylcoumarin fluorophore can interact with HDACs and alter the kinetics of deacetylation [23](as discussed later). Thus, the results obtained using this method may not accurately report the selectivity of HDACs for native peptides and proteins. Additionally, because the fluorophore is located immediately on the C-terminal side of the acetyl-lysine moiety, this method cannot be used to determine the preference for sequences downstream of the acetylated lysine. Many of these limitations were solved by a mass spectrometric assay developed by the Mrksich group; this assay uses MALDI mass spectrometry of peptides attached to a gold surface to observe the mass difference caused by deacetylation.[23] This assay is effective for peptide substrates and can be carried out in a high throughput manner, however a MALDI mass spectrometer is required. Additionally, this method requires the inclusion of a cysteine in the peptide sequence to conjugate the peptide to the plate via a maleimide linkage. HPLC separation of acetylated and deacetylated peptides on a C18 column is another method employed to determine deacetylation kinetics.[18] This assay quantifies product formation by absorbance (230 or 280 nm) and can measure the deacetylation of any peptide, yet it suffers from labor intensive techniques such as the determination of peak elution times making it not ideal for a high throughput format. Furthermore, it is not easily transitioned to assaying the deacetylation of proteins due to the difficulty of separating full length proteins differing by a single acetylated lysine. The assay presented in this paper provides an alternative that overcomes many of the limitations of the previous assays. This acetate assay is versatile; either fluorescence or absorbance can be measured using a cuvette or 96-well plate and in a stopped or continuous format. The continuous method is well-suited for high-throughput screening. Peptide substrates for the assay are inexpensive to purchase as they do not require conjugated fluorophores, and results can be quantified using an acetic acid standard curve. Furthermore, the assay can measure deacetylation of proteins (Figure S4). However, the acetate assay cannot be used with fluorescent peptides (i.e. methylcoumarin-conjugated peptides) that absorb and emit at wavelengths similar to NADH, nor can it be used on cell lysate extracts containing NADH, other metabolites, or various other enzymes. Overall, this assay provides a stable and sensitive platform for the measurement of deacetylation with few limitations. Finally, as this assay has been optimized to measure micromolar acetate concentrations, it can be used to assay other enzymes that produce acetate as a product and have KM values in the micromolar range, including other HDAC isozymes (Figure S3).</p><p>While most kinetic measurements of HDAC8 have been performed using the Fluor de Lys assay,[9, 11, 13-15] the Mrksich group used a mass spectrometric assay to demonstrate that a peptide mimicking the p53 transcription factor was deacetylated significantly slower than the same peptide containing a methyl-coumarin fluorophore.[23] However, the steady state kinetic parameters were not determined for these substrates. Using the optimized coupled assay that measures the formation of acetate, we demonstrated that substitution of the coumarin fluorophore with a carboxylate lowered the value of kcat/KM for Co(II)-HDAC8 by 50-fold, resulting from a 22-fold reduction in the value of kcat and a 2.3-fold increase in the KM value (Table 1). As the value of KM is relatively high (730 μM) and the value of kcat is relatively low (0.041 s−1) compared to other enzymes acting under diffusion control,[34] it is likely that substrate dissociation is faster than deacetylation, indicative of a rapid equilibrium substrate binding model. This assumption is further validated by the increase in kcat measured for trifluoro-acetyl lysine substrates, [18, 35] suggesting that deacetylation is the rate-limiting step for kcat. Based on this assumption, KM reflects KD for the peptide. Therefore, ΔΔGbinding, calculated from the alteration in the KM values5, indicative of the additional binding affinity conferred by the methylcoumarin fluorophore relative to a carboxylate at the C-terminus, is equal to ~0.45 kcal/mol. This alteration in binding energy is modest but within the range of energy due to the addition of a single pi-pi interaction[36-37] or hydrogen bond.[34] Crystal structures of a methylcoumarin-conjugated peptide bound to HDAC8 visualize interactions between the methylcoumarin and the side chain of Tyr100.[13, 38] These structures suggest that the enhanced binding energy for the methylcoumarin peptide results from a combination of interactions between the C-terminus of the peptide and the hydrophobic cavity formed by the L1, L7, and L8 loops, and interactions between the aromatic C-terminal residue of the peptide and Tyr100 on the L2 loop of HDAC8.</p><p>The steady state kinetic parameters for deacetylation catalyzed by HDAC8 demonstrate that the significant (50-fold) enhancement in Co(II)-HDAC8 kcat/KM for the Ac-KGGAKac-methylcoumarin peptide compared to the Ac-KGGAK-COO− peptides is largely due to an increase in the kcat value. The change in the stabilization of the transition state relative to the unbound ground state (ΔΔG‡)6 of 2.3 kcal/mol likely results from a combination of altered electrostatic and pi-pi interactions between the peptide and HDAC8 that enhance optimal positioning of the peptide and side chains in the active site to efficiently catalyze deacetylation. The effects of the C-terminal interactions of the peptide with Co(II)-HDAC8 on kcat and KM are consistent with data demonstrating that L2 loop residues are important for both binding and catalysis; mutations at Asp101 in HDAC8 lead to both higher KM and lower kcat values,[13] compared to the wildtype enzyme. However, these mutations do not lead to observable alterations in the crystal structure of inhibitor-bound HDAC8,[13, 38] suggesting that the activity decrease may be due to an alteration in the HDAC8 dynamics. The catalytic efficiency (kcat/KM) of Co(II)-HDAC8 with the Ac-KGGAKac-NH2 peptide is enhanced 9-fold (ΔΔG‡ = 1.6 kal/mol)6 compared to the Ac-KGGAKac-COO− peptide indicating that electrostatic interactions between the C-terminus and HDAC8 impair peptide binding and/or reactivity. Addition of a tryptophan (Ac-KGGAKacW-NH2) or methylcoumarin moiety to the peptide increases net transition state stabilization by 0.1 – 0.5 kcal/mol, providing an estimate of the enhancement of the catalytic efficiency by base stacking with Tyr100. Previous studies performed by the Mrksich lab show that HDAC8 catalyzes deacetylation of peptides containing a phenylalanine on the C-terminal side of the acetyl lysine faster than substrates containing any other amino acid, including tryptophan, at that position.[21, 23] These data suggest that both amino acid hydrophobicity and volume play a role in substrate preference. However, a direct comparison of the reactivity of peptides in this paper compared to those in Gurard-Levin et al. [21, 23] is complicated by differences in peptide length, the sequence at other positions of the peptide, and method of kinetic measurement.</p><p>Interestingly, Co(II)-HDAC8-catalyzed deacetylation of acetyl-lysine peptides have low values for kcat/KM(56 – 2800 M−1 s−1) compared to enzymes that are limited by diffusion,[34] suggesting either that these peptides are poor substrates for this enzyme or that the low activity is biologically relevant for control of enzyme activity.[39] The in vivo catalytic efficiency could be enhanced either by additional interactions with the protein substrates or by additional cofactors or binding partners. Furthermore, the measured rate constants for Co(II)-HDAC8-catalyzed deacetylation of Ac-KGGAKac-COO− and high concentrations of Ac-KGGAKac-NH2 peptides are similar to the rates measured for deacetylation catalyzed by various class II HDAC isozymes (HDAC7, 10)[17, 40] suggesting that the low activity of these isozymes in the Fluor de Lys assay may reflect decreased enhancement of reactivity by aromatic C-terminal moieties.</p>

PubMed Author Manuscript

A Redesigned Vancomycin Engineered for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria

The emergence of bacteria resistant to vancomycin, often the antibiotic of last resort, poses a major health problem. Vancomycin-resistant bacteria sense a glycopeptide antibiotic challenge and remodel their cell wall precursor peptidoglycan terminus from D-Ala-d-Ala to D-Ala-D-Lac, reducing the binding of vancomycin to its target 1000-fold and accounting for the loss in antimicrobial activity. Here, we report [\xce\xa6[C(=NH)NH]Tpg4]-vancomycin aglycon designed to exhibit the dual binding to D-Ala-D-Ala and D-Ala-D-Lac needed to reinstate activity against vancomycin-resistant bacteria. Its binding to a model D-Ala-D-Ala ligand was found to be only two-fold less than vancomycin aglycon and this affinity was maintained with a model D-Ala-D-Lac ligand, representing a 600-fold increase relative to vancomycin aglycon. Accurately reflecting these binding characteristics, it exhibits potent antimicrobial activity against vancomycin-resistant bacteria (MIC = 0.31 g/mL, VanA VRE). Thus, a complementary single atom exchange in the vancomycin core structure (O NH) to counter the single atom exchange in the cell wall precursors of resistant bacteria (NH O) reinstates potent antimicrobial activity and charts a rational path forward for the development of antibiotics for the treatment of vancomycin-resistant bacterial infections.

a_redesigned_vancomycin_engineered_for_dual_d-ala-d-ala_and_d-ala-d-lac_binding_exhibits_potent_anti

<p>Vancomycin (1) is the most widely recognized member of an important family of glycopeptide antibiotics.1 Clinical uses of vancomycin include its use in the treatment of patients on dialysis, and patients allergic to -lactam antibiotics.2 However, its most important use is in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, for which vancomycin is the drug of last resort.3,4 The prevalence of MRSA in US intensive care units (ICU, 60% of SA infections are MRSA, 2003)5 and the movement of MRSA from a hospital-acquired to a community-acquired infection have intensified the need to combat such resistant bacterial infections. Concurrent with the emergence of community-acquired MRSA, vancomycin-resistant strains of other bacteria are also on the rise with US ICU isolates of vancomycin-resistant Enterococcus faecalis (VRE) approaching 30% (2003),5 albeit in strains remaining sensitive to other antibiotics. Most feared is the emergence of MRSA strains now insensitive or resistant to vancomycin (VISA and VRSA) even in developed countries.6,7 This poses a major health problem and has stimulated efforts to develop vancomycin analogues8,9 or alternative antibiotics for the treatment of such vancomycin-resistant bacterial infections.7,10</p><p>Vancomycin inhibits bacterial cell wall synthesis by binding to the peptidoglycan peptide terminus D-Ala-D-Ala found in cell wall precursors,11 sequestering the substrate from transpeptidase and inhibiting cell wall cross-linking. The D-Ala-D-Ala complex with the antibiotic is stabilized by an array of hydrophobic van der Waals contacts and five hydrogen bonds (H-bonds) lining the vancomycin binding pocket (Fig. 1).12 Vancomycin-resistant bacteria (VanA and VanB) sense the antibiotic challenge13 and subsequently remodel their precursor peptidoglycan terminus from D-Ala-D-Ala to D-Ala-D-Lac.14,15 Normal D-Ala-D-Ala production continues despite the presence of vancomycin, but a late-stage remodeling to D-Ala-D-Lac ensues to avoid the action of the antibiotic. The substitution of a linking ester for the amide with the exchange of a single atom (NH O) reduces the binding to vancomycin 1000-fold and accounts for the 1000-fold higher MIC's seen in VRE clinical isolates.14 One key, but subtle insight to emerge from this characterization of vancomycin-resistant bacteria is that efforts to redesign vancomycin for their treatment should target compounds that not only bind D-Ala-D-Lac, but that also maintain binding to D-Ala-D-Ala.</p><p>The complex of vancomycin with D-Ala-D-Lac lacks the central H-bond of the D-Ala-D-Ala complex and suffers a repulsive lone pair interaction between the vancomycin residue 4 carbonyl and D-Ala-D-Lac ester oxygens (Fig. 1). We provided an experimental estimation of the magnitude of these two effects by examining the model ligands 2-4, revealing that it is the repulsive lone pair interactions (100-fold), not the H-bond loss (10-fold), that is responsible for the largest share of the reduced binding affinity (1000-fold).16 These observations had important ramifications on our redesign of vancomycin to bind D-Ala-D-Lac, suggesting that efforts could focus principally on removing the destabilizing lone pair interaction rather than reintroduction of a H-bond and that this may be sufficient to compensate for the majority of the binding affinity lost with D-Ala-D-Lac.</p><p>In conjunction with studies on the total synthesis of the glycopeptide antibiotics17-22 and concurrent with efforts probing systematic modifications to vancomycin itself,23 we initiated efforts on the redesign of vancomycin and its aglycon 5 to bind D-Ala-D-Lac.24 We focused our attention on [Φ[C(=NH)NH]Tpg4]-vancomycin aglycon (6), replacing the residue 4 amide with the corresponding amidine (Fig. 2). The key question addressed with 6 is whether the incorporation of the residue 4 amidine could accommodate D-Ala-D-Lac binding by removing the destabilizing electrostatic interaction and perhaps serving as a H-bond donor, while simultaneously maintaining affinity for D-Ala-D-Ala by virtue of serving as a H-bond acceptor (Fig. 2). Such binding characteristics of 6 were not easy to anticipate as it is not clear whether the ester oxygen of D-Ala-D-Lac could serve as a H-bond acceptor,25 or whether an amidine, which is likely protonated, might remain a good H-bond acceptor for D-Ala-D-Ala. Since the utility of an amidine as an amide isostere in peptides has been essentially unexplored,26-28 the projected binding properties of 6 were even more unclear. Key to the preparation of 6 herein is the use of [Φ[C(=S)NH]Tpg4]vancomycin aglycon (8), bearing a residue 4 thioamide, for single-step, site-specific amidine introduction. Among its many attributes, this strategy not only permits access to 6, but it also allows late stage synthetic access to related analogues including the interesting thioamide 8 itself, and alternative access to our prior methylene derivative 724 from a common late stage intermediate.</p><p>Treatment of the fully deprotected vancomycin aglycon thioamide 8, prepared by a total synthesis29 modeled on our preceding work,19-21,24 with silver acetate (AgOAc, 10 equiv) in methanol saturated with ammonia (NH3-MeOH) at 25 °C (12 h) directly provided the amidine 6 cleanly as a colorless solid that is stable to extensive handling (Fig. 2). It is considerably more polar than 5 and 8, likely reflecting amidine protonation. It is readily soluble in water (H2O) or H2O-MeOH, but insoluble in acetonitrile (MeCN), and it required addition of trifluoroacetic acid (TFA) to the sample before reverse-phase high-performance liquid chromatography (HPLC) purification. The simplicity of this transformation does not do justice to the efforts that went into its development. A number of instructive alternative approaches were examined, establishing the experience needed to conduct this reaction within the chemical and structural framework of a fully functionalized and fully deprotected vancomycin aglycon.</p><p>The results of the examination of 6 are summarized in Fig. 3 alongside those of vancomycin aglycon (5) and the synthetic methylene derivative 7,24 lacking the amide carbonyl. Both the C=N bond length of an amidine (1.30 vs 1.23 Å) and the van der Waals radii of nitrogen (1.55 vs 1.52 Å) closely approximate those of an amide carbonyl and oxygen atom, suggesting that an amidine may serve geometrically and sterically as an effective amide isostere. The binding affinity30 of 6 with the model D-Ala-D-Ala ligand 2 was found to be only approximately 2-fold less than the vancomycin aglycon itself and 15-fold greater than the methylene derivative 7, suggesting that the amidine functions well as a H-bond acceptor for the amide NH in the model ligand. Moreover, this binding affinity of 6 was maintained with the model D-Ala-D-Lac ligand 4, representing a nearly 600-fold increase in affinity relative to the vancomycin aglycon (5) and a more than 10-fold increase relative to the methylene derivative 7. Importantly, 6 displays effective, balanced binding affinity for both model ligands (Ka 2/4 = 1.05) at a level that is within 2-3 fold that exhibited by vancomycin aglycon (5) for D-Ala-D-Ala. Accurately reflecting these binding properties, 6 exhibited potent antimicrobial activity (MIC = 0.31 g/mL) against VanA resistant E. faecalis (VanA VRE, BM4166), the most stringent of vancomycin-resistant bacteria, being equipotent to the activity that vancomycin (1) and vancomycin aglycon (5) display against sensitive bacterial strains (MIC = 0.3-2 g/mL).</p><p>Although the behavior of 6 toward the D-Ala-D-Ala ligand 2 may not be too surprising, requiring the unprotonated amidine to function effectively as a H-bond acceptor for the ligand amide NH, its binding to the D-Ala-D-Lac ligand 4 is remarkable. There is no precedent on which to suggest that the residue 4 amidine could function as a H-bond donor to the ester oxygen of the D-Ala-D-Lac ligand sufficient to achieve this level of increased affinity. Rather, we suggest that this is additionally and largely the result of a now stabilizing electrostatic interaction between the protonated amidine and the ester oxygen lone pairs (Fig. 2). Thus, removal of the vancomycin carbonyl oxygen atom and its destabilizing electrostatic interaction with the D-Ala-D-Lac ester oxygen atom (lone pair/lone pair repulsion) and its replacement with a protonated amidine nitrogen and its complementary stabilizing electrostatic interaction reinstates essentially full binding affinity to the altered ligand. Beautifully, this represents a complementary single atom exchange in the antibiotic (O→NH) to counter the single atom exchange in the cell wall precursors of resistant bacteria (NH→O).</p><p>Although [Φ[C(=S)NH]Tpg4]vancomycin aglycon (8) was prepared as the immediate precursor to 6, it also proved especially interesting to examine. Since a thioamide is regarded as a weaker H-bond acceptor than an amide, the affinity of 8 for the D-Ala-D-Ala ligand 2 was anticipated to be reduced relative to the vancomycin aglycon, whereas its binding with the D-Ala-D-Lac ligand 4 was not as easily predicted. However, its behavior proved equally stunning, failing to bind either the model D-Ala-D-Ala or D-Ala-D-Lac ligand to any appreciable extent and being inactive as an antimicrobial agent. Most remarkable of these observations is the 1000-fold loss in affinity for the D-Ala-D-Ala ligand 2 relative to the vancomycin aglycon, indicating that this seemingly benign change in a single atom (O S) in going from the amide to thioamide is sufficient to completely disrupt binding. Although the weaker H-bonding ability of a thioamide is likely contributing to this lowered affinity, the magnitude of the loss indicates something more fundamental is responsible. We suggest that both the increased bond length of the thiocarbonyl (1.66 vs 1.23 Å) and the increased van der Waals radii of sulfur (1.80 vs 1.52 Å) are sufficient to sterically displace and completely disrupt the intricate binding of D-Ala-D-Ala. These contrasting observations further underscore the remarkable behavior of the amidine 6.</p><p>The clinical impact of such redesigned glycopeptide antibiotics is likely to be important, charting a rational approach forward in the development of antibiotics for the treatment of vancomycin-resistant bacterial infections. Since the single atom exchange described here is a deep-seated change that entails the selective transformation of one of seven amides in the vancomycin core structure, this was accomplished initially by total synthesis. In addition to semisynthetic approaches to 6 and 8 that now may be explored with the benefit of authentic samples in hand, a provocative ramification of the observations is the possibility that Nature also may have discovered this solution to the redesign of vancomycin for dual D-Ala-D-Ala and D-Ala-D-Lac binding in the form of related natural products yet to be isolated or characterized. In this respect, such residue 4 amidine derivatives possess the same nominal molecular weight as the corresponding amides, but are more polar, and it is possible they have been overlooked in screening efforts to date. Finally, we note that beyond the impact of unraveling the subtle details of the interaction of vancomycin with its biological target and their ramifications, the studies provide fundamental new insights into molecular recognition events, replacing a lost H-bond not with a reengineered reverse H-bond, but by replacing the resulting destabilizing electrostatic interaction with a stabilizing electrostatic interaction.</p>

PubMed Author Manuscript

From Zn to Mn: The Study of Novel Manganese-Binding Groups in the Search for New Drugs against Tuberculosis

In most eubacteria, apicomplexans, and most plants, including the causal agents for diseases such as malaria, leprosy and tuberculosis, the methylerythritol phosphate pathway is the route for the biosynthesis of the C5 precursors to the essential isoprenoid class of compounds. Owing to their absence in humans, the enzymes of the methylerythritol phosphate pathway have become attractive targets for drug discovery. This work investigates a new class of inhibitors against the second enzyme of the pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (MtDXR). Inhibition of this enzyme may involve the chelation of a crucial active site Mn ion, and the metal chelating moieties studied here have previously been shown to be successful in application to the zinc-dependent metalloproteinases. Quantum mechanics and docking calculations presented in this work suggest the transferability of these metal chelating compounds to Mn-containing MtDXR enzyme, as a promising starting point to the development of potent inhibitors.

from_zn_to_mn:_the_study_of_novel_manganese-binding_groups_in_the_search_for_new_drugs_against_tuber

INTRODUCTION<!>Quantum Mechanics Calculations<!>Generation of different Active Site Conformations of MtDXR<!>QM-polarized Docking Calculations<!>RESULTS<!>CONCLUSIONS

<p>Tuberculosis (TB) is a serious infectious disease caused by the Mycobacterium tuberculosis bacterium. It is a major cause of illness and death, and owing to a rise in HIV cases, the neglect of TB control programs and an increase in drug-resistance, the disease has resurged in recent years in well-developed countries and has exacerbated the TB problem in the lesser developed countries(1). Therefore, there is an urgent need for the development of new drugs and suitable therapeutic targets.</p><p>In most eubacteria, apicomplexans, and most plants, including the causal agents for diseases such as malaria, leprosy and tuberculosis, the methylerythritol phosphate pathway (MEP, also known as the DOXP or non-mevalonate pathway) is the route for the biosynthesis of isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP), the common C5 precursors to isoprenoids(2–5). Isoprenoids comprise a large and diverse family of compounds with numerous vital and diverse functions, with involvement in processes such as respiration, electron transport, hormone-based signaling and membrane stability(6, 7).</p><p>The MEP pathway comprises nine enzymes(8, 9), all of which have been identified as viable drug targets by genetic approaches(10, 11) and are of particular interest owing to their absence in humans, who use the alternative mevalonate pathway(10, 12). The 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) enzyme is the most studied of the pathway's enzymes to date. This enzyme is involved in the second stage of the pathway, mediating the reversible intramolecular rearrangement and NADPH-dependent reduction of 1-deoxy-d-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) in the presence of a divalent metal ion (for which Mn2+ has shown to be the most effective(13)).</p><p>Drugs, such as fosmidomcyin and its analogues, whose structure is similar to the natural substrate have been developed and shown to be efficacious against the E. coli(14) and P. falciparum(15, 16) DXR enzymes. As with the natural substrate, the inhibitors chelate the divalent metal ion present in the active site of the enzyme. However, as observed with the majority of antibiotics and chemotherapeutic agents, these inhibitors are ineffective in vivo against the M. tuber strain of the enzyme(17). In the case of fosmidomycin, the lack of potency has been attributed to the complex and hydrophobic nature of the mycobacterial cell wall and the absence of a cAMP dependent glycerol-3-phosphate transporter preventing the uptake of such a small and highly charged molecule(18). Even in the absence of these resistance issues, such as in the treatment against the P. falciparum pathogen, the late recrudescence observed in clinical trials precludes the drug as a monotherapy, with efficacious treatment requiring it to be administered with clindamycin(15, 16). Clinical studies have also shown that repeated and comparably high doses of the drug are required to achieve acceptable cure rates(15, 16). Furthermore, although the hydroxamate moiety of fosmidomycin exhibits attractive metal chelating properties, these compounds are associated with low availability, poor in vivo stability, and undesirable side effects, making them often undesirable in the manufacture of drugs(19).</p><p>In this study, we propose an alternative metal chelating group to hydoxamate, as a starting point for the development of a new class of inhibitors against the DXR enzyme. Cohen et al. have identified and synthesized a group of compounds, which are indicated to be successful alternatives to hydroxamate in the chelation of Zn2+, in the Zn-dependent matrix metalloproteinases (MMP)(20). The structures of the ligands featured in this study comprise hydroxypyridinones, hydroxpyridinethiones, pyrones and thiopyrones (Figure 1). The study by Cohen et al. found these ligands to share similarities to the hydroxamate moiety in terms of their bidentate-chelate formation properties, with improved hydrolytic stability and biological tolerance and proposed increase in affinity towards Zn.</p><p>In this work, the computational techniques of quantum mechanics (QM) and QM-polarized docking calculations were used to study the potential of these metal binding groups (MBGs) as potential Mn-binding moieties, as part of a search for a new class of inhibitors against the MtDXR enzyme. This study provides promising results, indicating these compounds to possess similar or improved binding affinities against the MtDXR enzyme, when compared to the reference hydroxamate-based compound, acetohydroxamic acid (AHA), therefore suggesting them to be good candidates for further development in an effort to produce potent inhibitors.</p><!><p>Modeling ligand binding can be problematic using conventional methods, with issues such as polarization and charge transfer being inadequately accounted for in the usual forcefields. This can be a hurdle in the design of novel and potent inhibitors, and is exacerbated with the presence of metal ions in the active site. In an attempt to determine whether the 12 proposed chelating moieties (Figure 1), taken from the study by Cohen et al. would form chelates with a Mn2+ ion, QM calculations were used to predict the binding affinity.</p><p>For this purpose, the affinity between the Mn2+ ion and the ligand was measured in the absence of the protein environment, using a Mn2+-containing scaffold molecule, based on a Mn+2 complex synthesized by Nabika et al. (Figure 2), [Mn(II) tris(3.5-diisopropyl-1-pyrazolyl) methane](21). This scaffold, and the 12 compounds shown in Figure 1 were constructed using a drawing program available in Maestro. The ligand preparation wizard was used to add hydrogen atoms to each of the 12 compounds, with the hydroxyl groups assumed to be deprotonated at the protein active site at a pH of 7. Prior to calculations, each of the compounds was positioned at the same distance from the Mn2+ ion, as observed in the crystal structure of the MtDXR enzyme when bound to fosmidomycin (PDB ID: 2JCZ).</p><p>For the geometry optimization, a BL3YP level of theory was applied, and for the energy calculations, LMP2/LACVP*+ level of theory was used, with the energies being counterpoise corrected.</p><!><p>Each of the 12 compounds shown in Figure 1 was computationally docked into the active site of monomer A of the MtDXR crystal structure (Table 2, PDB ID: 2JCZ), and three other conformations (labeled A–C in Table 2) selected from a prior study which examined the dynamics of the enzyme using an enhanced sampling MD technique (22). These three conformations were identified by clustering analysis as being the dominant conformations sampled throughout the simulations, together accounting for almost ~70 % of the total number of conformations sampled (see reference(22) for details). The active site of the crystal structure, which is bound with the fosmidomycin inhibitor (PDB ID: 2JCZ, monomer A) is observed to be the most closed of the four conformations shown here, with an important catalytic loop overhanging and enclosing the binding site to the greatest extent. Structures A and B represent the monomer in conformations intermediate between the closed crystal structure, and the most open form observed in Structure C (see Table 2), where the catalytic loop has moved away, exposing the active site.</p><!><p>In this study, the QM-polarized ligand-docking protocol of the Schrödinger suite(23) was utilized to dock the 12 MBGs into the active site of the four conformations of MtDXR. This protocol improves docking accuracy over the non-QM docking, through the use of QM calculations (QSite package in Maestro) to determine the partial charges on the ligand atoms in the field of the receptor molecule, thus accounting for charge polarization. The purpose of this docking study was to determine the orientation of the ligands within the active site, and predict any potential interactions with neighboring residues. The compound labeled 'AHA' (Figure 1) is the hydroxamate template ligand and is used as a reference compound for the docking of the 11 alternative metal-chelating groups studied here. The metal chelating properties of the hydroxamate group are well established, and crystal structures observe the MtDXR enzyme inhibitor, fosmidomycin, to form bidentate chelates between the inhibitor hydroxamate moiety and the active site metal ion(24–26).</p><p>Prior to the calculation, each of the protein and ligand structures was prepared, with hydrogens added according to the expected protonation at pH 7, using the protein preparation and ligand preparation wizards available within Maestro. A grid of an appropriate size, which fully incorporated the active site of the enzyme was applied as the target area for the docking calculations.</p><p>The QM-polarized docking protocol initially docked each of the ligands using Glide before deriving the charges of the five top ligand poses in the field of the receptor, using QM calculations (QSite). In the final stage of the protocol, these poses are redocked into the active site of the receptor with the new charges, and the top 10 scoring poses were recorded.</p><!><p>One of the most frequently applied strategies in the development of metalloproteinase inhibitors consists in the design of molecules that contain a metal-chelating group (MBG) and backbone fragments.(27) The backbones are drug-like structures that interact with active site binding pockets through non-covalent interactions, conferring selectivity towards a specific metalloprotein receptor. In recent years, Cohen and collaborators have expanded the library of MBGs for zinc-containing enzymes by introducing a new bioinorganic approach to design new matrix metalloproteinase inhibitors.(28–36) The main idea behind the bioinorganic approach is to use small molecule complexes to model the active site of metalloenzymes. The complex [(TpPh,Me)Zn(OH)] (TpPh,Me = hydrotris(3,5-phenylmethylpyrazolyl)borate) has been successfully applied to model the active site of zinc(II)-dependent matrix metalloproteases (MMP).(31, 34) The hypothesis is that, since the three pyrazole rings mimic the catalytic histidines bound to the zinc in the MMP active site, the characterized complexes formed between the MBG and [(TpPh,Me)Zn(OH)] reflect the binding mode of the chelating group. This assumption has been supported by comparing the complex of [(TpPh,Me)Zn(AHA)], where AHA stands for acetohydroxamic acid, with the available crystal structures of the complexes formed between MMPs and hydroxamate-based inhibitors.(31) In addition, by using this approach, the Cohen group was able to propose new classes of matrix metalloproteinase inhibitors exhibiting improved potency and novel selectivity relative to similar hydroxamic-based inhibitors against MMPs and anthax lethal factor.(36–38)</p><p>Although several zinc-binding groups have been proposed in the literature, hydroxamic acid functional groups are still by far the most used MBG in MMP inhibitors.(27) Similarly, fosmidomycin and its analogues also display the hydroxamate moiety in their structure. However, as aforementioned, hydroxamate-based MMP inhibitors are often associated with poor pharmacokinetics, poor oral bioavailability and toxicity.(39–41) Monoanionic bidentate zinc-chelators such as pyrones, hydroxypyridinones and their thione analogues (Figure 1) were the first heterocycles reported in the literature that showed significantly improved potency against MMPs when compared to acetohydroxamic acid.(32, 42) These compounds have been considered as very promising alternatives to hydroxamic acid due to their low toxicity and relatively high potency against MMPs in cell culture.(42)</p><p>Although, some hydroxypyrone and hydroxypyridinone MBGs can modulate selective inhibition of MMPs when linked to biphenyl backbone fragments,(43) it is usually assumed that the chelating group itself does not contribute significantly to selectivity. Since the main function of the MBG in the inhibitor molecules is to coordinate the catalytic metal, it has been proposed that the chelating group mainly contributes to the binding affinity of the inhibitor-metalloprotein complex. The lack of selectivity of acetohydroxamic acid for metal ions, such as Zn2+ and Mn2+, is also shown by the presence of a hydroxamic acid functional group in both DXR and MMP inhibitors.</p><p>To our knowledge, neither experimental nor theoretical work has investigated the use of pyrone-based molecules as chelating groups for enzymes besides zinc-dependent metalloproteins. In this work, we estimate the affinity of these newly identified metal chelating groups to Mn+2 through the use of QM calculations to evaluate their binding energies according to the scheme showed in Figure 2. Inspired by the bioinorganic approach of Puerta et al,(35) we use the Mn2+ complex with tris(3,5-diisopropyl-1-pyrazolyl)methane ligand as our model compound to mimic the active site of MtDXR.(44) To reduce computational cost, all isopropyl groups were substituted to hydrogen atoms. The relative affinity to Mn+2 (ΔMnaffinity) of each compound was defined according to Equation 1, where the energy difference between the bound and unbound states was calculated for each MBG, relative to AHA. Equation 1ΔMnaffinity=ΔEMBGΔEAHA where ΔE is defined by, Equation 2ΔEX=EX−Model CompoundComplex−(EX+EModel Compound) where X corresponds to a MBG structure in Figure 1.</p><p>Table 1 shows the binding affinity of each MBG, relative to AHA, obtained from the QM calculations performed on the model system displayed in Figure 2. These results indicate that MBGs 1–11 have very similar affinity to Mn2+ when compared to AHA. Our QM calculations suggest that substitution of the hydroxamate moiety by any of the MBGs 1–11 can be well tolerated without compromising the inhibitor potency against MtDXR. To further explore the use of MBGs 1–11 in the design of MtDXR inhibitors, we carried out docking studies to evaluate their binding mode, metal-chelation, and interactions with the MtDXR active site resides.</p><p>Each of the ligands numbered 1–11 in Figure 1, and the reference compound, AHA, were docked into the four varied active site conformations (shown in Table 2) of monomer A of the MtDXR enzyme, as described in the Methods section. The docking calculations showed that the majority of the compounds achieve a pose whereby the O, O (ligands 1 to 6) or S, O (ligands 7 to 11) donor atoms of the ligands form a bidentate chelate with the Mn ion, in a very similar orientation to what is observed with the hydroxamate moiety of the fosmidomycin inhibitor (and AHA reference compound) in the crystal structure. For a few of the ligands, a suitable docking pose was not achieved in all of the active site conformations, and this has been denoted by the absence of an entry in Table 2.</p><p>On docking each ligand into the four different MtDXR conformations, variation is observed in the docking scores and poses owing to differences in active site volume of the various conformations, and the orientations of residue side-chains in the vicinity of the ligand. Generally, for ligands possessing bulky methyl groups, the space restrictions presented by some of the conformations represented in this study cause them to achieve lower docking scores than the non-methyl containing ligands. This difference is most apparent when comparing the docking scores of methyl-containing ligands 2, 3, 8, 9 and 10, when docked into the crystal structure, which has the most limited space within the active site, and Conformation C, which has the most available space of the four active site conformations. The steric constraints imposed by the more closed active sites recognize orientations of the ligand around the metal ion, which possess less favorable interactions with the residues of the active site. An example is shown in Figure 3, where ligand 6 flips orientation from its observed pose in the crystal structure active site conformation, to that observed in Conformation C, where there is now available space to allow the methyl group to be positioned in a more hydrophobic neighborhood.</p><p>Of the 11 metal-chelating groups tested here, the non-methyl containing ligands tend to observe improved binding affinity in the active site. Their best scores are observed in Conformation A, where, the active site is less restricted compared with the crystal structure, but is still in a closed-type conformation, with the close packing of the active site residues providing a good environment for favorable interactions to exist. The consistently top ranked binding score of ligand 6 across the four MtDXR conformations indicates that an amide group at a position adjacent to the metal-chelating carboxylate group on the ring may provide a favorable contribution to the binding affinity.</p><p>All docked poses of the non-methyl containing ligands exhibit the catalytically important Trp loop residue to be in close proximity and stacked over the ring of the ligand, providing the possibility for π-π interactions to occur. The docking poses of these ligands vary significantly, and of all the conformations, their lowest docking scores are achieved with Conformation C. The reason for such differences in docking pose is illustrated in Figure 4, where the sidechains of the hydrophobic Trp203 loop residue and Met205 are shown to impinge on the docking space in the active site, sterically hindering the docking pose observed in the active sites of the crystal structure and Conformations A and C. This disrupts the favorable residue packing and opportunity for the π-π stacking between the ligand and Trp residue.</p><p>In the case of Conformation C, the docking scores of the non-methyl containing ligands decrease slightly compared with Conformation A owing to the active site being significantly more open, with less opportunity for the ligand to interact with the more distant active site residues.</p><p>In summary, the docking calculations reveal the 11 zinc-binding moieties to interact with the Mn ion to form a bidentate chelate, with a similar, or improved binding affinity compared with the hydroxamate reference compound. The results suggest the ligands without methyl substituents, and possessing a polar group (e.g. amide) at a position adjacent to the metal-chelating moiety on the ring would provide improved interactions with the protein. In addition, the quantum mechanics calculations showed that the binding energies between the Mn ion, and compounds 1–11, are very similar to the one obtained for the AHA reference compound.</p><p>Therefore, from the computational studies performed here, the compounds are indicated to be promising candidates for experimental testing as viable alternatives to the hydroxamate group for a new class of inhibitors against the DXR enzyme in M. tuberculosis.</p><!><p>The results of this study suggest a series of MBGs as promising candidates for the development of novel and potent inhibitors against the crucial MtDXR enzyme in Mycobacterium tuberculosis. These pyrone-based structures, originally developed by the Cohen group as metal chelating groups for the Zn-dependent metalloproteinases, are indicated by computational QM and docking studies, to be transferable to the Mn-containing MtDXR enzyme.</p><p>Quantum mechanics calculations demonstrated the binding affinity of each of the MBGs to Mn2+, as part of a scaffold structure, to be comparable to the reference AHA compound, which is known to chelate both Mn and Zn ions. QM-polarized docking calculations revealed the MBGs to form bidentate chelates with the Mn ion, as observed with fosmidomycin in the crystal structure, with similar, or improved binding affinity to the reference AHA compound. In addition to the crystal structure, this docking was performed using three dominant protein conformations, as identified by MD simulations, removing the bias generated by the fosmidomycin-preformed active site conformation of the crystal structure, and provides increased knowledge of favorable/unfavorable structural aspects of these MBGs, which maybe useful in the further development of these compounds into specific and potent inhibitors of MtDXR.</p>

PubMed Author Manuscript

A \xe2\x80\x98Magnetic\xe2\x80\x99 Gram Stain for Bacterial Detection

Magnetic stain. Bacteria are often classified into Gram-positive and Gram-negative strains by their visual staining properties using crystal violet (CV), a triarylmethane dye. Here we show, that bioorthogonal modification of crystal violet with transcyclooctene (TCO) can be used to render Gram-positive bacteria magnetic with magneto-nanoparticles-Tetrazine (MNP-Tz). This allows for class specific automated magnetic detection, magnetic separation or other magnetic manipulations.

a_\xe2\x80\x98magnetic\xe2\x80\x99_gram_stain_for_bacterial_detection

<p>Bacterial cell walls are made up of peptidoglycans (polysaccharides crosslinked by unusual peptides) in addition to other components.[1] Bacteria are often classified into Gram-positive and Gram-negative strains by their visual staining properties using crystal violet (CV), a triarylmethane dye.[2] Here we show that bioorthogonal modification of crystal violet with transcyclooctene can be used to render Gram-positive bacteria magnetic. This allows for class specific automated magnetic detection, magnetic separation or other magnetic manipulations.</p><p>The Gram stain is one of the most commonly used tools for detecting and differentiating bacteria. The method is routinely used for clinical diagnostic purposes, identification of a bacterial organism, as well as detecting them in environmental samples. The procedure involves staining bacterial samples with crystal violet, which binds to the peptidoglycan layer of Gram-positive and negative bacteria (Figure 1). Subsequent treatment with iodine solution results in crystal violet to form an insoluble complex. Gram-positive bacteria have a thick peptidoglycan layer, whereas Gram-negative bacteria only have a thin peptidoglycan layer covered by lipopolysaccharides and lipoproteins. Upon decolorization with alcohol or acetone, only Gram-positive bacteria remain purple, while Gram-negatives loose the purple color.[3–5] Despite the simplicity and robustness of the staining procedure, the final detection still relies on optical microscopy which is often susceptible to user-dependent sampling error. Strategies for quantitative and automated detection are highly desirable, especially for the diagnosis of infectious pathogens.</p><p>Magnetic, rather than optical, labeling and detection is advantageous because of its high sensitivity and ability to diagnose crude specimens without major purification.[6] For example, one could envision rapid and sensitive detection of bacterial samples in point-of-care settings by using a miniaturized micro-nuclear magnetic resonance (µNMR) device.[7,8] Direct bacterial detection by µNMR is a sensitive diagnostic method[9] and potentially allows the exclusion of culturing steps thus minimize the time required for diagnosis. Alternative magnetic detection devices include giant magnetoresistance,[10] or Hall sensors.[11] Furthermore, rendering bacteria magnetic also has implications for magnetic separation,[11,12] cell sorting,[13] magnetic force microscopy[14] or micromanipulation and force measurements using magnetic tweezers.[15]</p><p>We hypothesized that orthogonal triarylmethane dye derivatives could be used as affinity ligands to bioorthogonally couple magnetic nanomaterials onto Gram-positive bacteria. We thus developed a crystal violet modified with transcyclooctene (CV-TCO). We show that this reagent can be used for staining Gram-positive bacteria similar to the native crystal violet. Importantly, the CV-TCO can also serve as an anchor to attach tetrazine (Tz)-modified magnetic nanoparticles (or other Tz derivatized reporters). The developed magnetic Gram stain method was then used to enable highly sensitive detection of Gram-positive pathogens by µNMR.</p><p>Crystal violet (CV; 4,4',4"-dimethylaminotriphenylmethane), is a deep purple dye. We sought to develop a chromophore derivative where one of the anilino moieties is modified with a transcyclooctene (TCO) orthogonal group. We started the synthesis by the condensation of two equivalents of dimethylaniline with paranitrobenzaldehyde under microwave irradiation at 90°C for 4 min in the presence of a catalytic amount of aniline.[16] The aromatic nitro group was then reduced quantitatively by hydrogenolysis in presence of activated palladium affording the free amine 2 (Figure 2). However, the formed adduct instantaneously oxidizes in presence of air rendering purification and further conjugation difficult. The oxidation process is readily apparent since the oxidized compound has an intense purple color. To avoid oxidation to the cationic dye, the aniline was therefore derivatized twice to be stable under oxygen. We thus explored the synthesis of the disubstituted aniline 4 using multi-step one pot synthetic sequence. The nitro compound 2 was reduced by hydrogenation and reaction progress was followed by LC-MS (Figure S1). After completion of the reaction, the flask was purged with argon and the free aniline was engaged in a reductive amination with Boc-2-aminoacetaldehyde, sodium cyanoborohydride and acetic acid and stirred until completion. The secondary amine 3 was then engaged in a classic reductive amination condition with acetaldehyde, sodium cyanoborohydride and acetic acid for five hours yielding compound 4 in 71% yield over three steps. Structure and purity of 4 was confirmed by 1H NMR showing the characteristic chemical shift of the methylene proton at 5.30 ppm (see supplemental information). Compound 4 was then oxidized with tetrachloroquinone in refluxed ethyl acetate causing the formation of an intense blue indicating the formation of the cationic dye. After Boc deprotection under acidic conditions, compound 5 was isolated and purified on neutral alumina. Finally, the free amine 5 was treated with TCO-NHS, furnishing 6 (Crystal violet-TCO, CV-TCO) with an overall yield of 17% over seven steps (Figure 2A).</p><p>The molar extinction coefficient of CV-TCO 6 was ε592= 133013 L.mol−1.cm−1 as compared to unmodified CV which had ε592= 89146 L.mol−1.cm−1. These results suggest that the TCO linker modification only minimally affects the molar absorptivity of the triarylmethane dye and that the bioorthogonal compound can likewise be used for Gram staining (Figure S2). We then investigated the cycloaddition of 6 with a fluorescently labeled tetrazine, fluorescein-tetrazine (Fluo-Tz). After mixing the two compounds (0.25 mM), stirring for two minutes, the sample was analyzed by high performance liquid chromatography–mass spectrometry (HPLC-MS). HPLC-MS spectra confirmed rapid and quantitative conversion of Fluo-Tz to the cycloaddition-product without any side products (Figure 2B and Figure S3).</p><p>We next evaluated the efficacy of CV-TCO as a staining agent for Gram-positive bacteria. Three representative samples were prepared: Staphylococcus aureus (S. aureus; Gram-positive), Escherichia coli (E. coli; Gram-negative), and the mixture of both bacterial species. Bacterial smears on glass slides were stained with a solution of CV-TCO (1 mM) or CV for three minutes, followed by treatment with Gram's iodine solution for one minute, decolorization with 95% ethanol, and counterstaining with red Safranin solution. Microscopy revealed that only Gram-positive S. aureus remained purple, while Gram-negative E. coli was decolorized due to dissolution of the outer membrane (Figure 3A). The specificity of CV-TCO was further confirmed by UV-visible spectrometry; only gram-positive bacteria showed an intense absorption at 595 nm (Figure S4). Importantly, there was excellent correlation between CV and CV-TCO staining (r2 > 0.99; Figure 3B).</p><p>We further investigated if the bacteria stained with CV-TCO could be magnetically labeled via the TCO group. Bacteria stained with CV-TCO were incubated with magnetofluorescent nanoparticles modified with tetrazine (MFNP-Tz). Control samples were prepared by incubating unstained bacteria with MFNP-Tz. The T2 relaxation values of samples were measured using a miniaturized µNMR system. For comparative analyses, the absorption (at 595 nm) of the same samples was also measured. Cellular relaxivity (r2) was obtained by normalizing the measured 1/T2 values with bacterial concentration, and the r2 differences (Δr2) between targeted and control samples were calculated. We observed an excellent correlation (r2 > 0.9) between the extent of Gram-staining and the cellular relaxivity in Gram-positive species, which confirmed that CV-TCO on the bacterial surface was accessible for reaction with MFNP-Tz.</p><p>The labeling strategy was further applied to a panel of different bacterial species (Figure 4). Results showed that all Gram-positive species tested showed significantly higher cellular relaxivity values when compared to Gram-negative bacteria. Such magnetic labeling enabled the performance of highly sensitive and rapid detection of gram-positive bacteria. Titration measurements with serially diluted bacterial samples established that the detection limit with the current experimental setup was ~4,000 bacteria (Figure S5). This is significantly better than standard UV absorption detection, which has a detection limit of approximately 105 bacteria (Figure S6). It is likely that the sensitivity of the magnetic detector could be improved to the level of single-cells by 1) further miniaturizing the µNMR detection coils, 2) implementing fluidic systems for bacterial enrichment (e.g., membrane filters, magnetic separation steps), and 3) employing different types of magnetic readers (e.g., Hall-effect sensors, giant magnetoresistive sensors).[7–9]</p><p>Bioorthogonally labeled bacteria were also analyzed by confocal microscopy using MFNP-Tz (Figure 5A). CV-TCO stained bacteria showed uniform and high fluorescence signals in the bacterial cell wall, while the control experiments without CV-TCO showed no signal (Figure S7). Similarly, transmission electron microscopy was performed in CV-TCO treated bacteria but which were incubated with tetrazine modified gold nanoparticles. Gold nanoparticles were used instead of magnetic nanoparticles to obtain higher contrast. Gold nanoparticles were found distributed throughout the bacterial surface treated with CV-TCO, while bacteria without CV-TCO labeling showed a smooth surface devoid of nanoparticles (Figure 5B).</p><p>By modifying the above procedure, the detection strategy can be applied to detect both Gram-positive and Gram-negative bacteria. Performing the staining without the decolorization process would result in labeling both Gram-positive and negative species, since the Gram-negative species would also retain the CV-TCO (Figure S8A). This is in analogy to the conventional Grams stain where the first staining step "colors" all bacteria and the second decolorization step allows differentiation between the two Gram classes. µNMR measurements showed that before decolorization, both Gram-positive and negative bacteria could be magnetically labeled and detected, while after decolorization, only Gram-positive species retained their signals (Figure S8B). Through these sequential measurements, it is thus possible to obtain total bacterial counts (i.e. detection before decolorization) as well as their Gram-negative and Gram-positive composition (i.e. detection after decolorization).</p><p>In summary, we show that an orthogonal CV can be used to detect and broadly classify bacteria in biological samples. Staining bacteria with CV-TCO using the standard Gram stain procedure, followed by labeling with MFNP-Tz allows the detection and characterization of bacteria both by µNMR as well as by optical imaging. The "magnetic Gram stain" could be potentially implemented into automated point-of-care diagnostics, bacterial enrichment for subsequent analysis, as well as into therapeutic applications that utilize the antibacterial, antifungal, and antihelminthic properties of CV. The method could also be used to label bacteria in vivo for various imaging applications.[9] Moreover, the staining strategy presented could be further extended to other small molecule affinity ligands (e.g., bioorthogonal carbol fuchsin or trehalose for Mycobacterial species) to enable either universal or specific detection of other bacterial targets. This ability will not only facilitate the clinical diagnosis of a range of bacterial infections but will also promote advances in basic microbiological research.</p>

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NS-187 (INNO-406), a Bcr-Abl/Lyn Dual Tyrosine Kinase Inhibitor

Protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl groups of protein side chains, and they play critical roles in regulating cellular signal transduction and other biochemical processes. They are attractive targets for today’s drug discovery and development, and many pharmaceutical companies are intensively developing various kinds of protein kinase inhibitors. A good example is the recent success with the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec™) in the treatment of chronic myeloid leukemia. Though imatinib has dramatically improved the treatment of Bcr-Abl-positive chronic myeloid leukemia, resistance is often found in patients with advanced-stage disease. Several mechanisms have been proposed to explain this resistance, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA, increased drug efflux mediated by P-glycoprotein, and activation of the Src-family kinase (SFK) Lyn. We set out to develop a novel drug whose affinity for Abl is higher than that of imatinib and whose specificity in inhibiting Lyn is higher than that of SFK/Abl inhibitors such as dasatinib (Sprycel™) or bosutinib (SKI-606). Our work has led to the development of NS-187 (INNO-406), a novel Abl/Lyn dual tyrosine kinase inhibitor with clinical prospects. To provide an overview of how a selective kinase inhibitor has been developed, this review presents chemical-modification studies carried out with the guidance of molecular modeling, the structural basis for the high potency and selectivity of NS-187 based on the X-ray structure of the NS-187/Abl complex, and the biological profiling of NS-187, including site-directed mutagenesis experiments.

ns-187_(inno-406),_a_bcr-abl/lyn_dual_tyrosine_kinase_inhibitor

Introduction<!>Structural Analysis of Kinases<!>Chemical Modification<!>X-ray structure of NS-187 bound to human Abl<!>Effects of the CF3 group of NS-187<!>NS-187 blocks wild-type Bcr-Abl signaling<!>Antiproliferative activity of NS-187 against cells bearing wild-type or mutated Bcr-Abl<!>Mechanisms of NS-187-mediated cell death in Bcr-Abl+ leukemic cells<!>Inhibition of phosphorylated Abl by NS-187<!>Selectivity of NS-187 for Abl<!>Activity of NS-187 in mouse tumor models<!>Activity of NS-187 against central nervous system leukemia<!>Phase I clinical study of NS-187 (INNO-406)<!>Summary and Conclusions

<p>Protein kinases play critical roles in regulating cellular signal transduction and other biochemical processes by catalyzing the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl groups of protein side chains. They are therefore attractive targets for today's drug discovery and development, and many pharmaceutical companies are intensively developing kinase inhibitors that may have therapeutic value (Cohen, 2002). A good example is imatinib mesylate (Gleevec™), a specific inhibitor of breakpoint cluster region—Abelson tyrosine kinase (Bcr-Abl TK) (Buchdunger et al. 1996). Imatinib (Fig. 1) is efficacious in the treatment of Philadelphia-chromosome—positive (Ph+) leukemias such as chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (Goldman et al. 2003; Kimura et al. 2006). Philadelphia chromosome is a specific chromosomal abnormality resulting from a reciprocal translocation between chromosomes 9 and 22. This translocation fuses the c-abl proto-oncogene to bcr, leading to the production of a Bcr-Abl fusion protein that constitutively activates multiple signaling pathways. Because most patients with chronic myeloid leukemia have this abnormality, Bcr-Abl tyrosine kinase is a promising target for treating Ph+ leukemias (Sawyers, 1999).</p><p>Within a few years of its introduction to the clinic, imatinib had dramatically altered the first-line therapy for chronic myeloid leukemia, because most patients newly diagnosed with this disease in the chronic phase achieve durable responses when treated with imatinib (O'Brien et al. 2003). However, a small percentage of these patients, as well as most patients with advanced-phase chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia, relapse on imatinib therapy (Druker et al. 2002; Ottmann et al. 2002). Several mechanisms have been proposed to explain the cases of refractory disease and relapse, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA (Gorre et al. 2001; Hofmann et al. 2002; Nardi et al. 2004; Deininger et al. 2005), increased drug efflux from the target cells mediated by P-glycoprotein (P-gp) (Hegedus et al. 2002), and activation of Lyn, a Src-family protein kinase (SFK) (Donato et al. 2003; Dai et al. 2004; Ptasznik et al. 2004).</p><p>To overcome imatinib resistance, higher doses of imatinib and combination therapy with other agents have been used, with some efficacy. However, these strategies are limited in their application and effectiveness, especially for patients with mutations in the Abl kinase domain (Cortes et al. 2003; Kantarjian et al. 2004; O'Brien et al. 2003b). Therefore it is necessary to develop more-effective Abl TK inhibitors. Several SFK inhibitors from various chemical classes, including PD166326 (Wisniewski et al. 2002), SKI-606 (Golas et al. 2003), AP23464 (O'Hare et al. 2004), and dasatinib (Sprycel™; formerly BMS-354825) (Shah et al. 2004) have been reported to be 100–300 times more effective than imatinib in blocking Bcr-Abl TK autophosphorylation, and this inhibition of autophosphorylation extends to point mutants of Bcr-Abl. However, while imatinib binds only to the inactive form of Bcr-Abl, these SFK/Abl inhibitors bind also to the active form, which shares considerable conformational similarity with the active forms of diverse kinases, including the SFKs (Nagar et al. 2003). This characteristic of SFK/Abl inhibitors has some advantage with respect to Lyn kinase, because overexpression of Lyn may be associated with imatinib resistance (Donato et al. 2003; Dai et al. 2004; Ptasznik et al. 2004). However, the effects of lower specificity against SFKs are not yet fully understood, because these kinases play many important roles in vivo (Cary et al. 2002; Davis et al. 2003; Tanaka et al. 1996; Touyz et al. 2001). In addition to these SFK/Abl inhibitors, nilotinib (Tasigna™; formerly AMN107) has been developed as a novel Abl TK inhibitor. The in vitro inhibitory effect of nilotinib is 10–30 times greater than that of imatinib, but it is weaker than that of SFK/Abl inhibitors (Weisberg et al. 2005). Therefore, we set out to develop a drug whose affinity for Abl is higher than that of imatinib and whose specificity in inhibiting Lyn at clinically relevant concentrations without affecting the phosphorylation of other SFKs is greater than that of other SFK/Abl inhibitors.</p><!><p>Protein kinases are attractive targets for drug discovery programs in many disease areas, and most kinase inhibitors under development act by directly competing with ATP at the ATP-binding site of kinases. However, there are more than 500 protein kinases (Manning et al. 2002), and the ATP-binding site is highly conserved among them. Selectivity is therefore an essential requirement for clinically effective drugs targeted against protein kinases, and it is crucial to understand the structural characteristics of the ATP-binding site. Because kinase inhibitors on the market and currently under development often lack a portion to interact with the phosphate-binding region of the ATP-binding site, the term "ligand-binding site" will be used hereinafter instead of the term "ATP-binding site".</p><p>X-ray crystallography is a promising method for understanding the structural and physicochemical characteristics of the ligand-binding sites of protein kinases, and we have closely examined the X-ray structure of the imatinib/Abl complex. We first explored the ligand-binding site by using the 3D atomic coordinates of Abl kinase (Nagar et al. 2002), and then we calculated the surface properties of the binding site with a molecular modeling suite of MOE (Chemical Computing Group, Inc.). In Figure 2, the spheres indicate the predicted locations of an inhibitor's atoms, and imatinib is shown for reference. The spheres are classified as either hydrophilic (red) or hydrophobic (white) depending on whether or not they are in good hydrogen-bonding locations. Green, blue, and red express the hydrophobic, hydrophilic, and exposed nature of the surface, respectively. A large part of the surface is shown in green, indicating the hydrophobic nature of the ligand-binding site of Abl.</p><p>The five rings in imatinib were labeled A through E as shown in Figure 1, and the region of the binding site around the A and B rings is shown in Figure 2A. The positions and properties of the spheres in this region correspond well to those of the A and B rings of imatinib, and there is limited space for chemical modification. There is also limited space around the C ring. In contrast, much space is available around the D ring, suggesting the feasibility of an intensive program of chemical modification of this part of the molecule (Fig. 2B). The surface color of the part of the binding site adjacent to the terminal E ring of imatinib is blue, indicating that hydrogen-bonding interactions are probably important for the binding of the E ring (Fig. 2C).</p><p>Though X-ray crystallography is very helpful for understanding the structural and physicochemical characteristics of ligand-binding sites, a sufficient number of X-ray crystallographic structures for exhaustive comparison of the binding sites of various kinases is not yet available. The only data generally available for any kinase is the amino acid sequence, and, accordingly, sequence similarity is widely used for classifying proteins and predicting biological activities (Hank et al. 1988; Hank et al. 1991). However, it is difficult to elucidate the structural characteristics of a ligand-binding site from the amino acid sequence alone.</p><p>We have developed a procedure to overcome this difficulty by using physicochemical descriptors of amino acids in conjunction with neural network modeling (Niwa, 2006). The physicochemical properties of amino acids were expressed by hydrophobic, steric and structural descriptors. Kinases are classified into four major groups based on sequence similarity, AGC (PKA, PKG, and PKC families), CaMK (calcium/calmodulin-dependent protein kinases), CMGC (CDK, MAPK, GSK3, and CLK families), and TK (Hank et al. 1988). Abl kinase belongs to the TK group, so we aimed to elucidate which amino acids and which properties characterize the ligand-binding sites of TK, and to visualize the results by molecular graphics. TK ligand-binding sites are characterized by the branched nature of the side chains of the amino acids at positions 313 and 315. The methyl group of the C ring of imatinib and similar tyrosine kinase inhibitors, known as the "flag methyl", makes a large contribution to both their inhibitory activity and their selectivity (Zimmermann et al. 1997). The C ring and the flag methyl are located close to the amino acids at positions 313 and 315. Another characteristic feature of the TK ligand-binding site is the short side chain of the amino acid at position 322, which forms a hydrogen bond with Tyr253 and helps to stabilize the inactive conformation of Bcr-Abl kinase. Based on the above results, we developed guidelines for the chemical modification of Abl kinase ligands (Fig. 3). Though they are rather rough guidelines, they have helped us to understand the structural characteristics of the binding site of Abl kinase. In addition, they helped us to decide at the beginning of the project which kinds of chemical modifications were likely to be useful.</p><!><p>To guide our chemical-modification studies, we used the reported X-ray structure of the imatinib/Abl complex (Nagar et al. 2002). When we closely examined the structure, we found a hydrophobic pocket formed by amino acids Ile293, Leu298, Leu354 and Val379 around the phenyl (D) ring of imatinib. To improve the antiproliferative activity of imatinib against Bcr-Abl-positive (Bcr-Abl+) leukemia cell lines, we focused on this hydrophobic pocket and introduced various hydrophobic substituents on the phenyl (D) ring (Asaki et al. 2006). We found that 3-halogenated and 3-trifluoromethylated derivatives have significantly increased inhibitory activity compared to unsubstituted imatinib (Table 1).</p><p>To compensate for the increase in hydrophobicity caused by the introduction of the hydrophobic trifluoromethyl group in 5e, the distal pyridine (A) ring was selected for further modification. In the crystal structure of the imatinib/Abl complex, Tyr253 is located very close to the A ring, and their interaction helps to stabilize the inactive form of the kinase. Therefore, a structural modification that increases the bulk in this region would be expected to be unfavorable. The pyridine ring was therefore replaced by the more hydrophilic pyrimidine ring. Pyrimidine derivative 9a displayed activity (IC50 = 4 nM) similar to the original pyridine derivative 5e, showing that pyrimidinyl substitution is compatible with the retention of inhibitory activity.</p><p>The crystal structure of the complex reveals that the piperazine moiety (E) of imatinib interacts with the carbonyl oxygen atoms of Ile360 and His361 through hydrogen bonding. Taking account of this important interaction, we replaced the piperazine moiety in 9a with other cyclic amines. The optically pure 3-(dimethylamino) pyrrolidine derivatives 9b and 9c, the 3-(dimethylaminomethyl)pyrrolidine derivatives 9d and 9e, and the 3-(dimethylamino)azetidine derivative 9f, all of whose E-ring systems had the potential to function as piperazine isosteres, were synthesized (Fig. 4). The pyrrolidine derivative 9c exhibited excellent potency (IC50 = 4 nM), comparable to 9a. Compound 9b, the enantiomer of 9c, and other pyridine derivatives (9d and 9e) had lower, though still excellent, antiproliferative activity (IC50 = 11, 11 and 9 nM, respectively). Azetidine 9f had lower potency still (IC50 = 17 nM). These results reveal that the antiproliferative activity is somewhat affected by the position of the terminal dimethylamino function. In other words, the differences in activity among these compounds may be attributed to subtle differences in the distance between the dimethylamino function and the carbonyl oxygen atoms of Ile360 and His361.</p><p>At the beginning of our project, the involvement of Lyn kinase in imatinib resistance was unknown. In 2003, Donato et al. reported the association of the overexpression of Lyn kinase with imatinib resistance (Donato et al. 2003; Dai et al. 2004; Ptasznik et al. 2004). Thereafter, we tried to develop Abl/Lyn dual inhibitors, and found that NS-187 (9b; INNO-406) and its derivatives also inhibit Lyn kinase. To investigate why this series of compounds act as dual Bcr-Abl/Lyn kinase inhibitors, we determined their inhibitory activities against Abl and Lyn kinases and studied their structure-activity relationships (Horio et al. 2007). All compounds tested show more-potent inhibitory activity against Abl and Lyn than does imatinib (Table 1), and the inhibitory activities of 3-substituted benzamides against Abl and Lyn are highly correlated (r = 0.982 when the activity is expressed as pIC50).</p><p>Judging from its overall characteristics, including its pharmacokinetics and toxicity as determined in animal studies, we selected 9b (NS-187) as a candidate for clinical development (Kimura et al. 2005; Asaki et al. 2006). NS-187 is now under investigation in a Phase I clinical trial with Ph+ leukemia.</p><!><p>We recently determined the X-ray structure of NS-187 bound to human Abl (Horio et al. 2007) shown in Figure 5B (Abl, blue; NS-187, yellow). For comparison, the X-ray structure of imatinib bound to Abl (Abl, cyan; imatinib, white) is shown in Figure 5A. Only the amino acids within 4 Å of NS-187 or imatinib are depicted for clarity. The two X-ray structures resemble each other very closely, with only slight differences in the positions of the ligands and the side chains and backbones of the kinases. Therefore it is clear that NS-187 and imatinib interact with Abl in very similar ways. This finding validates our use of the X-ray structure of the imatinib/Abl complex to guide our chemical-modification studies.</p><p>We checked whether our strategy for chemical modification was appropriate by analyzing the X-ray structure of the NS-187/Abl complex (Fig. 5C). The trifluoromethyl (CF3) group is well placed to interact with the hydrophobic pocket formed by Ile293, Leu298, Leu354, and Val379, shown in magenta. Tyr253 is located close to the pyrimidine (A) ring, so that our use of a pyrimidine instead of a pyridine ring does not appear to alter the important role of Tyr253 in stabilizing the inactive form of the kinase. Hydrogen-bonding interactions are shown as broken white lines in Figure 5C, and it can be seen that the nitrogen atom of the dimethylamino group is well placed to interact with the carbonyl oxygen atoms of Ile360 and His361 through hydrogen bonding. Our strategy for chemical modification was thus validated.</p><!><p>The 3-substituents (R1) on the D ring greatly enhance the inhibitory activity against both Abl and Lyn kinases (Table 1). To elucidate this effect, we quantitatively analyzed the effect of the 3-substituent on the inhibitory activity of the compounds against Abl and Lyn kinases by using various physicochemical parameters of the 3-substituents. We found that the inhibitory activity is highly correlated with the hydrophobic substituent parameter π (correlation coefficient r = 0.958 for Abl and r = 0.977 for Lyn) (Horio et al. 2007). This means that the inhibitory effect increases with the hydrophobicity of the 3-substituent.</p><p>To understand this effect more clearly, we examined the molecular surfaces of Abl and Lyn kinases near the 3-substituent (Fig. 5D–F). It is apparent that there remains room to accommodate chemical modification of the D ring in the hydrophobic pocket formed by the amino acids Ile293, Leu298, Leu354 and Val379, shown in magenta in the X-ray structure of the imatinib/Abl complex (Fig. 5D). However, in the X-ray structure of the NS-187/Abl complex, the CF3 group occupies this hydrophobic pocket well (Fig. 5E). The modeled structure of the NS-187/Lyn complex, which is based on the X-ray structure of the NS-187/Abl complex, is depicted in Figure 5F. Close to the 3-substituent there are four hydrophobic amino acids, Leu293, Leu298, Ile354 and Ile379, shown in magenta (Horio et al. 2007). Although the identities of three of the four amino acids differ between Abl and Lyn, they are all hydrophobic amino acids. Therefore it is likely that the enhanced inhibitory activity of the modified compounds against both Abl and Lyn can be explained by increased hydrophobic interactions. It is reasonable that the hydrophobic effect of the 3-substituent, as expressed by π, significantly enhances the inhibitory activity.</p><p>The inhibitory activities of the compounds are also linearly correlated with the Sterimol parameter B1, which expresses the minimum width of the 3-substituent (r = 0.988 for Abl and r = 0.991 for Lyn) (Horio et al. 2007); that is, the inhibitory effect increases with the size of the 3-substituent. Since the 3-substituent is located adjacent to the terminal dimethylaminopyrrolidine ring (E), it would be expected to hinder the rotation of the terminal ring. When we calculated the rotational barrier of the terminal ring by using the MMFF94x force field with MOE, we indeed found a restricted rotation about the bond connecting the D and E rings of NS-187 (Fig. 6). The CF3 group not only reduces the flexibility of rotation of the terminal ring, but also helps NS-187 to adopt its bound (active) conformation (Kimura et al. 2006b). Because of the reduced loss of entropy upon binding, the inhibitory activity of the ligand would be expected to increase as the probability that it will adopt its active conformation increases. In addition, the reduced conformational flexibility could reduce the probability of binding to other proteins, thereby reducing the probability of adverse side effects.</p><p>Though kinase inhibitors bearing a CF3 group are not rare, those with a CF3 group adjacent to another group are rare. The adjacent location of these groups is a very characteristic structural feature of NS-187. The increased hydrophobicity and reduced conformational flexibility of NS-187 relative to imatinib cooperate to enhance its inhibitory activity against both Abl and Lyn kinase and reduce the probability of binding to off-target proteins.</p><!><p>We compared the ability of NS-187 and imatinib to inhibit the phosphorylation of Bcr-Abl and other tyrosine kinases at the cellular level (Kimura et al. 2005). The IC50 values of NS-187 against wild-type Bcr-Abl in human erythroleukemia K562 cells and human embryonic kidney 293T cells are 11 and 22 nM, respectively, while the corresponding values for imatinib are 280 and 1200 nM. NS-187 is therefore 25 to 55 times more potent than imatinib in blocking Bcr-Abl autophosphorylation. NS-187 suppresses the phosphorylation of platelet-derived growth factor receptor (PDGFR) and c-Kit with a potency similar to that of imatinib. However, while the potency ranking for imatinib is PDGFR > c-Kit > Bcr-Abl, the potency ranking for NS-187 is Bcr-Abl > PDGFR > c-Kit, so that the specificity of NS-187 for Bcr-Abl is greater than that of imatinib. Because inhibition of PDGFR or c-Kit could cause unpredictable adverse effects, specific inhibition of Bcr-Abl is desirable. Examination of the intracellular phosphorylation status of CrkL and ERK, downstream mediators of the effects of Bcr-Abl, revealed that NS-187 inhibits the phosphorylation of these proteins in K562 cells at much lower concentrations than does imatinib. This inhibition of phosphorylation is also observed in the mouse ProB cell line BaF3 expressing wild-type Bcr-Abl (BaF3/wt). Taken together, these findings indicate that NS-187 is much more potent and specific than imatinib in blocking the effects of Bcr-Abl.</p><!><p>More than 40 point mutations within the Abl kinase domain have been reported (Hochhaus and Rosee, 2004). NS-187 at physiologically obtainable concentrations inhibits the phosphorylation of Bcr-Abl bearing the M244V, G250E, Q252H, Y253F, E255K, E255V, F317L, M351T, E355G, F359V, H396P, or F486S mutations, but it does not inhibit the phosphorylation of the T315I mutant (Kimura et al. 2005). Against all mutants except T315I, NS-187 is at least five times as potent as imatinib (Table 2).</p><p>NS-187 suppresses the growth of the Bcr-Abl+ cell lines K562, KU812 and BaF3/wt much more potently than does imatinib, but neither drug affects the proliferation of the Bcr-Abl-negative cell line U937 (Kimura et al. 2005). NS-187 exhibits a concentration-dependent antiproliferative effect against BaF3 cell lines expressing the Bcr-Abl mutants M244V, G250E, Q252H, Y253F, E255K, M351T or H396P, but has no effect on BaF3 cells expressing the T315I mutant. Bcr-Abl/wt, Q252H and M351T are especially sensitive to NS-187. Imatinib, meanwhile, is much less active against all cell lines tested (Naito et al. 2006). NS-187 therefore potently inhibits both the intracellular phosphorylation of most mutated Bcr-Abl kinases and the proliferation of cells expressing these kinases.</p><!><p>NS-187 augments the activity of pro-apoptotic Bcl-2 homology domain 3 (BH3)-only proteins and induces apoptosis in Bcr-Abl+ leukemic cells, as evidenced by DNA fragmentation, caspase-3 activation, and the loss of mitochondrial-outer-membrane permeabilization (Kuroda et al. 2007). ABT-737, an inhibitor of Bcl-2 and Bcl-XL, enhances the apoptosis induced by NS-187, even in cells with mutated Bcr-Abl that are less sensitive to NS-187, suggesting that Bcl-2-family-regulated, intrinsic apoptosis occurs through caspase activation. Even in the presence of the pan-caspase inhibitor zVAD-fmk, NS-187 still induces apoptosis in some cells, indicating the additional involvement of NS-187 in a caspase-independent apoptotic pathway. The observation of an increased number of cells showing the hallmarks of autophagy suggests that autophagy participates in the response against Bcr-Abl blockade. Inhibition of autophagy by chloroquine significantly enhances NS-187-induced cell death. These results may be useful in the design of a rational therapeutic approach for efficiently eradicating Bcr-Abl+ leukemic cells.</p><!><p>Imatinib inhibits the kinase activity of the Tyr393-unphosphorylated form of the Abl kinase domain with an IC50 value of 35 nM but has little effect on the phosphorylated form. In contrast, NS-187 effectively inhibits the kinase activity of both Tyr393-phosphorylated and Tyr393-unphosphorylated forms of Abl with respective IC50 values of 72 nM and 30 nM, suggesting that NS-187 may have sufficiently high affinity for Bcr-Abl to enable it to bind even to an unfavourable conformation of the kinase (Naito et al. 2006).</p><!><p>A panel of 79 tyrosine kinases, including the five Src-family proteins Blk, Src, Fyn, Lyn and Yes, was assayed in the presence and absence of NS-187 or imatinib (Kimura et al. 2005). Concentrations of 0.1 μM NS-187 and 10 μM imatinib were used because these concentrations give equal inhibition of Abl. At 0.1 μM, NS-187 strongly inhibits only three of the 79 tyrosine kinases, that is, Abl, Arg and Lyn (Fyn is less strongly inhibited). At this concentration, NS-187 does not inhibit PDGFRα, PDGFRβ, Blk, Src or Yes. In contrast, 10 μM imatinib inhibits nine tyrosine kinases, that is, Abl, Arg, Blk, Flt3, Fyn, Lyn, PDGFRα, PDGFRβ and p70S6K. NS-187 therefore inhibits Abl more selectively than does imatinib. The IC50 values of NS-187 for Abl, Src and Lyn are 5.8, 1700 and 19 nM, respectively, while those of imatinib are 106, >10,000 and 352 nM, respectively. These findings suggest that NS-187 acts as an Abl/Lyn dual inhibitor while otherwise remaining highly specific.</p><!><p>The ability of NS-187 to suppress tumor growth was tested in two murine tumor models (Kimura et al. 2005). In one model, Balb/c-nu/nu mice were injected subcutaneously with KU812 cells on Day 0 and given NS-187 or imatinib orally twice a day from Day 7 to Day 17. At 20 mg/kg/day, imatinib inhibits tumor growth slightly, while at 200 mg/kg/day, it inhibits tumor growth almost completely. NS-187, meanwhile, significantly inhibits tumor growth at only 0.2 mg/kg/day, while at 20 mg/kg/day it completely inhibits tumor growth without adverse effects. When mice were treated with NS-187 at 0.2 or 20 mg/kg/day, the estimated Cmax was 4 or 400 nM, respectively, comparable to the concentrations at which the in vitro effects of NS-187 are obtained. NS-187 is therefore at least 10-fold more potent than imatinib in vivo with complete inhibition of tumor growth as the end-point and at least 100-fold more potent with partial inhibition as the endpoint. NS-187 was well tolerated by the mice.</p><p>In the other model, Balb/c-nu/nu mice intravenously injected with BaF3/wt cells were given NS-187 or imatinib orally for 11 days starting on Day 1. All seven untreated mice had died by Day 23 due to leukemic cell expansion, while all mice treated with 400 mg/kg/day imatinib had died by Day 25. NS-187, in contrast, significantly prolonged the survival of the mice in a dose-dependent manner compared with untreated mice.</p><p>To investigate the efficacy of NS-187 in a mouse model of leukemia, we tested its ability to block the growth of BaF3 cells expressing mutated Bcr-Abl in Balb/c-nu/nu mice (Naito et al. 2006). Mice bearing BaF3 cells expressing M244V, G250E, Q252H, Y253F, E255K, T315I, M351T or H396P were treated with NS-187 or imatinib. Mice bearing BaF3 cells expressing wild-type Bcr-Abl or any mutant form of Bcr-Abl except T315I show significant prolongation of survival when they receive NS-187 at a dosage of 200 mg/kg/day, without any apparent signs of toxicity (Fig. 7). These in vivo results are consistent with the in vitro results. Imatinib, even at a dosage of 400 mg/kg/day, is much less effective. NS-187 results in the highest observed percentage increase in mean survival in mice bearing BaF3 cells expressing wild-type Bcr-Abl, Q252H or M351T, in good agreement with the in vitro results. More-over, the rank-order of the IC50 values for cell growth inhibition is inversely correlated with the percentage increase in the mean survival of mice treated with NS-187. Thus, the efficacy of NS-187 in the mouse leukemia model mirrors its in vitro activity, a result which suggests that NS-187 will be clinically effective.</p><!><p>Because the penetration of imatinib into the central nervous system (CNS) is poor, the CNS can become a sanctuary site of relapse in patients on prolonged imatinib therapy. P-gp plays an important role in limiting the distribution of imatinib to the CNS, and it is well known that imatinib is a substrate for P-gp. Our preliminary pharmacokinetic study (Yokota et al. 2007) showed that the intracranial concentration of NS-187 is only 10% of its serum concentration, suggesting the involvement of P-gp. However, even though NS-187 is a substrate for P-gp, it still inhibits the proliferation of leukemic cells in the brain, whereas imatinib does not. NS-187 significantly prolongs the survival of mice in a dose-dependent manner in two CNS leukemia murine models compared with imatinib. Furthermore, cyclosporine A, a P-gp inhibitor, augments the in vivo activity of NS-187 against CNS Ph+ leukemia, as shown by whole-brain fluorescence imaging (Fig. 8A–D) and survival curves (Fig. 8E and F). These findings indicate that NS-187 is a promising agent for the treatment of CNS Ph+ leukemia.</p><!><p>A phase I study of NS-187 (INNO-406) in 21 patients with Ph+ leukemia who were resistant to or intolerant of imatinib is in progress (Kantarjian et al. 2007).</p><!><p>Using X-ray crystallographic information and computer modeling, we have developed a highly potent and selective Abl/Lyn dual tyrosine kinase inhibitor, NS-187 (INNO-406). Its characteristic structural features are a trifluoromethyl group on the D ring that occupies a hydrophobic pocket of the Abl ligand-binding site and an adjacent dimethylaminopyrrolidine E ring whose rotation is restricted by the trifluoromethyl group. These features not only enhance inhibitory activity against Abl but also increase selectivity by reducing binding to off-target proteins. NS-187 has higher potency in inhibiting Abl than does imatinib and higher selectivity in inhibiting Lyn than do other SFK/Abl inhibitors. NS-187 is less sensitive to point mutations in the Abl kinase domain than are other inhibitors such as imatinib, while maintaining a high selectivity for Abl and Lyn. NS-187 may be effective in the treatment of chronic myeloid leukemia with possible application to CNS leukemia and it may also be less liable to cause unfavorable side effects than are therapeutic agents that target multiple kinases, such as SFK inhibitors.</p>

PubMed Open Access

A Unified Strategy to Access Trans-Syn-Fused Drimane Meroterpenoids: Chemoenzymatic Total Syntheses of Polysin, N-Acetyl-Polyveoline and the Chrodrimanins

Trans-syn-fused drimane meroterpenoids are unique natural products that arise from contra-thermodynamic polycyclizations of their polyene precursors. Herein we report the first total syntheses of four trans-syn-fused drimane meroterpenoids, namely polysin, N-acetyl-polyveoline, chrodrimanin C and verruculide A in 7-18 steps from sclareolide. The trans-syn-fused drimane unit is accessed through an efficient acid-mediated C9 epimerization of sclareolide. Subsequent applications of enzymatic C-H oxidation and contemporary annulation methodologies install the requisite C3 hydroxyl group and enable rapid generation of structural complexity to provide concise access to these natural products.

a_unified_strategy_to_access_trans-syn-fused_drimane_meroterpenoids:_chemoenzymatic_total_syntheses_

<!>While indole hydrogenation typically requires high H2

<p>Meroterpenoids are a highly diverse class of natural products that arise in nature from hybrid terpenoid/non-terpenoid biosynthetic pathways. 1 One highly prevalent motif in many meroterpenoids is the C3-oxidized drimane substructure, which forms the central core of many families including the 3,5-dimethylorsellinic acid-derived fungal meroterpenoids 2 and the sesquiterpenyl indoles. 3 Biosynthetically, this motif is produced through the cyclization of a linear polyisoprene-derived epoxide precursor by various terpene cyclases. Compelling literature evidence 3,4,5 has suggested that these enzymes are capable of pre-organizing their respective substrates in specific conformations to generate products with unique ring topologies and stereoconfigurations, which ultimately contribute to the immense structural diversity of the meroterpenoids. Among the possible polycyclization product topologies, the alltrans configuration is the most favored thermodynamically as it allows the fused cyclohexane rings to adopt an all-chair conformation. However, there exists a subset of drimane-containing meroterpenoids that possess alternative ring fusions (Figure 1A), such as the trans-syn-cis-fusion found in polysin 6 (1) and N-acetyl-polyveoline 7 (2) and the trans-syn-trans-fusion found in the chrodrimanins 8,9 (e.g., 3 and 4). Access to thermodynamically disfavored ring fusions in these meroterpenoids is made possible by the ability of the respective cyclases to generate the less stable, boat-like transition state during their reactions (Figure 1B). Such conformational requirements have proven to be prohibitive for synthetic recapitulation as attempts to effect biomimetic cyclizations to prepare trans-synfused drimanes have been met with limited to no success. 10,11 A synthetic approach towards polyveoline featuring an indoleterminated polyene cyclization failed to overcome the innate thermodynamic preference of the substrate and resulted in exclusive formation of the undesired all-trans product. 10 Though the use of substrates with alternative olefin placement or geometry has garnered some success, 12,13 these approaches have resulted in either sub-optimal diastereoselectivity or low yields for the desired products. 2. A. Synthetic strategy to access polysin, N-acetyl-polyveoline and the chrodrimanins from 9, which could be obtained via C9 epimerization of sclareolide (7). B. Screening of P450BM3 variants in our collection for the C3 hydroxylation of 9. C. Screening of P450BM3 variants in our collection for the C3 hydroxylation of 12. See Supporting Information for the identities of the variants tested. Reaction conditions for enzymatic hydroxylation were: 9 or 12 (5.0 mM), NADP + (1.0 mM), NaHPO3 (100 mM), clarified lysate of E. coli BL21(DE3) expressing the appropriate P450BM3 variant and Opt13 (suspension in 50 mM kPi (pH 8.0) and pre-lysis at an optical density of 30, measured at a wavelength of 600 nm) for 20 h at 20 °C. *additional regioisomers were detected in the product mixture.</p><p>In parallel with the above efforts, several groups have sought to harness the power of terpene cyclases to biocatalytically access trans-syn-fused terpenoids. Since van Tamelen's landmark study on a cyclase from rat liver, 14 several reports have demonstrated the feasibility of this approach. Virgil and coworkers were able to use an unnatural oxidosqualene derivative in an enzymatic polycyclization to access the isomalabaricane tricyclic core 15 and more recently, a collaborative work by the Porco and Abe laboratories 5 showcased the utility of several fungal cyclases in constructing unnatural meroterpenoids with unusual ring fusions from synthetic substrates. These demonstrations notwithstanding, the approach suffers from low material throughput arising from the inefficiency of the enzymatic reaction and the difficulty in obtaining large quantities of the membrane-bound enzymes.</p><p>In the context of target-oriented chemical synthesis, only three total syntheses of trans-syn-fused drimane terpenoids have been reported thus far (Figure 1C). Two of these syntheses 16,17 pertain the brasilicardin natural products and involved lengthy synthetic sequences to generate the key trans-syn-trans-fused tricyclic intermediates. More recently, a landmark synthesis of the isomalabaricanes (e.g., stellettin E, 5) by the Sarlah group has enabled initial structure-activity relationship studies on the cytotoxicity of the scaffold. 18,19 To complement the aforementioned approaches, we sought to develop an alternative strategy to collectively prepare trans-syn-fused drimane meroterpenoids through the use of a chiral pool approach. This report discloses the development of a unified strategy to access both trans-syn-cisand trans-syn-trans-fused drimane meroterpenoids from sclareolide that culminates in the first total syntheses of polysin, N-acetyl-polyveoline, chrodrimanin C and verruculide A. To the best of our knowledge, this is the first reported de novo constructions of trans-syn-cis-fused perhydrobenz[e]indene and trans-syn-trans-fused dodecahydro-1Hbenzo[f]chromene frameworks. This work was made possible by the use of an underexplored epimerization reaction on sclareolide, 20 which was combined with enzymatic C-H oxidations and efficient ring annulations to complete the divergent syntheses. We anticipate that the strategy delineated herein will find a broad range of applications in the preparation of other drimane terpenoids with unusual ring fusions.</p><p>As noted above, our synthetic strategy was predicated upon the ability of sclareolide (7) to undergo facile epimerization at its C8 and C9 positions. Prior report from Ohloff 20 (Figure 2A, inset) showed that treatment of 7 with mineral acid at room temperature could readily afford the C8-epimerized product 12 (8). Alternatively, the C9-epi product (9) was observed as the major product at elevated temperature, likely via elimination to the corresponding C8-C9 olefin, followed by re-protonation from the b-face at C8 and quenching of the C9 carbocation from the a-face by the pendant carboxylic acid. In our hands, this transformation could be routinely conducted on multigram scale with 95% yield. With the C9 stereocenter established, access to polysin and N-acetyl-polyveoline could be accomplished through selective C-H oxidation at C3 and the appendage of a pendant indole unit (Figure 2A). We envisioned introducing the former through enzymatic C-H hydroxylation 21,22 and the latter through ring synthesis by leveraging the C12 carbonyl as a chemical handle. Adaptation of this idea to access the chrodrimanin series would necessitate the invention of a synthetic sequence to construct the C-ring pyran while also inverting the stereochemistry at C8. While the general pyran structure could be prepared via a one-carbon homologation, the stereoinversion at C8 was expected to be non-trivial as it would result in an A/B/C-ring connectivity that forces the B-ring to adopt the energetically-unfavored twist-boat conformation. Nevertheless, if this transformation could be realized, an efficient synthesis of chrodrimanin C would ensue through subsequent use of the C-ring lactone as a chemical handle in an aromatic annulation sequence. Finally, enzymatic conversion of 3 to 4 through a series of in vitro reactions has previously been reported by Matsuda, Abe and co-workers. 23 Scheme 1. Chemoenzymatic total synthesis of polysin (1) and N-acetyl-polyveoline (2) via enzymatic C-H oxidation of 9.</p><p>In light of our previous work in the synthesis of a-pyrone meroterpenoids from 7, a route involving enzymatic C3 oxidation of 7 with variants of P450BM3, 22 followed by epimerization at C9 was initially considered. However, preliminary forays into this route showed that the C3 alcohol is incompatible with strong acids, even in its protected form. As a workaround, we decided to investigate the feasibility of performing enzymatic C-H oxidation on lactones 9 and 12, which was prepared in seven steps from 9 (vide infra). Despite the high structural similarities of 9 and 12 to 7, it is widely accepted that even minor alterations in substrate structure could result in dramatic changes in reactivity in enzymatic transformations. Gratifyingly, initial screening of a subset of our P450BM3 library revealed a few variants with C3 hydroxylation activity on 9 (Figure 2B). Variant KSA15, 24 previously developed by Reetz and co-workers for steroid hydroxylation, showed the highest conversion (54%) among all the library members tested. Following an analogous screening with lactone 12, variant MERO1 L75A, previously developed in our laboratory for the synthesis of oxidized meroditerpenoids, 22 was identified to be the optimal enzyme to hydroxylate 12 at C3 (Figure 2C). While variant KSA15 provided higher conversion (95%) in its reaction with 12, additional product regioisomers could be detected. Thus, we elected to perform subsequent C-H oxidation scale-up with MERO1 L75A.</p><p>With the above results in hand, we set our sights establishing a concise access to polysin and N-acetyl-polyveoline (Scheme 1). Preparative scale enzymatic hydroxylation of 9 provided alcohol 13 with 67% yield, which was subjected to a Smith-modified Madelung indole synthesis 25 to provide a mixture of two adducts, 14 and 15. Treatment of this mixture with p-toluenesulfonic acid (PTSA) effected complete formation of the indole nucleus with concomitant dehydration of the tertiary alcohol at C8. In light of its potential incompatibility with acidic conditions needed for the subsequent cyclization step, oxidation of the C3 alcohol at this stage was deemed strategic and was accomplished using Albright-Goldman protocols. 26 Following an extensive screening of Lewis acids (see Supporting Information Table S4), we arrived at the use of MK-10 under microwave heating to generate a mixture of Friedel-Crafts adducts. As the C-cyclized product was observed to be unstable, an in-situ capping approach with Ac2O was devised to deliver a mixture of enol ethers 17 and 18 in 40% and 25% yields respectively under telescoped procedure. Routine saponification of 18 completed the synthesis of polysin (1) in seven steps from 7. Conversely, 17 was subjected to hydrogenation in the presence of palladium on carbon, followed by a diastereoselective reduction with K-selectride to complete the synthesis of N-acetyl-polyveoline (2) in eight steps from sclareolide (7). S5). Unfortunately, no marked increase in diastereoselectivity was observed in all conditions tried and under the best set of conditions, a diastereomeric ratio of 1:1 at C8 was obtained. At this stage, the desired tertiary alcohol diastereomer 22 was saponified and converted to trans-syn-trans-fused lactone 12 through the use of Yamaguchi's reagent. To improve material throughput, the unwanted diastereomer 21 could be recycled into the sequence by simple methyl ester formation to regenerate 20 along with its olefin regioisomer. After three cycles, a combined 60% isolated yield of 22 could be achieved. Enzymatic hydroxylation of 12 with P450BM3 variant MERO1 L75A was next conducted on preparative scale to provide alcohol 11 in 82% yield. The structure of this compound was verified by Xray diffraction analysis, which prominently revealed the twistboat configuration of the B-ring. Drawing inspiration from the syntheses of arene-containing terpenoids by Li and co-workers, 28,29 we sought to construct the central arene ring of the chrodrimanins through a 6p electrocyclization of the corresponding triene precursor. Toward this goal, the C3 alcohol of 11 was temporarily protected as the trimethylsilyl (TMS) ether and the C-ring lactone was converted to the corresponding vinyl triflate (compound 23). Sonogashira coupling of 23 with alkyne 24, synthesized in 6 steps from (R)-methyl 3-hydroxybutanoate (see Supporting Information), delivered dienyne 25 in 67% yield over 3 steps from alcohol 11. With the goal of introducing a suitable functional handle for subsequent phenol formation, an alkyne hydrosilylation approach was pursued. Previous work by Ferreira and co-workers 30 showed that the regioselectivity of alkyne hydrosilylation is predominantly dictated by electronic effects whereby hydride delivery would take place at the sp carbon that is further away from electron-withdrawing group. Indeed, treatment of alkyne 25 with Et3SiH in the presence of catalytic Pt(DVDS) successfully provided the desired hydrosilylation product 26 as a single regioisomer.</p><!><p>Following precedent by Li, Nicolaou and co-workers, 31 a 6p electrocyclization/aromatization sequence could be effected to generate arene 27. In agreement with their work, the use of CuOTf as a Lewis acid promoter was found to improve the yield of the reaction (73% isolated yield) while also effecting a concomitant hydrolysis of the TMS ether at C3. While oxidation of the C3 alcohol to the corresponding ketone proceeded uneventfully, attempts to effect a Fleming-Tamao oxidation 32 to convert 27 to the corresponding phenol were met with failure. Similar outcomes were obtained when alternative silanes at C4' were tested in the reaction. Earlier iterations of the route featuring a late-stage sp 2 C-H oxidation at C4' using Ru catalysis 33 or peroxide-based reagents 34,35 also failed to deliver the desired product. As a workaround, silane 27 was first subjected to desilylative iodination with NIS to provide 28. Following screening of several reported conditions for haloarene hydroxylation, access to chrodrimanin C (3) could be realized through the use of Cu(acac)2 and N,N'-bis(4-hydroxyl-2,6-dimethylphenyl)oxalamide (BHMPO) on 28. 36 This method, initially reported by Ma and co-workers, proved superior to Pd-based hydroxylation methods 37,38 and with slight modifications to the originally reported conditions, the desired phenol product could be obtained in 83% isolated yield. A-ring desaturation of 3 proceeded uneventfully under standard Saegusa conditions to deliver verruculide A (29) in 82% yield. Interestingly, TMS ether formation at the phenolic OH was not observed in this reaction, likely due to the presence of an intramolecular hydrogen bonding with the neighboring lactone. Overall, this sequence provided a 16-step synthesis of chrodrimanin C (3) and a 18-step synthesis of verruculide A (29) from sclareolide (7), respectively. As noted earlier, the biosynthetic pathway towards the chrodrimanins was recently elucidated by Matsuda, Abe and co-workers 23 and we anticipate that future work involving incorporation of some of the enzymes from the pathway would allow for a rapid chemoenzymatic diversification of the scaffold to provide a wider range of synthetic chrodrimanins.</p><p>This work reports the development of a chiral-pool-based strategy for the asymmetric synthesis of trans-syn-fused drimane meroterpenoids. Two enabling features in the synthesis are the strategic use of an acid-mediated C9-epimerization of sclareolide to generate the general trans-syn-fused architecture of these natural products and the ability to perform regioselective C-H oxidations on different key synthetic intermediates at their C3 position by relying on a small pool of P450BM3 biocatalysts. By combining these features with contemporary annulation methodologies, the first total syntheses of polysin, N-acetyl-polyveoline, chrodrimanin C and verruculide A could be realized. The route disclosed herein lays the foundation for future synthetic access to other unusually-cyclized meroterpenoids and their unnatural derivatives to facilitate a more thorough investigation into their pharmacology.</p>

ChemRxiv

TRPswitch \xe2\x80\x94 a step function chemo-optogenetic ligand for the vertebrate TRPA1 channel

Chemo-optogenetics has produced powerful tools for optical control of cell activity, but current tools suffer from a variety of limitations including low unitary conductance, the need to modify the target channel, or the inability to control both on and off switching. Using a zebrafish behavior-based screening strategy, we discovered \xe2\x80\x9cTRPswitch\xe2\x80\x9d, a photoswitchable non-electrophilic ligand scaffold for the transient receptor potential ankyrin 1 (TRPA1) channel. TRPA1 exhibits high unitary channel conductance, making it an ideal target for chemo-optogenetic tool development. Key molecular determinants for the activity of TRPswitch were elucidated and allowed for replacement of the TRPswitch azobenzene with a next-generation azoheteroarene. The TRPswitch compounds enable reversible, repeatable, and nearly quantitative light-induced activation and deactivation of the vertebrate TRPA1 channel with violet and green light, respectively. The utility of TRPswitch compounds was demonstrated in larval zebrafish hearts exogenously expressing zebrafish Trpa1b, where heartbeat could be controlled using TRPswitch and light. Therefore, TRPA1/TRPswitch represents a novel step-function chemo-optogenetic system with a unique combination of high conductance, high efficiency, activity against an unmodified vertebrate channel, and capacity for bidirectional optical switching. This chemo-optogenetic system will be particularly applicable in systems where a large depolarization current is needed or sustained channel activation is desirable.

trpswitch_\xe2\x80\x94_a_step_function_chemo-optogenetic_ligand_for_the_vertebrate_trpa1_channel

Introduction<!>Zebrafish behavior-based chemical screening identifies TRPswitch-A<!>TRPswitch-A structure activity relationship analysis<!>TRPswitch is a reversible photoswitch ligand for Trpa1b<!>TRPswitch\xe2\x80\x99s mechanism of action<!>Trpa1b/TRPswitch allows cellular activation in non-neuronal cells<!>Discussion<!>Zebrafish<!>Chemical libraries and treatments<!>Behavioral assay<!>Behavioral analysis<!>Electrophysiology analysis<!>Heart experiments<!>UV-Vis measurement<!>Statistical Analyses

<p>Optogenetics has proven to be a transformative technology for various fields of basic research, particularly in neuroscience. It allows for a non-invasive, localized, and temporally selective optical modulation of selected cells within an animal. Optogenetic technologies hold great promise for clinical applications. For example, preclinical animal models have demonstrated potential utility in treating retinitis pigmentosa with AAV-delivered channelrhodopsin2 (ChR2).1 However, high expression of the light-gated ion channel ChR2, originating from green algae, has cytotoxic effects.2 It remains unclear whether or not expression of ChR2 in humans will result in immune rejection or inflammation. Therefore, to advance the utility of optogenetics in clinical applications, development of optogenetic actuators that are vertebrate in origin or endogenous to humans, as well as those based on ion channels with high unitary conductance, might be advantageous. The use of vertebrate protein actuators might reduce the risk of immunological reactions due to long-term expression of exogenous proteins. High conductance actuators should also decrease the amount of channel expression needed for sufficient light-controlled activity. Some progress has been made toward these goals, including the development of an endogenous protein-targeting photoswitch that confers light-sensitivity on endogenous neuronal ion channels, proposed to have the potential to restore vision to the blind.3–5</p><p>Discovery of new optogenetic actuators that possess novel and unique properties will undoubtedly enhance our ability to dissect biological systems such as the complex neuronal networks of the brain. For example, optogenetic experiments that require long activation periods would benefit greatly from "step function" optogenetic tools that allow stable, bi-directional on and off switching of channels. Step function opsins (SFOs) are engineered versions of ChR2 that persist in their open state after illumination ceases.6–8 As such, they are useful in modifying the spontaneous firing rate of neurons, as well as in applications where behavioral analysis without continuous optic fiber tethering is desired.9–10 However, as mentioned above, the non-vertebrate origin of these opsins present potential challenges in clinical applications. As a complementary approach, chemo-optogenetic tools that combine chemicals and optogenetics have been under rapid development. Using a chemical design approach, photochromic soluble ligands (PCLs) for various wild-type ion channels have been synthesized to allow for light-controlled channel activity.11–15 These photoswitchable PCLs block and unblock their corresponding ion channel either in their E or Z configuration. Examples include PCLs for the TRPV1 channel,11 NMDA receptor,16–17 and kainate receptor.18–19</p><p>TRPA1 is a member of the Transient Receptor Potential (TRP) channel family. It is a non-selective cation channel that plays an important role in inflammatory and neuropathic pain, itch, and respiratory diseases.20–22 We have previously reported photoactivable ligands for TRPA1, such as optovin, and demonstrated their ability to act as chemo-optogenetic tools.23–24 TRPA1 has a channel conductance of approximately 100 pS,25 1000 times greater than ChR2, making it ideal for applications where high conductance or low expression levels are desired. Importantly however, while photochemical stimulation of optovin-class chemo-optogenetic ligands activate TRPA1 rapidly (in low millisecond time scales), channel deactivation depends upon spontaneous (i.e. non-photochemical) reversal of ligand action, which occurs on the time scale of seconds. In other words, the approach is somewhat akin to photodecaging, where a 'protected' ligand is activated ('deprotected') following a light stimulus,26 but where deactivation requires spontaneous and often slow dissipation of the ligand. It would be a great advancement to develop a chemo-optogenetic system that preserves the high conductance of TRPA1 and the rapid activation of optovin-class ligands, but enables rapid, light-controlled channel deactivation: a photoreversible/photoswitchable system.</p><p>In this study, we used a customized, light-responsive chemical library coupled with a behavior-based screening assay with zebrafish larvae to discover 'TRPswitch' azoarene photoswitchable ligands for the TRPA1 channel. Our TRPswitch molecules allow for optical control of both the activation and deactivation of the TRPA1 channel. Our analysis suggests that the zebrafish Trpa1b channel is necessary and sufficient for TRPswitch light-induced activity. Channel activation and deactivation can be controlled by violet light and green light illumination, respectively. The TRPswitch/light-induced TRPA1 channel activity is reversible and repeatable in vivo, and sustained channel activation is achieved after only a short pulse of light illumination. To our knowledge, this is the first time a photoswitchable TRPA1 system has been described. Our data show that this TRPA1/TRPswitch system is a robust chemo-optogenetic tool that can be applied to both neuronal and non-neuronal cells. The TRPA1/TRPswitch system's step function properties, along with its high unitary conductance, make it a complementary alternative to existing chemo-optogenetic tools.</p><!><p>With the discovery of photochromic soluble ligands (PCLs) as a means to control ion channel function using light,11–15 we reasoned that molecular photoswitches for TRPA1 could be discovered using small molecule screening. To achieve this goal, we developed a modified version of the behavioral assay that we previously utilized to identify photoactivable, but non-photoreversible, ligands of TRPA1.23 This medium-throughput, semi-automated screening assay was performed using a 96-well plate format where three larvae per well were incubated with small molecules. We screened a library of 1,000 structurally diverse small molecules enriched for molecular photoswitch moieties such as acylhydrazone,27 azobenezene,28–31 azoheteroaryl32–33 and stilbene.31, 34 We particularly focused on molecular photoswitches that operate via a reversible E/Z isomerization using different wavelengths of light. In this new assay, each well of a 96-well plate is illuminated with a series of different wavelengths of light (450-500 nm, 415 – 455 nm, 352-402 nm and white light) for one second to induce isomerization of the photoswitchable compounds (Fig. 1a). Each illumination event is separated by a dark period of 5 s and the motion of larvae during this light illumination sequence is recorded and analyzed (Fig. 1b). Wild-type 3-days-post-fertilization larvae (dpf) were used, as they have a relatively developed central nervous system and show no motion response to light exposure at this stage of development. Zebrafish larvae express Trpa1b in a subset of trigeminal and Rohon-Beard sensory neurons.35 Activation of Trpa1b induces a reproducible and robust motion response.23–24,35–36 A light-induced motion response, due to the presence of a photoactivated ligand for ion channels such as Trpa1b, is used as the readout for the assay. DMSO treated larvae on each screening plate served as negative controls. Using this screening assay, we identified TRPswitch-A, an azobenzene containing small molecule with no previously annotated biological activity. The presence of TRPswitch-A led to a light-induced motion response across multiple wavelengths, with the most activity in a bandwidth between 415 nm – 455 nm (WL2 in Fig. 1c). Using whole-cell patch-clamp recordings of HEK293T cells expressing zebrafish Trpa1b, we characterized the photocurrents elicited by the Trpa1b/TRPswitch-A pair upon light stimulation. Violet light stimulation of TRPswitch-A-primed Trpa1b channels generated high amplitude currents when compared to baseline measurements (Fig. 1d). When the same cell was subsequently stimulated with green light, Trpa1b photocurrent recovered to its baseline current magnitude (Fig. 1d). This reversible light response corresponds to the reversible E/Z isomerization of TRPswitch-A observed upon illumination with violet and green light, as judged by UV-Vis absorbance measurements (Fig. 1e). Expression of functional Trpa1b channels in HEK293T cells was confirmed by the observance of allyl isothiocyanate (AITC)-activated currents (Fig. 1f), as AITC is a potent TRPA1 agonist. We also observed desensitization of AITC-induced current over time, further confirming the activity of zTrpa1b channels (Fig. 1f, S1). Overall, these data indicate that TRPswitch-A is a light-dependent and light-reversible activator of Trpa1b.</p><!><p>The TRPswitch-A chemical structure contains an azobenzene and a 2-furamide group in both the ortho and para positions of one of the benzene rings (Fig. 2, compound 1). It is clear that while TRPswitch-A undergoes reversible E/Z isomerization upon illumination with violet and green light, the Z-E photoswitching event is incomplete (Fig. 1e). We sought to determine the chemical features responsible for TRPswitch-A's biological activity and how photoswitch performance correlates to the biological effects observed. We therefore designed, synthesized and analyzed the structure activity relationships of key derivatives of TRPswitch-A. We found that the para amide is needed for Trpa1b activation as suggested by the reduced activity of compound 2 (Fig. 2). Substituting the furan for thiophene was tolerated (Fig. 2, compound 3), albeit with a slightly lower photoresponse which might be due to the overall chemical structural change. Derivatives bearing para electron-withdrawing groups – to increase the "push-pull" character 37–38 – were prepared (compounds 4, 5, 6, 7) and found to be far less active in the assay (Fig. 2). This poor activity may, in part, be ascribed to the incomplete E-Z photoswitching of these compounds (Fig. S2 and Supp. Table 1). We have previously found that azoarene performance can be improved/tuned by substituting one of the benzene rings in a conventional azobenzene for a 5-membered heteroaromatic ring.32, 39–40 Specifically, we have found the azopyrazoles show near quantitative photoswtiching in both directions and exhibit long Z-isomer thermal half-lives. Replacement of the phenyl ring in compound 1 (TRPswitch-A) and compound 3 with a trimethylpyrazole generates compounds (8 and 9) that undergo quantitative photoswitching in both directions (Fig. 3f) and that have long thermal half-lives. The thermal Z-isomer half-life of compound 9 (TRPswitch-B), in DMSO-d6, is 17 hours (Fig. 3a). While we observed some aqueous solubility issues for compound 8, which likely limits its biological response, compound 9 (TRPswitch-B) has a comparable biological response to TRPswitch-A.</p><p>To further characterize the thermal half-lives of TRPswitches in conditions that more closely resemble those used in biological experiments, we measured their thermal half-lives at higher water contents (30% water:DMSO solutions). We found that the thermal half-lives of TRPswitch-A and -B are 43 min and 1 hour, respectively. When measurements were made in DMSO- d6, the half-lives for TRPswitch-A and -B were 11 and 17 hours, respectively.</p><p>To further characterize the photochemical properties of TRPswitches, the photostationary state (PSS) of TRPswitches in 30% water:DMSO solutions were examined via UV-Vis. By extrapolating a pure Z spectrum, using methodology outlined in previous publications,32, 39, 41 we approximate the PSS compositions to be 86% Z at 365 nm and 73% E at 495 nm for TRPswitch-A; and 92% Z at 365 nm and 100% E at 495 nm for TRPswitch-B (Table 1). In addition, we also examined the PSS of TRPswitches in DMSO-d6 at 365, 420 and 495 nm (Supp. Table 2, Figs. S11–16). From our analysis, TRPswitch-B demonstrates superior, near quantitative photoswitching compared to the azobenzene analogue, at least under the conditions tested. These data suggest that photoswitch performance, particularly Z-isomer half-life, contributes to the activity and that both azobenzene (TRPswitch-A) and azoheteroaryl (TRPswitch-B) moieties can be used as PCLs of Trpa1b, with the latter appearing to have an improved intrinsic photoswitch performance. Additionally, we have obtained the X-Ray crystal structure of TRPswitch-B (Fig. S3). Notably, this compound crystallizes in a chiral space group, with the azo bridge orientated to take advantage of a favorable hydrogen bonding interaction from the ortho amide.</p><!><p>We previously identified optovin as a photoactivable ligand for the Trpa1b channel, and confirmed that its activity is abolished in Trpa1b mutant zebrafish.23–24 To determine if Trpa1b is necessary for TRPswitch's biological activity, we performed a behavioral assay using Trpa1b mutant larvae.35 When Trpa1b mutant larvae were used, the TRPswitch light-induced motion response was abolished (Fig. 3b). This suggests that Trpa1b is required for the activity of TRPswitch in vivo, consistent with our electrophysiology analysis (Fig. 1d). Both TRPswitch-A- and TRPswitch-B-treated larvae showed a higher probability of light-induced motion response as the light stimulation duration increased (Fig. 3c). The probability for larvae to respond to light reached its maximum with a <1 s light-pulse length. The biological response triggered by TRPswitch/Trpa1b activation was very rapid (Supp. Movie 1). The latency to motion response from the introduction of light was in the range of milliseconds and decreased with increasing light intensity (Fig. 3d). A positive correlation was observed between the activity of TRPswitch and an increase in compound concentration, with maximum activity achieved at concentrations between 10 – 20 μM (Fig. 3e). Similar to TRPswitch-A, TRPswitch-B undergoes reversible E/Z isomerization upon illumination with violet and green light. However, unlike TRPswitch-A, the Z-to-E conversion by green light leads to complete return to the pre-illumination "off" state (Fig. 3f).</p><p>Next, we turned to characterize the kinetics of Trpa1b channel activity. However, in the behavioral screening assay, the light-induced motion response does not require sustained channel activation, i.e., the duration of motion does not directly correlate with the half-life of the TRPswitch Z state. To characterize the stability and kinetics of Trpa1b/TRPswitch-dependent photocurrents, we recorded zebrafish Trpa1b activity in HEK293T cells. The light-induced activation and deactivation of Trpa1b/TRPswitch was triggered with violet and green light pulses, respectively (Fig. 3g and 3h). Importantly, the photocurrents were sustained after the initial short pulse of violet light illumination and did not require continuous illumination. The photocurrents could be converted back to baseline using a subsequent pulse of green light. The current density fold increments of TRPswitch-A and TRPswitch-B upon violet light illumination were 2.36 ± 0.50 and 2.12 ± 0.47 at +100 mV and 4.35 ± 1.44 and 2.27 ± 0.38 at −100 mV, respectively. A subsequent pulse of green light reduced the current density fold increments of TRPswitch-A and TRPswitch-B to 1.15 ± 0.17 and 1.26 ± 0.33 at +100 mV and 3.71 ± 1.31 and 1.41 ± 0.38 at −100 mV, respectively. Together, these data indicate that TRPswitch-A and TRPswitch-B are novel, reversible photoswitches that act on the Trpa1b channel. Overall, the light-activated currents with TRPswitches are comparable to those induced by the canonical TRPA1 agonist AITC (Fig. 3i).</p><p>To test the potential cross activity of TRPswitch-B on the zebrafish ohnolog Trpa1a and on mammalian orthologs, we transiently re-expressed zebrafish Trpa1a, mouse TRPA1 or human TRPA1 in the Rohon-beard neurons of mutant Trpa1b−/− zebrafish and performed light-induced motion response experiments in the presence of TRPswitch-B. Only re-expression of Trpa1b rescued the light-induced motion response of Trpa1b−/−mutants (Table 2). Our results suggest that TRPswitch-B is specific to the zebrafish Trpa1b channel.</p><!><p>Most known activators of the TRPA1 channel are electrophilic ligands that covalently modify cysteines in TRPA1's cytoplasmic domain.42 Optovin, our previously identified photoactivable TRPA1 ligand, reacts with those cysteines through a photochemical reaction involving the generation of singlet oxygen species. DABCO, a singlet oxygen quencher and triplet energy acceptor can completely suppress the optovin response.23 To determine whether or not TRPswitch is also photoactivated in a singlet oxygen-based mechanism, we tested the ability of DABCO to suppress TRPswitch activity and found that it did not (Fig. 3j). These data suggest that light-induced generation of singlet oxygen is not necessary for TRPswitch's behavioral effect. As the TRPswitches contain either azobenzene or azopyrazole groups, it is likely that their activity is due to the E/Z isomerization of the compounds upon light illumination (Fig. 1e and 3f), without the production of radicals. These observations suggest that both TRPswitch-A and -B are reversible photoswitches for the Trpa1b channel and have a distinct mechanism of activation as compared to electrophilic ligands.</p><!><p>To demonstrate the practical utility of TRPswitch in vivo, we performed a heartbeat interruption experiment using a zebrafish transgenic line expressing Trpa1b in cardiomyocytes, Tg(cmlc2:Trpa1b-2A-EGFP), and by applying pulses of violet and green light to the zebrafish heart. Since proper calcium handling is important for normal heartbeat, we used heartbeat as the biological readout for the activation and deactivation of Trpa1b channels. During regular heartbeat cycles, an increase in intracellular calcium is required for cardiomyocyte contraction as calcium binding to troponin C leads to a conformational change that displaces tropomyosin from the actin binding sites. Calcium levels must then decrease for cardiomyocyte relaxation as calcium prevents tropomyosin from returning to its original conformation. We hypothesize that a sustained elevation of intracellular calcium, such as with TRPA1 activation, will result in a sustained tetanic contraction of the heart and interrupt rhythmic beating. In transgenic larvae treated with either TRPswitch-A or TRPswitch-B, we found that the ventricle heartbeat could be stopped and would persist in a sustained systolic state (tetany) after a brief 1 s illumination with violet light (Fig. 4a–d, Supp. Movie 2). Normal ventricular rhythm was restored after briefly illuminating the heart with 1 s of green light (Fig. 4a–d, Supp. Movie 2). These data suggest that the reversible E/Z isomerization of the TRPswitches by violet and green light induced in vivo activation and deactivation of Trpa1b channels, respectively. The ability for TRPswitch/light to induce tetany depends on the number of cardiomyocytes with Trpa1b expression. In larvae with transient mosaic expression of Trpa1b in cardiomyocytes treated with TRPswitch-B, ventricle tetany can only be induced when at least around 15% of cardiomyocytes in the ventricle have Trpa1b expression (Fig. S4). Furthermore, there was no significant difference in heart rate among transgenic larvae treated with DMSO control or either TRPswitch in the dark (Fig. S5a), suggesting that the TRPswitches have no effect on Trpa1b channels without light illumination. Treating larvae that lack exogenous expression of Trpa1b in cardiomyocytes with TRPswitch-B and light illumination resulted in similar negative results (Fig. S5b), indicating that the switching off and on of heartbeat with violet and green light illumination was due to TRPswitch isomerization and the subsequent effect on Trpa1b channel activity.</p><p>Photoswitchable control of Trpa1b activity by the TRPswitches was repeatable. Cyclical rounds of illumination using violet and green light induced the stopping and restarting of the ventricle heartbeat (Fig. 4c and 4d; Supp. Movie 3 and 4). Additionally, channel activation after a brief pulse of violet light was sustained on a timescale of minutes, as measured in the gradual relaxation of ventricle width over time (Fig. 4e). As a further demonstration of the utility of the heterologously expressed zTrpa1b/TRPswitch system, we expressed zTrpa1b in human colonic fibroblast cells, CCD-18Co, which express human TRPA1 endogenously.43 The expression of human TRPA1 channel in CCD-18Co cells was verified with RT-PCR (data not shown). CCD-18Co cells responded to AITC, confirming the functional expression of human TRPA1 channel (Fig. S6a). As expected, TRPswitch showed no light induced photocurrent in CCD-18Co cells without the expression of zTrpa1b (Fig. S6b). When CCD-18Co cells were transfected with zTrpa1b and incubated with TRPswitch-B, they exhibited violet light-induced photocurrent and green light-induced decrease in photocurrent (Fig. S6c). These data provide further evidence that TRPswitch-B has specific activity on zTrpa1b and does not cross react with endogenously expressed human TRPA1 channel. Taken together, these data suggest that the TRPA1/TRPswitch pair constitutes a reversible and repeatable chemo-optogenetic system that is compatible with use in zebrafish and mammalian cells.</p><!><p>These studies have identified TRPswitch A and B, two photoswitchable small molecules that enable optical control of currents in Trpa1b expressing cells in vivo. Our data suggest that the TRPswitches specifically target Trpa1b channel and enable repeatable optical control of both neuronal and non-neuronal cells. This is the first example of TRPA1 channel activation by a photoswitchable compound. Importantly, the TRPswitches allow for sustained channel activation after only a brief pulse of violet light illumination, but the channel can also be rapidly deactivated with green light illumination. As only short pulses of light are required to control the activity of the TRPA1 channel, cells subjected to the TRPA1/TRPswitch chemo-optogenetic system are less prone to photo-toxicity. This requirement of a short photoactivation period for target activation is a unique and advantageous feature among current PCLs for unmodified ion channels11–15 and receptors.44</p><p>The TRPA1 channel's high conductance combined with the step function property of the TRPA1/TRPswitch chemo-optogenetic system offers certain new opportunities for basic research. As TRPswitch activity is specific to zebrafish Trpa1b, TRPswitch can be used in heterologous applications by expressing zTrpa1b in animals or cells with endogenous mammalian TRPA1 expression, without interference from the endogenous channel. This new tool will be particularly beneficial in applications where a large depolarization current is needed, such as in large primary motor neurons, or when sustained channel activation is desirable. In addition, tools for manipulating TRPA1 activity are relevant for medical research since TRPA1 is involved in inflammatory and neuropathic pain, itch, and respiratory diseases.20–21, 45 TRPswitch may prove useful as a research tool to help dissect the mechanism of TRPA1-related disease, as well as to identify disease-modifying agents.</p><p>The TRPA1/TRPswitch chemo-optogenetic system offers several advantages over existing tools, and we have shown it to be robust and easy to use in cultured mammalian cells and in zebrafish. Nevertheless, its suitability for other in vivo and clinical applications remains unknown. TRPswitch is a freely diffusible small molecule, and exposing zebrafish larvae by incubating them in a solution containing TRPswitch is sufficient for robust activity. However, its uptake and distribution in rodents and larger mammals remains to be determined. In zebrafish larvae incubated with TRPswitch for several hours, no obvious adverse physiological phenotypes were observed. The TRPswitches show minimal activity on TRPA1 before photoactivation, high-lighting the specificity of the light-induced effect of TRPswitch. On the other hand, as the TRPswitches display a wide range of spectral activity, it may be difficult to combine a TRPswitch with other optogenetic tools or fluorescence-based biosensors due to potential spectral overlap. The use of violet light for TRPswitch activation may also limit the tissue depth of its activation; however, future studies could explore the possibility for multi-photon 46 or longer near infrared (NIR) light excitation.47</p><p>Our analysis suggests that both TRPswitch-A and TRPswitch-B have thermal half-lives in the scale of hours in DMSO. However, the half-life of the biological response to these compounds was measured to be in the timescale of minutes during in vivo cardiac experiments (Fig. 4e). Several factors may account for the shorter half-life observed in vivo. Firstly, the thermal half-life of TRPswitch would be different under the conditions of the biological assays, for example in aqueous solution with only 1% DMSO. In fact, the thermal half-life of TRPswitches become shorter when measured in 30% water:DMSO (43 min and 1 hour for TRPswitch-A and -B, respectively). Secondly, although we found that TRPA1/TRPswitch light-induced activity is reversible and repeatable, TRPswitch does not covalently bind to TRPA1 and is therefore free to diffuse away from the channel over time. Thirdly, there might be biological adaptation during TRPA1 activation, such as receptor internalization, that leads to a shorter time of biological effect compared to the half-life measured by UV-Vis in vitro.</p><p>Most of the known TRPA1 ligands are electrophiles that activate TRPA1 via the covalent modification of cysteine residues present in the channel's cytoplasmic ankyrin repeat domain.48 This includes our previously identified TRPA1 photoactivatable ligand, optovin.23–24 Although both optovin and TRPswitch target TRPA1 and are activated by violet light, they are unique in their mechanism of action, reversibility, and kinetics. Unlike optovin and its derivatives, which act though light-induced covalent modification, TRPswitch's photoreversible TRPA1 activity depends on differential activity of its E and Z isomers. How isomerization triggers opening and closing of the TRPA1 channel remains unknown. Mechanistically, the fifth transmembrane (TM5) domain of TRPA1 has been shown to determine channel sensitivity to non-electrophilic agonists such as menthol and anethole.49–50 Considering that menthol and anethole are structurally distinct molecules, we speculate that TRPA1's TM5 may act as a general binding site for non-electrophilic agonists and may therefore be a candidate site for the binding of TRPswitch. Alternatively, a peptidergic scorpion toxin (WaTx) was recently discovered that activates TRPA1 by binding to the same allosteric nexus that is covalently modified by electrophilic irritants.51 It is possible that TRPswitch binds to this allosteric nexus on TRPA1 channel as well.</p><p>The development of PCLs for endogenous targets offers theoretical advantages in clinical applications, since the introduction of exogenous gene products is not needed. The common approach for PCL discovery is to modify a known biologically active molecule with photoswitchable functionality, such as 'azologization'.52 Although this approach has had some success,11–15 it is limited by the need for already identified active molecules for specific targets, which have a defined mechanism of activation and that contain a chemical structure amenable to incorporation of a photoswitch. The screening strategy described here offers an alternative, phenotypic approach for the discovery of photoswitchable ligands of novel endogenous targets.53 Since screening is performed using intact animals, hit compounds identified are, by definition, biologically active and likely to have minimal general toxicity. In the case of TRPswitches, it is note-worthy that they are specific for the zebrafish Trpa1b channel and are inactive at the mammalian channels tested. It remains to be seen whether modification of the TRPswitch scaffold can yield ligands with different species specificity, but the current specificity for zebrafish Trpa1b has significant advantages, including the ability to use the TRPswitch/Trpa1b system in mammalian cells without unwanted cross-reactivity with endogenous channels.</p><p>It should also be noted that despite having excellent and tunable switching properties, heteroaryl azo motifs are still significantly underexplored compared to their azobenzene counterparts in photoaddressible applications including as PCLs.54 Our discovery that the azopyrazole-containing TRPswitch-B exhibits a longer half-life and more efficient photoswitching than the azobenzene-containing TRPswtich-A is a good indication that heteroaryl azo photoswitches are good alternatives for photoswitch optimization. Indeed, this is the first application of the high performance arylazopyrazoles 32, 40 for in vivo photopharmacology, showcasing the potential of this scaffold for future studies.</p><!><p>Zebrafish (Danio rerio) wild-type TuAB or trpa1b mutant 35 larvae were used for all experiments. Zebrafish embryos were produced using group mating of adult zebrafish. Larvae were raised in E3 media and maintained in an incubator with a 14 h light/10 h dark cycle at 28.5°C until experiments. The maintenance of adult animals, obtaining of embryos and larvae, and all experimental procedures were carried out according to protocols approved by the University of Utah's Institutional Animal Care and Use Committee (IACUC).</p><!><p>A total of 1,000 compounds were selected from the Chem-Bridge Corp. catalog. Compounds were dissolved in DMSO at a stock concentration of 1 μg/μl (~1.5 mM). The library was screened at a 1:100 dilution in E3 solution for a final concentration of ~15 μ.M. Negative controls were treated with an equal volume of DMSO. Groups of 3 larvae at 3 days post fertilization (dpf) were distributed into the wells of 96-well clear bottom black microplates (07-200-567; Fisher Scientific) in 150 μl E3 before the addition of small molecules. Stock solutions of compounds were added directly to zebrafish in the wells of a 96 well plate (Corning 3631), mixed, and allowed to incubate for 1 h in the dark at 28.5°C prior to behavioral evaluation in the behavioral assay. Ordering information: 1 (5533696; ChemBridge); 3 (5538408; ChemBridge). Other compound synthesis and characterization information are described in the supplementary note.</p><!><p>Larvae in each of the wells were exposed to four 1 second flashes of light stimulus in the following order: 450-500 nm (WL1), 415 – 455 nm (WL2), 352-402 nm (WL3), white light (WL4), with a 5 second inter-stimulus interval. Digital video recording was performed for 5 seconds before the first light stimuli and continued throughout the stimulation sequence. Band pass filters (Semrock FF02-475/50, FF02-435/40, FF01-377/50) were used to restrict the excitation light to the indicated wavelengths. Light-induced motion response was used as the assay readout as DMSO treated control larvae do not respond to light stimulus. 290 frames of digital video were recorded per well at 10 FPS using an EMCCD camera (C9100; Hamamatsu) mounted on an inverted compound microscope (AxioObserver A1; Zeiss) with a NA 0.03 1.25x objective and a barrier with a 0.7 mm diameter opening to restrict light scattering to the sample. MetaMorph software (Molecular Devices) was used to control the execution of TTL signals and camera capture using the built-in stream acquisition with trigger function. Each video was saved for review. Light stimuli were generated with an ozone free 300 Watt xenon bulb housed in a Lambda LS illuminator (Sutter Instruments). A dichroic mirror (T510LPXRXT; Chroma) was used for appropriate excitation of samples and bright field acquisition. Schott longpass absorption glass (RG610; Chroma) was added in the transmitted light path to reduce unwanted excitation to the well and to provide sufficient light for video recording. All behavioral experiments were conducted at room temperature. For Fig. 3c and 3d, experiments were performed using an inverted compound microscope (AxioObserver A1; Zeiss) equipped with an EMCCD camera (C9100; Hamamatsu), a violet LED light source (415 nm) with a CW 310 mW maximum output power source (BLS-LCS-0415-03-22; Mightex) which was controlled by a BioLED light source control module (BLS-SA02-US; Mightex), and a pulse master multi-channel stimulator (A30; World Precision Instruments). Bright field time-lapse was captured for 500 frames at ~103 FPS. A light pulse was applied at frame 20. For Fig. 3c, percentage responding was quantified based on whether there was any motion in the entire acquisition period. For Fig. 3d, response time is the duration from the beginning of the light pulse to the first movement of the larvae. Light intensity was measured using a hand-held laser meter (LaserCheck, Coherent 1098293). For Trpa1b ohnolog and paralog rescue experiments, Trpa1b−/− embryos were injected with ngn1:zTrpa1b-2A-mCherry, ngn1:zTrpa1a-2A-mCherry,ngn1:hTRPA1-2A-EGFP or ngn1:mTRPA1-2A-mCherry at the 1-cell stage for mosaic Rohon-beard neuron expression. Embryos were screened at 2 dpf for fluorescent expression in Rohon-beard neurons, incubated with 20 μM TRPswitch-B for 1 hr and decapitated posterior to the eyes right before experiments. NA 0.25 5x objective and 1 s WL3 light illumination was used.</p><!><p>To analyze digital video recordings, custom MetaMorph software scripts were used to automatically threshold the video to identify the area of larvae in each frame. Threshold images of the frames after each light activation event were overlaid to calculate a new combined threshold area. The motion index was calculated as the percentage change between the threshold area in the frame right before light activation and the new combined threshold area. This motion index correlates with the overall amount of motion in the well.</p><!><p>Immortalized HEK293T cells or human colonic fibroblast cells (CCD-18Co; ATCC) were used. HEK293T cells were plated on 12 mm cover slips and transiently transfected with 10 μg of Trpa1b plasmid (pCMV-zTrpa1b-FLAG) and 5 μg of mNeonGreen cDNA (Allele Biotechnology), then grown in a 6 cm plate for 24-48 h. CCD-18Co were cultured in Eagle's minimal essential medium with 10% FBS, 1x penicillin and streptomycin. CCD-18Co cells were plated on 12 mm cover slips and transiently transfected with 5 μg of zTrpa1b plasmid (pCMV-zTrpa1b-FLAG) together with 5 μg of mNeonGreen cDNA (Allele Biotechnology), then grown in a 10 cm plate for 48-72 h. After this time the cover slips were transferred to a recording chamber containing an extracellular solution composed of (in mM): 145 NaGluconate, 4 KCl, 3 MgCl2, 10 D-glucose, 10 HEPES; pH 7.4 adjusted with NaOH. The internal solution contained (in mM): 122 Cesium methane sulfonate, 1.8 MgCl2, 9 EGTA, 14 creatine phosphate (Na-salt), 4 MgATP, 0.3 NaGTP, 10 HEPES, pH 7.2 adjusted with CsOH. Borosilicate glass pipettes with a resistance of 3–5 MΩ were used. Whole cell currents were measured on an Axopatch 200B amplifier (Molecular Devices) under the control of pClamp software. Signals were digitized through a Digidata1550B interface (Molecular Devices). Currents were filtered at 5 kHz (lowpass, Bessel) and sampled at 10 kHz prior to analysis with Clampfit software (Molecular Devices). All the plots and statistical tests were performed on Excel (Microsoft corp.). Basal Trpa1b whole cell currents were measured with a ramp protocol (−100 to +100 mV, at a holding potential of 0 mV). Photoactive compounds were perfused into the recording chamber and a fluorescent light generated by mercury vapor short arc (U-HGLGPS, OLYMPUS) filtered through a ET-ECFP 434/17 nm filter and through a ET-mCherry 560 nm filter (Chroma Technology) was switched ON and OFF for 10 s. All experiments were performed at room temperature. The p values were calculated using a two-tailed, paired Student's t-test.</p><!><p>Heartbeat interruption experiments were performed in vivo on Tg(cmlc2:Trpa1b-2A-EGFP)24 larvae or Tol2-cmlc2-Trpa1b-2A-EGFP plasmid24 (Addgene plasmid #106405) injected larvae in a Trpa1b−/− background. Expression of the plasmid was obtained by microinjecting 2 nL of a solution containing 7 ng/μl of DNA plasmid and 6.5 ng/μL in vitro transcribed (Ambion) Tol2 transposase mRNA into the cytoplasm of one-cell stage embryos. At 2 dpf, larvae were pretreated with 20 μM of TRPswitch-A or TRPswitch-B in E3 at a final concentration of 1% DMSO for 1 h in the dark at 28.5°C before experimental manipulation. Immediately prior to the onset of experiments, E3 solution was replaced with 0.2 mg/mL tricaine (Sigma, A5040) in E3 for anesthetizing the larvae. Violet light (352-402 nm) and green light (500-600 nm) were used for E to Z and Z to E isomerization, respectively. Violet and green light excitation were achieved using a band pass filter FF01-377/50 and an et500lp long pass barrier filter, respectively, together with a t600plxxr dichroic mirror and a 300 Watt xenon bulb light source (Sutter Instrument). Experiments were performed using a NA 0.6 40x air objective at room temperature. The zebrafish heart in the whole field of view was illuminated with 1 s violet light followed by various lengths of green light as indicated. Ventricle width measurement was performed using the ImageJ 'measure' function on the widest outer ventricle width every 100 ms.</p><!><p>Compounds were dissolved in DMSO at 250 μM and 260 μM for TRPswitch-A and TRPswitch-B, respectively and UV-Vis absorbance was measured using a NanoDrop 1000 (Thermo Scientific).</p><!><p>All results are expressed as means ± SEM. Unless otherwise indicated, a two-tailed unpaired student's t test with Mann-Whitney post-test was used to determine p values. The criterion for statistical significance was p < 0.05. Statistical analysis was performed using Prism (GraphPad Software).</p>

PubMed Author Manuscript

Chemical Methods for the Simultaneous Quantitation of Metabolites and Proteins from Single Cells

We describe chemical approaches for integrated metabolic and proteomic assays from single cells. Quantitative assays for intracellular metabolites, including glucose uptake and 3 other species, are designed as surface-competitive binding assays with fluorescence readouts. This enables integration into a micro-array format with functional protein immunoassays, all of which are incorporated into the microchambers of a single-cell barcode chip (SCBC). Using the SCBC, we interrogate the response of human-derived glioblastoma cancer cells to EGFR inhibition. We report, for the first time, on both the intercellular metabolic heterogeneity as well as the baseline and drug-induced changes in the metabolite-phosphoprotein correlation network.

chemical_methods_for_the_simultaneous_quantitation_of_metabolites_and_proteins_from_single_cells

<p>The emergence of powerful single-cell genomic, transcriptomic, and proteomic tools over the past decade has yielded exciting approaches towards resolving the heterogeneity of complex biological systems.1–3 To date, most single cell tools have focused on transcriptome or proteome analysis, or on the sequencing of specific sets of genes. Quantitative single cell metabolic assays have proven more challenging, although there mass spectrometric methods are promising.4–6 No reports on the integration of metabolite assays with other classes of biomolecules from the same single cells have emerged. The challenge is that different classes of biomolecules require unique assay formats that are typically not compatible. However, such integration might deliver unique information that is not readily available from traditional assays. For the case of metabolites and functional proteins, such measurements could directly resolve connections between two important classes of oncology biomarkers: the elements of the protein signaling networks that are implicated in tumor maintenance and growth, and the small molecule metabolites that provide energy sources for cell growth, or participate in metabolic signal transduction. We report on chemical methods that permit microchip-based quantitative, multiplex assays of metabolites and proteins from statistical numbers of single cells.</p><p>Quantitative measurements (generating copy numbers per cell) of intracellular proteins can be accomplished using calibrated, sandwich-type immunofluorescence assays. Such assays require a surface-bound capture antibody and a fluorophore-labeled detection antibody, and yield an optical readout that correlates with protein copy number. These assays can be miniaturized and multiplexed through spatial addressing using the single cell barcode chip (SCBC) format. Metabolites are small molecules, and so cannot be similarly detected by antibody pairs. We report on three types of spatially-addressable competition assays designed to measure the absolute or relative levels of 4 small molecule metabolites, in a manner that allows those assays to be integrated into SCBC (or other) proteomic assays.</p><p>The SCBC platform, the metabolite competition assays, and calibration and validation data are provided in Figure 1. The SCBC (Fig 1a) consists of 310 1.5 nanoliter microchambers into which cells are loaded, and each of which contains a full barcode array. Each microchamber has a companion lysis buffer reservoir separated by a programmable valve (Supporting Figure S1).7,8 For protein assays, specific stripes in the barcode represent a spatial address upon which a sandwich immunofluorescence assay for a specific protein is executed. Each barcode stripe is initially patterned with a unique ssDNA oligomer, and the barcode is converted into an antibody array using the DNA-encoded antibody library (DEAL) approach (Supporting Fig S2).9 Unlike antibody staining assays, such assays can be calibrated in absolute terms, and each individual assay can be analyzed for cross-reactivity against all other assays. The demonstrated measurement error for the protein assays is <10%, as shown in our previous reports.3,8 For the metabolites, the basic challenge is to design assays that are also localized to a particular barcode stripe, yield a fluorescent output, and may be automatically executed using steps that are compatible with the protein assays. The competitive binding assays we implemented (Figure 1b, c) borrow concepts from certain commercial kits used for measuring metabolites from bulk cell culture.</p><p>For proof of principle, we chose two second messengers that are closely related to metabolic activities and intracellular signaling: cyclic adenosine monophosphate (c-AMP) and cyclic guanosine monophosphate (c-GMP). We also demonstrate the detection of glutathione (GSH), which is an important molecule for assessing cellular redox stress. For these metabolites, commercial capture antibodies exist, and those can be integrated into specific stripes of the barcode array using DNA-encoded antibody libraries (DEAL). The GSH assay (Fig 1b, ii) was designed around an anticipated intracellular concentration of GSH in the millimolar range (thus not requiring amplification), while assays for the lower-abundance c-GMP and c-AMP (Fig 1b, iii) were designed with signal amplification in mind. We prepared a variant of GSH labeled with a single Alexa Fluor 647 (AF647) dye at a specific site to avoid interference with antibody capture (Supporting Figure S3) Similarly, c-GMP and c-AMP were labeled with horse radish peroxidase at non-competitive sites. A known amount of these three labeled molecules is mixed with the lysis buffer. Upon cell lysis, these reagents compete with the target analytes released from the cell for the antibody binding sites. The fluorescence intensities recorded from the respective barcode addresses for these analytes inversely correlate with the intracellular concentrations of the target analytes. Fig 1d shows the standard calibration curves of the metabolites, in which the coefficient of variation (CV) across the entire detection range is less than 10%. The dynamic ranges of the assays can be tuned by varying the concentrations of the labeled competitors. This offers flexibility for adapting this detection scheme to different biospecimens of interest.</p><p>For measuring the level of glucose uptake, we developed a glucose-biotin conjugate (Gluc-Bio) as a glucose analog (Fig 1c, Supporting Fig S4). Similar to the clinically adapted glucose analog 18F-fluorodeoxyglucose (18F-FDG), Gluc-Bio molecules were actively taken up by cells as evidenced by showing that the uptake of Gluc-Bio could be inhibited by increasing the extracellular glucose concentration and also by decreasing the temperature (Supporting Fig S5). We additionally showed, using enzyme kinetics studies, that Gluc-Bio serves as a substrate for hexokinase (HK) (Supporting Fig S6) (similar to glucose and 18F-FDG). This allows the Gluc-Bio molecules to accumulate inside the cell in a similar fashion to 18F-FDG, and ensures the validity of using Gluc-Bio as a probe for glucose influx. To quantify the amount of Gluc-Bio cellular uptake, a Förster resonance energy transfer (FRET)-based competitive binding assay was employed (Fig 1c). Cells are first incubated in the medium containing Gluc-Bio and then washed to remove Gluc-Bio from the supernatant. Following cell lysis, the intracellular Gluc-Bio is released and binds to Alexa Fluor 555-labeled (AF555) streptavidin. Subsequently, the unoccupied binding sites on the streptavidin are filled using a Biotin-BHQ2 conjugate (Supporting Fig S7), which quenches the fluorescence of AF555 through an FRET process. Thus, the fluorescence intensity readout positively correlates to the amount of Gluc-Bio uptaken and released from the cell. Fig 1 d shows the standard curve of the Gluc-Bio (with <10% CV). The dynamic range of this assay can be tuned via varying the streptavidin concentration.</p><p>To further verify the validity of this Gluc-Bio assay, we performed side-by-side comparison with the gold standard 18F-FDG radioassay, analyzing bulk numbers of cells from a patient-derived glioblastoma (GBM) neurosphere tumor model (GBM39) for these measurements. GBM39 expresses the epidermal growth factor receptor (EGFR) variant(v)III oncogene, which renders signaling through the EGFR pathway constitutively activated and sensitizes it to EGFR inhibitors, such as erlotinib.10 EGFR inhibition reduces the consumption of glucose in tumor cells. In Fig 1e we plot the kinetic changes of glucose uptake in GBM39 cells in response of erlotinib inhibition, for both the Gluc-Bio competition assay and the 18F-FDG radioassay. The agreement between the assays supports the use of the Gluc-Bio assay for measuring glucose uptake. In related measurements, Gluc-Bio uptake correlated nicely the cellular abundance of the hexokinase 2 (HK2) enzyme, further confirming that Gluc-Bio is a substrate for hexokinases (Supporting Fig S8). Similar validations were performed on the other metabolites against commercial kits (Supporting Fig S9). Note that while the GSH, cAMP, and cGMP assays can yield absolute quantitation (i.e. copy numbers per cell), the Gluc-Bio assay, like 18F-FDG, only yields relative quantitation of glucose influx.</p><p>To demonstrate an SCBC that simultaneously quantitates metabolites and functional proteins, we interrogated single cells separated from the GBM39 neurosphere tumor model before and following 24 hours of erlotinib treatment. The assayed panel included the 4 metabolites and 7 metabolism-related proteins and phosphoproteins (Figure 2). A typical SCBC single-chip dataset generates ~100 single cell assays and ~100 0-cell (empty chamber) assays. The 0-cell assays provide an assessment of background signal levels and 2-cell assays provide signal validation (Supporting Fig S10). Two SCBC chips were used for each condition to improve statistics. Fig 2 shows one-dimensional scatter plots of the single cell data for each analyte and each condition investigated. Average values for each plot are indicated by the black horizontal lines. The single cell data are consistent with the immunofluorescence bulk assays on bulk GBM39 neurosphere tumor models, confirming that erlotinib significantly inhibits EGFR phosphorylation (Figure 2 insert), suppresses glucose uptake and hexokinase activities (Supporting Fig S8).</p><p>The SCBC datasets provide 3 independent sets of observables: the average analyte levels (Fig 2), the variances in distributions of those levels (Fig 2), and the correlations (or anti-correlations) between any two analytes (Fig 3a). For example, an average analyte level may be comparable before and after the drug perturbation in GBM39, while the statistical distributions could be altered (e.g. PKM2). Similarly, the levels of 2 uncorrelated proteins may be repressed by a drug, but the correlations between those proteins can increase (e.g. p-ACAC and HK2). Collectively, these three observables can be associated with the heterogeneity of cellular responses.11 For example, the identification of metabolic outliers or distinct metabolic phenotypes might provide clues for identifying cell populations with differential responses to drugging.12 In the untreated sample, we identified strong correlations between cAMP and cGMP,13 between glucose uptake and HK2,14 and between glucose uptake and GSH.15 Additionally, the disappearance of positive correlations between PDK and PKM2 in the treated sample implies a reduction of glycolysis, presumably due to the down regulation of p-Akt under erlotinib treatment.18. These observations are consistent with the cited literature, and so provide a validation of the platform.</p><p>Anti-correlations are more difficult to identify using bulk assays, but are clearly resolved between the second messengers cAMP, cGMP and both glucose uptake and HK2. In fact, an unsupervised clustering analysis of the entire SCBC data set for the untreated GBM39 resolves that two metabolic phenotypes dominate the measured cellular heterogeneity (Fig 3b): 80% of the cells exhibit high glucose uptake and low cAMP and cGMP, while 20% of the cells exhibit high cAMP and cGMP, but low glucose uptake (Supporting Fig S11a). Following 24h erlotinib treatment, the levels of glucose uptake and GSH, as well as the activities of HK2 were sharply reduced (Fig 3a).16,17, but the same two metabolic phenotypes, corresponding to the same fractions of the total population, are still resolved (Supporting Fig S11b). This opens up biological questions that will be pursued elsewhere, but also points to the value of such integrated proteomic/metabolite single cell assays for uncovering new biology.</p><p>We sought a final verification of the gluc-bio assay by comparing the measured erlotinib-induced changes in glucose uptake that were seen in the in vitro GBM39 neurosphere tumor model with in vivo 18F-FDG PET imaging of a GBM39 flank xenograft mouse model (Fig 3c, Supporting Fig S12). After 24 hrs erlotinib treatment, both assays reflected a reduction in glucose uptake. Thus, while the in vitro gluc-bio assay cannot be directly translated into an in vivo imaging assay, it appears to faithfully reproduce the 18F-FDG PET molecular imaging radioassay for in vivo glucose uptake within a closely related tumor model.</p><p>The chemical methods reported here provide an integrated approach towards quantitatively co-analyzing two important classes of biomarkers: proteins (including phosphoproteins), and metabolites from statistical numbers of single cells. The levels of those biomarkers, the metabolic heterogeneity, as well as the correlative interactions between metabolites and signaling proteins can be readily resolved with high accuracy, yielding rich information in cellular metabolic signal regulations and their response to drug perturbations. Although only 4 metabolites and 7 proteins were included in the demonstrations, the numbers of both classes of analytes can be significantly increased through minor variations of the SCBC platform used here.</p><p> ASSOCIATED CONTENT </p><p> Supporting Information </p><p>Synthetic schemes, experimental details and statistical methods. This material is available free of charge via the Internet at http://pubs.acs.org.</p><p>The authors declare no competing financial interests.</p>

PubMed Author Manuscript

A model for optical gain in colloidal nanoplatelets

Cadmium chalcogenide nanoplatelets (NPLs) and their heterostructures have been reported to have low gain thresholds and large gain coefficients, showing great potential for lasing applications. However, the further improvement of the optical gain properties of NPLs is hindered by a lack of models that can account for their optical gain characteristics and predict their dependence on the properties (such as lateral size, concentration, and/or optical density). Herein, we report a systematic study of optical gain (OG) in 4monolayer thick CdSe NPLs by both transient absorption spectroscopy study of colloidal solutions and amplified spontaneous emission (ASE) measurement of thin films. We showed that comparing samples with the same optical density at the excitation, the OG threshold is not dependent of the NPL lateral area, while the saturation gain amplitude is dependent on the NPL lateral area when comparing samples with the same optical density at the excitation wavelength. Both the OG and ASE thresholds increase with the optical density at the excitation wavelength for samples of the same NPL thickness and lateral area. We proposed an OG model for NPLs that can successfully account for the observed lateral area and optical density dependences. The model reveals that OG originates from stimulated emission from the bi-exciton states and the OG threshold is reached when the average number of excitons per NPL is about half the occupation of the band-edge exciton states. The model can also rationalize the much lower OG thresholds in the NPLs compared to QDs. This work provides a microscopic understanding of the dependence of the OG properties on the morphology of the colloidal nanocrystals and important guidance for the rational optimization of the lasing performance of NPLs and other 1-and 2-dimensional nanocrystals.

a_model_for_optical_gain_in_colloidal_nanoplatelets

Introduction<!>Sample characterization<!>Lateral area independent optical gain threshold<!>Optical density dependent OG threshold<!>Model of the optical gain threshold<!>Conclusions<!>Conflicts of interest

<p>Cadmium chalcogenide nanoplatelets (NPLs), CdX (X ¼ Se, S, Te), and their heterostructures have shown many novel properties, such as large absorption cross-sections, uniform 1D quantum connement, long biexciton Auger lifetimes, and giant oscillator strength. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] These materials have attracted intense interest for lasing applications due to the reported large gain coefficients and low optical gain (OG) threshold. [15][16][17][18][19][20][21][22][23][24][25] For example, the reported threshold of the amplied spontaneous emission (ASE) of the CdSe NPLs is as low as 6 mJ cm À2 , 15 which is over an order of magnitude lower than that in cadmium chalcogenide quantum dots (QDs) or QD heterostructures. 26,27 Although OG models for QDs are well understood, it is unclear whether they are applicable to 1D nanorods (NRs) and 2D NPLs because of the fundamental differences in their exciton properties. In 0D QDs, the excitons are conned in all three dimensions, whereas in 2D NPLs and 1D NRs, the excitons are free to move in the plane and along the long axis, respectively, which increases the degree of degeneracy of the band edge exciton states and may alter their gain mechanism. So far, there is not an OG model for 2D NPLs or 1D NRs, and the reasons for the superior OG properties in NPLs remain unclear. In addition, many other interesting differences between the NPLs and QDs may also contribute to their different OG properties. For example, because of the atomically precise thickness, the NPLs have uniform quantum connement energy and narrow exciton transition linewidth, which should reduce the overlap between the stimulated absorption (loss) and emission (gain) transitions. 3 It has also been argued that the exciton transition oscillator strength in NPLs may be enhanced by the coherent delocalization of the exciton center of mass in the lateral direction, which should affect the stimulated emission cross section. 3 The biexciton Auger recombination lifetimes in the NPLs are much longer than QDs 6,17 and have been shown to increase linearly with their lateral size. [28][29][30] It has been proposed that the low OG thresholds in NPLs can be attributed to their longer Auger lifetime. 17,19 These observations would suggest that one of the key differences between NPLs and QDs is the possibility of tuning their OG performance through their lateral size. Olutas et al. have reported that the ASE threshold of CdSe NPLs increases with their lateral area. 18 However, She et al. reported a lateral areaindependent ASE threshold of the same materials. 19 These contradictory observations and a lack of understanding of the OG mechanisms in NPLs suggests the need for a systematic study and a model for optical gain in these materials.</p><p>Herein, we report a systematic study of the dependence of OG on the lateral area and optical density at pump wavelength in 4 monolayer (ML) CdSe NPLs. We investigate the OG characteristics by femtosecond transient absorption (TA) spectroscopy of the colloidal NPL samples and ASE measurements of the NPL lms at room temperature. We show that the OG thresholds are independent of the lateral area of the NPLs, whereas the saturation OG amplitude increases linearly with the area when comparing samples of the same optical density at the excitation wavelength. For NPLs of the same size, their OG and ASE thresholds increase with the optical density at the excitation wavelength. We propose a biexciton gain model that can satisfactorily account for the experimental observations in the NPLs and explain the origin for their much lower OG thresholds compared to QDs. We believe that this model should also be applicable to other 2D nanosheets and 1D nanorods.</p><!><p>4 ML CdSe NPLs (with 5 Cd layers, 4 Se layers, and a thickness of $1.8 nm) were synthesized according to reported procedures with slight modications. 3 The lateral size of the NPLs was tuned by changing the synthesis temperature and reaction time. The detailed synthesis procedures are described in the ESI. † The NPL samples with different lateral sizes are named NPLa to NPLd with increasing lateral size. The same batch of samples have been used in a previous study of the lateral size dependence of the biexciton Auger lifetime in NPLs. 28 S1 †). The absorption spectra of NPLa to NPLd (solid lines, Fig. 1b) show A ($512 nm) and B ($480 nm) exciton peaks that correspond to the electron-heavy hole (e-hh) and electron-light hole (e-lh) transitions, respectively. All the NPL samples of different lateral sizes have the same A and B exciton transition energy. The static photoluminescence (PL) spectrum of NPLb (blue dashed line in Fig. 1b) shows a sharp band edge (e-hh) emission peak at $518 nm with a full width at half maximum (FWHM) of $38 meV. The PL spectra of NPLa to NPLd are compared in Fig. S3, † showing that the band edge emission is independent of the lateral size.</p><!><p>To determine the optical gain threshold, we carried out a pump uence dependent TA spectroscopic study of NPLa to NPLd in hexane at room temperature. In a TA measurement, the optical density of the samples under illumination is given by DA(l,t) + A 0 (l), where DA(l,t) is the pump-induced absorbance change shown in the transient absorption spectra and A 0 (l) is the static absorbance prior to excitation. Thus optical gain is achieved when DA(l,t) + A 0 (l) < 0. Because the gain threshold is dependent on the optical density of the sample (see below), to enable the comparison of the NPL samples of different lateral areas, their optical density at pump wavelength (400 nm) was controlled to the same value to ensure the same number of absorbed photons (Fig. 1b). The TA spectra of NPLc at the lowest pump uence (3 mJ cm À2 , Fig. S4a †) show long-lived bleach signals of A ($512 nm) and B ($480 nm) excitons. According to our previous work on CdSe NPLs, both A and B exciton bleaches can be attributed to state-lling on the rst electron level in the conduction band (CB), and the contribution of the hole state-lling in the valence band (VB) is negligible due to degeneracy and strong mixing between the denser hole levels in the VB, 14,30,31 similar to cadmium chalcogenide quantum dots and nanorods. [32][33][34] Fig. S4b † shows the TA spectra of NPLc at the highest pump uence (629 mJ cm À2 ) when the bleach amplitudes of the A and B exciton states at an early delay time have saturated. Compared to those at low pump uence, these spectra show an additional broad negative peak, DA(l,t) < 0, at energy lower than the A exciton ($520-560 nm), which can be attributed to the optical gain (OG) signal, 19 similar to that reported in CdSe quantum dots (QDs). 27 The gain spectra (ÀDA(l,t) À A 0 (l)) at 3-4 ps of NPLc at different pump uences are shown in Fig. 2a and an expanded view of the gain spectra (Fig. 2a inset) shows a broad OG peak with a maximum at $528 nm. The kinetics of the gain signal of NPLc at the OG peak wavelength ($528 nm) and with different pump uences are compared in Fig. 2b. All the kinetics show a negative signal around time zero (<1 ps), which reects a red-shied exciton absorption caused by exciton-exciton interaction. 14,20,[28][29][30][31] Aer 1 ps, the OG amplitude remains negative, indicating no OG, for pump uences below 27 mJ cm À2 . The OG amplitude increases with increasing pump uence and becomes positive, indicating As shown in Fig. 2b and S4, † the OG amplitude of all the samples reaches the highest value at a delay time of 3-4 ps, aer which the OG signals decay due to multiple exciton Auger recombination. 28 A plot of the maximum OG amplitudes (at 3-4 ps) as a function of the pump uence (Fig. S7b †) shows that for all the NPL samples, the OG reaches saturation at high pump uences, but the saturation OG value increases linearly with lateral area (Fig. 2c). To facilitate comparison of the gain threshold, we have scaled the OG of different samples to the same saturation amplitude and plotted the normalized OG as a function of the pump uence in Fig. 2d. The comparison shows that the normalized OG of all the NPL samples exhibits the same dependence on the pump uence: a linear increase of OG with pump uence between 15-150 mJ cm À2 and reaching saturation between 150-500 mJ cm À2 . As shown in the inset of Fig. 2d, the intercept of the OG amplitude on the x-axis yields the same OG threshold of 54.6 AE 1.8 mJ cm À2 for all the four samples under our experimental conditions (optical density of 0.31 AE 0.01 at 400 nm pump), independent of their lateral area.</p><!><p>To investigate how the OG threshold changes with sample optical density, we carried out TA study of the NPLc samples in hexane solution with different concentrations, named NPLc1 to NPLc4 in the order of increasing NPL concentration (NPLc3 is the sample used in Fig. 2). The absorption spectra of NPLc1 to NPLc4 (Fig. 3a) show that the optical density at 400 nm increases from 0.12 to 0.49 from sample NPLc1 to NPLc4. These samples were investigated using the same pump uence dependent TA measurement and analysis method described above. Their OG kinetics as a function of the pump uence are shown in Fig. S4. † Their peak OG amplitudes at 3-4 ps and $528 nm are plotted as a function of the pump uence in Fig. 3b. The intercept of these data on the x-axis yields OG thresholds of 43.0 AE 1.6, 52.5 AE 1.7, 54.6 AE 1.8, and 63.5 AE 2.2 mJ cm À2 for NPLc1 to NPLc4, respectively. As shown in Fig. 3c, these OG threshold values increase with the optical density at pump wavelength (400 nm). Fig. 3d shows that the saturation OG amplitude increases linearly with optical density at 400 nm, indicating more gain at saturation if more photons are absorbed. Similar optical density dependent ASE thresholds using NPLc lms prepared by spin-coating of NPLc solutions with different concentrations on a glass substrate were also observed (Fig. S5 †).</p><!><p>To explain the experimental results described above, we propose a model for OG in NPLs. The details of this OG model can be found in the ESI † and only the key aspects are summarized here. This model is an extension of the previous gain model proposed for QDs, 26 which, because of the connement in all three dimensions, can only accommodate two band edge excitons. In this model, we assume that because of the large (unconned) lateral dimension of NPLs, the number (N s ) of band edge (or A) excitons can exceed 2, increasing the complexity of the number of transitions associated with single and multiple band edge exciton states, as shown in Fig. 4 (for an example of N s ¼ 4). This assumption is based on our previous observations of NPLs, 20 and the 2D hydrogen-like exciton model in 2D structures. 35,36 On the basis of the redshi of OG and ASE from NPL emission, it has been proposed that OG or ASE in NPLs can be attributed to stimulated emission from band edge bi-exciton states, [17][18][19] similar to QDs. 27 Therefore, our model only considers band edge exciton states with 0, 1, . N s band edge excitons, which are labeled as 0, X, XX, . states, respectively, and their population probabilities are indicated by N i (i ¼ 0 to N s ). Each exciton state (i) can undergo stimulated absorption (upward arrows in Fig. 4) or emission (downward arrows in Fig. 4) with partial cross-sections per NPL of A i (i ¼ 0 to N s À1) and A * i (i ¼ 1 to N s ), respectively, given by eqn (1) and (2):</p><p>i from 0 to N s À 1;</p><p>(1)</p><p>In eqn ( 1) and ( 2), h is the Planck's constant, n r is the refractive index, and c is the speed of light. A T is the transition strength of the band edge excitons (e-hh) per NPL, which is proportional to the NPL lateral area, A QW . 2g i and 2g * i are the full width at half maximum of the absorption and emission spectra of the N i species, respectively. E i and E * i are the stimulated absorption and emission peak energy for the N i species, respectively. We set both g 0 and g * 1 to $19 meV for both single band edge exciton absorption and emission according to Fig. 1b. We assume both g iÀ1 and g * i (i from 2 to N s ) to be the same as the broad OG spectra ($50 meV) shown in Fig. 2a. E 0 and E * 1 are 2.42 eV and 2.39 eV, respectively, according to the A exciton absorption (512 nm) and emission (518 nm) wavelengths in Fig. 1b. The energy of bi-exciton absorption (E 1 ) and emission ðE * 2 Þ is assumed to be the optical gain energy, E OG , which is set to the OG peak value (2.35 eV, 528 nm) according to Fig. 2a. The inhomogeneous distribution of E OG is ignored due to the uniform 1D quantum connement of the NPLs. The shi (from E OG ) of the transition energies for the tri and higher exciton states is assumed to be much smaller than the transition line width:</p><p>This assumption is based on the broad transition width for the tri and higher exciton states and Coulomb screening of the multiexcitons reported in other 2D materials, 37 although these values have not been observed directly in our NPLs.</p><p>The absorption coefficient of the NPL ensemble at OG energy is:</p><p>where N en is the number of NPLs in the ensemble, which is proportional to the NPL molar concentration, C m . The population probability of the NPL species (N i ) is assumed to follow</p><p>Poisson distribution: P n ðmÞ ¼ m n e Àm n! , which represents the possibility of nding NPLs with n excitons when the average number of excitons per NPL is m. The optical gain threshold is achieved when a(E OG ) ¼ 0. Solving eqn (3) numerically under this condition leads to m th (N s ), the average number of excitons per NPL at the OG threshold, of 0.49 (AE0.01) N s (see Fig. S6 and Table S2 †). The result suggests that OG is reached when about half of the band edge exciton states are occupied. Under this condition, the gain (emission from excited states) equals the loss (absorption from ground states). Within the limits of QDs (N s ¼ 2), m th $ 1, which is consistent with previous ndings on QDs. 26 Because m is proportional to the pump uence (I) and the optical density at pump wavelength following Beer's law, the m th value can be converted to threshold pump uence, i.e. the OG threshold (I th ), according to eqn (4).</p><p>In eqn (4), OD ¼ 3zA QW C m L, hn is the pump photon energy (3.1 eV), N A is Avogadro's constant, A X ¼ A QW /N s , 3 is the molar absorption coefficient per unit NPL volume, z is the NPL thickness ($1.8 nm), L is the light path of the cuvette (1 mm) and m th /N s ¼ 0.49 AE 0.01. The details of the derivation can be found in the ESI. † According to eqn (4), when comparing NPL samples of the same thickness, their OG thresholds are independent of the NPL lateral area as long as the optical densities at pump wavelength are the same. This prediction is consistent with the experimental result shown in Fig. 2d. Moreover, the observed OD dependent OG and ASE thresholds can be well tted by eqn (4), as shown in Fig. 3c and S5f †, respectively, providing further support for our OG model.</p><p>At the limit of large m, the optical gain reached saturation with the gain amplitude given by eqn (5).</p><p>This predicts that the saturation gain amplitude increases linearly with both the lateral area (proportional to N s ) and optical density (OD) of the NPL (Fig. S7a †), both of which are consistent with the experimental ndings as shown in Fig. 2c and 3d, respectively.</p><p>Finally, our model (eqn (3)) also predicts how OG increases with the pump uence. The observed OG amplitude as a function of m can be reasonably well tted by our model (Fig. S7h †), although the simulated OG saturates at a lower value of m compared to the experimental results for NPLs with large N s (N s > 3). The origin of this deviation is not well understood, but it indicates that some loss factors are not fully accounted for in our model. This is likely due to the lack of consideration of a transition width distribution from higher exciton states in our model, which have not been experimentally observed.</p><p>There have been two contradicting reports on whether the ASE threshold depends on the NPL lateral area. 18,19 In ref. 19, the optical density at pump wavelength of different NPL samples was controlled to similar values, and the lateral areaindependent ASE thresholds were observed, 19 which is consistent with our experimental results and OG model. In ref. 18, the lateral area dependent ASE threshold was observed, but it is unclear whether the optical density at pump wavelength for samples of different NPL areas was controlled to the same values. 18 Our result suggests that optical gain is achieved when the average number of excitons per NPL is close to half (0.49) of the band edge exciton states, which is similar to the OG requirement in QDs. Despite this similarity, the optical gain thresholds in QDs have been reported to be more than an order of magnitude higher than those in NPLs. 15,26,27 According to our model, the lower OG threshold of the NPLs can be attributed to the following reasons. First, the intrinsic absorption cross section of NPLs, i.e. the absorption coefficient per unit volume (3), is larger than that of QDs, which according to eqn (4) leads to lower OG threshold. Recently, Achtstein et al. have reported that the intrinsic absorption coefficient of CdSe NPLs is over 3 folds larger than that in CdSe QDs due to the larger aspect ratio of NPLs. 38 Second, the ratio of biexciton binding energy ($40 meV) and transition linewidth ($38 meV), ðE * 1 À E * 2 Þ=2g * 1 , in NPLs ($1.0) is larger than that in QDs (<0.3) whose biexciton binding energy is <30 meV and transition linewidth is $100 meV. 26 This reduces the overlap between the gain and loss transitions, decreasing the OG threshold in NPLs. The latter can be attributed to the atomically precise uniform thickness of NPLs, which reduces the inhomogeneous broadening of the exciton transition energy. Such sharp transitions are difficult to achieve in QDs because of the broad size distribution and large inhomogeneous distribution of transition energies. In addition, the symmetry of the NPLs dictates that both the electric eld of the exciton and the dipole moments lie within the lateral plane, 39 which may account for the observed large Stark effect induced shi of transition energy between the bi-exciton and single exciton states.</p><!><p>In summary, we have systematically studied the dependence of the OG properties of CdSe NPLs on their lateral area and the optical density at pump wavelength using TA spectroscopy and ASE measurements. We show that the OG threshold is lateral area independent when comparing samples of the same optical density at the excitation wavelength, although the saturation OG amplitude increases with the lateral area. Furthermore, for samples of the same NPL size, the OG and ASE threshold increases with their optical density at pump wavelength. To account for these observations, we proposed an optical gain model for 2D CdSe NPLs. This model assumes that the number of band edge excitons scales with the NPL lateral area (and can exceed 2) and optical gain results from the stimulated emission from biexciton states. Our model successfully explains the experimental observations. The model also reveals that OG is achieved when the average number of excitons reaches $49% of the band edge exciton states. This OG requirement is similar to that in QDs, despite the observed OG threshold in NPLs being an order of magnitude smaller than that in QDs. According to our model, the lower OG threshold of NPLs can be attributed to their unique 2D morphology, which leads to a larger intrinsic absorption coefficient, narrower transition linewidth, and larger shi between the bi-and single-exciton state. This work provides not only important insights on how the crystal morphology affects the OG properties of the colloidal nanocrystals, but also guidance on the rational improvement of the OG and ASE in NPL materials for lasing applications. Finally, we believe that this OG model should be applicable to other 2D and 1D nanocrystals.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Efficacy of depatuxizumab mafodotin (ABT-414) monotherapy in patients with EGFR-amplified, recurrent glioblastoma: results from a multi-center, international study

PurposePatients with recurrent glioblastoma (rGBM) have a poor prognosis. Epidermal growth factor receptor (EGFR) gene amplification is present in ~ 50% of glioblastomas (GBMs). Depatuxizumab mafodotin (depatux-m), formerly ABT-414, is an antibody–drug conjugate that preferentially binds cells with EGFR amplification, is internalized and releases a potent antimicrotubule agent, monomethyl auristatin F (MMAF). Here we report the safety, pharmacokinetics, and efficacy of depatux-m monotherapy at the recommended Phase 2 dose (RPTD) in patients with EGFR-amplified, rGBM.MethodsM12-356 (NCT01800695) is an open-label study with three escalation and expansion cohorts. Sixty-six patients with EGFR-amplified, rGBM were treated with depatux-m monotherapy at 1.25 mg/kg intravenously every 2 weeks. Adults with measurable rGBM, who were bevacizumab-naïve, with EGFR amplification were eligible.ResultsAmong 66 patients, median age was 58 years (range 35–80). All patients were previously treated with radiotherapy/temozolomide. The most common adverse events (AEs) were eye related (91%), including blurred vision (65%), dry eye (29%), keratitis, and photophobia (27% each). Grade 3/4 AEs occurred in 42% of all patients, and ocular Grade 3/4 AEs occurred in 33% of patients overall. One patient (2%) had a Grade 4 ocular AE. Ocular AEs were manageable and usually resolved once treatment with depatux-m ceased. The objective response rate was 6.8%, the 6-month progression-free survival rate was 28.8%, and the 6-month overall survival rate was 72.5%.ConclusionDepatux-m monotherapy displayed frequent but mostly Grade 1/2 ocular toxicities. A PFS6 of 28.8% was observed in this rGBM population, warranting further study.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-017-3451-1) contains supplementary material, which is available to authorized users.

efficacy_of_depatuxizumab_mafodotin_(abt-414)_monotherapy_in_patients_with_egfr-amplified,_recurrent

Introduction<!>Materials and methods<!>Patients<!>Study design<!>Treatment regimen<!>Pharmacokinetics<!>Tumor molecular characterization<!>Statistical analysis<!>Patient characteristics<!><!>Safety of depatux-m<!><!>Safety of depatux-m<!><!>Safety of depatux-m<!>Resolution of ocular side effects<!><!>Pharmacokinetics<!>Efficacy of depatux-m<!><!>Efficacy of depatux-m<!>Discussion<!>

<p>Glioblastoma (GBM) is the most common malignant brain cancer with an incidence of 2–3 of every 100,000 adults per year. Patients afflicted with GBM have a poor prognosis, with a median survival of 14–16 months from original diagnosis [1, 2]. Many patients will experience recurrent disease (rGBM), and treatment options are limited, with survival under 12 months and rare responses [3]. Six-month progression-free survival rates exceeding 20–25% are considering promising in this setting [4].</p><p>Given the dismal survival rates in rGBM, there is an urgent need to develop effective novel therapies. Amplification of the Epidermal Growth Factor Receptor (EGFR) gene, observed in 50% of GBMs [5–7], creates a tumor-specific target for experimental treatment. About 50% of GBMs with EGFR amplification also harbor the EGFRvIII deletion variant [8]. Of note, EGFR amplification usually remains unchanged at the time of tumor recurrence [9]. Several types of targeted therapies have been used to target EGFR in GBM. Tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib have been found to increase PFS in non-small cell lung cancer [10] but have not proven efficacious in GBM, [11–16]. Antibodies that target and bind the extracellular domain of EGFR, such as cetuximab, have shown decreased tumor growth and increased survival in mouse xenograft models [17] but again, did not demonstrate a survival benefit in patients [18]. There are several explanations for the failures of these agents, in particular the absence of the EGFR exon 19 deletion and exon 21 mutations that are correlated with activity in NSCLC [19]. Immunotherapy has improved outcomes for many cancers, and the vaccine rindopepimut, which targets EGFRvIII, showed promising results in early stage testing in GBM. However, a Phase 3 trial was recently discontinued due to a lack of survival benefit [20]. Numerous ongoing studies are evaluating the potential activity of various immunotherapy agents, including vaccines and immune checkpoint inhibitors, in newly diagnosed and recurrent GBM.</p><p>Depatuxizumab mafodotin (depatux-m), formerly ABT-414, is an antibody–drug conjugate (ADC) composed of the EGFR-directed monoclonal antibody, depatuxizumab (depatux), formerly ABT-806, conjugated to the potent antimicrotubule agent monomethyl auristatin F (MMAF, now mafodotin) via a non-cleavable maleimidocaproyl linker [21, 22]. EGFR amplification leads to a unique conformation of the EGFR protein that exposes a tumor-specific binding site for depatux-m. This epitope is also exposed in the EGFRvIII deletion variant. Once depatux-m enters the cell, MMAF is released, leading to cell death. Depatux-m has limited binding to EGFR in normal tissues and thus does not lead to other toxicities typically associated with other EGFR-targeted therapies, usually dermatological [23]. Preclinical data suggest that depatux-m has potent anti-tumor activity in GBM cell lines and xenograft models [6].</p><p>Recently published results from this study show that depatux-m in combination with either chemoradiation or TMZ in both newly diagnosed and recurrent GBM has a tolerable safety and pharmacokinetics (PK) profile [24, 25]. Here, we present efficacy data, including objective response rate (ORR) and PFS6, for depatux-m monotherapy at the recommended Phase 2 dose (RPTD) in patients with recurrent, EGFR-amplified GBM.</p><!><p>Study M12-356 (NCT01800695) was a multi-center, Phase 1, open-label study designed to evaluate the safety, preliminary efficacy and PK of depatux-m alone or in combination with other treatments in patients with GBM. The trial had three treatment arms: Arm A, depatux-m with radiation therapy (RT) and temozolomide (TMZ) in newly diagnosed GBM; Arm B, depatux-m with TMZ after RT in newly diagnosed or recurrent GBM; and Arm C, depatux-m monotherapy in rGBM. Each arm was composed of a dose escalation and dose expansion cohort [24]. This study was performed in accordance with the 1964 Declaration of Helsinki and its later amendments. All patients provided written informed consent prior to enrollment according to national regulation; the study design was approved by the Institutional Review Board/Ethics Committees of participating institutions.</p><!><p>This analysis encompassed 66 patients from Arm C who had EGFR-amplified, rGBM and received at least one dose of depatux-m at the RPTD of 1.25 mg/kg. Inclusion and exclusion criteria were as described previously [24]. Only patients with rGBM and centrally confirmed EGFR amplification were included. More specifically, patients had Response Assessment in Neuro-Oncology (RANO) defined [26] disease progression which included either: (1) measurable progressive or rGBM as seen by contrast-enhancing MRI and an interval of at least 12 weeks from completion of RT to study entry; (2) progression outside the radiation field; or (3) biopsy or surgically proven disease progression. An MRI with contrast was required within 14 days of Study Day 1, and patients were required to be on a stable or decreasing dose of corticosteroids for at least 5 days prior to the scan. Patients were ineligible if: they had received bevacizumab as prior treatment for rGBM, had a secondary GBM, or had been exposed to prior EGFR therapy for GBM, including EGFRvIII-specific immunotherapies.</p><!><p>Study design of M12-356 has been described previously [24, 25]. The primary objective was to determine the ORR [partial response (PR) + complete response (CR)]. The secondary objectives were to determine the PFS6, PFS, OS, and safety and tolerability of depatux-m.</p><!><p>The RPTD of depatux-m monotherapy was determined previously as 1.25 mg/kg via intravenous (IV) infusion every 2 weeks [25]. All patients received 1.25 mg/kg of depatux-m via intravenous infusion over 30–40 min on Days 1 and 15 of a 28-day cycle (Supplementary Fig. 1). Radiographic assessment of disease progression was performed before every other cycle. Treatment was intended to continue until either intolerable toxicity or disease progression as assessed locally by the investigator using RANO criteria [26]. Central review was not performed. Depatux-m dosing could be reduced to 1.0 or 0.5 mg/kg for Grade 3/4 toxicities. Re-escalation was permitted.</p><!><p>Serum samples for the determination of depatux-m concentrations were collected before and immediately after depatux-m infusions on Day 1 of Cycles 1 and 2, and before depatux-m infusions on Day 15 of Cycles 1 and 2. Serum samples for the determination for anti-drug antibody (ADA) were collected biweekly before each depatux-m infusion up to Day 1 of Cycle 3 and once every four weeks before depatux-m infusion in the subsequent cycles. For patients who were able to return to the clinic for the follow-up visit, ADA samples were also collected approximately 35 days after the last depatux-m infusion.</p><p>Depatux-m serum concentrations and ADA titers were determined using validated electrochemiluminscence immunoassays [24]. The depatux-m concentrations in the Arm C expanded cohort were compared to those in the Arm C dose escalation cohort only with intensive pharmacokinetic sampling [24].</p><!><p>Molecular characterization of archival tumor tissue, including testing performed to determine EGFR expression, amplification, and EGFRvIII mutation status before protocol therapy was performed as described previously [24]. Briefly, fluorescence in situ hybridization (FISH) was used to detect locus-specific EGFR amplification. Two probes were employed: Vysis Locus Specific Identifier (LSI) EGFR SpectrumOrange Probe, and Vysis Chromosome Enumeration Probe (CEP) 7 SpectrumGreen Problem (Abbott Laboratories, Abbott Park, IL, USA). To call a tumor EGFR amplified, the sample should show ≥ 15% tumor cells with an EGFR/CEP 7 ratio ≥ 2.</p><!><p>Descriptive statistics were provided for patient demographic variables. Safety/toxicity summaries were provided for all patients who received at least one dose of depatux-m. Frequencies of adverse events (AEs) were tabulated by the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE, version 4.1) and listed by MedDRA (version 19) system organ class and preferred term. Responses were assessed per RANO criteria. The primary efficacy endpoint was objective response rate (ORR, complete response (CR) and partial response (PR)) and was determined for patients with measurable disease at baseline. The secondary endpoints included PFS6, PFS, OS, safety and tolerability. PFS was defined as the time period from the first dose of depatux-m to RANO-defined disease progression or date of death, if disease progression did not occur. OS was determined from the time of first dose of depatux-m to death from any cause. Ninety-five percent confidence interval (CI) was constructed for the estimated ORR (determined from the exact binomial distribution), PFS, and OS. The Greenwood formula was used to calculate the confidence limits for the quartiles of survival distribution (PFS and OS).</p><!><p>As of 15 March 2017, enrollment was completed with 66 patients. The median age was 58 years. Forty-one percent were women and 59% were men. All patients had EGFR-amplified rGBM, and were previously treated with RT/TMZ (Table 1). Thirty-one patients (47%) received depatux-m as the first treatment after initial RT/TMZ. Thirty-one (47%) had tumors which harbored an EGFRvIII mutation (Table 1), which is similar to previously reported mutation rates of 50% in patients with EGFR-amplified GBM [8].</p><!><p>Patient demographics</p><p>aNot enough tissue available for testing</p><!><p>All patients received depatux-m at the RPTD of 1.25 mg/kg [25]. Sixty-four of 66 patients experienced at least one AE (Table 2). Nearly, all patients (91%) experienced at least one ocular AE. The most frequent included blurred vision (65%) and dry eye (29%). The most common non-ocular event was fatigue (33%).</p><!><p>All adverse events (AEs)</p><!><p>Forty-two percent of patients experienced a Grade 3/4 AE, with ocular Grade 3/4 adverse events (AEs) due to microcystic keratopathy being the most common (35%, Table 3). Ocular-related Grade 3/4 AEs included keratitis (17%), corneal epithelial microcysts (8%), blurred vision (5%), and reduced visual acuity (5%). Non-ocular Grade 3/4 AEs occurred in 15% of patients. A further breakdown of all ocular AEs by grade (1/2 vs. 3/4, Supplementary Table 1) showed that the majority were Grade 1/2. Only 1 Grade 4 AE of reduced visual acuity was observed. A serious AE was observed in 36% of patients (Supplementary Table 2), with seizure (9%) occurring most frequently. Two serious AEs were assessed by the investigator as having a reasonable possibility as being attributable to depatux-m. These included one case of seizure and one case of cerebrovascular accident, which are not uncommon in patients with rGBM.</p><!><p>Grade 3/4 AEs having a reasonable possibility as being depatux-m-related</p><p>Per investigator assessment</p><!><p>All patients had discontinued depatux-m at the time of analysis, the majority for disease progression (85%). Two patients discontinued for an AE related to progression, and eight patients (12%) discontinued for an AE unrelated to disease progression. These included four patients with ocular side effects, two with thrombocytopenia, one with proteinuria, and one with left-sided muscle weakness. Interruption of depatux-m dosing occurred in 33/66 patients (50%), with the most common reason for interruption due to ocular side effects in 25/66 patients (38%). Finally, 21 patients (32%) underwent a dose reduction of depatux-m due to an AE, with ocular AEs again the most common reason for reduction in 19/66 patients (29%). Fifty-six patients (85%) died during the course of the study.</p><!><p>As mentioned, ocular side effects were very common in patients. The type and severity of ocular AEs varied, but all were attributed to generalized microcystic keratopathy, which is observed with some types of ADCs (see Discussion). Although the ocular side effects were common, they resulted in treatment discontinuation in only 4 patients (6%). The median time to onset of any ocular side effect was 3.2 weeks (95% CI 2.6, 3.6), as determined from all 66 patients. There was a trend toward reversibility of ocular side effects (Fig. 1); however, a precise definition of a median time to resolution could not be established, due to confounding factors including time on study.</p><!><p>Kaplan–Meier curve of time to resolution of ongoing ocular AEs (all grades) in patients after discontinuation of depatux-m. Time to resolution was defined as the number of days from the last dose date to the last end date of all ocular AEs</p><!><p>The PK of depatux-m in the Arm C expanded cohort was consistent with that in the Arm C dose escalation cohort [25] for both Cycle 1 and Cycle 2 (Supplementary Fig. 2). No ADA was detected or confirmed in any sample during therapy (n = 60 patients with at least one post-treatment ADA result) or at final follow-up visit (n = 13 patients with ADA results at final follow-up, which was not mandated).</p><!><p>Sixty of 66 patients had at least one post-baseline assessment allowing determination of change in tumor size (Fig. 2). Per RANO criteria, a best response of stable disease (SD) was observed in 27/66 patients (41%) and 34/66 patients (52%) had a best response of progressive disease (PD, Fig. 3). Of patients with measurable disease at baseline, the ORR was 6.8% (1/59 CR, 3/59 PR, 95% CI 1.9%, 16.5%). The median duration of response in 66 patients was 6.7 months (95% CI 1.6, 8.1).</p><!><p>The percent change in target lesion from baseline are shown for 60/66 patients who had at least one post-baseline measurement. Best tumor percent change is defined as the maximum reduction/minimum increase from baseline in tumor size. Values were determined per investigator measurements</p><p>The best responses as determined by the investigator using RANO criteria and time on depatux-m therapy are shown for 65/66 patients with available data. One patient had a baseline assessment but discontinued before the first follow-up, and is not included in this analysis</p><!><p>The PFS6 was 28.8% (95% CI 18.5, 39.9%) and median PFS was 1.7 months (95% CI 1.4, 3.3). The OS6 was 72.5% (95% CI 60.0, 81.7%) and median OS was 9.3 months (95% CI 6.6, 11.7). For patients with EGFRvIII mutation (n = 29 patients with available response data), the PFS6 was 17.2% (95% CI 6.3%, 32.7%) and median PFS was 1.6 months (95% CI 1.4, 3.3).</p><!><p>Depatux-m monotherapy administered at the RPTD of 1.25 mg/kg in patients with EGFR-amplified rGBM demonstrated a PFS6 of 28.8% and OS6 of 72.5%, benchmarks [27] that suggest this drug could show benefits above current standard of care agents. These results, combined with a similar PFS6 of 27.1% in patients with EGFR-amplified rGBM treated with depatux-m alone or with TMZ (n = 126, which includes patients from this analysis and patients from Arm B) [28], suggest that further investigation of depatux-m in this population is warranted. Ten patients remained on treatment for more than 9 months (Fig. 3), suggesting that despite the ocular side effects, treatment was tolerated for a prolonged period of time. All 10 patients experienced typical ocular AEs, which were mainly Grade 1/2. Six of the ten had Grade 3 keratitis, one had Grade 3 corneal microcysts, and one had Grade 3 reduced visual acuity. One patient had proteinuria and one patient had significant neutropenia, both of which were managed by depatux-m dose interruption and the latter also by dose reduction.</p><p>The occurrence of microcystic keratopathy is a very predictable side effect in treatment with ADCs, particularly those with the MMAF toxin [29, 30]. It is not clear why certain ADC payloads induce such specific eye sensitivity, but it could be related to drug accumulation within various ocular tissues. Ocular side effects have been observed in the other arms of this study as well [24, 25]. Ocular side effects were generally manageable with dexamethasone eye drops, corneal bandages, dose reductions, and delays. Other prophylactic measures are being investigated, but have not yet been fully evaluated. Although ocular side effects were frequent, the majority of patients (56%, Supplementary Table 1) experienced only Grade 1/2 side effects, and only 6% of patients discontinued due to an ocular AE. Based on the details of the ten patients who had SD for more than 9 months mentioned above, the severity of ocular side effects did not correlate with time on therapy. Side effects did improve upon treatment discontinuation; however, the median time to resolution was unreliable based on data for a limited number of patients and is thus confounded by competing risks. These include patients who discontinued follow-up for progressive disease, initiated alternative therapies, or died, all of which led to a high censoring rate. Of note, six patients experienced complete resolution of ocular side effects before completion of study treatment.</p><p>Additionally, in this EGFR-amplified population, EGFRvIII mutation did not seem to further differentiate responders from non-responders, or patients likely to experience a PFS event within 6 months. Increased patient numbers are required to further understand the impact that depatux-m may have on the EGFRvIII vs. EGFR wild-type-amplified populations.</p><p>To conclude, we observed in this multicenter, dose expansion study that depatux-m monotherapy administered at the RPTD in patients with EGFR-amplified, rGBM demonstrated promising efficacy and manageable toxicity, indicating that further study of this novel targeted therapy in GBM is justified. Two other global, randomized trials are ongoing: depatux-m or placebo + RT/TMZ in EGFR-amplified, newly diagnosed GBM (INTELLANCE 1, NCT02573324); and depatux-m vs. depatux-m + TMZ vs. TMZ/lomustine in EGFR-amplified, rGBM has completed accrual with results expected in late 2017 (EORTC 1410-BTG, INTELLANCE 2, M14-483, NCT02343406).</p><!><p>Below is the link to the electronic supplementary material.</p><p>Supplementary material 1 (PPTX 62 KB)</p><p>Supplementary material 2 (DOCX 17 KB)</p><p>Electronic supplementary material</p><p>The online version of this article (doi:10.1007/s00280-017-3451-1) contains supplementary material, which is available to authorized users.</p>

PubMed Open Access

A Sensitive and Efficient Method for Determination of Capecitabine and Its Five Metabolites in Human Plasma Based on One-Step Liquid-Liquid Extraction

Colorectal cancer is the most common critical disease both in the developed and developing countries. Capecitabine, which has served in clinical practice at least for 10 years, is a first-line antidigestive tract cancer drug for its better efficacy, patient compliance, and lower side effects. An ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been developed and completely validated for simultaneous determination of capecitabine and its five metabolites in human plasma from colorectal cancer patients after administration of capecitabine tablet. One-step liquid-liquid extraction was successfully applied using ethyl acetate and isopropanol (19 : 1, V : V) for sample pretreatment. Chromatographic separation was achieved within 5 min based on an Atlantis T3-C18 column (3.0 µm, 2.1 × 100 mm) with gradient elution using mobile phases consisting of 0.0075% formic acid in water (pH 4) and in acetonitrile, and the flow rate was 0.3 mL/min. Linear range was approximately 20.0–5000.0 ng/mL for all analytes. Linear correlation coefficients were >0.99 for all regression curves. The intraday and interday accuracy and precision of the method were within ±15.0% and less than 15.0%, respectively. The mean recovery and matrix effect as well as stability of all the analytes ranged from 59.27% to 90.15% and from 74.84% to 114.48% as well as within ±15.0%. This simple, rapid, and sensitive method was successfully applied in 42 sparse clinical samples to verify its practicability.

a_sensitive_and_efficient_method_for_determination_of_capecitabine_and_its_five_metabolites_in_human

1. Introduction<!>2.1. Chemicals and Reagents<!>2.2. LC-MS/MS Instrumentation<!>2.3. Liquid Chromatographic Conditions<!>2.4. Mass Spectrometry Conditions<!>2.5. Preparation of Standard and Quality Control Samples<!>2.6. Sample Pretreatment<!>2.7. Human Sample Collection<!>2.8. Method Validation<!>3.1. Chromatography Condition Optimization<!>3.2. Sample Pretreatment<!>3.3.1. Specificity<!>3.3.2. Linearity of Calibration Curves and LLOQ<!>3.3.3. Inter- and Intraprecision and Accuracy<!>3.3.4. Matrix Effect and Recovery<!>3.3.5. Stability<!>3.4. Application in Determination of Clinical Samples<!>4. Conclusion

<p>Colorectal cancer is the leading cause of death in both developed countries and developing countries, and heavy social and economic burden have been brought by this malignant disease. According to the GLOBOCAN2012, colorectal cancer ranked third in new cancer cases worldwide, and second in the developed countries, with estimated 1.4 million cases and 693,900 deaths occurred in 2012 worldwide [1]. In China, there were 310,244 new colorectal cancer cases, which ranked fourth in all new cancer cases (9.20%), and the people who died of colorectal cancer was 149,722 (7.09%), ranking fifth in all cancer death, according to the Chinese Cancer Registry Annual Report. From 1998, the morbidity and mortality of colorectal cancer has been gradually increasing in China [2]. Capecitabine (Cap), which was approved in 2005 by the FDA for the treatment of Dukes' C stage colorectal cancer in adjuvant setting, has turned into the cornerstone for anticolorectal cancer as a prodrug of 5-FU in recent years [3]. As an oral administration prodrug, three metabolic steps were needed to catabolize itself to the active agent 5-FU both in liver and target cells. After administration, Cap was almost totally absorbed in the intestine as an intact molecule and then transferred to the liver by circulatory blood. In liver, Cap was metabolized by carboxylesterase to 5'-deoxy-5-fluorocytidine (5'-DFCR), and then, cytidine deaminase transforms the 5'-DFCR to doxifluridine (5'-DFUR), and this step could also be detected in other normal or tumor tissues. 5'-DFUR would be preferably metabolized to 5-FU in tumor tissue by activity-enhanced thymidine phosphorylase with more than 10 times higher concentration of product compared with other normal tissue [4]. This partial targeted drug delivery was believed to improve the treatment efficacy and tolerance of Cap. It had been reported that approximately 80% of 5-FU is catabolized to inactive product dihydrofluorouracil (FUH2) by the rate-limiting enzyme dihydropyrimidine dehydrogenase (DPD) in liver, and declined activity of DPD often caused a longer retention of 5-FU [5–7]. Finally, FUH2 would be excreted along with urine as α-fluoro-β-alanine. The antitumor activity of 5-FU usually worked through three metabolic pathways: the first pathway was the anabolism of 5-FU to fluorouridine triphosphate and incorporating the product into RNA, and finally damage the structure of RNA; the second pathway was similar with the first mechanism and deoxyfluorouridine triphosphate was produced to damage the DNA structure; the third way was anabolism of fluorodeoxyuridine monophosphate from fluorouridine monophosphate or 5-fluoro-2'-deoxyuridine (2'-DFUR), and then a ternary complex would be formed with thymidylate synthetase and folic acid to suppress the synthesis of thymidine. Thus, it blocked the in vivo synthesis pathway of DNA (Figure 1). Due to a long metabolic pathway and the interindividual genetic variations, the pharmacokinetic parameters of Cap and its metabolites showed significant differences in patients, and therapeutic drug monitoring of Cap and its metabolites was not applied in clinical practice because of its specific regimen, lack of associations between plasma exposure and clinical response and/or toxicity, and the difficulties of simultaneously quantifying Cap and its metabolites. It is difficult for clinicians to precisely evaluate the exposure level of Cap and its metabolites without proper pharmacokinetic parameters. Several LC-MS/MS methods had been developed to solve this problem [8–14], but the relative high requirement of instruments and/or complicated operations and/or long analytical time has delayed their clinical application (Table 1). Thus, the aim of this study was to develop a simple, rapid, and sensitive method for simultaneous determination of Cap and its five metabolites in human plasma and verify its clinical application.</p><!><p>All analytes including Cap (Lot: J0713AS), 5'-DFCR (Lot: J1112AS), 5'-DFUR (Lot: J0713A), 2'-DFUR (Lot: J0620A), 5-FU (Lot: A0930AS), fludarabine (Fdb) (Lot: M0501AS) and 5-chlorouracil (5-ClU) (Lot: J1204A) but excepting FUH2 (Lot: 5-PTR-167-1) were purchased from Meilun Biotech Co., Ltd (Dalian City, China). FUH2 was supplied by Toronto Research Chemicals (Toronto, Canada). HPLC-grade acetonitrile was obtained from Merck (Merck Company, Darmstadt, Germany). HPLC-grade formic acid, dimethyl sulfoxide (DMSO), and ammonium acetate were purchased from Tedia Company Inc (Tedia, Fairfield, OH, USA). Ultrapurified water (0.22 µm filter membrane) was self-made in laboratory on a Milli-Q Reagent Water System (Millipore, MA) and was used throughout. HPLC-grade isopropanol and ethyl acetate were bought from Thermo fisher (Thermo Fisher Scientific, Waltham, MA) and Caledon laboratories (Caledon Laboratories Ltd, Georgetown, Canada). Human blank plasma was donated by Shanghai Red Cross Blood Center (Shanghai, China).</p><!><p>All experiments were performed on Agilent 1260 series UHPLC system, which included an online degasser, a binary pump, an autosampler, and column oven and interfaced to an Agilent 6460A triple-quadrupole mass spectrometer equipped with an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA). All raw data were acquired and analyzed using Agilent Masshunter data processing software (version B.06.00; Agilent Technologies, Santa Clara, CA, USA).</p><!><p>The optimized chromatographic conditions were completed on an Atlantis T3-C18 analytical column (3.0 µm, 2.1 × 100 mm; Waters Co, Milford, CT, USA). The column was equilibrated and eluted under gradient phases containing 0.0075% formic acid in water (phase A, pH4) and in acetonitrile (phase B), and the flow rate was 0.3 mL/min. The mobile phases were degassed automatically using the online degasser system. The gradient variation started with 0% phase B. Within 0.5 min, the phase B escalated to 10% and maintained until 2 min and then sharply rose to 90% at 3 min and maintained until 5 min. The total run time was 5 min. The column temperature was maintained at 35°C. The injection volume was 5 µL with a 3-second needle wash using 5% methanol aqueous solution.</p><!><p>The mass detection was achieved using electrospray ionization both in the positive and negative modes with the capillary voltage set at 4000 V. Nitrogen was utilized as drying gas, nebulizer gas, and sheath gas. Drying gas was heated to 300°C and delivered at 10 L/min. The temperature of sheath gas was set at 300°C, and the flow rate was 12 L/min. Nebulizer pressure was set at 45 psi. High purity nitrogen served as collision gas at a pressure around 0.2 MPa. Data acquisition was performed in the multiple reaction monitoring (MRM) mode (Figure 2). Table 2 shows the optimized MRM parameters for Cap and its five metabolites. The peak widths of precursors and product ions were maintained at 0.7 amu at half-height of peak in the MRM mode, and the dwell time for all analytes was 80 ms.</p><!><p>The stock solutions of Cap and its metabolites 5'-DFCR, 5'-DFUR, 2'-DFUR, 5-FU, and FUH2 as well as internal standards (IS) 5-ClU and Fdb were individually prepared in methanol. 2.04, 2.00, 2.04, 2.20, 2.06, and 2.02 mg of Cap, 5'-DFCR, 5'-DFUR, 2'-DFUR, 5-FU, and FUH2 were accurately weighed and dissolved in methanol. Several drops of DMSO were added into the FUH2 solution, and the final volumes of all stock solutions were 2.00 mL. All stock solutions were subpackaged and stored at −80°C. The stock solution of each analyte was further diluted with 10% methanol to obtain a series of work solutions at the following concentrations: 204.0, 510.0, 1020.0, 5100.0, 10200.0, 25500.0, and 51000.0 ng/mL for Cap and 5'-DFUR; 200.0, 500.0, 1000.0, 5000.0, 10000.0, 25000.0, and 50000.0 ng/mL for 5'-DFCR; 220.0, 550.0, 1100.0, 5500.0, 11000.0, 27500.0, and 55000.0 ng/mL for 2'-DFUR; 206.0, 515.0, 1030.0, 5150.0, 10300.0, 25750.0, and 51500.0 ng/mL for 5-FU; and 202.0, 505.0, 1010.0, 5050.0, 10100.0, 25250.0, and 50500.0 ng/mL for FUH2. Calibration standards were prepared by 10 times dilution of the corresponding combined working solutions with blank human plasma to obtain final concentrations in the range of 20.4–5100 ng/mL for Cap and 5'-DFUR; 20.0–5000.0 ng/mL for 5'-DFCR; 22.0–5500.0 ng/mL for 2'-DFUR; 20.6–5150.0 ng/mL for 5-FU; and 20.2–5050.0 ng/mL for FUH2. Quality control (QC) samples were also prepared in the same way (20.4, 51.0, 1020.0, and 2550.0 ng/mL for Cap and 5'-DFUR; 20.0, 50.0, 1000.0, and 2500.0 ng/mL for 5'-DFUR; 22.0, 55.0, 1100.0, and 2750.0 ng/mL for 2'-DFUR; 20.6, 51.5, 1030.0, and 2575.0 ng/mL for 5-FU; 20.2, 50.5, 1010.0, and 2525.0 ng/mL for FUH2). The QC samples were stored at −20°C and brought to room temperature (25°C) for thaw before pretreatment. The IS stock solutions were prepared in the same way. 2.32 mg Fdb and 1.84 mg 5-ClU were individually dissolved in 2 mL methanol, and finally combined IS solution was freshly prepared in the extraction solvent at a concentration of 30 ng/mL for both Fdb and 5-ClU before use. The Fdb was utilized as IS for Cap and 5'-DFCR, 5'-DFUR, 2'-DFUR, and 5-ClU was the IS for 5-FU and FUH2.</p><!><p>For all analytes, sample pretreatment was performed by a one-step liquid-liquid extraction procedure. A 100 µL aliquot of sample was transferred to a 10 mL glass centrifuge tube prior to adding 3 mL extraction solvent (ethyl acetate : isopropanol = 19 : 1, vol : vol). After 3 min of vortex-mixing, tubes were centrifuged at 1710 × g for 10 min at room temperature. Then, 2.7 mL of organic phase was drawn and transferred to 5 mL plastic centrifuge tubes and evaporated by a SCANVAC Freeze Dryer (Labogene, Shanghai, China) at 1000 × g, 73 bar, and room temperature for 30 min. The residual was reconstituted with 100 µL of 10% methanol aqueous solution, and 5 µL of the reconstituted solution was injected directly to the UHPLC-MS/MS system for analysis.</p><!><p>This research protocol was approved by the Ethical Committee of Changzheng Hospital (Shanghai, China) and performed in the Changzheng Hospital. Informed consent was signed by all recruited patients. Samples were solely utilized to validate this method, and sparse sampling points were applied to collect clinical samples. Venous blood samples were collected in EDTA-3K collecting tubes and gently shaken and then immediately centrifuged at 1710 × g for 10 min. The plasma was collected and stored at −80°C until analysis.</p><!><p>Method validation, including specificity, linearity, inter- and intraprecision and accuracy, matrix effect, recovery, carryover, and stability, was performed according to the Chinese pharmacopeia (version 2015).</p><p>For specificity, comparisons of responses in spiked and blank samples from at least six different lots were performed. The responses of interferents not more than 20% of LLOQ sample and 5% of IS were acceptable.</p><p>Matrix effect and recovery were assessed in six replicates at two (low and high) concentration levels for all analytes, and 50 and 2500 ng/mL were chosen as the low and high concentration levels. The matrix effect was the ratios of peak area in the spiked postextraction samples to the peak area in solvent-substituted samples in the same concentration, and the recovery was the ratio of peak area in the spiked samples to the peak area in spiked postextraction samples in the same concentration. The IS was assessed for the matrix effect and recovery at 1000 ng/mL.</p><p>Inter- and intraprecision and accuracy were assessed in five replicates at four concentration levels (LLOQ, low, middle, and high). Samples were analyzed in three analytical lots in separate days (at least 2 days), and the RSD% for inter- and intraday precision not more than 15% were rational (for LLOQ, not more than 20%). For intra- and interday accuracy, RE% (relative error) within ±15% (for LLOQ, within ±20%) were considered to be acceptable.</p><p>Linearity of each analyte was evaluated in three analytical lots in separate days (at least 2 days) along with the precision and accuracy, and at least three calibration curves were assessed in each analytical lot for one analyte. Calibration curves were regressed from IS-adjusted peak area versus corresponding concentrations in at least six calibration standards using a 1/χ 2 weighted linear least-squares regression model. The LLOQ was the lowest point in the calibration curve. For each concentration point, the deviation of back-calculation in the corresponding calibration curve should be within ±15% (RE%), and the deviation of back-calculation for LLOQ should not go beyond ±20%. Carryover of all analytes was also tested by injecting the highest calibration standard sample prior to injecting a blank sample, and the response of analyte in blank samples not more than 20% of the LLOQ and 5% of the IS was considered to be rational.</p><p>Stability, including long-term stability (3 months), short-term stability (24 h in autosampler), and three frozen-thaw cycles stability, was evaluated using QC samples at two concentration levels (low and high). The corresponding calibration curve for each analyte was employed to obtain the measured concentrations, and the deviation from nominal concentration within ±15% (RE%) conformed to the criterion.</p><!><p>The physicochemical properties of Cap and its metabolites have little in common with each other. Cap had a long carbon chain and showed lipophilicity. After metabolized gradually by the enzyme, the residual structure increased its polarity and showed hydrophilicity. It had reported that only some specific columns could retain simultaneously Cap and its metabolites, for instance, Hypercarb column [10] and Atlantis T3 column [11]. However, Hypercarb column needed to cooperate with complex quaternary mobile phases, including water, acetonitrile, 2-propanol, and tetrahydrofuran, and took a long elution time (12 min). Also an early reported method based on Atlantis T3 column took a long time (14 min) for analytes separation and did not contain the FUH2. Because of these disadvantages, we developed an optimized method with shorter analytical time (5 min) based on the Altantis T3 column and binary mobile phase system. The Altantis T3 column, which is optimized for retention polar compounds, can stand 100% water in separation process, and this characteristic was utilized in this method to increase the retention time of 5-FU and FUH2 to 2 min. In addition, some universal columns containing ZORBAX SB-C18, Xselect BEH, Xbridge BEH, and Eclipse PLUS-C18 were also tested for their retention and separation ability. Unfortunately, enough retention and separation could not be obtained from these universal columns. 0.0075% formic acid in water (pH 4) and in acetonitrile was utilized to elute the analytes and suppress tailing, and optimized retention time and separation were gained after testing different ratio and kinds of acids (formic acid, acetic acid, trifluoroacetic acid, heptafluorobutyric acid; ratio: 0.5%, 0.1%, 0.05%, 0.01%, 0.0075%) in mobile phase. Ammonium acetate as additive in mobile phase could suppress signal response, and methanol decreased the symmetry of peaks.</p><!><p>Owing to the great difference of physicochemical property between Cap and its metabolites, liquid-liquid extraction by ethyl acetate and isopropanol (19 : 1, vol : vol) was chosen as the extraction solvent which gave optimal extraction recovery and matrix effect in pretreatment procedure. Before that, we tested different proportions and combinations of organic solvents, for example, ethyl acetate, isopropanol, dichloromethane, trichloromethane, methyl tertiary-butyl ether, and cyclohexane. During the pretreatment method development, protein precipitation and solid phase extraction were temporarily utilized as the pretreatment method. For protein precipitation, methanol(1 : 3, vol : vol), acetonitrile(1 : 2, vol : vol), acetone(1 : 2, vol : vol), and 10% trichloroacetic acid(1 : 1, vol : vol) were tested for their ability of deproteinization and removing interferential matrix. The results showed a recovery less than 10% for all the analytes, and then solid phase extraction for all the analytes using Osis HLB, MCX, and MAX cartridge (Waters Co., Milford, CT) were tried. The 5-FU and FUH2 could not be retained in these cartridges, and ion-exchange solid phase extraction was also utilized based on the Plexa PCX cartridge (Agilent Technologies, Santa Clara, CA) to extract all analytes according to the instructions. But this might be restricted by the chemical structure of Cap and its metabolites. There were several nitrogen atoms in both Cap and its metabolites, which would carry out ion-exchange process in given chemical circumstance, but the ringlike structure and steric hindrance might hinder the formation of ammonium and succedent ion-exchange. Finally, the Cap and its metabolites could not be retained adequately in the ion-exchange cartridge.</p><!><p>Comparisons of chromatograms from blank, IS spiked, LLOQ, and real samples (Figure 3) indicated that there were not any significant interferences at the same retention times of the analytes and IS.</p><!><p>Calibration curves were constructed by calculating the peak area ratios (analyte/IS) of calibration standards versus measured concentrations. Seven calibration standards were obtained from spiked samples, and the best linearity and least-squares residuals for the calibration curves were achieved with a 1/χ2 weighing factor. The linear correlation coefficients were more than 0.99 for all analytes. Typical regression equations for the calibration curves are summarized in Table 3. The LLOQs were all around 20 ng/mL in human plasma matrix, which were also in accordance with the accuracy within ±20% and precision less than 20%. These LLOQs were more sensitive than some previously reported method and sufficient for monitoring of Cap and its metabolites in clinical practice.</p><p>Carryover between samples often caused confusing results to the lower ones. In this method, three cycles of highest-blank samples were injected orderly to assess responses in the blank samples. The results showed that responses in blank sample were less than 20% of the LLOQ and 5% of the IS (Figure 4).</p><!><p>Four levels of QC samples (LLOQ, low, middle, and high) were chosen to analyze the inter- and intraprecision and accuracy. The results showed a good precision and accuracy with intra- and interprecision less than 10.45% and accuracy within ±15% (LLOQ within ±20%). Table 4 summarizes the inter- and intraday precision and accuracy for the six analytes.</p><!><p>The liquid-liquid extraction commonly could remove the endogenous interferents at the greatest extent, while the protein precipitation left the most serious matrix interference [15]. Researchers had reported the severe ion suppression for the downstream products of Cap, such as FUH2 and 5-FU, using the protein precipitation as sample pretreatment method [12]. Severe ion suppression often caused by the coeluted interferents, for example, lipids and some polar small molecular compounds, and liquid-liquid extraction with ethyl acetate and isopropanol in this method could eliminate more polar small molecular compounds. So, the results showed an immensely declined matrix effect which ranged from 74.84% to 114.48% compared with other reported methods, and the recovery ranged from 59.27% to 90.15%. The IS-normalized matrix and recovery factor were calculated following the acquisition of matrix effect and recovery, and the results showed that the RSD (%) of matrix and recovery factors was not more than 15% (Table 5). The matrix effect and recovery were stable and conformed to the criterion for all the analytes and IS.</p><!><p>The stability of analytes containing long-term stability, short-term stability, and three frozen-thaw cycles was investigated at two concentration levels (low and high). The analytes were found to be stable in human plasma for 3 months at −80°C and in autosampler at 4°C for 24 h (<10% reduction). After three freeze-thaw cycles, no obvious deviations (within ±15%) were observed for all analytes (Table 5).</p><!><p>Totally, 42 sparse samples were collected from 36 colorectal cancer patients who were treated with 1000 mg/m2 Xeloda tablet at the Changzheng Hospital. Thirty-one samples were collected on 31 patients, and the time points ranged from 0.5 to 9 h after administration. Nine samples were harvested from 3 patients at 1, 2.5, and 4 h and the other 2 samples originated from 1 patient at 1 and 4 h after administration. The analytes were quantitatively measured by this fully validated UHPLC-MS/MS method in the plasma. As a result, apparent differences of drug concentrations in plasma were found in Cap and its metabolites in absorption and metabolism processes, and several studies also reported these differences [16–20]. The drug exposure in vivo has a close relationship with the treatment efficacy and/or side effects, and it was still the promising biomarker for the clinical prognosis. The AUC of 5-FU was reported to associate with the myelosuppression and mucositis as well as the treatment response [21]. A timely reflection of the drug exposure might be vital for a better treatment outcome and alleviation of side effects. As the samples were collected in sparse points, no definite pharmacokinetic parameters were gained in this validation process (Supplementary Table S1).</p><!><p>A simple, rapid, and sensitive UHPLC-MS/MS method was successfully developed and validated, which was suitable for simultaneous determination of Cap and its five metabolites in human plasma from colorectal cancer patients. The LLOQ was approximately 20 ng/mL, and the analytical time was 5 min for all analytes after optimizing the chromatography separation and mass spectrometer detection conditions. With a simple sample pretreatment, this method was suitable for clinical therapeutic drug monitoring of Cap and its metabolites to get a better treatment outcome.</p>

PubMed Open Access

Cycloparaphenylene-Phenalenyl Radical and Its Dimeric Double Nanohoop

We report the first example of a neutral spin-delocalized carbon-nanoring radical, achieved by integration of an open-shell graphene fragment phenalenyl into cycloparaphenylene (CPP). We show that spin distribution in this hydrocarbon partially extends from the phenalenyl onto the CPP segment as an interplay of steric and electronic effects. The resulting geometry is reminiscent of a diamond ring, with pseudo-perpendicular arrangement of the radial and the planar π-surface. Remarkably, this geometry gives rise to a steric effect that governs a highly selective dimerization pathway, yielding a giant double nanohoop. Its π-framework made of 158 sp 2 -carbon atoms was unambiguously elucidated by single-crystal X-ray diffraction, which revealed a three-segment CPP-peropyrene-CPP structure. This nanocarbon shows a fluorescence profile characteristic of peropyrene, regardless of which segment gets excited. These results in conjunction with DFT suggest that adjustment of the size of the CPP segments in this double nanohoop could deliver true donor-acceptor systems.Open-shell nanographenes [1] characterized by delocalized spin density are synthetic targets of fundamental interest to chemists. They are investigated for their amphoteric redox properties, [2] magnetism [ 3 ] and conductivity, [ 4 ] and potential applications in spintronics [ 5 ] and quantum computing. [6] The prototype of such class of compounds is phenalenyl (Phen; Figure 1a), a spin-1/2 odd-alternant neutral hydrocarbon and the smallest triangular graphene fragment. [ 7 ] Its characterization by electron paramagnetic resonance (EPR) spectroscopy in 1950s confirmed the delocalized nature of its open-shell electronic structure, [ 8 ] where one of the 13 π-electrons is unpaired and evenly distributed between the six α-positions of the triangular core (Figure 1a). In

cycloparaphenylene-phenalenyl_radical_and_its_dimeric_double_nanohoop

<p>solution, phenalenyl exists in equilibrium with its σ-dimer, [9] which in the presence of oxygen undergoes a reaction cascade to yield peropyrene as the final product. [ 10 ] The reactivity of phenalenyl can be controlled by steric effects of substituents [11] or electronically by π-extension [12] that affects spin distribution, which can lead to a selective reactivity [13] or suppress it. [12] The known π-extended spin-1/2 derivatives of phenalenyl are restricted to planar [1] and helical examples. [13a,14] To explore new structural platforms, we turned our attention to a cylindrical cycloparaphenylene (CPP) framework, [15] which offers unique opportunities to control spin distribution and reactivity by a combination of steric and electronic effects. CPPs are structural models [16] and synthetic templates [17] for armchair carbon nanotubes. On account of their curved cyclic π-conjugation and hollow interior, they are attractive synthetic targets in material science and supramolecular chemistry. [18] Their curved geometry weakens the overlap of p orbitals, which gives rise to a partial quinoidal character that increases with decreasing diameter. [19] Quinoidal structures favor delocalization of unpaired electrons, as it does not involve the loss of aromatic stabilization energy, which has been observed in radical cations [20] and anions [21] of CPPs but not in neutral species. Changing the linkage mode of one phenylene ring from para to meta results in a geometry, where the meta-phenylene ring lies within the CPP plane perpendicularly to the neighboring para-phenylene rings, which disrupts their π-communication. [22] To utilize the potential of CPPs as a platform to control spin distribution and reactivity, we began to explore hybrid nanocarbon systems, comprising open-shell nanographenes integrated within the CPP nanorings.</p><p>Herein, we present the prototype of such systems, a CPP-based neutral radical CPP-Phen (Figure 1b), in which one phenylene ring of the CPP loop is replaced by a phenalenyl unit connected in a pseudo-meta-fashion via two α-positions (α3 and α6). This linkage mode results in a geometry reminiscent of a diamond ring, where the phenalenyl unit lies in the CPP plane, that is, almost perpendicularly to the connecting phenylene rings. Consequently, (1) the majority of spin density resides on the phenalenyl unit and only a part of it extends onto the CPP segment, and (2) the dimerization pathway through the α3-α6-positions of the phenalenyl unit is suppressed. This steric effect leads to a highly selective dimerization via α1-and α2-postions, yielding a double-nanohoop peropyrene (CPP-PP; Figure 1c) as the exclusive dimeric product. Notably, CPP-PP represents a fully π-conjugated hydrocarbon framework made of 158 sp 2 -carbon atoms as a novel member of CPP architectures that feature multiple nanoring-cycles. [23,24,25,26,27] The pivotal intermediate in our synthetic strategy was nanoring 1 (Scheme 1), formed by one dihydrophenalenone unit and 11 phenylene rings. It was synthesized by means of the Suzuki-Miyaura cross-coupling macrocyclization of dihydrophenalenone 2 and C-shaped linker 3. Precursor 2 was prepared in multiple steps starting from trisubstituted naphthalene 4, which first underwent a selective Kumada cross-coupling with 4-(trimethylsilyl)phenylmagnesium bromide to afford 5 in 76% yield. The subsequent Heck coupling and the reduction of the C-C double bond followed by hydrolysis afforded 6 in 71% yield. In the final step, ICl-promoted iodination, acyl chloride formation and Friedel-Crafts acylation provided 2 in 65% yield. The structure of this key intermediate was determined by single-crystal X-ray diffraction (SC-XRD; Figure S2). The directing angle of the corner linker plays a crucial role in the construction of the cyclic scaffold in the CPP chemistry. [15] After screening various candidates, C-shaped linker 3 was found ideal to unite with 2 and afford nanoring 1 in 15% yield upon reductive aromatization. The structure of 1 was unequivocally validated by SC-XRD. The inspection of the solid-state structure (Figure S3) revealed that the dihydrophenalenone unit is non-coplanar with the neighboring phenylene rings, with the dihedral angles (64° and 67°) significantly larger than those between any two neighboring phenylene rings of the CPP segment (28° on average). Finally, 1 was subjected to the reduction and dehydration to afford CPP-1H-phenalene intermediate, the direct precursor of CPP-Phen.</p><p>Upon the addition of p-chloranil in toluene at room temperature under a nitrogen atmosphere, the pale-yellow solution of the hydroprecursor rapidly changes color to yellow, then green, and after some time, a dark brown-orange mixture is observed. This observation suggests the "decomposition" pathway of phenalenyl to peropyrene, which involves σ-dimer formation as shown in Scheme 1. Kubo and co-workers identified [10b] all intermediates of this reaction sequence (Scheme S3), which involves three oxidation steps starting from hydroprecursor (-6H). This means that at least three equivalents of p-chloranil had to be used for the full conversion of CPP-1H-phenalene to CPP-PP. The use of less than three equivalents of p-chloranil results in a mixture of all intermediates -these conditions were used for the EPR spectroscopic characterization of CPP-Phen. The dimeric product CPP-PP was separated and its formation was confirmed with MALDI-TOF MS, which showed ionized species with an m/z value of 2226.91 (Figure S1). CPP-PP exhibited a poor solubility in common organic solvents due to its large and rigid structure. It is noteworthy that the total isolated yield of CPP-PP starting from 1 is 32% (~80% per step), surpassing that of peropyrene from a predimerized dihydrophenalenone precursor (<22%). [10b] This facile π-skeletal expansion from CPP-Phen to CPP-PP via an oxidative dimerization cascade represents a novel synthetic tactic to achieve double nanohoop architectures. It is distinct from the conventional synthesis of lemniscate CPPs, where the bridging building block is constructed prior to the macrocyclization. [23] In addition, to better understand the photophysical properties of CPP-PP (vide infra), two segments of it, namely, CPP and PP (Scheme 1b), were synthesized and their structures were fully characterized including SC-XRD (see the Supporting Information). The paramagnetic nature of CPP-Phen was probed by EPR spectroscopy. A diluted toluene solution of CPP-1H-phenalene and p-chloranil (1:1, ~10 −3 M) gave a well-resolved 16-peakmultiplet EPR spectrum at 307 K (Figure 2d) with a g value of 2.0039, which is typical for delocalized spin-1/2 hydrocarbon radicals. The measured EPR spectrum was simulated [28] as a quartet of a pentet (qp) using proton hyperfine coupling constants (hcc) of 6.10 G (four α-protons) and 1.95 G (three β-protons). [29] This splitting pattern is in an excellent agreement with the results of DFT calculations, which reveal that the majority of spin density is localized on the phenalenyl unit (Figure 2b,c). The simulated hcc values agree well with the calculated ones (Figure 2a, ∼6.5 G for α-and ∼2.4 G for β-protons), which are slightly higher. Based on DFT, the protons of the neighboring two phenylene rings possess dramatically smaller hcc values (0.27-0.58 G), which are not resolved in the experimental spectrum. This spin distribution can be rationalized by a pseudoperpendicular arrangement between the phenalenyl unit and the neighboring phenylene rings, which is favored for units with a meta-linkage (angle of 120°). [22] The steric preference for perpendicular arrangement acts against the electronic preference for a coplanar arrangement, which is ideal for spin-delocalization onto the phenylene rings. This "clash of forces" is supported by the calculated dihedral angles (~64° on average, Figure S10), which are lower compared to CPP-PP (~80° on average, Figure 3a), and by comparing CPP-Phen to its strain-free linear analog (Figures S5 and S12), which displays a higher degree of spin density on the phenylene rings. Interestingly, in the lowest-energy geometry, the spin density in both systems is not delocalized evenly on both sides. Single crystals of CPP-PP were obtained by slow vapor diffusion of acetonitrile into a CHCl3 solution at room temperature. The XRD analysis unambiguously confirmed the fully π-conjugated framework of CPP-PP with a C2 symmetry, where the peropyrene segment serves as a rigid bridge between two CPP segments. The dihedral angles at the four linkages are ~80° on average (Figure 3a, green), implying an unprecedented perpendicular alignment of three π-conjugated segments (radial-planar-radial). The overall length reaches 4.20 nm and the two cavities have an oval shape with a short axis of 1.52 nm. The distance between the protons of the peropyrene segment and the opposite propoxylated phenylene rings is 1.45 nm, which implies that the cavities of CPP-PP might be suited for the encapsulation of fullerenes such as C60 or C70. [30] No evidence of binding C60 or C70, however, was observed, which suggests that the cavities are not an ideal fit. The herringbone packing structure is similar to that of most CPP molecules [30a] that arrange in staggered layers (Figure 3c). The photophysical properties of CPP, PP and CPP-PP were investigated in CHCl3 solutions (Figure 4). CPP showed only one intense absorption band at 342 nm with an extinction coefficient (ε) of 1.49 × 10 5 M −1 cm −1 , which is nearly identical to that of parent [12]CPP. [31] According to timedependent (TD)-DFT calculations, the HOMO→LUMO (S0→S1) transition is weakly allowed with f = 0.283, and the major contributions for the observed band are HOMO−1→LUMO (S0→S2, f = 2.30) as well as HOMO−2→LUMO and HOMO→LUMO+1 (S0→S3, f = 3.26) transitions (Figure S17). PP exhibited three main absorption bands: the most intensive one at 345 nm with ε = 0.77 × 10 5 M −1 cm −1 and two less intensive bands at 447 and 477 nm, which are characteristic of the peropyrene backbone (HOMO→LUMO (S0→S1, f = 1.47) transition, Figure S18). [32] CPP-PP showed an absorption spectrum that represents a superposition of those of CPP and PP, except a minor shift (Figure 4a). The dominant absorption band at 338 nm (ε = 2.24 × 10 5 M −1 cm −1 ) is primarily derived from the absorption of two CPP segments, and it can be ascribed to multiple HOMO−n→LUMO+m transitions (n = 0-7, m = 0-11; S0→S5, f = 4.46; S0→S7, f = 4.43; Figure S19), including CPP-localized (e.g., HOMO−3→LUMO+2) and charge-transfer transitions (e.g., HOMO→LUMO+5). In addition, the two typical absorption bands of the peropyrene segment were observed at 434 nm and 463 nm (HOMO→LUMO (S0→S1, f = 2.15) peropyrene-localized transition), blue-shifted by 14 nm compared to those of PP. This blue shift is presumably due to the weaker conjugation between the peropyrene and CPP segments, as indicated by the significantly larger dihedral angles at the four linkages (avg. 80°, Figure 3a) compare to those of PP (avg. 57°, Figure S4).</p><p>The profile of the fluorescence spectrum of CPP-PP is almost identical to that of PP, except a blue shift of 18 nm, without any contribution from CPP (Figure 4). For a comparison, a 1:1 mixture of CPP and PP shows a fluorescence profile, which represents the superposition of the individual spectra of both components. The fluorescence quantum yields for CPP, PP and CPP-PP in CHCl3 are 0.63, 0.76 and 0.75, respectively (excitation of 350 nm, Figure S7). These results indicate an efficient internal conversion from the Sn (n > 1) states to the S1 state in CPP-PP.</p><p>To conclude, we synthesized the first example of a neutral spin-delocalized carbon-nanoring radical and demonstrated that the radially π-conjugated CPP framework is a unique structural platform to control spin distribution and reactivity of spin-delocalized systems such as phenalenyl. By means of SC-XRD, EPR and UV-Vis spectroscopy, and DFT calculations, we provided the first insight into the interplay of steric and electronic effects that govern spin distribution in this new type of open-shell nanocarbon hybrids. Our results lay the ground-work for future research towards the understanding of spin-delocalization through a radially π-conjugated backbone. For example, systems with a smaller ring size of the CPP segment are expected to favor a quinoidal over a benzenoid structure [19] and thus lead to a more extended spin-delocalization. These concepts can be applied to understand through-space or guest-mediated spin interactions in multi-spin-unit and hostguest CPP systems, respectively. The highly selective dimerization of the nanoring radical to the double nanohoop represents a novel synthetic approach towards CPP architectures with unusual arrangements of π-surfaces such as the perpendicularly alternating "CPP-peropyrene-CPP" array mode. Our photophysical and DFT studies suggest that decreasing the size of the CPP segments in our double nanohoop, and thus lowering the HOMO-LUMO gap, could deliver true donor-acceptor systems. We believe that investigation of other members of this novel family of non-planar openshell hydrocarbon radicals will have implications in the fields of material science, spintronics and supramolecular chemistry. This work is ongoing in our laboratory.</p>

ChemRxiv

Myocardial relaxation in human heart failure: Why sarcomere kinetics should be centerstage.

Myocardial relaxation is critical for the heart to allow for adequate filling of the ventricles prior to the next contraction. In human heart failure, impairment of myocardial relaxation is a major problem, and impacts most patients suffering from end-stage failure. Furthering our understanding of myocardial relaxation is critical in developing future treatment strategies. This review highlights processes involved in myocardial relaxation, as well as governing processes that modulate myocardial relaxation, with a focus on impairment of myocardium-level relaxation in human end-stage heart failure.

myocardial_relaxation_in_human_heart_failure:_why_sarcomere_kinetics_should_be_centerstage.

Introduction<!>Human versus small rodent myocardium<!>Human myocardium and contractile strength<!>Impaired relaxation in human myocardium<!>Determinants of myocardial relaxation<!>Intracellular calcium decline.<!>Thin filament de-activation<!>Cross-bridge detachment<!>Sarcomere lengthening<!>Regulation of relaxation<!>Myofilament properties, the sarcomere takes stage

<p>Roughly one in nine deaths in the US is due to heart failure, with an annual cost of patient care exceeding 30 billion US$, and those costs are expected to increase to 53 billion US$ by the year 2030(23). Heart failure is a debilitating disease that is often characterized by a heart that is not strong enough to pump sufficient amounts of blood. The weakness of the heart is actually only one of the potential underlying causes of end-stage heart failure, since even in end-stage failure the heart pumps enough blood to keep the patient alive. However, when patients exert themselves by, for instance, climbing stairs, cardiac pathology becomes more evident as seen by the inability of the heart to increase its cardiac output. Over the past decades, we have come to realize that the majority of patients suffer not necessarily from impaired contractile function, but from impaired relaxation, and for a large part of this heart failure patient population this impaired relaxation is the major pathological problem.</p><p>Myocardial relaxation is a complex process that involves multiple distinct rate- limiting steps, including calcium transient decline, thin filament de-activation, and crossbridge cycling kinetics/detachment(6). Each of these processes must take place to allow for relaxation to occur. In addition, each of these processes is further critically modulated by mechanical factors such as pre-load, after-load, and the rate and direction in which the sarcomeres move(7, 9, 28). Understanding of this process has proven extremely difficult, since the vast amount of players and processes that determine and regulate myocardial relaxation necessitate an inclusive systems approach to study these processes, as they do not work in isolation. The role of the involved processes is not only complex and interactive, but during different segments of a heartbeat they each exert distinct levels of involvement and regulatory importance. Relaxation of the heart is therefore akin to an emerging systems property, and a failure of this system is a complex process to dissect and understand. The goal of this review is to point out the central role the sarcomere and its components play in the relaxation process, and why they are crucial to further our understanding of human heart failure.</p><!><p>The study of human myocardial relaxation is complicated by the fact that the kinetics of this process are about 10 times slower than in the most commonly used and powerful model in cardiovascular research, the laboratory mouse(25), and this hampers unambiguous translation of findings from mice to humans. The resting murine heart beats ~10 times faster than a resting human heart, and despite a very similar anatomy of the myocytes, sarcomere, and nearly identical afterload/blood pressure, the vast majority of "players" that are involved in determining myocardial relaxation are much different in the mouse(36). These differences are found at all levels; ion channels, EC- coupling proteins, and myofilament isoforms all significantly differ between mice and humans, as they need to be working in a much different frequency range. Sometimes the molecular differences are very small, but even small differences, for instance in the myosin isoform in small rodents, can have very significant functional consequences(24). The species differences are further complicated by the fact that in the mouse the kinetics of contraction and relaxation are only very modestly modified upon exercise. Whereas the mouse only changes cardiac output by ~20–30%, in the healthy human this change in cardiac output is easily 200–300%, and even substantially more in extreme cases. Thus, although the laboratory mouse has, is, and will advance our knowledge on the molecular and regulatory level of many processes of the heart, it is often unsuitable to study quantifiable determinants of relaxation as they relate to human physiology and disease(25).</p><!><p>Several laboratories have used isolated human myocardium in an in vitro/ex vivo setting to assess contraction and relaxation properties. Over the past decades, studies have investigated contractile properties of human myocardium to further understand the working of the human heart and how and to what extent it malfunctions. Because the human population has a much more widespread genotypical and phenotypical variety, the variability in experimental outcome of measured contractile parameters has a significantly larger spread than data obtained in in-bred animal models. Although theoretically this can be easily overcome statistically by simply increasing the n-number of subjects in the study, this has practically proven to be difficult. Working with human myocardium, and especially freshly isolated myocardium that allows for the assessment of dynamic contractile parameters, typically hampers studying large groups of subjects, unless it involves studies conducted over a longer timespan. As a result of the conflict between the need for large n-numbers and the scarceness of available tissue, many past experiments on human myocardium were hampered by a lack of statistical power, sometimes resulting in conflicting results (8, 14–22, 35, 41–43, 45). However, on average these previous studies found no or little difference in developed contractile strength of viable myocardium at rest. A recent study conducted over 6 years (10) on >80 human hearts confirmed that there was indeed no significant difference inactive developed force of viable right ventricular trabeculae between failing and non-failing myocardium at rest. This may seem at odds with our view of heart failure as predominantly a weak heart, but this outcome is actually expected given the fact that the failing heart at rest, and the non-failing heart at rest, produce a very similar cardiac output. Indeed, heart failure typically manifests phenotypically under conditions where cardiac output is elevated, such as with exercise. When human myocardium in vitro is subject to the conditions of exercise, causing an elevation in heart rate, contractile deficits clearly become obvious in the end-stage failing myocardium. Healthy human myocardium increases developed force when heart rate is elevated, failing myocardium either increases less, but more often does not increase at all or even decreases when heart rate is elevated (10, 16, 40).</p><!><p>Although at rest force of contraction is largely unaffected, impaired human myocardial relaxation is a long-known prominent pathological component in the majority of patients suffering from heart failure(13). In most, but not all, past human myocardium studies, slower kinetics of contraction and relaxation have been observed. Again, given the need for large n-numbers, and the generally hard to procure human tissue, some of the past results were hampered by lack of statistical power. Our recent study on 80+ human hearts determined that in end-stage heart failure, at least in the right ventricular myocardium of end-stage failing hearts, a significant deficit in relaxation kinetics is indeed present. This impaired relaxation is present at a resting heart rate, and this impaired relaxation was more prominent in myocardium of ischemic etiological origin compared to failing myocardium from non-ischemic etiology(10).</p><!><p>The large amount of players and processes that determine, influence, and regulate myocardial relaxation do not work in isolation, but rather in a complex concerted effort. First and foremost, they are tuned to work together in the heart rate range of the species in question. Although most species will share many of the proteins involved in various aspects of contraction and relaxation, for each species small but impactful changes of these individual proteins allow for their tuning with the other components to work in the desired heart rate range. Although individual changes in a protein, enzyme, and/or a biochemical- or mechanical action can be studied in isolation, understanding their role in the concerted relaxation process necessitates the assessment of their role within this concerted and tuned environment. In isolation, the functional role of a protein cannot be completely understood in relation to its contribution to the ultimate emerging systems property that encompassed myocardial relaxation. From decades of research, we identified several major processes that are involved in the cardiac relaxation process, as they are each necessary for the muscle to relax.</p><!><p>The intracellular calcium level that is increased to initiate contraction needs to decline to allow for relaxation to occur. In humans, about 30% of the calcium transient decline is accomplished by the activity of the Na+-Ca2+ exchanger, with the remaining 70% of the cycled calcium taken up back into the sarcoplasmic reticulum (SR) by the SR Ca2+-ATPase. In sharp contrast, mice almost exclusively depend on SR re-uptake, as transmembrane transport via Na+-Ca2+ exchanger is limited to only a few % of the total cycled Ca2+ ions during a contraction-relaxation cycle(5, 36). There is however not necessarily a given level of calcium that needs to be reached to allow for relaxation, since other factors too play a role in calcium-initiated myofilament (de-)activation. In fact, once the calcium transient has declined below the threshold for further activation, the rate of decline of the calcium transient appears to be no longer a regulatory factor for relaxation. Most experimental work has shown that the quantitative rate of decline of the calcium transient is typically slower than the rate of force relaxation(3, 39). Thus, even though a decline to a certain Ca2+ level is essential for relaxation to occur, the rate and regulation of relaxation appears mainly independent of the rate of intracellular calcium decline. Thus, one cannot infer an improved relaxation solely from an increased rate of calcium decline, as they are not directly related.</p><!><p>A second factor in relaxation is that the thin filament activation needs to cease in order to prevent additional myosin head binding to the thin filament(31). The calcium dissociation from Troponin-C is governed by the concentration of Ca2+ in the myofilament matrix, and by the dissociation constant of TnC for Ca2+ ions. It is often assumed that the calcium concentration reflects the amount of TnC that has calcium bound, i.e. it is assumed that calcium binding and release of TnC is in a rapid equilibrium. That is however not the case, the association and dissociation rates of calcium for TnC are highly dependent on the "level of organization". In isolation, i.e. in solution, the TnC calcium kinetics are extremely fast, but in the cellular setting, with all other proteins present, these rates are much, much slower, to the extend that they can be quite similar to the rate of force relaxation, and thus can become rate-limiting(11). This implies that even when the calcium concentration is very low, a substantial amount of Ca2+ can still be bound to TnC. Moreover, TnC-Ca2+ dissociation is indirectly modulated by the contraction itself. When the ventricle ejects blood, sarcomere length decreases, and with the decrease in sarcomere length a decrease in calcium sensitivity is induced(1, 12). The length-dependent activation, which is for a large part due to calcium sensitization of the myofilaments is now reversed, causing an ejection- dependent deactivation. Thus, TnC-Ca2+ dissociation kinetics can play a major role in determining the rate of relaxation.</p><!><p>A third "must-happen" factor for myocardial force to decline is that cross-bridges have to detach from the thin filament. The detachment rate of cross-bridges is generally seen as the rate-limiting step of the cross-bridge cycle. Most of the data on crossbridge cycling has been obtained from perturbations from a steady-state activation, often in skinned muscles, fibers, or myofibrils. In the beating heart, no steady-state contraction is reached, and since cycling behavior is generally dependent on the level of activation, the cycling behavior of cross-bridges in intact, contracting myocardium is still only partially resolved and understood. Assessment, or approximation of cross-bridge cycling behavior in human myocardium has been done mainly in skinned preparations, typically at cold temperatures. Extrapolation of such data to body temperature, as well as recent studies on intact human myocardium in which the rate of tension redevelopment was assessed(35), shows that the cycling rate of cross-bridges in human myocardium is rather slow, with time constants in the same range as TnC calcium dissociation, and relaxation of force. This implies that a single given crossbridge likely only cycles once or twice during a heart-beat, and that the overall detachment rate of cross-bridges could encompass a critical rate limiting step, specifically in late relaxation where relaxation of force is often mono-exponential.</p><!><p>A fourth important modulator of relaxation is found not in the proteins that regulate the above processes, but in the direction and amplitude the sarcomeric proteins moves. When a sarcomere is stretched near or right after the end of systole, either by a single stretch(9), by application of an external vibration(28, 29), or by stretch by a neighboring sarcomere(47), relaxation has been shown to greatly accelerate. A recent study by Chung and coworkers(9) showed that in load-clamped twitch contractions, it was end systolic strain rate, and not solely afterload, that accelerated the myocardial relaxation by accelerating the detachment of the cross-bridges. Earlier studies showed that application of a low amplitude sinusoidal vibration at the peak of an isometric contraction greatly accelerated relaxation(28), and this acceleration impact could be observed throughout the relaxation process(29). In general, a lengthening sarcomere likely gives the myosin head a much lesser chance of binding to the actin binding site than in an isometric or shortening sarcomere. This "direction-dependent detachment" phenomenon also helps explain why slow isovolumic relaxation correlates with impaired relaxation during early filling in patients with diastolic dysfunction(50). As soon as the myocardium start to relax, and sarcomeres lengthen, it would provide a mechanical way of cessation of additional cross-bridge formation, despite a potentially activated thin filament. If this lengthening does not happen, or happens less or slower, cross-bridges can still attached, and/or remain attached longer, impairing filling. In addition to an absolute change in sarcomere length, the prevailing absolute sarcomere length can too impact the myocardial relaxation rate. The regulatory protein MyBP-C is located on a distinct part of the thick filament. Since MyBP-C has been shown to modulate the cross-bridge cycling kinetics(32, 34, 44) and is one of the few proteins that can change contraction-relaxation coupling (26, 27), not only the amount and direction of relengthening is important, but likely also the specific prevailing sarcomere length can impact myocardial relaxation.</p><!><p>In the human heart, the rate of relaxation is modulated to allow the heart to perform over a wide range of heart rates, with relaxation rates accelerating up to 30–45% from rest to peak heart rate. Such modulation barely occurs in the mouse, where relaxation changes by only 5–10% from rest to peak heart rate(25). The 3 most prominent regulators of contractile strength are volume (sarcomere length), heart rate (frequency), and β-adrenergic stimulation (the flight-fright-fight response). Each of these three regulatory mechanisms impact not only contractile strength, but also impact the kinetics of contraction and relaxation. First, with increased volume (i.e. increase muscle or sarcomere length), relaxation becomes slower(2). This is mainly due to myofilament properties, as calcium transients change only slightly, and more so, in the opposite direction; the calcium transients decline slightly accelerated when sarcomeres are stretched, while force relaxation slowed down(2, 39). This later finding again argues against the rate of calcium transient decline being a prominent determinant of relaxation, since calcium decline and force decline are uncoupled. The slower force decline with increased sarcomere length is predominantly attributed to increase myofilament calcium sensitivity(l), and may additionally involve post-translational modification of myofilament proteins(38). Second, when heart rate increases, relaxation accelerates(26). The calcium transients both gain in amplitude and accelerate in decline rate, while in addition the myofilaments desensitize to activator calcium(48), allowing for faster relaxation to occur, despite the increased intracellular diastolic calcium levels. Rise of intracellular calcium with frequency show that diastolic calcium levels at high rates can even exceed systolic levels at low rates(46), necessitating a substantial frequency-dependent myofilament desensitization to maintain force development(48, 49). Third, β-stimulation causes a similar impact as the elevation of heart rate alone (note that heart rate elevation is mainly due to an increase in β- stimulation), with additional phosphorylation targets activated that overall increase contractile strength, and aid in acceleration of relaxation(33).</p><!><p>The collective proteins, processes, and regulatory parameters in the myocardium discussed above govern the relaxation of force development as an integrated system. One could view the myocardial relaxation process as an emerging systems property, with no clear single molecular target, pathway, process, or regulator that is the main driver. That said, the proteins and processes that are most likely to be the major players have in common that they are an integral part of the sarcomere. Although EC- coupling is a critical part of the initiation of contraction, the determinants of kinetics of contraction, and those of relaxation, are located and regulated in and by the sarcomere as a whole(4, 30). Troponin-C as the critical Ca2+-sensor does not work in isolation to activate the thin filament; an integrated process that involves many other proteins feed back and co-determine the apparent association and dissociation constants for calcium(11). In addition, cross-bridge cycling kinetics play a major part in the kinetic regulation of the heart(37). Lastly, the external and internal loads that the sarcomere both carries and generates resulting in sarcomere length changes impacts TnC-Ca2+ binding as well as cross-bridge cycling properties. In human heart failure, the kinetic rate of contraction and relaxation are prominently involved in the manifestation of dysfunction, even at rest(10), possibly more often so than a weak contraction, and a further understanding of the sarcomere as an integrated system will be needed to strategize sarcomere-based treatment for this sarcomere-based dysfunction.</p>

PubMed Author Manuscript

Regioselective Simmons–Smith-type cyclopropanations of polyalkenes enabled by transition metal catalysis

A [ iÀPr PDI]CoBr 2 complex (PDI ¼ pyridine-diimine) catalyzes Simmons-Smith-type reductive cyclopropanation reactions using CH 2 Br 2 in combination with Zn. In contrast to its non-catalytic variant, the cobalt-catalyzed cyclopropanation is capable of discriminating between alkenes of similar electronic properties based on their substitution patterns: monosubstituted > 1,1-disubstituted > (Z)-1,2disubstituted > (E)-1,2-disubstituted > trisubstituted. This property enables synthetically useful yields to be achieved for the monocyclopropanation of polyalkene substrates, including terpene derivatives and conjugated 1,3-dienes. Mechanistic studies implicate a carbenoid species containing both Co and Zn as the catalytically relevant methylene transfer agent.

regioselective_simmons–smith-type_cyclopropanations_of_polyalkenes_enabled_by_transition_metal_catal

Introduction<!>Results and discussion<!>Conclusions

<p>Cyclopropanes are common structural elements in synthetic and natural biologically active compounds. 1 The Simmons-Smith cyclopropanation reaction was rst reported over half a century ago but remains today one of the most useful methods for converting an alkene into a cyclopropane. 2 As compared to diazomethane, which is shock sensitive and must be prepared from complex precursors, CH 2 I 2 is both stable and readily available, making it an attractive methylene source. Additionally, the stereospecicity of the Simmons-Smith reaction allows diastereomeric relationships in cyclopropanes to be established with a high degree of predictability. Several advances have addressed many of the limitations of the initial Simmons-Smith protocol. For example, Et 2 Zn can be used in the place of Zn to more reliably and quantitatively generate the active carbenoid reagent. 3 Acidic additives, such as CF 3 CO 2 H 4 and substituted phenols, 5 have been found to accelerate the cyclopropanation of challenging substrates. Finally, Zn carbenoids bearing dialkylphosphate anions 6 or bipyridine ligands 7 are sufficiently stable to be stored in solution at low temperatures (Fig. 1).</p><p>Despite the many notable contributions in Zn carbenoid chemistry, a persistent limitation of Simmons-Smith-type cyclopropanations is their poor selectivity when attempting to discriminate between multiple alkenes of similar electronic properties. For example, the terpene natural product limonene possesses a 1,1-disubstituted and a trisubstituted alkene. Friedrich reported that, under a variety of Zn carbenoid conditions, the two alkenes are cyclopropanated with similar rates, resulting in mixtures of monocyclopropanated (up to a 5 : 1 ratio of regioisomers) and dicyclopropanated products. 8 This issue is exacerbated by the challenge associated with separating the two monocyclopropane regioisomers, which only differ in the position of a non-polar CH 2 group. In general, synthetically useful regioselectivities in Simmons-Smith reactions are only observed for substrates containing directing groups. 9</p><p>In principle, catalysis may provide an avenue to address selectivity challenges in Simmons-Smith-type cyclopropanations; however, unlike diazoalkane transfer reactions, which are catalyzed by a broad range of transition metal complexes, 9b,10 there has been comparatively little progress toward the development of robust catalytic strategies for reductive cyclopropanations. 11 Lewis acids in substoichiometric loadings have been observed to accelerate the Simmons-Smith reaction, but in many cases, this rate effect is restricted to allylic alcohol substrates. 12,13 Recently, our group described an alternative approach to catalyzing reductive cyclopropanation reactions using a transition metal complex that is capable of activating the dihaloalkane reagent by C-X oxidative addition. A dinickel catalyst was shown to promote methylene 14 and vinylidene 15 transfer using CH 2 Cl 2 and 1,1-dichloroalkenes in combination with Zn as a stoichiometric reductant. Here, we describe a mononuclear [PDI]Co (PDI ¼ pyridine-diimine) catalyst 16 that imparts a high degree of steric selectivity in the cyclopropanation of polyalkene substrates. Mechanistic studies suggest that the key intermediate responsible for methylene transfer is a heterobimetallic conjugate of Co and Zn.</p><!><p>4-Vinyl-1-cyclohexene contains a terminal and an internal alkene of minimal electronic differentiation and thus provided a suitable model substrate to initiate our studies (Table 1). 8,17 Under standard CH 2 I 2 /Et 2 Zn conditions (entry 1), there is a modest preference for cyclopropanation of the more electronrich disubstituted alkene (rr ¼ 1 : 6.7) with increasing amounts of competing dicyclopropanation being observed at higher conversions (entries 2 and 3). Other modications to the conditions, including the use of a Brønsted acid 4 (entry 4) or a Lewis acid additive 12b,18 (entry 5), did not yield any improvements in selectivity. Likewise, an Al carbenoid generated using CH 2 I 2 and AlEt 3 afforded a similar preference for cyclopropanation of the endocyclic alkene (entry 6). 19 In a survey of transition metal catalysts, the [ iÀPr PDI]CoBr 2 complex 1 was identied as a highly regioselective catalyst for the cyclopropanation of 4-vinyl-1-cyclohexene, targeting the less hindered terminal alkene (Table 2). CH 2 Br 2 and Zn alone do not afford any background levels of cyclopropanation (entry 1); however, the addition of 6 mol% [ iÀPr PDI]CoBr 2 (1) provided monocyclopropane 3 (81% yield) with a >50 : 1 rr and <1% of the dicyclopropane product (entry 5). The steric prole of the catalyst appears to be critically important for yield. For example, the mesityl-(entry 6) and phenyl-substituted variants (entry 7) of the ligand provided only 58% and 4% yield respectively under the same reaction conditions. Related N-donor ligands similarly afforded low levels of conversion (entries 8-12) as did the use of other rst-row transition metals, including Fe (entry 14) and Ni (entry 15), in the place of Co.</p><p>In order to dene the selectivity properties of catalyst 1, we next conducted competition experiments using alkenes bearing different patterns of substitution (Fig. 2). Reactions were carried out using an equimolar amount of each alkene and run to full conversion of the limiting CH 2 Br 2 reagent (1.0 equiv.). Monosubstituted alkenes are the most reactive class of substrates using 1 but are not adequately differentiated from 1,1-disubstituted alkenes (3 : 1). By contrast, terminal alkenes are signicantly more reactive than internal alkenes, providing synthetically useful selectivities ($31 : 1). Furthermore, a model Z-alkene was cyclopropanated in preference to its E-alkene congener in a 33 : 1 ratio. Using catalyst 1, trisubstituted alkenes are poorly reactive, and no conversion is observed for tetrasubstituted alkenes.</p><p>The synthetic applications of the catalytic regioselective cyclopropanation were examined using the terpene natural products and derivatives shown in Fig. 3. In all cases, the selectivity properties follow the trends established in the competition experiments. Substrates containing ether or free alcohol functionalities (e.g., 7, 10, and 11) exhibit a strong directing group effect under classical Simmons-Smith conditions; however, catalyst 1 overrides this preference and targets the less hindered alkene. Additionally, the presence of electron-decient a,b-unsaturated carbonyl systems (e.g., 9, 13, and 14) do not perturb the expected steric selectivity. Vinylcyclopropanes are a valuable class of synthetic intermediates that engage in catalytic strain-induced ring-opening reactions. 20 The monocyclopropanation of a diene represents an attractive approach to their synthesis but would require a catalyst that is capable of imparting a high degree of regioselectivity and avoiding secondary additions to form dicyclopropane products. 21 These challenges are addressed for a variety of diene classes using catalyst 1 (Fig. 4). Over the substrates that we have examined, the selectivities for cyclopropanation of the terminal over the internal double bond of the diene system are uniformly high. Additionally, the catalyst is tolerant of vinyl bromide (15) and vinyl boronate (23) functional groups, which are commonly used in cross-coupling reactions.</p><p>Like the non-catalytic Simmons-Smith reaction, 2c the cyclopropanation using 1 is stereospecic within the limit of detection, implying a mechanism in which the two C-C s-bonds are either formed in a concerted fashion or by a stepwise process that does not allow for single bond rotation. For example, cyclopropanation of the Z-alkene 24 affords the cis-disubstituted cyclopropane 25 in 95% yield as a single diastereomer (Fig. 5a). Furthermore, the vinylcyclopropane substrates 26 and 28, commonly used as tests for cyclopropylcarbinyl radical intermediates, react without ring-opening to afford products 27 and 29 (Fig. 5b). a Reaction conditions: 4-vinylcyclohexene (0.14 mmol), THF (1.0 mL), 24 h, 22 C. Yields and ratios of regioisomers were determined by GC analysis against an internal standard. Under standard catalytic conditions, the reaction mixtures using 1 adopt a deep violet color, which persists until complete consumption of the alkene. The UV-vis spectrum of the catalytic mixture at partial conversion is consistent with a Co(I) resting state (Fig. 6a). The authentic [ iÀPr PDI]CoBr complex (30) can be prepared by stirring the [ iÀPr PDI]CoBr 2 complex 1 over excess Zn metal. 22 Cyclic voltammetry data (Fig. 6b) indicates an E 1/2 for the Co(II)/Co(I) redox couple of À1.00 V vs. Fc/Fc + . The large peak-to-peak separation (0.96 V in 0.3 M [n-Bu 4 N] [PF 6 ]/THF) is characteristic of a slow bromide dissociation step following electron transfer. The second Co(I)/Co(0) reduction event is signicantly more cathodic at À1.93 V and is inaccessible using Zn.</p><p>In order to decouple the cyclopropanation steps of the mechanism from catalyst turnover, we conducted stoichiometric reactions with the isolated [ iÀPr PDI]CoBr complex in the absence of Zn (Fig. 6c). The reaction of 30 with 4-vinylcyclohexene and CH 2 Br 2 generates the [ iÀPr PDI]CoBr 2 complex 1 within 24 h at room temperature but forms cyclopropanated products in a relatively low combined yield of 26%, which is not commensurate with the efficiency of the catalytic process. Furthermore, the regioselectivity is only 3 : 1, whereas the catalytic cyclopropanation achieves a >50 : 1 selectivity for this substrate. When the same stoichiometric reaction is conducted in the presence of ZnBr 2 , the yield and selectivity of the catalytic process is fully restored.</p><p>The Co-containing product (31) of the stoichiometric reaction in the presence of ZnBr 2 is green, which is notably distinct from the tan color of the [ iÀPr PDI]CoBr 2 complex 1. This green species is NMR silent but may be crystallized from saturated solutions in Et 2 O to afford 31 (Fig. 6f). The solid-state structure reveals the expected [ iÀPr PDI]CoBr 2 fragment in a distorted square pyramidal geometry (s 5 ¼ 0.36) with a Zn(THF/Et 2 O)Br 2 Lewis acid coordinated to one of the Br ligands. This interaction induces an asymmetry in the structure, causing the Co-Br1 distance (2.557(1) Å) to be elongated relative to the Co-Br2 distance (2.358(2) Å).</p><p>Collectively, these studies suggest that both Co and Zn are present in the reactive carbenoid intermediate, and that ZnBr 2 may interact with the [ iÀPr PDI]Co complex through Lewis acidbase interactions. There is a notable similarity between the observed Co/Zn effect and previous studies of Lewis acid acceleration in the Simmons-Smith cyclopropanation. For example, Zn carbenoid reactions are known to be accelerated by the presence of ZnX 2 , 12c which is generated as a byproduct of the reaction. DFT calculations conducted by Nakamura have suggested that the origin of this rate acceleration may be due to the accessibility of a ve-membered ring transition state, which requires the presence of an additional Zn equivalent to function as a halide shuttle. 23</p><!><p>In summary, transition metal catalysis provides a pathway to accessing unique selectivity in reductive carbenoid transfer reactions. A [ iÀPr PDI]CoBr 2 complex functions as a robust catalyst for Simmons-Smith type cyclopropanation using a CH 2 Br 2 /Zn reagent mixture. This system exhibits the highest regioselectivities that have been observed in reductive cyclopropanations based solely on the steric properties of the alkene substrate. Accordingly, a range of terpenes and conjugated dienes may be converted to a single monocyclopropanated product. Ongoing studies are directed at exploring the applications of transition metal catalysts to other classes of carbenoid transfer reactions.</p>

Royal Society of Chemistry (RSC)

Discovery and Optimization of Salicylic Acid-Derived Sulfonamide Inhibitors of the WD Repeat-Containing Protein 5 (WDR5)\xe2\x80\x93MYC Protein\xe2\x80\x93Protein Interaction

The treatment of tumors driven by overexpression or amplification of MYC oncogenes remains a significant challenge in drug discovery. Here, we present a new strategy towards the inhibition of MYC via the disruption of the protein-protein-interaction between MYC and its chromatin cofactor WDR5. Blocking the association of these proteins is hypothesized to disrupt the localization of MYC to chromatin, thus disrupting the ability of MYC to sustain tumorigenesis. Utilizing a high-throughput screening campaign and subsequent structure-guided design, we identify small molecule inhibitors of this interaction with potent in vitro binding affinity, and report structurally related negative controls that can be used to study the effect of this disruption. Our work suggests that disruption of this protein-protein interaction may provide a path toward an effective approach for the treatment of multiple tumors, and anticipate that the molecules disclosed can be used as starting points for future efforts toward compounds with improved drug-like properties.

discovery_and_optimization_of_salicylic_acid-derived_sulfonamide_inhibitors_of_the_wd_repeat-contain

INTRODUCTION<!>Identification and validation of hits<!>X-ray co-crystal structure of WDR5 bound with a WBM-site hit<!>Early SAR studies<!>Optimization to salicylic acid motif<!>Salicylic Acid Replacement<!>Phenol substitutions<!>Pharmaceutical properties<!>WDR5 stabilization by thermal shift<!>Confirmation of target engagement in cell lysates<!>Chemistry<!>CONCLUSIONS<!>Protein Expression and Purification.<!>HTS Screening.<!>Hit Validation by NMR.<!>Protein Crystallization, Data Collection, and Structure Refinement.<!>Cloning and plasmids.<!>Cell culture.<!>WDR5 Immunoprecipitation.<!>Antibodies.<!>Pharmaceutical property determinations<!>General Chemistry<!>General Procedure A: Sulfonamide coupling<!>General Procedure B: Hydrogenation<!>General Procedure C: Nitration<!>General Procedure D: BBr3 mediated demethylation<!>General Procedure E: Ester hydrolysis<!>General Procedure F: Methyl amide synthesis<!>General Procedure G: Benzyl protection<!>5-Bromo-N-(5-chloro-2-hydroxyphenyl)-2-methoxybenzenesulfonamide (1)<!>N-(5-Chloro-2-hydroxyphenyl)-2-methoxybenzenesulfonamide (5a)<!>5-Chloro-N-(5-chloro-2-hydroxyphenyl)-2-methoxybenzenesulfonamide (5b)<!>N-(5-Chloro-2-hydroxyphenyl)-5-iodo-2-methoxybenzenesulfonamide (5c)<!>3-Bromo-N-(5-chloro-2-hydroxyphenyl)benzenesulfonamide (5d)<!>5-Bromo-N-(2-hydroxyphenyl)-2-methoxybenzenesulfonamide (5e)<!>Step A: 5-Bromo-2-hydroxybenzenesulfonyl chloride (18a)<!>Step B: 5-Bromo-N-(5-chloro-2-hydroxyphenyl)-2-hydroxybenzenesulfonamide.<!>5-Bromo-N-(4-hydroxy-[1,1\xe2\x80\x99-biphenyl]-3-yl)-2-methoxybenzenesulfonamide (5g)<!>5-Bromo-N-(5-(tert-butyl)-2-hydroxyphenyl)-2-methoxybenzenesulfonamide (5h)<!>Step A: 1-(Benzyloxy)-4-bromo-2-nitrobenzene (22)<!>Step B: 1-(Benzyloxy)-4-cyclopropyl-2-nitrobenzene<!>Step C: 2-Amino-4-cyclopropylphenol (23)<!>Step D: 5-Bromo-N-(5-cyclopropyl-2-hydroxyphenyl)-2-hydroxybenzenesulfonamide (5i)<!>Step A. 2-Amino-4-(trifluoromethoxy)phenol (20a)<!>Step B. 5-Bromo-2-hydroxy-N-(2-hydroxy-5-(trifluoromethoxy)phenyl)benzenesulfonamide (5j)<!>Step A: 2-Nitro-4-(pentafluorosulfanyl)phenol<!>Step B: 2-Amino-4-(pentafluorosulfanyl)phenol (20b)<!>Step C: 5-Bromo-2-hydroxy-N-(2-hydroxy-5-(pentafluorosulfanyl)phenyl)benzenesulfonamide (5k)<!>Step A: 2-(4-(Benzyloxy)-3-nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane<!>Step B: 1-(Benzyloxy)-2-nitro-4-(3,3,3-trifluoroprop-1-en-2-yl)benzene (25)<!>Step C: 1-(Benzyloxy)-2-nitro-4-(1-(trifluoromethyl)cyclopropyl)benzene<!>Step D: 5-Bromo-2-hydroxy-N-(2-hydroxy-5-(1-(trifluoromethyl)cyclopropyl)phenyl)benzenesulfonamide (5l)<!>Step A: 1-(4-Methoxy-3-nitrophenyl)cyclopropane-1-carbonitrile (28a)<!>Step B: 1-(4-Hydroxy-3-nitrophenyl)cyclopropane-1-carbonitrile<!>Step C: 1-(3-amino-4-hydroxyphenyl)cyclopropane-1-carbonitrile (29a)<!>Step D: 5-Bromo-N-(5-(1-cyanocyclopropyl)-2-hydroxyphenyl)-2-hydroxybenzenesulfonamide (5m)<!>Step A: 2-(4-hydroxy-3-nitrophenyl)acetonitrile<!>Step B: 2-(4-(benzyloxy)-3-nitrophenyl)acetonitrile<!>Step C: 1-(4-(benzyloxy)-3-nitrophenyl)cyclobutane-1-carbonitrile<!>Step D: 1-(3-amino-4-hydroxyphenyl)cyclobutane-1-carbonitrile (29b)<!>Step E: 5-Bromo-N-(5-(1-cyanocyclobutyl)-2-hydroxyphenyl)-2-hydroxybenzenesulfonamide (5n)<!>Step A: 1-(4-(benzyloxy)-3-nitrophenyl)cyclopentane-1-carbonitrile<!>Step B: 1-(3-amino-4-hydroxyphenyl)cyclopentane-1-carbonitrile (29c)<!>Step C: 5-Bromo-N-(5-(1-cyanocyclopentyl)-2-hydroxyphenyl)-2-hydroxybenzenesulfonamide (5o)<!>Step A: 1-(4-(benzyloxy)-3-nitrophenyl)cyclohexane-1-carbonitrile<!>Step B: 1-(3-amino-4-hydroxyphenyl)cyclohexane-1-carbonitrile (29d)<!>Step C: 5-Bromo-N-(5-(1-cyanocyclohexyl)-2-hydroxyphenyl)-2-hydroxybenzenesulfonamide (5p)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-chlorobenzoic acid (6a)<!>Step A: Methyl 3-((5-bromo-2-methoxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate<!>Step B: 3-((5-Bromo-2-methoxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoic acid (6b)<!>Step A: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate<!>Step B: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoic acid, 6c<!>Step A: 5-Bromo-3-chloro-2-hydroxybenzenesulfonyl chloride (18b)<!>Step B: Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate<!>Step C: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoic acid (6d)<!>Step A: Methyl 5-bromo-2-hydroxy-3-nitrobenzoate<!>Step B: Methyl 2-(benzyloxy)-5-bromo-3-nitrobenzoate<!>Step C: Methyl 2-(benzyloxy)-5-cyclopropyl-3-nitrobenzoate<!>Step D: Methyl 3-amino-5-cyclopropyl-2-hydroxybenzoate<!>Step E: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-2-hydroxybenzoate<!>Step F: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-2-hydroxybenzoic acid (6e)<!>Step A: Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-2-hydroxybenzoate<!>Step B: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-2-hydroxybenzoic acid (6f)<!>Step A: Methyl 2-fluoro-6-hydroxybenzoate (41)<!>Step B: Methyl 3-bromo-2-fluoro-6-hydroxybenzoate (42)<!>Step C: Methyl 3-bromo-2-fluoro-6-hydroxy-5-nitrobenzoate (43)<!>Step D: Methyl 2-(benzyloxy)-5-bromo-6-fluoro-3-nitrobenzoate (44)<!>Step E: Methyl 2-(benzyloxy)-5-cyclopropyl-6-fluoro-3-nitrobenzoate<!>Step F: Methyl 3-amino-5-cyclopropyl-6-fluoro-2-hydroxybenzoate (45)<!>Step G: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoate<!>Step H: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoic acid (6g)<!>Step A: Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoate<!>Step B: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoic acid (6h)<!>Step A: Methyl 3-bromo-5-(trifluoromethoxy)benzoate<!>Step B: Methyl 3-amino-5-(trifluoromethoxy)benzoate<!>Step C: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(trifluoromethoxy)benzoate<!>Step D: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(trifluoromethoxy)benzoic acid (6i)<!>Step A: Methyl 2-hydroxy-5-(trifluoromethoxy)benzoate<!>Step B. Methyl 2-hydroxy-3-nitro-5-(trifluoromethoxy)benzoate<!>Step C: Methyl 3-amino-2-hydroxy-5-(trifluoromethoxy)benzoate<!>Step D: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoate<!>Step E: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoic acid (6j)<!>Step A. Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoate<!>Step B. 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoic acid, 6k<!>Step A: Methyl 3-bromo-5-(pentafluorosulfanyl)benzoate<!>Step B: Methyl 3-amino-5-(pentafluorosulfanyl)benzoate<!>Step C: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoate<!>Step D: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoic acid (6l)<!>Step A: Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoate<!>Step B: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoic acid (6m)<!>Step A: Phenyl 2-methoxy-5-(pentafluorosulfanyl)benzoate<!>Step B: Phenyl 2-hydroxy-5-(pentafluorosulfanyl)benzoate (35)<!>Step C: 2-Hydroxy-5-(pentafluorosulfanyl)benzoic acid<!>Step D: 2-Hydroxy-3-nitro-5-(pentafluorosulfanyl)benzoic acid<!>Step E: Methyl 2-hydroxy-3-nitro-5-(pentafluorosulfanyl)benzoate<!>Step F: Methyl 3-amino-2-hydroxy-5-(pentafluorosulfanyl)benzoate (36)<!>Step G: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfaneyl)benzoate<!>Step H: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoic acid (6n)<!>Step A: Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoate<!>Step B: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoic acid (6o)<!>Step A: Methyl 3-bromo-5-(cyanomethyl)benzoate<!>Step B: Methyl 3-bromo-5-(1-cyanocyclobutyl)benzoate<!>Step C: Methyl 3-amino-5-(1-cyanocyclobutyl)benzoate (53)<!>Step D: Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)benzoate<!>Step E: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)benzoic acid (6p)<!>Step A: 1-(3-Bromo-4-methoxyphenyl)cyclobutane-1-carbonitrile<!>Step B: Phenyl 5-(1-cyanocyclobutyl)-2-methoxybenzoate<!>Step C: Phenyl 5-(1-cyanocyclobutyl)-2-hydroxybenzoate<!>Step D: Phenyl 5-(1-cyanocyclobutyl)-2-hydroxy-3-nitrobenzoate<!>Step E: Phenyl 3-amino-5-(1-cyanocyclobutyl)-2-hydroxybenzoate<!>Step F: Phenyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate<!>Step G: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoic acid (6q)<!>Step A: Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate<!>Step B: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoic acid (6r)<!>Step A: 1-(3-Bromo-4-methoxyphenyl)cyclohexane-1-carbonitrile<!>Step B: Phenyl 5-(1-cyanocyclohexyl)-2-methoxybenzoate<!>Step C: Phenyl 5-(1-cyanocyclohexyl)-2-hydroxybenzoate<!>Step D: Phenyl 3-amino-5-(1-cyanocyclohexyl)-2-hydroxybenzoate<!>Step E: Phenyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoate<!>Step F: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoic acid, 6s<!>Step A: Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoate<!>Step B: 3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoic acid (6t)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxy-N-methylbenzamide (7a)<!>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxy-N-methylbenzamide (7b)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxy-N-methylbenzamide (7c)<!>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxy-N-methylbenzamide (7d)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-N-methyl-5-(pentafluorosulfanyl)benzamide (7e)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-N-methyl-5-(pentafluorosulfanyl)benzamide (7f)<!>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-N-methyl-5-(pentafluorosulfanyl)benzamide (7g)<!>Step A: 3-Amino-5-(1-cyanocyclobutyl)-N-methylbenzamide<!>Step B: 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-N-methylbenzamide, 7h<!>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-N-methylbenzamide (7i)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxy-N-methylbenzamide (7j)<!>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxy-N-methylbenzamide (7k)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxy-N-methylbenzamide (7l)<!>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxy-N-methylbenzamide (7m)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxy-N-(1H-1,2,4-triazol-3-yl)benzamide (7n)<!>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxy-N-(2-methoxyethyl)benzamide (7o)<!>Step A: 3-Chloro-5-(methylsulfonyl)aniline<!>Step B: 5-Bromo-N-(3-chloro-5-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide (7p)<!>5-Bromo-3-chloro-N-(3-chloro-5-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide (7q)<!>Step A: 4-Chloro-2-(methylthio)-6-nitrophenol<!>Step B: 4-Chloro-2-(methylsulfonyl)-6-nitrophenol<!>Step C: 2-Amino-4-chloro-6-(methylsulfonyl)phenol<!>Step D: 5-Bromo-N-(5-chloro-2-hydroxy-3-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide (7r)<!>5-Bromo-3-chloro-N-(5-chloro-2-hydroxy-3-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide (7s)<!>Step A: Methyl 3-(methylsulfonyl)-5-(pentafluorosulfanyl)benzoate<!>Step B: 3-(Methylsulfonyl)-5-(pentafluorosulfanyl)benzoic acid<!>Step C: 3-(Methylsulfonyl)-5-(pentafluorosulfanyl)aniline<!>Step D: 5-Bromo-2-hydroxy-N-(3-(methylsulfonyl)-5-(pentafluorosulfanyl)phenyl)benzenesulfonamide (7t)<!>5-Bromo-3-chloro-2-hydroxy-N-(3-(methylsulfonyl)-5-(pentafluorosulfanyl)phenyl)benzenesulfonamide (7u)<!>Step A: 2-Bromo-4-(pentafluorosulfanyl)phenol<!>Step B: 2-(Methylthio)-4-(pentafluorosulfanyl)phenol<!>Step C: 2-(Methylsulfonyl)-4-(pentafluorosulfanyl)phenol<!>Step D: 2-(Methylsulfonyl)-6-nitro-4-(pentafluorosulfanyl)phenol<!>Step E: 2-Amino-6-(Methylsulfonyl)-4-(pentafluorosulfaneyl)phenol<!>Step F: 5-Bromo-2-hydroxy-N-(2-hydroxy-3-(methylsulfonyl)-5-(pentafluorosulfanyl)phenyl)benzenesulfonamide, 7v<!>5-Bromo-3-chloro-2-hydroxy-N-(2-hydroxy-3-(methylsulfonyl)-5-(pentafluorosulfanyl)phenyl)benzenesulfonamide (7w)<!>Step A: 3-Methyl-5-(methylsulfonyl)aniline<!>Step B: tert-Butyl (3-methyl-5-(methylsulfonyl)phenyl) carbamate<!>Step C: tert-Butyl (3-(bromomethyl-5-(methylsulfonyl)phenyl) carbamate<!>Step D: tert-Butyl (3-(cyanomethyl-5-(methylsulfonyl)phenyl) carbamate<!>Step E: tert-Butyl (3-(1-cyanocyclobutyl)-5-(methylsulfonyl)phenyl) carbamate<!>Step F: 1-(3-Amino-5-(methylsulfonyl)phenyl)cyclobutene-1-carbonitrile hydrochloride<!>Step G: 5-Bromo-3-chloro-N-(3-(1-cyanocyclobutyl)-5-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide<!>Step A: 1-(4-Methoxy-3-(methylthio)phenyl)cyclobutane-1-carbonitrile<!>Step B: 1-(4-Methoxy-3-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile<!>Step C: 1-(4-Hydroxy-3-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile<!>Step D: 1-(4-Hydroxy-3-(methylsulfonyl)-5-nitrophenyl)cyclobutane-1-carbonitrile<!>Step E: 1-(3-Amino-4-hydroxy-5-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile<!>Step F: 5-Bromo-N-(5-(1-cyanocyclobutyl)-2-hydroxy-3-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide (7y)<!>5-Bromo-3-chloro-N-(5-(1-cyanocyclobutyl)-2-hydroxy-3-(methylsulfonyl)phenyl)-2-hydroxybenzenesulfonamide (7z)<!>5-Bromo-N-(5-chloro-2-hydroxy-3-(methylsulfonyl)phenyl)-2-methoxybenzenesulfonamide (8)<!>3-((5-Bromo-2-methoxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxy-N-methylbenzamide (9)<!>6-Bromo-N-(5-chloro-2-hydroxy-3-(methylsulfonyl)phenyl)quinoline-8-sulfonamide (10)<!>6-Bromo-N-(5-(1-cyanocyclobutyl)-2-hydroxy-3-(methylsulfonyl)phenyl)quinoline-8-sulfonamide (11)<!>Step A: Phenyl 3-((6-bromoquinoline)-8-sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate<!>Step B: 3-((6-Bromoquinoline)-8-sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoic acid (12)<!>3-((6-Bromoquinoline)-8-sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxy-N-methylbenzamide (13)<!>Step A: Methyl 3-((6-bromoquinoline)-8-sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoate<!>Step B: 3-((6-Bromoquinoline)-8-sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoic acid (14)<!>3-((6-Bromoquinoline)-8-sulfonamido)-2-hydroxy-N-methyl-5-(pentafluorosulfanyl)benzamide (15)

<p>MYC oncogenes encode a family of related transcription factors (C-, L- and N-MYC) that are overexpressed in a significant number of malignancies.1,2 The oncogenic effects of these MYC proteins are caused by their action as sequence-specific DNA binding transcription factors that drive the expression of genes required for cell growth, proliferation, metabolism, genome instability, and apoptosis.3 MYC proteins are intrinsically disordered, with minimal secondary structure; as such, they lack traditional binding sites for direct inhibition and have been considered to be 'undruggable.'4 Despite significant effort and investment toward the identification of compounds that can directly affect MYC function, few successes have been reported to date.5,6 New approaches to MYC-targeted therapies may focus on the inhibition of the protein-protein interactions (PPI's) of MYC with critical partner proteins.6,7 For example, much work on the MYC-MAX heterodimer has been reported,8–12 and the direct interactions of MYC with other proteins, such as Aurora A, have recently garnered interest.13–15 We have previously reported on the identification of WD repeat-containing protein 5 (WDR5) as a crucial partner in the facilitated recruitment of MYC to chromatin,16 thus making it a critical co-factor for MYC-driven tumorigenesis. Further we have defined the WDR5-MYC interaction site as a potentially tractable target for small molecule inhibition of MYC-driven tumors.16,17</p><p>WD-repeat proteins are a ubiquitous family of scaffolding proteins, containing β-propeller domains that form donut-shaped structures which participate in many multi-protein complexes.18,19 In yeast, WD-repeats are reported to participate in more interactions than any other protein domain.20 The direct targeting of these multi-protein complexes make the WD-repeat family an interesting, although clinically unvalidated, target class in drug discovery.21 WDR5 is one such regulatory scaffolding protein that has reported involvement in protein complexes related to chromatin structure and epigenetic machinery.22 The protein has two major binding sites, previously referred to as the WDR5 Binding Motif (WBM) and WDR5 Interaction Motif (WIN) sites (Figure 1A and B, respectively), that have each been demonstrated to be key interfaces for its role in complex formation.23–25</p><p>We previously described that the association of WDR5 with MYC occurs at the WBM site, and confirmed by co-immunoprecipitation that WDR5 binds to the central-portion of MYC via the evolutionarily conserved 'MYC box' MbIIIb motif. The binding surface is a shallow, hydrophobic cleft containing multiple basic residues, that binds to a 10-amino acid chain containing a "EEIDVV" motif that is conserved in all extant MYC proteins. This same binding cleft has been shown to also bind other proteins; those reported to-date include RBBP5 in the MLL1/SET complex,26,27 and KANSL2, a protein critical for the histone acetyltransferase (HAT) activity of the NSL complex.28 These proteins (MYC, RBBP5, KANSL2) contain a highly similar binding motif (IDVV, LDVV, VDVT respectively) that engage WDR5 in a structurally homologous way. On the opposite face of the protein is the WIN-binding site, which is well studied and contains a deep binding pocket that binds mixed lineage leukemia 1 (MLL1) via a conserved arginine containing motif known as the WIN-motif.23–25 The ability of MLL1 to act as a histone methyltransferase (HMT) and catalytically methylate histone H3 lysine 4 (H3K4) has been demonstrated to be reliant on the formation of a four-protein complex, including WDR5, RBBP5, ASH2L and DPY30 (WRAD complex).26,29,30 Multiple small molecules that disrupt this complex by binding to WDR5 at the WIN binding site have now been published, including both peptidomemetics31–33 and small molecule inhibitors.34–38</p><p>Due to the tertiary structure of the protein, targeting the WIN site is expected to be much more straightforward than discovering potent small molecules against the WBM site. It has been demonstrated that targeting the central cavity of WD-repeat proteins leads to inhibitors that have significantly higher potency than compounds that occupy cavities on the outer surfaces,39 highlighting the challenge presented in targeting this MYC-binding site. For example, CDC20 and Cdc4 are WD-repeat proteins for which inhibitors are reported that bind at such 'external' sites with affinities of >10 μM and 6.2 μM, respectively.40,41 Interestingly, the Cdc4 inhibitor, SCF-12, is reported to occlude substrate binding into the central cavity through a long-range allosteric effect.40 To date, however, there are no reports of a small molecule probe that directly binds to the WBM cleft used by MYC and RBBP5.</p><p>Here, we describe the discovery of the small molecules that bind to the WBM-binding cleft. Utilizing structure-based design approaches based upon our previously published structure of WDR5, we have improved the affinity of the screening hits. Further, we characterize the utility of the lead compounds for their ability to disrupt binding of WDR5 to MYC and RBBP5, and demonstrate that they are capable of functional inhibition of the WRAD complex.</p><!><p>A high-throughput screen (HTS) was performed using a competition fluorescence polarization-based (FP) assay to assess interruption of the binding of a FITC-labeled peptide probe derived from the MYC MbIII protein sequence to WDR5 (aa. 23–334).16 Compounds from the Vanderbilt Discovery and VICB collections (~250,000 total) were screened at 50 μM, and 410 preliminary 'hit' compounds that demonstrated greater than 15% inhibition of the interaction were identified (a hit rate of 0.16%). After duplicate screening, 110 compounds were confirmed as hits; each of these was subsequently evaluated using a ten-point dose-response protocol to calculate binding affinity from fresh DMSO compound dilutions. 76 compounds afforded a measurable Kd value and were taken forward as MYC-site hits (0.03% confirmed hit-rate).</p><p>As the HTS was based upon a competition assay, we also validated the hits in an orthogonal biophysical assay, collecting SOFAST 1H-15N heteronuclear multiple quantum coherence (HMQC) spectra of WDR5 to determine direct binding of the compounds. Peak shifts that correspond to binding at the WBM-binding site were identified by obtaining an HMQC spectrum of uniformly 15N labeled WDR5 with the unlabeled MYC-derived peptide. Identification of this chemical shift perturbation (CSP) pattern allowed us to monitor compound binding to this site by NMR. Screening hits which did not cause any CSP to the NMR spectra of WDR5 were removed from consideration for follow-up chemistry. Validated hits induced either peak shift (Figure 2A) or peak broadening (Figure 2B). These findings suggest that different HTS hits presumably exhibited an intermediate and fast exchange respectively, based upon the NMR chemical shift timescale. These exchange regimes suggest Kd values in the single-digit micromolar (intermediate exchange) or high micromolar (fast exchange), range consistent with the primary FP assay, ranging from 8–175 μM. Multiple confirmed hits contain a bi-aryl sulfonamide motif, with many of the examples also containing an acidic motif that may interact with the charged residues that surround the MYC binding site of WDR5 (Exemplar hits shown in Figure 3).</p><!><p>We obtained an X-ray co-crystal structure of the leading hit, 1, bound to WDR5 by co-crystallization. The overlay of this structure with those of published peptides confirmed that the small molecule bound into the WBM site that mediates the binding of WDR5 to MYC and RBBP5 (Figure 4A). The chlorine atom on the aniline ring is well positioned to mimic the hydrophobic side-chain of the isoleucine of the IDVV motif, while the methoxy group provides a potential vector for compound growth. Key interactions responsible for compound binding include: 1) a hydrogen bond from an oxygen of the sulfonamide to the backbone NH of ASN225; and 2) a halogen bonding interaction from the aromatic bromine to the carbonyl of TRP273 (Figure 4B). These interactions appear to cause a shift of the ASN225 containing loop towards the ligand.</p><!><p>After confirmation of the hit compounds, the focus of our initial synthetic efforts was to investigate the two halogen substituents, the aniline ring chlorine and the sulfonyl ring bromide. The bromine atom forms a key halogen bonding interaction in the X-ray structure. Indeed, when this bromine atom is removed (5a, Table 1) all binding affinity for WDR5 is lost, while replacement with either a chlorine or an iodide both affords a compound with reduced Kd (5b, 5c). Interestingly, removing any one of the four substituents that decorate the biaryl-sulfonamide substructure proved highly deleterious (5d, 5e), demonstrating that this substitution pattern shows good shape complementarity for this binding surface. Similarly, we observed a consistent, significant improvement in affinity when switching from a methoxy R3 group to a phenol (5f).</p><p>Early in the project we observed compounds that were able to bind below the lower limit of detection of the HTS FP assay that used the FITC-labeled MYC peptide (to Kd < 2.5 μM). The data for hits shown in Figure 3 corresponds to this peptide-based probe used throughout the screening campaign. We later designed and synthesized fluorescein-labeled small-molecule probes with higher affinity for WDR5 compared to the peptide, and thus offered lower limits of detection in the FP assay and correspondingly our interpretation of SAR. For clarity, data shown in Tables 1–3 utilize only a single generation of small-molecule fluorescein probe (see structure in Supplemental Figure S1), hence the difference in Kd value of 1 reported in Figure 3, from the high throughput screening campaign, and Table 1.</p><p>On the aniline ring, a chlorine atom occupies the same region as the isoleucine of the native IDVV motif, and as such the chlorine was substituted for a range of hydrophobic groups that were hypothesized to more optimally occupy this region of the protein. Replacement of the halogen with phenyl was unfavorable and resulted in a loss of binding affinity (5g), while the tert-butyl analog (5h) has similar affinity to the parent chlorine. X-ray crystallography demonstrated that larger groups tended to sit in the same space in the binding pocket, but doing so forces the rest of the molecule to shift upward (Figure 5A); smaller aliphatic groups complement this pocket much better (5i, 5j). Pentafluorosulfanyl groups have been reported as metabolically inert tert-butyl analogs,42 and their use herein affords the most potent example of this phenolic class, 5k, with ~5-fold improved binding affinity in comparison to the trifluoromethyl cyclopropane, 5l.43</p><p>The cyano-spirocyclic examples (5m–5p) were initially designed to act as a handle for further growth of the compounds along the edge of this pocket, with the hydrocarbon chain hypothesized, using molecular docking studies, to complement the hydrophobic region. However, we observed by X-ray crystallography that the cyano moiety of 5n is instead oriented toward the protein, albeit with no obvious directional interaction (Figure 5C). The 3- and 5-membered ring examples, 5m and 5o, have slightly diminished affinity in comparison to the 4- and 6-membered rings. We hypothesize that the shape of C3- and C5-carbocycles produce a less favorable orientation of the nitrile, although we do not have crystallographic evidence of such an altered binding pose.</p><!><p>The X-ray co-crystal structure of 1 allowed us to employ structure-based design for the development of further optimized compounds, although a significant challenge to this effort relates to the shallow nature of this binding interface, which contains few areas that may traditionally be referred to as a 'pocket.'44 As well as the early optimization of the hydrophobic moiety in the 'ILE' region of the peptide binding site, a further breakthrough came from the addition of a carboxylic acid directed toward Q289 affording salicylic acid derivative 6b (Kd = 0.43 μM, Table 2). This engagement of Q289 afforded an increase in binding affinity compared to 1, and the positive interaction was confirmed by X-ray crystallography (Figure 5B). Similarly, a significant potency increase was obtained by the addition of a chlorine ortho to the phenol of the sulfonyl ring (6d, Table 2); gaining additional hydrophobic contacts with WDR5 in the interface occupied by a valine side chain of Myc and RBBP5 (IDVV).</p><p>The cyclopropyl derivatives (6e & 6f) demonstrated an improvement versus the parent chloro-containing compound, and the addition of an ortho-fluoro group afforded a further increase (6g). This enhanced affinity could be explained by the good shape complementarity of this compound to the binding site as demonstrated crystallographically (Figure 5E). In line with the SAR trends of Table 1, the hydrophobic chloro-replacement trifluoromethoxy, pentafluorosulfanyl, and cyano-spirocycle derivatives afforded low nanomolar potency with improvements 5 to 15-fold upon the addition of the acid. Generally, we observed a significant decrease in binding affinity when removing the phenol of the salicylic acid moiety, 6a, 6i, 6p; however, this decrease was not observed for the -SF5 analogs (6l, 6m to 6n, 6o).</p><!><p>With this series of salicylic acid-based compounds, we were able to reach low-nanomolar levels of potency. However, we suspected inherent issues related to permeability and plasma protein binding due to the presence of the carboxylic acid, limiting their use in whole cell mechanism of action (MOA) studies. To avoid these problems, we explored the replacement of the carboxylic acid with a methyl amide (Table 3). Within this salicylamide series, a reduction in potency compared to the direct acid analogs was observed; however, by combining optimal SAR observations in the ILE262 region we were able to identify analogs which retained binding affinities below 100 nM. The hydrophobic ring of the cyclobutyl-cyano moiety (7k), adopts a similar binding pose as the cyclopropane of 6g (Figure 5D), bolstering our hypothesis of improved affinity when designing analogs that grow from the ipso carbon. Interestingly, while in the salicylic acid series, the cyclohexyl derivatives 6s, 6t demonstrated high binding affinity, this trend did not hold for the amide derivatives 7l, 7m.</p><p>During this exercise we explored alternate amide groups, including 7n, 7o, but, with the exception of 7n, we observed very flat SAR when exploring different amine partners (data not shown). We hypothesize that the amide group is directed toward solvent and does not make positive interactions with the protein, which led to the choice of this vector for attaching the small molecule FITC-probes used for the assay (structure of probe shown in Supplemental Figure S1). In a separate salicylate replacement effort, we were able to demonstrate that the acid/amide moiety could be exchanged with a sulfone; for example, methyl sulfone containing compounds 7p-7z were synthesized. Their design was driven in part to enhance physicochemical properties which might overcome some of the limitations observed in the salicylic acid subseries. Incorporation of the best-in-class pieces led to compounds such as 7w and 7z that bind with high potency to WDR5 and are comparable to analogs in the acid and amide series. Indeed, examples lacking the aniline-ring phenol were superior to the corresponding amide (compare 7b vs 7q, 7e vs 7t). Co-crystallization of WDR5 with 7x demonstrates that the sulfone also engages Q289 via a hydrogen bond (Figure 5F). Similar to the amides, the methyl group is directed toward bulk solvent offering a potential vector for future derivatization to tune the physicochemical properties.</p><!><p>While we were able to discover small molecules that bind to WDR5 with excellent affinity, considering the shallow nature of the binding site, we recognize that these molecules retain functionality, such as phenols, that would likely occlude their development. Tables 2 and 3 already detail the synthesis of selected compounds with the aniline-ring phenol removed. Separately, we explored several strategies to remove or replace the para-bromophenol of the sulfonyl ring. A number of phenol and bromine replacement strategies failed (not shown), however, we were able to show, using optimized aniline-ring groups "capping" the phenol as a methoxy (e.g. 8, 9) can lead to inhibitors with sub-micromolar affinity in selected cases (Figure 6). Further, combining the best-in-class aniline groups to generate 6-bromoquinoline-8-sulfonamides afforded nanomolar analogs that do not contain the para-bromophenol motif. In this class, we observe that the sulfone and acid derivatives retain promising affinity in comparison to their direct ortho-chlorophenol analogs. Interestingly, the reduction in affinity observed when replacing the bromophenol appears more pronounced for the methyl amides (compare 7j, 7k, vs 9, 13), while the sulfone examples 8, 10 and 11 remain similar to 7x-7z. Notably, 15 is an exception.</p><!><p>We explored the tier 1 DMPK profile of a number of compounds within this class (Table 4). These data demonstrate that all tested analogs have good solubility, and the amide and sulfone series have significantly improved MDCK permeability in comparison to their salicylic acid counterparts. There was no evidence of P-gp mediated efflux with the tested analogs. A limitation of this compound class is the severe plasma protein binding exhibited by all tested compounds; indeed, salicylic acid itself is known to be a substrate for human serum albumin.45,46 Efforts to overcome this hurdle to develop an improved chemical probe are ongoing.</p><!><p>In order to further demonstrate effective, on-target binding of our inhibitors to WDR5, we used differential scanning fluorimetry (DSF) to examine a subset of compounds (Figure 7). For the salicylic acid subseries (Figure 7A), we observe that the most potent examples by FPA, (6o, 6r, 6t) also impart the largest ΔTm, and thereby stabilization of WDR5. A negative control compound, 6-NC, contains a regioisomeric bromine designed such that the key halogen bonding bromine atom is moved around the phenyl ring, causing a significant decrease in potency (see Supplemental Figure S1.) This is a direct regioisomer of 6q, and in this assay 6-NC has minimal effect (ΔTm = 0.42 °C). When comparing a compound sub-set that all contain the spirocyclobutane moiety (Figure 7B), the addition of the carboxylic acid (6q vs 5n) offers a significant increase in binding affinity by FPA, but only a marginal improvement in thermal shift is observed. However, the additional hydrophobic interaction brought by the addition of the chlorine on the sulfonyl-ring affords compounds with an increased ΔTm (6r, 7k). Figure 7C highlights that the binding of 7k demonstrates a concentration-dependent response. A summary of the ΔTm values is displayed in Figure 7D, and the effect of further tested examples is shown in Supplemental Table S1. We have also confirmed that the binding of the 7k in the absence of WDR5 protein does not have any effect on the background fluorescence at any of these concentrations (Supplemental Figure S2), and that these compounds do not display significant binding at the 'WIN' binding site (Supplemental Figure S4).38</p><!><p>To assess whether compounds disrupt the association between WDR5 and interacting partners at the WBM-site, (MYC and RBBP5), we immunoprecipitated WDR5 from cell lysates treated with compounds. We first chose a representative selection of compounds from the acid and amide subseries with a range of binding affinities to WDR5 as measured by FPA. Initially, we treated cell lysates with 50 μM compound, and observed varying degrees of disruption between both WDR5-c-MYC and WDR5-RBBP5 as compared to both the vehicle control and the representative negative control compounds that bind with markedly lower affinity to WDR5 from both acid and amide subseries (Figure 8A, B). As anticipated, the degree of disruption between WDR5-c-MYC and WDR5-RBBP5 was similar for each compound. Furthermore, the degree of disruption also corresponded to the compound's respective binding affinity to WDR5, where compounds with a higher affinity to WDR5 disrupted the WBM-site protein interactions to a greater degree, further highlighting the efficacy of the acid compound subseries. Similarly, exemplar compounds from the sulfone series, as well as those with phenol replacements can also disrupt binding of c-MYC to WDR5 in cellular lysates (Supplemental Figure S3), in a manner that is associated with the affinity of their binding to WDR5.</p><p>Further, to assess the effects of lower compound concentrations on the disruption of the WDR5-c-MYC and WDR5-RBBP5 associations, we also conducted a dose response treatment with compounds 6r and 7k, alongside their corresponding negative controls 6-NC and 7-NC (Figure 8C). We observed a dose-dependent disruption of the association between both WDR5-c-MYC and WDR5-RBBP5, as compared to the vehicle control and to the respective negative control compounds from each subseries. The dose-dependency was most clearly demonstrated in response to 7k treatment when assessing the association between WDR5 and RBBP5. The improved efficacy of the acid compound 6r was also evident, as a marked disruption between both WDR5-c-MYC and WDR5-RBBP5 was clearly observed even at the lowest concentration tested.</p><p>Overall, these compounds demonstrate biochemical binding affinity caused by the displacement of a FITC-labeled probe from recombinant, purified WDR5, and binding to WDR5 in cell lysates by thermal shift and co-immunoprecipitation. We are also able to demonstrate a functional effect of this binding, as these compounds inhibit the biochemical histone methyltransferase (HMT) activity of MLL-1 in the full WRAD complex (Supplemental Table S2). The results of this assay suggest that compounds disrupting the WDR5:RBBP5 interface of the multi-protein WRAD complex may offer an alternative strategy for the targeting of tumors driven by abrogation of MLL-proteins, avoiding the WIN binding site. Interestingly, we observe that the potency of functional inhibition in the HMT assay is much closer to the biochemical binding affinity of inhibitors at the WBM site, versus previously disclosed inhibitors of WIN site from both our group and others.32,33,38 The inherent weaker affinity of the WDR5:RBBP5 interaction may present a significantly lower barrier for a small molecule disruptor to overcome in comparison to that of the WDR5:MLL interface.47</p><!><p>All compounds were synthesized in modular fashion using a sulfonamide coupling to join elaborated anilines and sulfonyl chlorides. This coupling used pyridine as a base, which favors N- versus O-linked reaction. For examples 1, 5a–e, 5g, 5h, both coupling partners were commercially available, allowing for rapid initial SAR exploration (Scheme 1A), while the more elaborate aniline pieces often required multi-step synthesis. Chlorosulfonation of the 4-bromophenol derivatives afforded corresponding sulfonyl chlorides, 18a,b (Scheme 1B). Examples 5j and 5k both originated from corresponding phenols, which were nitrated and reduced to the required aniline intermediates 20. The Suzuki-Miyaura coupling using cyclopropylboronic acid was reliant on protection to form intermediate 23. The phenol was benzyl protected, and the aniline masked as the corresponding nitro group. After cross-coupling the subsequent hydrogenation cleanly affords the anilino-phenol, 23 (Scheme 1D). The trifluoromethylcyclopropane example was synthesized using methodology from Merck,48,49 cross-coupling first to the CF3-styrene, 21, and cyclopropanation using a sulfonium ylide as a diazomethane equivalent (Scheme 1E). As previously described, deprotection by hydrogenation affords the desired intermediate 26.</p><p>The cyano-spirocycles were all synthesized by cyclization of O-protected intedmediates 27 with the corresponding dibromoalkane using sodium hydride in DMSO and the common bromide intermediates 28 were used to furnish all of the required aniline intermediates (Scheme 2). Toward products 5m-p, the bromide was displaced with tert-butyl carbamate to furnish the desired aniline, 29a-d, after deprotection. Intermediates 28 could instead be converted to the respective phenyl ester using palladium-mediated carbonylation to generate 30 or to the methyl sulfone by palladium-mediated coupling with sodium methanethiolate and then oxidation with potassium peroxymonosulfate to 32. The synthesis of products 6q, 6r and 12 was completed by heating the phenyl ester, 31, with lithium hydroxide. The salicylamide formation to 7j-o, 9 and 13 proceeded by heating the ester directly with the corresponding amine; such amide formations did not require prior hydrolysis.</p><p>Pentafluorosulfanyl analogs were synthesized using a similar chemical approach as previously used for the cyano-spirocycles (Scheme 3). The -SF5 group in all instances was incorporated in the commercially available intermediatedes. 34 underwent same palladium-mediated carbonylation and demethylation to 35, but the nitration of the phenyl ester was unsuccessful despite a condition screen. However, nitration of the benzoic acid and subsequent esterification to the methyl ester afforded access to intermediate 36 in good yield (Scheme 3A). The corresponding sulfones were introduced via palladium-cross coupling of the aryl bromide with dimethyl disulfide, then oxidation to 38; nitration with a nitric acid followed by hydrogenation furnished 39.</p><p>The synthesis of the densely substituted analogs 6g, 6h, 7c, and 7d is outlined in Scheme 4. The readily available acid 40 was esterified, and the subsequent bromination was found to be largely selective para- to the phenol. The inseparable, minor dibrominated byproduct was separable after subsequent nitration to 43. As described above, protection of the phenol was required for cross-coupling of cyclopropylboronic acid, and a similar sequence of hydrogenation, sulfonamide coupling, and hydrolysis or amidation affords the screened compounds.</p><p>The synthesis of analogs that lack a phenol on the aniline ring is shown in Scheme 5. The sulfone intermediates are all synthesized by palladium-mediated cross coupling with sodium methanesulfinate (Scheme 5A – C). Toward 7t and 7u, a Curtis rearrangement of the acid intermediate was utilized to generate aniline 49. Spiro-cyclobutanes were synthesized from toluene-derived starting materials, which were brominated, and the nitrile introduced using TMSCN. Cyclisation with 1,3-dihalopropanes affords intermediates 51 and 53, that were used for sulfonamide formation.</p><!><p>WD-repeat proteins are an emerging class of targets for a range of therapeutic areas, although there has been, to date, no clinical validation of these proteins with approved drugs. Inhibitors of WDR5 are being sought as highly attractive options for the treatment of multiple cancer types. To date, there have been several inhibitors published that bind to WDR5 at the 'WIN' binding site, but no inhibitors of the 'WBM' site have been disclosed. An inhibitor of this site directly prevents WDR5 from being able to bind to MYC, RBBP5, and KANSL2 among others, and thus could be of therapeutic interest in cancers driven by oncogenic effects of these proteins. Further, this approach may offer an alternative pathway to affect the HMT activity of MLL1 while not directly preventing the binding of other protein partners into the MLL1 binding region.</p><p>We performed a high throughput screen of the Vanderbilt library and obtained a series of biaryl sulfonamide hits that were unambiguously confirmed as inhibitors of this binding site by NMR and X-ray crystallography. Using rational design, we optimized hit compound 1, yielding multiple analogs binding at less than 30 nM. To our knowledge, the data reported herein represents the first published examples of a small molecule inhibitor binding to an external cleft of a WD-repeat protein with such levels of affinity. Binding to the protein was confirmed by secondary assays, including NMR and DSF. Biochemical activity at this cleft was demonstrated in a secondary HMT assay, whereby the small molecule was able to successfully disrupt the WRAD multi-protein complex, and thus the methyltransferase function of MLL1. These compounds bind on the opposite face of WDR5 to the MLL1 interaction. We observe no major changes in the protein X-ray structure that would suggest an allosteric modification in the MLL1 binding pocket, yet these compounds are still able to prevent the arginine methylation of this complex. Intriguingly, the difference in activity observed between the binding and HMT assays is significantly less than that previously reported for direct WIN-site inhibitors.37 Further, some SAR correlations are evident with subseries of compounds, even in this limited dataset, prompting significant interest and further studies within our laboratories.</p><p>These inhibitors were demonstrated to be highly soluble, and permeability was significantly improved by switching from a salicylic acid to salicylamide or methyl sulfone motif, all of which are able to form a H-bond interaction with GLN289. Compounds from this series are able to disrupt the interaction of WDR5 with both c-MYC and RBBP5 by co-immunoprecipitation in cellular lysates in a manner that is correlated with binding affinity. However, the low free fraction shown by most analogs limits their utility in whole cells. Ongoing efforts seek to improve the drug-like character of these compounds to discover molecules that can be used as probes of the interruption of the WBM-site.</p><!><p>Truncated WDR5 (a.a. 22–334) was cloned into a pET vector with a 6xHis-SUMO tag fused at the N-terminus. The plasmids WDR5 was transformed into E. coli BL21 (DE3) cells. The overnight culture was used to start a 10 L fermentation (BioFlo 415, New Brunswick Scientific) grown at 37 °C. For NMR samples, uniformly 15N-labeled protein was produced in minimal M9 medium, where 15NH4Cl (Cambridge Isotope Laboratories) and D-glucose were used as sole nitrogen and carbon sources. When the cell density reached OD600 = 2.5, the temperature was lowered to 30 °C. The protein was expressed overnight with 1mM isopropyl-β-D-thiogalactoside (IPTG). Myc peptide (DEEEIDVVSVE) was ordered (Genscript) as HPLC purified synthetic polypeptide. It was dissolved in DMSO for further use.</p><p>Cell pellets were dissolved in lysis buffer (1XPBS plus 300 mM NaCl, 20 mM imidazole, 5 mM BME, and10% glycerol), and broken by homogenization (APV-2000, APV). The lysate was cleared by centrifugation and filtering, and then applied to an affinity column (140 mL, ProBond, Invitrogen). Bound protein was eluted by an imidazole gradient. The His-SUMO-tag was removed by SUMO protease cleavage during dialysis and the subsequent subtractive second nickel-column. WDR5 protein was then purified by size-exclusion chromatography (HiLoad 26/60, Superdex 75, GE Healthcare) using NMR or crystallization buffer.</p><!><p>The previously described Myc peptide,16 was labelled with FITC and used as the probe for FPA assays. The probe was ordered form Genscript. 5 μM WDR5 protein and 5 μM probe were used in the HTS, and the buffer condition was 1XPBS plus 300 mM NaCl, pH 6.0, 0.5mM TCEP, 0.1% CHAP, and 5% DMSO. Vanderbilt Discovery Collection (VDC) and VICB collection compounds (~250,000) were tested at 50 μM concentration, and the compounds was put into 781 386-well plates with necessary positive and negative controls in each plate. After adding all reagents, the plates were shaken for ~2 minutes, and incubated for 60 minutes before first plate to be read on plate reader (Biotek). The reading settings of the plate reader were 50 flashes, low lamp energy, and 7.75 read height. All the plates were screened, and those plates (totaling 17) with Z' less than 0.3 were repeated. Compounds, which showed >15% inhibition, were selected for the confirmation screen. Confirmed compounds then were used for a 10-point dose response study.</p><!><p>NMR samples contained 2 mg/mL (~67 μM) 15N-labeled WDR5 in 25 mM phosphate buffer, pH=6.0, 100 mM NaCl, and 1 mM DTT. HTS hits were used at 100 μM. Nuclear magnetic resonance screening was conducted using a Bruker Avance III 600 MHz NMR spectrometer equipped with a 5mm single-axis z-gradient cryoprobe and a Bruker Sample Jet sample changer. Two-dimensional, gradient-enhanced 1H-15N heteronuclear multiple-quantum coherence (SOFAST-HMQC)51 spectra were collected at 25 °C and used to track chemical shift perturbation upon compound binding. Spectra were processed and visualized using Topspin (Bruker BioSpin).</p><!><p>WDR5 was concentrated to 10 mg/mL (~300 μM) in the buffer of 20 mM HEPES, pH 7.0, 250 mM NaCl, and 5 mM DTT. Co-crystals were obtained at 18 °C using the hanging drop method. The crystallization condition was 0.1 M Bis-Tris pH 6.0, 0.2 M ammonium acetate, 28% to 32% PEG3350. Soaking was also applied to some compounds using crystals of WDR5 that contain an MLL-site binder. Crystals were flash frozen in liquid nitrogen directly.</p><p>Diffraction data were collected on the Life Sciences Collaborative Access Team (LS-CAT) 21-ID-D and G beamlines at the Advanced Photon Source (APS), Argonne National Laboratory. Data were indexed, integrated, and scaled with HKL2000.52 Molecular replacement was achieved with Phaser44 as implemented in CCP4.4553 using a previously determined WDR5 structure (PDB code 3EG6). Refinement of the structural models was conducted with PHENIX54 and included rounds of manual model building in COOT.55 All structure images were prepared with PyMOL.56</p><!><p>Full length human WDR5 with an N-terminal FLAG tag was cloned into the NgoMIV and SalI sites as a NgoMIV/XhoI fragment. pBabe-GFP was constructed by PCR amplifying the GFP fragment from pEGFP-C2 (Clontech) and cloning the product into the EcoRI and BamHI sites of pBabe Puro. Full length human c-MYC with a C-terminal double HA tag was cloned into the BamHI and EcoRI sites of pBabe-IGH.16 HEK293 cells stably expressing MYC2HA were made by retroviral transduction followed by selection in Hygromycin (50 μg/mL). The mixed population was then infected with pBabe-Puro expressing GFP or WDR5 with selection in puromycin (1 μg/mL). For retroviral transductions, HEK293T cells were transfected with the appropriate pBabe vector, the pCL10A packaging vector, and pMax-GFP to estimate transfection efficiency. Viral supernatant was collected and used to infect HEK293 class over three days.</p><!><p>HEK293 cells were maintained in DMEM supplemented with 10% FBS. Hygromycin B (50 μg/mL) and puromycin (100 ng/mL) were added to media to maintain plasmid expression. Both cell lines were tested and confirmed negative of mycoplasma using the VenorGem PCR test kit (Sigma Aldrich). After thawing from liquid nitrogen, cells were passaged at least twice before use in experiments, and passaged for a maximum of 25 times.</p><!><p>HEK293 cells were harvested and lysates were prepared on ice in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X100 and supplemented with protease and phosphatase inhibitors). Equal amounts of protein lysate were subject to immunoprecipitation with M2 agarose overnight (for WDR5-c-MYC) or for 2 hours with magnetic M2 beads (for WDR5-RBBP5) at 4 °C. Immune complexes were recovered, washed in lysis buffer, and resolved by SDS-PAGE. Immunoblotting was performed using the indicated primary antibodies, incubated with labeled secondary antibodies and membranes were scanned using the Odyssey imager (LiCor).</p><!><p>The following primary antibodies were used for this study: α-c-MYC (#5605), α-RBBP5 (#13171), α-WDR5 (#13105), α-FLAG (#8146) all purchased from Cell Signaling.</p><!><p>All experiments performed at Q2 Solutions Ltd. (https://www.q2labsolutions.com) using commercially available, standard assay formats. Kinetic solubility determinations: Method QUI-SOL-001. MDCK permeability determinations: Method QUI-PERM-003. Protein binding assessment: Method QUI-PERM-002.</p><!><p>All chemical reagents and reaction solvents were purchased from commercial suppliers and used as received. Hydrogenation reactions are performed using an atmospheric balloon. Normal phase flash silica gel-based column chromatography is performed using ready-to-connect cartridges from ISCO, on irregular silica gel, particle size 15–40 μM using a Teledyne ISCO Combiflash Rf system. Preparative reversed-phase HPLC was performed on a Gilson instrument equipped with a Phenomenex Kinetex C18 column, using gradients of MeCN in H2O and 0.1% TFA. Compounds that are obtained as a TFA salt after purification were afforded as free base, by dissolving the salt in EtOAc and washing with sat. aq. K2CO3. Proton nuclear magnetic resonance (1H NMR) spectra were recorded at either 400 MHz or 600 MHz on a Bruker spectrometer, as stated. For 1H NMR spectra, chemical shifts are reported in parts per million (ppm) and are reported relative to residual non-deuterated solvent signals. Coupling constants are reported in hertz (Hz). The following abbreviations (or a combination, thereof) are used to describe splitting patterns: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; br, broad. All compounds were of 95% purity or higher, unless otherwise noted, as measured by analytical reversed-phase HPLC. Analytical HPLC was performed on an Agilent 1200 series system with UV detection at 214 and 254 nm, along with evaporative light scattering detection (ELSD). Low-resolution mass spectra were obtained on an Agilent 6140 mass spectrometer with electrospray ionization (ESI). LCMS experiments were performed with the following parameters: Phenomenex Kinetex 2.6 μm XB-C18 100 Å, LC column 50 × 2.1 mm; a gradient of 5%–95% MeCN in H2O, and 0.1% TFA, over a 1.2 min or 2.0 min gradient. HRMS experiments were conducted at the University of Notre Dame Mass Spectrometry and Proteomics Facility.</p><!><p>The corresponding aniline (1 eq) and sulfonyl chloride (1.2–1.5 eq) were combined in CH2Cl2 (at 0.2 M]) at r.t., and pyridine (3 eq) was added; the mixture was stirred for 1–16 h. The mixture was concentrated in vacuo and purified by ISCO flash chromatography, unless stated.</p><!><p>Corresponding intermediate (1 eq) was dissolved in MeOH, EtOAc was added to aid dissolution where required. Pd/C (5% C by wt., 10 mol%) was added and the mixture stirred under a H2 atmosphere for 16 h. The mixture was filtered through celite, concentrated, and purified by ISCO flash chromatography if required.</p><!><p>Corresponding intermediate (1 eq) was dissolved in DCM(at [0.4 M]) and cooled to 0 °C in an ice/water bath. To this was added a pre-mixed solution of HNO3 (conc., 1.5 eq) and H2SO4 (conc., 1.5 eq) drop wise. The mixture was stirred for 1–16 h, allowing to warm to r.t., then poured over ice and extracted with DCM. The crude material was purified by ISCO flash chromatography.</p><!><p>The intermediate (1 eq) was dissolved in anhydrous DCM (at [0.4 M]) and cooled to −78 °C in an acetone/dry ice bath under an inert atmosphere. To this was added BBr3 ([1.0 M in DCM], 2 eq) drop-wise, and the mixture stirred for 1 h at −78 °C, then allowed to warm to r.t.. The mixture was poured over an ice-water slurry and extracted with EtOAc, washing with water, brine. Crude product was purified by ISCO flash chromatography.</p><!><p>The ester intermediate (1 eq) was stirred with LiOH (2 M, 5 eq) in THF (at 0.2 M]) for 1–16 h at 65 °C, unless stated at r.t.. The mixture was acidified with hydrochloric acid, extracted with EtOAc and washed with brine. The crude material was purified by preparative HPLC.</p><!><p>To the salicylate ester intermediate (1 eq) was added methylamine ([2.0M in THF], 10 eq) and the mixture heated at 65 °C for 1–16 h. The mixture was concentrated in vacuo and purified by preparative HPLC.</p><!><p>The intermediate phenol (1 eq), benzyl bromide (1.05 eq) and potassium carbonate (1.1 eq) were combined in acetonitrile and heated to reflux for 18 h. Upon cooling the mixture was concentrated in vacuo, re-dissolved in EtOAc and washed with water, brine. Crude material was purified by ISCO flash chromatography.</p><!><p>2-Amino-4-chlorophenol (144 mg, 1.0 mmol) was reacted with 5-bromo-2-methoxybenzenesulfonyl chloride, 16a (428 mg, 1.5 mmol) following General Procedure A, affording the title compound as a colorless solid (283 mg, 0.72 mmol, 72%). 1H NMR (400 MHz, MeOH-d4) δH 7.83 (d, J = 2.5 Hz, 1H), 7.66 (dd, J = 8.9, 2.5 Hz, 1H), 7.31 (d, J = 2.5 Hz, 1H), 7.08 (d, J = 8.9 Hz, 1H), 6.88 (dd, J = 8.6, 2.6 Hz, 1H), 6.67 (d, J = 8.6 Hz, 1H), 3.91 (s, 3H); LCMS tR = 1.44 min, m/z = 393.7 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+H]+ calculated for C13H12BrClNO4S 393.9353, found 393.9344.</p><!><p>2-Amino-4-chlorophenol (14 mg, 0.10 mmol) was reacted with 2-methoxybenzenesulfonyl chloride (31 mg, 0.15 mmol) following General Procedure A, affording the title compound as a colorless solid (25 mg, 0.08 mmol, 80%). 1H NMR (400 MHz, CDCl3) δH 7.79 (dd, J = 7.9, 1.7 Hz, 1H), 7.57 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.99 (dd, J = 8.7, 2.5 Hz, 1H), 6.86 – 6.79 (m, 3H), 6.58 (s, 1H), 4.07 (s, 3H); LCMS tR = 1.54 min, m/z = 313.9 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Amino-4-chlorophenol (14 mg, 0.1 mmol) was reacted with 5-chloro-2-methoxybenzenesulfonyl chloride (36 mg, 0.15 mmol) following General Procedure A, affording the title compound as a colorless solid (21 mg, 0.06 mmol, 60%). 1H NMR (400 MHz, CDCl3) δH 8.50 (s, 1H), 7.47 (dd, J = 6.6, 2.6 Hz, 2H), 7.34 (dd, J = 8.9, 2.6 Hz, 1H), 7.11 (dd, J = 8.8, 2.5 Hz, 1H), 7.04 (s, 1H), 6.89 (d, J = 8.8 Hz,1H), 6.70 (d, J = 8.8 Hz, 1H), 3.65 (s, 3H); LCMS tR = 1.03 min, m/z = 377.3 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Amino-4-chlorophenol (14 mg, 0.1 mmol) was reacted with 5-iodo-2-methoxybenzenesulfonyl chloride (50 mg, 0.15 mmol) following General Procedure A, affording the title compound as a pale brown solid (5 mg, 0.01 mmol, 11%). 1H NMR (400 MHz, MeOH-d4) δ 7.98 (d, J = 2.3 Hz, 1H), 7.82 (dd, J = 8.8, 2.3 Hz, 1H), 7.27 (d, J = 2.6 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 6.86 (dd, J = 8.6, 2.6 Hz, 1H), 6.67 (d, J = 8.6 Hz, 1H), 3.90 (s, 3H); LCMS tR = 1.03 min, m/z = 440.1 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Amino-4-chlorophenol (17 mg, 0.12 mmol) was reacted with 3-bromobenzenesulfonyl chloride (46 mg, 0.18 mmol) following General Procedure A, affording the title compound as a colorless solid (22 mg, 0.72 mmol, 51%). 1H NMR (400 MHz, MeOH-d4) δH 7.92 (t, J = 1.8 Hz, 1H), 7.75 – 7.66 (m, 2H), 7.37 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 2.6 Hz, 1H), 6.95 (dd, J = 8.7, 2.6 Hz, 1H), 6.66 (d, J = 8.7 Hz, 1H); LCMS tR = 1.50 min, m/z = 363.6 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Aminophenol (17 mg, 0.16 mmol) was reacted with 16a (68 mg, 0.24 mmol) following General Procedure A, affording the title compound as a colorless solid (22 mg, 0.06 mmol, 38%). 1H NMR (400 MHz, CDCl3) δH 7.88 (d, J = 2.5 Hz, 1H), 7.63 (dd, J = 8.8, 2.5 Hz, 1H), 7.07 (ddd, J = 8.9, 7.4, 1.6 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.91 (dd, J = 8.0, 1.4 Hz, 1H), 6.86 (dd, J = 8.0, 1.6 Hz, 1H), 6.83 (s, 1H), 6.73 (td, J = 7.7, 1.4 Hz, 1H), 6.39 (br s, 1H); LCMS tR = 1.41 min, m/z = 359.3 [M+H]+; Purity (AUC) ≥95%.</p><!><p>4-Bromophenol, 17a (1.73 g, 10 mmol) was added portion wise to ice-cold chlorosulfonic acid (4.77 mL, 70 mmol) and the mixture stirred for 16 h, warming to r.t. The mixture was carefully poured over a slurry of ice, DCM and brine, and extracted with DCM. Purification by flash chromatography affords title compound as a pale brown oily solid (1.50 g, 5.53 mmol, 55%). 1H NMR (400 MHz, CDCl3) ∂H 7.95 (d, J = 2.4 Hz, 1H), 7.71 (dd, J = 8.9, 2.4 Hz, 1H), 7.04 (d, J = 8.9 Hz, 1H).</p><!><p>2-Amino-4-chlorophenol (14 mg, 0.1 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording title compound as a colorless solid (21 mg, 0.06 mmol, 55%). 1H NMR (400 MHz, CDCl3) δH 7.66 (d, J = 2.5 Hz, 1H), 7.53 (dd, JJ = 8.8, 2.5 Hz, 1H), 7.08 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H); LCMS tR = 1.53 min, m/z = 377.8, 379.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>3-Amino-[1,1'-biphenyl]-4-ol (24.3 mg, 0.13 mmol) was reacted with 16a following General Procedure A to afford title compound as a colorless solid (28 mg, 0.06 mmol, 49%). 1H NMR (400 MHz, CDCl3) δH 7.98 (t, J = 1.9 Hz, 1H), 7.57 (dd, J = 8.8, 2.5 Hz, 1H), 7.40 – 7.35 (m, 5H), 7.31 – 7.27 (m, 1H), 7.18 (dd, J = 8.4, 2.2 Hz, 1H), 6.88 (t, J = 7.7 Hz, 2H), 3.96 (s, 3H); LCMS tR = 1.644 min, m/z = 435.3 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Amino-4-(tert-butyl)phenol (22 mg, 0.13 mmol) was reacted with 16a following General Procedure A to afford title compound as a colorless solid (26 mg, 0.06 mmol, 48%). 1H NMR (400 MHz, CDCl3) δH 7.86 (d, J = 2.5 Hz, 1H), 7.62 (dd, J = 8.8, 2.5 Hz, 1H), 7.07 (dd, J = 8.6, 2.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.76 (d, J = 2.5 Hz, 2H), 6.27 (s, 1H), 4.05 (s, 3H), 1.10 (s, 9H); LCMS tR = 1.70 min, m/z = 415.3 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Nitro-4-bromophenol, 21 (10.9 g, 50 mmol) was reacted according to General Procedure G, to afford 22 as a yellow solid (14.2 g, 46.1 mmol, 92%). LCMS tR = 1.25 min, m/z = 307.2 [M+H]+.</p><!><p>1-(benzyloxy)-4-bromo-2-nitrobenzene, 22 (1.54 g, 5.0 mmol), cyclopropylboronic acid (515 mg, 6.0 mmol), Pd(OAc)2 (56 mg, 0.25 mmol), PCy3.HBF4 (184 mg, 0.50 mmol), K3PO4 (2.65 g, 12.5 mmol) were taken in toluene (25 mL) and water (5 mL) and stirred at 110 °C in a sealed tube for 16 h. The cooled mixture was filtered through celite, diluted with EtOAc and washed with water. Purification by flash chromatography affords a yellow solid (1.22 g, 4.53 mmol, 91%). LCMS tR = 1.27 min, does not ionize by ESI.</p><!><p>1-(Benzyloxy)-4-cyclopropyl-2-nitrobenzene (539 mg, 2.0 mmol) was reacted following General Procedure B to afford 23 as a colorless solid (262 mg, 1.75 mmol, 88%). 1H NMR (400 MHz, MeOH-d4) δH 6.59 (d, J = 8.2 Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H), 6.36 (dd, J = 8.2, 2.2 Hz, 1H), 1.80 – 1.70 (m, 1H), 0.87 – 0.78 (m, 2H), 0.58 – 0.49 (m, 2H); LCMS tR = 1.00 min, m/z = 150.3 [M+H]+.</p><!><p>2-Amino-4-cyclopropylphenol, 23 (15 mg, 0.1 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording title compound as a colorless solid (17 mg, 0.04 mmol, 44%). 1H NMR (400 MHz, CDCl3) δH 8.40 (s, 1H), 7.61 (d, J = 2.5 Hz, 1H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 6.90 (dd, J = 8.4, 2.2 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 2.2 Hz, 1H), 6.56 (s, 1H), 5.53 (s, 1H), 1.80 – 1.68 (m, 1H), 0.91 – 0.81 (m, 2H), 0.46 (dt, J = 6.6, 4.7 Hz, 2H); LCMS tR = 1.56 min, m/z = 383.8, 385.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Nitro-4-(trifluoromethoxy)phenol (900 mg, 4.03 mmol) was reacted following General Procedure B, affording a crude brown solid (693 mg, 3.59 mmol, 89%) that was taken forward without purification. LCMS tR = 0.83 min, m/z = 194.1 [M+H]+.</p><!><p>2-amino-4-(trifluoromethoxy)phenol (19 mg, 0.1 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording a colorless solid (4 mg, 0.009 mmol, 9%). 1H NMR (400 MHz, MeOH-d4) δH 7.80 (d, J = 2.4 Hz, 1H), 7.58 (d, J = 2.2 Hz, 1H), 7.51 (dd, J = 8.8, 2.5 Hz, 1H), 7.43 (d, J = 2.3 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF −60.3; LCMS tR = 1.57 min, m/z = 429.2 [M+H]+; Purity (AUC) ≥95%.</p><!><p>4-Hydroxyphenyl sulfur pentafluoride (1.1 g, 5 mmol) was dissolved in DCM (25 mL) and cooled to 0 °C before the addition of nitronium tetrafluoroborate (863 mg, 6.5 mmol) and stirred for 18 h. The mixture was neutralized with sat. aq. NaHCO3 and extracted with DCM to afford a crude yellow oil (479 mg, 1.81 mmol, 36%) which was used without further purification. 1H NMR (400 MHz, CDCl3) δH 10.78 (s, 1H), 8.57 (d, J = 2.7 Hz, 1H), 7.96 (dd, J = 9.2, 2.7 Hz, 1H), 7.27 (d, J = 9.2 Hz, 1H); 19F NMR (376 MHz, CDCl3) δF 82.43 (d, J = 151 Hz, 1F), 63.76 (d, J = 151 Hz, 4F); LCMS tR = 1.03 min, does not ionize by ESI.</p><!><p>2-Nitro-4-(pentafluorosulfanyl)phenol (479 mg, 1.81 mmol) was reacted following General Procedure B, affording a brown gum (99 mg, 0.42 mmol, 23%). LCMS tR = 0.55 min, m/z = 236.2 [M+H]+.</p><!><p>2-Amino-4-(pentafluorosulfanyl)phenol, 20b (24 mg, 0.1 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording 5k as a colorless solid (12 mg, 0.026 mmol, 26%). 1H NMR (400 MHz, CDCl3) δH 7.63 (d, J = 2.6 Hz, 1H), 7.59 – 7.54 (m, 1H), 7.57 – 7.52 (m, 1H), 7.31 (d, J = 2.6 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 6.70 (br s, 2H); 19F NMR (376 MHz, CDCl3) δF 84.3 (p, J = 151 Hz, 1F), 64.0 (d, J = 151 Hz, 4F); LCMS tR = 1.66 min, m/z = 1.66 min, m/z = 471.2 [M+H]+; Purity (AUC) ≥95%.</p><!><p>4-Hydroxy-3-nitrophenylboronic acid, pincaol ester, 24 (980 mg, 3.7 mmol), was reacted following General Procedure G. Purification by flash chromatography affords yellow solid (1215 mg, 3.4 mmol, 92%). MS (ESI) m/z = 373.1 [M+NH4]+.</p><!><p>2-(4-(Benzyloxy)-3-nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (710 mg, 2.0 mmol), 2-bromo-3,3,3-trifluoroprop-1-ene (830 μL, 8.0 mmol), Pd(dppf)Cl2.DCM (163 mg, 0.2 mmol) and K2CO3 (553 mg, 4.0 mmol) were taken in dioxane:water (10:1, 10 mL) and heated to 70 °C for 3 h under an inert atmosphere. The cooled mixture was diluted with EtOAc and washed with water, brine. Purification by flash chromatography affords a straw-colored oil (433 mg, 1.34 mmol, 67%). 1H NMR (400 MHz, CDCl3) δH 7.96 (d, J = 2.4 Hz, 1H), 7.61 – 7.55 (m, 1H), 7.48 – 7.32 (m, 5H), 7.13 (d, J = 8.8 Hz, 1H), 6.01 (d, J = 1.6 Hz, 1H), 5.80 (d, J = 1.6 Hz, 1H), 5.28 (s, 2H); 19F NMR (376 MHz, CDCl3) δF −65.1; LCMS tR = 1.26 min, does not ionize by ESI.</p><!><p>To a mixture of 1-(benzyloxy)-2-nitro-4-(3,3,3-trifluoroprop-1-en-2-yl)benzene (65 mg, 0.20 mmol) and Ph2MeSBF4 (115 mg, 0.40 mmol) in anhydrous THF at −78 °C, was added LiHMDS [1.0 M in THF] (0.8 mL, 0.8 mmol). The mixture was stirred for 12 h, warming to r.t., then diluted with EtOAc and washed with water. Purification by flash chromatography affords a pale-yellow gum (32 mg, 0.09 mmol, 47%). 1H NMR (400 MHz, CDCl3) δH 7.96 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 8.7, 2.3 Hz, 1H), 7.51 – 7.35 (m, 5H), 7.11 (d, J = 8.7 Hz, 1H), 5.27 (s, 2H), 1.45 – 1.39 (m, 2H), 1.09 – 1.01 (m, 1H); 19F NMR (376 MHz, CDCl3) δF −70.3; LCMS tR = 1.26 min, does not ionize by ESI.</p><!><p>1-(benzyloxy)-2-nitro-4-(1-(trifluoromethyl)cyclopropyl)benzene (32 mg, 0.09 mmol) was reacted following General Procedure B, affording a crude colorless solid (20 mg, 0.09 mmol) that was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording a colorless solid (13 mg, 0.029 mmol, 31%). 1H NMR (400 MHz, MeOH-d4) δH 7.72 (d, J = 2.5 Hz, 1H), 7.49 (dd, J = 8.8, 2.5 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.3, 2.2 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.3 Hz, 1H), 1.30 – 1.20 (m, 2H), 0.93 – 0.87 (m, 2H); 19F NMR (376 MHz, CDCl3) δF −71.9; LCMS tR = 1.67 min, m/z = 451.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>1-(4-Methoxyphenyl) cyclopropanecarbonitrile, 27 (1 mL, 6.3 mmol) was reacted following General Procedure C, affording a yellow solid (1178 mg, 5.40 mmol, 86%). 1H NMR (400 MHz, CDCl3) δH 7.69 (d, J = 2.5 Hz, 1H), 7.60 (dd, J = 8.8, 2.5 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 3.97 (s, 3H), 1.83 – 1.71 (m, 2H), 1.45 – 1.36 (m, 2H); LCMS tR = 0.87 min, does not ionize by ESI.</p><!><p>1-(4-methoxy-3-nitrophenyl)cyclopropane-1-carbonitrile (218 mg, 1.0 mmol) was reacted following General Procedure D to afford a cream solid (161 mg, 0.79 mmol, 79%). 1H NMR (400 MHz, CDCl3) δH 10.54 (s, 1H), 8.01 (d, J = 2.5 Hz, 1H), 7.60 (dd, J = 8.8, 2.5 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 1.87 – 1.71 (m, 2H), 1.48 – 1.32 (m, 2H). LCMS tR = 0.86 min, does not ionize by ESI.</p><!><p>1-(4-hydroxy-3-nitrophenyl)cyclopropane-1-carbonitrile (102 mg, 0.5 mmol) was reacted following General Procedure B, affording a cream solid (74 mg, 0.43 mmol, 85%). 1H NMR (400 MHz, CDCl3) δH 6.73 (d, J = 2.2 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 6.57 (dd, J = 8.2, 2.2 Hz, 1H), 4.72 (s, 1H), 3.71 (s, 2H), 1.65 – 1.58 (m, 2H), 1.32 – 1.27 (m, 2H); m/z (ESI) = 175.3 [M+H]+.</p><!><p>1-(3-amino-4-hydroxyphenyl)cyclopropane-1-carbonitrile (27 mg, 0.16 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording a colorless solid (47 mg, 0.12 mmol, 72%). 1H NMR (400 MHz, CDCl3) δH 8.51 (s, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 8.9, 2.4 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 6.92 – 6.85 (m, 3H), 6.80 (s, 1H), 1.67 – 1.61 (m, 2H), 1.26 – 1.20 (m, 2H); LCMS tR = 1.42 min, m/z = 408.9, 410.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>4-Hydroxybenzylacetontrile (2.66 g, 20 mmol) was reacted following General Procedure C to afford a yellow-orange solid (3.42 g, 19.2 mmol, 96%). 1H NMR (400 MHz, CDCl3) δH 10.57 (s, 1H), 8.10 (d, J = 2.4 Hz, 1H), 7.57 (dd, J = 8.7, 2.4 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 3.76 (s, 2H).</p><!><p>2-(4-hydroxy-3-nitrophenyl)acetonitrile (1.78 g, 10 mmol) was reacted following General Procedure G to afford a yellow-orange solid (2.31 g, 8.6 mmol, 86%). 1H NMR (400 MHz, CDCl3) δH 7.82 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 8.7, 2.4 Hz, 1H), 7.47 – 7.30 (m, 6H), 7.15 (d, J = 8.7 Hz, 1H), 5.26 (s, 2H), 3.74 (s, 2H).</p><!><p>To a solution of 2-(4-(benzyloxy)-3-nitrophenyl)acetonitrile (134 mg, 0.5 mmol) and 1,3-dibromopropane (61 μL, 0.6 mmol) in DMSO (5 mL) was added carefully added NaH (60% in mineral oil, 60 mg, 1.5 mmol) and the mixture stirred for 16 h. The mixture was diluted with EtOAc:Et2O (1:1, 50 mL) and washed with water (3 × 100 mL), brine (100 mL). Purification by flash chromatography affords a yellow-brown oil (80 mg, 0.26 mmol, 52%); m/z (ESI) = 309.0 [M+H]+.</p><!><p>1-(4-(benzyloxy)-3-nitrophenyl)cyclobutane-1-carbonitrile (80 mg, 0.26 mmol) was reacted following General Procedure B to afford a colorless oil (26 mg, 0.14 mmol, 53%). 1H NMR (400 MHz, MeOH-d4) δH 6.84 (d, J = 2.3 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.66 (dd, J = 8.2, 2.3 Hz, 1H), 2.77 – 2.66 (m, 2H), 2.66 – 2.56 (m, 2H), 2.42 – 2.28 (m, 1H), 2.13 – 2.00 (m, 1H); m/z (ESI) = 189.1 [M+H]+.</p><!><p>1-(3-amino-4-hydroxyphenyl)cyclobutane-1-carbonitrile, 29b (26 mg, 0.14 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording a colorless solid (22.5 mg, 0.05 mmol, 38%). 1H NMR (400 MHz, CDCl3) δH 7.65 (d, J = 2.4 Hz, 1H), 7.56 (dd, J = 8.8, 2.4 Hz, 1H), 7.22 (dd, J = 8.5, 2.4 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.71 (s, 1H), 2.82 – 2.71 (m, 2H), 2.52 – 2.33 (m, 2H), 2.08 – 2.01 (m, 2H); LCMS tR = 1.60 min, m/z = 439.8, 441.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>To a solution of 2-(4-(benzyloxy)-3-nitrophenyl)acetonitrile (134 mg, 0.5 mmol) and 1,4-dibromobutane (72 μL, 0.6 mmol) in DMSO (5 mL) was added carefully added NaH (60% in mineral oil, 60 mg, 1.5 mmol) and the mixture stirred for 16 h. The mixture was diluted with EtOAc:Et2O (1:1, 50 mL) and washed with water (3 × 100 mL), brine (100 mL). Purification by flash chromatography affords a yellow-brown oil (103 mg, 0.32 mmol, 64%). 1H NMR (400 MHz, CDCl3) δH 7.89 (d, J = 2.5 Hz, 1H), 7.62 (dd, J = 8.8, 2.5 Hz, 1H), 7.48 – 7.31 (m, 5H), 7.13 (d, J = 8.8 Hz, 1H), 5.26 (s, 2H), 2.55 – 2.44 (m, 2H), 2.14 – 1.88 (m, 6H); LCMS tR = 1.96 min, m/z = 323.1 [M+H]+.</p><!><p>1-(4-(benzyloxy)-3-nitrophenyl)cyclopentane-1-carbonitrile (103 mg, 0.32 mmol) was reacted following General Procedure B to afford a colorless oil (50 mg, 0.25 mmol, 78%). 1H NMR (400 MHz, MeOH-d4) δH 6.86 (t, J = 1.4 Hz, 1H), 6.68 (d, J = 1.4 Hz, 2H), 2.44 – 2.29 (m, 2H), 2.13 – 2.00 (m, 2H), 2.00 – 1.89 (m, 4H); m/z (ESI) = 203.2 [M+H]+.</p><!><p>1-(3-amino-4-hydroxyphenyl)cyclopentane-1-carbonitrile, 29c (50 mg, 0.25 mmol) was reacted with 18a (41 mg, 0.15 mmol) following General Procedure A, affording a colorless solid (12 mg, 0.04 mmol, 11%). 1H NMR (400 MHz, CDCl3) δH 7.61 (d, J = 2.5 Hz, 1H), 7.50 (dd, J = 8.8, 2.5 Hz, 1H), 7.17 (dd, J = 8.5, 2.3 Hz, 1H), 6.98 (d, J = 2.3 Hz, 2H), 6.90 (d, J = 5.2 Hz, 1H), 6.87 (d, J = 5.2 Hz, 1H), 2.40 – 2.26 (m, 2H), 2.01 – 1.81 (m, 6H); LCMS tR = 1.65 min, m/z = 453.8, 455.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>To a solution of 2-(4-(benzyloxy)-3-nitrophenyl)acetonitrile (134 mg, 0.5 mmol) and 1,5-dibromopentane (82 μL, 0.6 mmol) in DMSO (5 mL) was added carefully added NaH (60% in mineral oil, 60 mg, 1.5 mmol) and the mixture stirred for 16 h. The mixture was diluted with EtOAc:Et2O (1:1, 50 mL) and washed with water (3 × 100 mL), brine (100 mL). Purification by flash chromatography affords a yellow-brown oil (91 mg, 0.27 mmol, 54%). LCMS tR = 1.98 min, m/z = 337.0 [M+H]+.</p><!><p>2221-(4-(Benzyloxy)-3-nitrophenyl)cyclohexane-1-carbonitrile (91 mg, 0.27 mmol) was reacted following General Procedure B to afford a colorless oil (39 mg, 0.18 mmol, 67%). 1H NMR (400 MHz, MeOH-d4) δH 6.91 (d, J = 1.9 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H), 6.71 (s, 1H), 2.12 – 2.02 (m, 3H), 1.92 – 1.69 (m, 6H), 1.43 – 1.26 (m, 1H); m/z (ESI) = 217.1 [M+H]+.</p><!><p>1-(3-Amino-4-hydroxyphenyl)cyclohexane-1-carbonitrile, 29d (39 mg, 0.18 mmol) was reacted with 18a (54 mg, 0.20 mmol) following General Procedure A, affording a colorless solid (24 mg, 0.05 mmol, 30%). 1H NMR (400 MHz, MeOH-d4) δH 7.79 (d, J = 2.5 Hz, 1H), 7.51 (dd, J = 8.8, 2.5 Hz, 1H), 7.37 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 1.98 (dt, J = 12.8, 1.9 Hz, 2H), 1.91 – 1.63 (m, 9H), 1.43 – 1.27 (m, 1H); LCMS tR = 1.72 min, m/z = 467.8, 469.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-5-chlorobenzoate (143 mg, 0.5 mmol) was reacted with 18a following General Procedure A, which without purification was hydrolyzed following General Procedure E, to afford title compound as a colorless solid (86 mg, 0.21 mmol, 42% over two steps). 1H NMR (400 MHz, MeOH-d4) δH 7.83 (d, J = 2.5 Hz, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.55 (d, J = 2.6 Hz, 1H), 7.53 (dd, J = 8.7, 2.5 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H); LCMS tR = 1.31 min, m/z = 427.8, 429.7 [M+Na]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-5-chloro-2-hydroxybenzoate (202 mg, 1.0 mmol) was reacted with 16a following General Procedure A, to afford a colorless solid (370 mg, 0.82 mmol, 82%). 1H NMR (DMSO-d6) δH 7.81 – 7.76 (m, 2H), 7.55 (d, J = 2.7 Hz, 1H), 7.49 (d, J = 2.6 Hz, 1H), 7.19 (dt, J = 8.6, 1.0 Hz, 1H), 3.88 (s, 3H), 3.76 (s, 3H); LCMS tR = 1.77 min, m/z = 450, 452 [M+H]+.</p><!><p>Methyl 3-((5-bromo-2-methoxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate (370 mg, 0.82 mmol) was reacted following General Procedure E, to afford a colorless solid (260 mg, 0.59 mmol, 72%). 1H NMR (DMSO-d6) δH 9.52 (s, 1H), 7.80 – 7.75 (m, 2H), 7.52 (d, J = 2.7 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.18 (d, J = 9.6 Hz, 1H), 3.77 (s, 3H); LCMS tR = 1.55 min, m/z = 436.0, 438.0 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+Na]+ calculated for C14H11BrClNNaO6S 457.9071, found 457.9075.</p><!><p>Methyl 3-amino-5-chloro-2-hydroxybenzoate (605 mg, 3.0 mmol) was reacted with 18a following General Procedure A, to afford a colorless solid (918 mg, 2.1 mmol, 70%). LCMS tR = 1.15 min, m/z = 437.8 [M+H]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate (918 mg, 2.10 mmol) was reacted following General Procedure E, to afford a colorless solid (425 mg, 1.0 mmol, 48%). 1H NMR (400 MHz, MeOH-d4) δH 7.81 (d, J = 2.5 Hz, 1H), 7.62 (d, J = 2.6 Hz, 1H), 7.55 (d, J = 2.6 Hz, 1H), 7.53 (dd, J = 8.7, 2.5 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H); LCMS tR = 1.53 min, m/z = 421.7, 423.7 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+Na]+ calculated for C13H9BrClNNaO6S 443.8915, found 443.8909.</p><!><p>Chlorosulfonic acid (4.66 mL, 70 mmol) was cooled to 0 °C and 2-chloro-4-bromophenol (2.08 g, 10 mmol) was added portion-wise and stirred for 24 h. The mixture was carefully poured over a slurry of ice, DCM and brine, and extracted with DCM. Purification by flash chromatography affords title compound as a colorless solid (2.19 g, 7.15 mmol, 72%). 1H NMR (400 MHz, CDCl3) δH 7.92 (1H, d, J = 2.4 Hz), 7.86 (1H, d, J = 2.4 Hz).</p><!><p>Methyl 3-amino-5-chloro-2-hydroxybenzoate (192 mg, 0.95 mmol) was reacted with 5-bromo-3-chloro-2-hydroxybenzenesulfonyl chloride, 18b following General Procedure A, to afford a colorless solid (374 mg, 0.91 mmol, 91%). 1H NMR (400 MHz, CDCl3) δH 11.03 (s, 1H), 7.71 – 7.68 (m, 2H), 7.64 (d, J = 2.5 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H), 3.95 (s, 3H); LCMS tR = 1.22 min, m/z = 471.6 [M+H]+.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate (298 mg, 0.63 mmol) was reacted following General Procedure E, to afford a colorless solid (231 mg, 0.51 mmol, 80%). 1H NMR (400 MHz, CDCl3) δH 7.76 (s, 2H), 7.63 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 2.6 Hz, 1H); LCMS tR = 1.68 min, m/z = 457.6 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 2-hydroxy-4-bromobenzoate (9.24 g, 40 mmol) was reacted following General Procedure C, to afford nitrated product as a crude yellow solid (10.3 g, 37.3 mmol, 93%), which was used without purification. 1H NMR (400 MHz, CDCl3) δH 8.27 (d, J = 2.5 Hz, 1H), 8.24 (d, J = 2.5 Hz, 1H), 4.03 (s, 3H); LCMS tR = 1.02 min, m/z = 276, 278 [M+H]+.</p><!><p>Methyl 5-bromo-2-hydroxy-3-nitrobenzoate (8.9 g, 32.2 mmol) was reacted following General Procedure G. Purification by flash column chromatography affords a yellow solid (10.6g, 28.8 mmol, 89%).</p><!><p>Methyl 2-(benzyloxy)-5-bromo-3-nitrobenzoate (1.1 g, 3.0 mmol), cyclopropylboronic acid (515 mg, 6 mmol), Pd(OAc)2 (33.7 mg, 0.15 mmol), PCy3.HBF4 (110 mg, 0.30 mmol) and K3PO4 (1.59 g, 7.5 mmol) were taken in Toluene:H2O (10:1, 15 mL) and heated to 80 °C for 16 h. The cooled mixture was diluted with EtOAc and washed with water, brine. Purification by flash chromatography affords a brown oil (895 mg, 2.73 mmol, 91%). 1H NMR (400 MHz, CDCl3) δH 7.77 (d, J = 2.4 Hz, 1H), 7.64 (d, J = 2.5 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.46 – 7.35 (m, 3H), 3.91 (s, 3H), 2.03 – 1.94 (m, 1H), 1.13 – 1.07 (m, 2H), 0.83 – 0.74 (m, 2H).</p><!><p>Methyl 2-(benzyloxy)-5-cyclopropyl-3-nitrobenzoate (895 mg, 2.73 mmol) was reacted following General Procedure B, to afford an off-white solid (495 mg, 2.39 mmol, 88%). 1H NMR (400 MHz, CDCl3) δH 10.78 (s, 1H), 7.15 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 3.96 (s, 3H), 1.83 (tt, J = 8.4, 5.1 Hz, 1H), 0.96 – 0.86 (m, 2H), 0.68 – 0.59 (m, 2H); LCMS tR = 1.28 min, m/z = 208.2 [M+H]+.</p><!><p>Methyl 3-amino-5-cyclopropyl-2-hydroxybenzoate (207 mg, 1.0 mmol) and 18a were reacted following General Procedure A to afford a cream solid (395 mg, 0.89 mmol, 89%). 1H NMR (400 MHz, MeOH-d4) δH 7.76 (d, J = 2.5 Hz, 1H), 7.51 (dd, J = 8.8, 2.5 Hz, 1H), 7.36 (d, J = 2.3 Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 3.93 (s, 3H), 1.84 (tt, J = 8.4, 5.1 Hz, 1H), 0.98 – 0.90 (m, 2H), 0.54 (dt, J = 6.5, 4.7 Hz, 2H); LCMS tR = 1.67 min, m/z = 442.1, 444.1 [M+H]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-2-hydroxybenzoate (44 mg, 0.10 mmol) was reacted following General Procedure E to afford a colorless solid (33 mg, 0.078 mmol, 78%). 1H NMR (400 MHz, DMSO-d6) δH 11.25 (s, 1H), 8.85 (s, 1H), 7.68 (d, J = 2.6 Hz, 1H), 7.59 (dd, J = 8.7, 2.6 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 1.84 (tt, J = 8.4, 5.1 Hz, 1H), 0.97 – 0.77 (m, 2H), 0.51 – 0.37 (m, 2H); LCMS tR = 1.51 min, m/z = 427.8, 429.8 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+H]+ calculated for C16H15BrNO6S 427.9798, found 427.9796.</p><!><p>Methyl 3-amino-5-cyclopropyl-2-hydroxybenzoate (41 mg, 0.20 mmol) and 18b were combined following General Procedure A, to afford a colorless solid (76 mg, 0.16 mmol, 80%). 1H NMR (400 MHz, DMSO-d6) δH 10.62 (s, 1H), 9.42 (s, 1H), 7.93 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 7.34 (d, J = 2.3 Hz, 1H), 7.09 (d, J = 2.3 Hz, 1H), 3.88 (s, 3H), 1.89 (ddt, J = 13.5, 8.5, 4.3 Hz, 1H), 1.03 – 0.76 (m, 2H), 0.61 – 0.32 (m, 2H); LCMS tR = 1.76 min, m/z = 475.8, 477.7 [M+H]+.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-2-hydroxybenzoate (24 mg, 0.05 mmol) was reacted following General Procedure E to afford a colorless solid (19 mg, 82%). 1H NMR (400 MHz, MeOH-d4) δH 7.61 (dd, J = 2.4, 1.7 Hz, 1H), 7.53 (dd, J = 10.1, 2.4 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 1.85 (tt, J = 8.4, 5.1 Hz, 1H), 0.98 – 0.90 (m, 2H), 0.59 – 0.52 (m, 2H); LCMS tR = 1.53 min, m/z = 462.8, 464.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Fluoro-6-hydroxybenzoic acid, 40 (5.0 g, 32 mmol) was dissolved in THF (160 mL) and MeOH (32 mL) and cooled to 0 °C. TMS diazomethane [2.0 M in hexanes] (17.6 mL, 35.2 mmol) was added over 20 mins and the mixture stirred for 3 h at 0 °C. The mixture was concentrated, redissolved in EtOAc, and washed with water and brine. Purification by flash chromatography affords a colorless liquid (3.8 g, 22.3 mmol, 70%). 1H NMR (400 MHz, CDCl3) δH 11.26 (s, 1H), 7.40 – 7.36 (m, 1H), 6.79 (br d, 1H), 6.63 – 6.58 (m, 1H), 3.99 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −105.1; LCMS tR = 1.33 min, m/z = 171.1 [M+H]+.</p><!><p>41 (3.8 g, 22.3 mmol) was dissolved in MeCN (144 mL) and cooled to −10 °C in a salt/ice bath. HBF4.Et2O (3.37 mL, 24.6 mmol) was added, followed by N-bromosuccinimide (4.8 g, 26.8 mmol) portion wise, then stirred for 1 h at −10 °C. The mixture was concentrated in vacuo, then redissolved in EtOAc and washed with sat. aq. Na2S2O8, sat. aq. NaCl and dried (MgSO4). Purification by flash chromatography affords a cream solid (2.49 g, 10 mmol, 45%). 1H NMR (400 MHz, CDCl3) δH 11.26 (s, 1H), 7.58 (dd, 1H, 9.1, 7.5 Hz), 6.76 (dt, 7.5, 0.9 Hz), 4.01 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −86.7; LCMS tR = 1.59 min, m/z = 249, 251 [M+H]+; Note: Material contains di-brominated by-product which is inseparable by chromatography, but is readily removed after Step C.</p><!><p>42 (2.49 g, 10 mmol) was reacted following General Procedure C, to afford a pale-yellow solid (1.92 mg, 65%). 1H NMR (400 MHz, CDCl3) δH 8.44 (d, 1H, J = 7.0 Hz), 4.03 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −88.1; LCMS tR = 1.55 min, m/z = 294, 296 [M+H]+.</p><!><p>43 (588 mg, 2.0 mmol) was reacted following General Procedure G to afford a pale-yellow solid (615 mg, 80%). 1H NMR (400 MHz, CDCl3) δH 8.25 (d, 1H, J = 7.0 Hz), 7.42 – 7.38 (m, 5H), 5.16 (s, 2H), 3.88 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −86.8; LCMS tR = 1.75 min, m/z = 406, 408 [M+Na]+.</p><!><p>Methyl 2-(benzyloxy)-5-bromo-6-fluoro-3-nitrobenzoate (103 mg, 1.2 mmol), cyclopropylboronic acid (155 mg, 1.8 mmol), Pd(OAc)2 (13.5 mg, 0.06 mmol), PCy3.HBF4 (44.2 mg, 0.12 mmol) and K3PO4 (637 mg, 3.0 mmol) were taken in Toluene:H2O (10:1, 6 mL) and heated to 80 °C for 16 h. The cooled mixture was diluted with EtOAc and washed with water, brine. Purification by flash chromatography affords a cream solid (150 mg, 0.43 mmol, 43%). 1H NMR (400 MHz, CDCl3) δH 7.59 (d, 1H, J = 7.6 Hz), 7.45 – 7.37 (m, 5H), 5.12 (s, 2H), 3.88 (s, 3H), 2.09 – 2.06 (m, 1H), 1.11 – 1.07 (m, 2H), 0.81 – 0.77 (m, 2H); 19F NMR (376 MHz, CDCl3) δF −108.7; LCMS tR = 1.85 min, m/z = 363.1 [M+H]+.</p><!><p>Methyl 2-(benzyloxy)-5-cyclopropyl-6-fluoro-3-nitrobenzoate (145 mg, 0.42 mmol) was reacted following General Procedure B, to afford an off-white solid (66 mg, 70%). 1H NMR (400 MHz, CDCl3) δH 11.17 (s, 1H), 6.46 (d, J = 7.1 Hz, 1H), 4.01 (s, 3H), 2.12 – 1.92 (m, 1H), 1.07 – 0.82 (m, 2H), 0.70 – 0.50 (m, 2H); 19F NMR (376 MHz, CDCl3) δF −125.0; LCMS tR = 1.04 min, m/z = 226.1 [M+H]+.</p><!><p>45 (45 mg, 0.2 mmol) and 18a were reacted following General Procedure A to afford a pale-brown solid (69 mg, 0.15 mmol, 75%). 1H NMR (400 MHz, MeOH-d4) δH 11.34 (s, 1H), 10.13 (s, 1H), 9.00 (s, 1H), 7.61 (dd, J = 8.7, 2.6 Hz, 1H), 7.57 (d, J = 2.5 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 3.82 (s, 3H), 1.90 – 1.78 (m, 1H), 0.95 – 0.77 (m, 2H), 0.39 – 0.24 (m, 2H); LCMS tR = 1.82 min, m/z = 460, 462 [M+H]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoate (23 mg, 0.05 mmol) was reacted following General Procedure E, to afford colorless solid (9 mg, 40%). 1H NMR (400 MHz, MeOH-d4) δH 7.71 (d, J = 2.5 Hz, 1H), 7.53 (dd, J = 8.8, 2.5 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 3.96 (s, 3H), 2.01 – 1.93 (m, 1H), 0.99 – 0.91 (m, 2H), 0.59 – 0.51 (m, 2H); 19F NMR (376 MHz, MeOH-d4) δF −116.6; LCMS tR = 1.53 min, m/z = 467.7, 469.7 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-5-cyclopropyl-6-fluoro-2-hydroxybenzoate (45 mg, 0.2 mmol) and 18b were reacted following General Procedure A to afford a pale-brown solid (69 mg, 0.15 mmol, 75%). 1H NMR (400 MHz, MeOH-d4) δH 7.73 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.14 (d, J = 7.4 Hz, 1H), 3.97 (s, 3H), 2.03 – 1.94 (m, 1H), 1.01 – 0.93 (m, 2H), 0.63 – 0.55 (m, 2H); 19F NMR (376 MHz, MeOH-d4 δF −115.5; LCMS tR = 1.94 min, m/z = 494.2, 496.2 [M+H]+.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoate (23 mg, 0.05 mmol) was reacted following General Procedure E, to afford a colorless solid (10 mg, 42%). 1H NMR (400 MHz, DMSO-d6) δH 7.94 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 2.5 Hz, 1H), 6.78 (d, J = 7.7 Hz, 1H), 1.89 (td, J = 8.4, 4.2 Hz, 1H), 1.00 – 0.83 (m, 2H), 0.48 – 0.37 (m, 2H); 19F NMR (376 MHz, DMSO-d6) δF −116.3; LCMS tR = 1.74 min, m/z = 477.7, 479.7 [M+H]+; Purity (AUC) ≥95%. HRMS (ESI/TOF) [M+Na]+ calculated for C16H12BrClFNNaO6S 501.9133, found 501.9121.</p><!><p>3-Bromo-5-(trifluoromethoxy)benzoic acid (2.0 g, 7.0 mmol), was dissolved in THF (35 mL) and MeOH (7 mL) and cooled to 0 °C. TMS diazomethane [2.0 M in Et2O] (3.85 mL, 7.7 mmol) was added drop-wise, then the mixture stirred for 16 h. The mixture was concentrated and purified by flash chromatography to afford a colorless solid (1.82 g, 6.09 mmol, 82%). 1H NMR (400 MHz, CDCl3) δH 8.12 (t, J = 1.6 Hz, 1H), 7.83 (t, J = 2.4 Hz, 1H), 7.57 (t, J = 1.6 Hz, 1H), 3.95 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −57.98; m/z (ESI) = 300.1 [M+H]+.</p><!><p>Methyl 3-bromo-5-(trifluoromethoxy)benzoate (1.79 g, 5.98 mmol) tert-butyl carbamate (841 mg, 7.18 mmol), Pd2dba3 (110 mg, 0.12 mmol), Xantphos (208 mg, 0.36 mmol) and Cs2CO3 (5.85 g, 17.9 mmol) were combined in Toluene (24 mL) in a sealed tube and stirred at 90 °C for 16 h. The mixture was filtered through celite, concentrated and the residue re-dissolved in DCM (50 mL). TFA (10 mL) was added and the mixture stirred at r.t. for 2 h. The mixture was washed with sat. aq. Na2CO3, concentrated and purified by flash chromatography to afford a brown solid (1.18 g, 5.02 mmol, 84%). m/z (ESI) = 236.1 [M+H]+.</p><!><p>Methyl 3-amino-5-(trifluoromethoxy)benzoate (118 mg, 0.5 mmol) and 18a were reacted following General Procedure A to afford a pale-brown solid (179 mg, 0.38 mmol, 76%). 1H NMR (400 MHz, CDCl3) δH 7.72 – 7.69 (m, 1H), 7.68 (d, J = 2.5 Hz, 1H), 7.64 (t, J = 1.7 Hz, 1H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 7.27 (s, 1H), 6.89 (d, J = 8.9 Hz, 1H), 3.93 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −58.0; LCMS tR = 1.11 min, m/z = 486.9, 488.9 [M+NH4]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(trifluoromethoxy)benzoate (91 mg, 0.19 mmol) was reacted following General Procedure E to afford a colorless solid (64 mg, 0.14 mmol, 72%). 1H NMR (400 MHz, MeOH-d4) δH 7.86 (d, J = 2.5 Hz, 1H), 7.79 (dd, J = 2.1, 1.4 Hz, 1H), 7.53 – 7.49 (m, 2H), 7.35 (dq, J = 2.1, 1.1 Hz, 1H), 6.85 (d, J = 8.7 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF −59.7; LCMS tR = 1.34 min, m/z = 477.7, 479.7 [M+Na]+; Purity (AUC) ≥95%.</p><!><p>2-Hydroxy-5- (trifluoromethoxy)benzoic acid (2.0 g, 9.00 mmol) was dissolved in DCM (30 mL) and MeOH (30 mL) and cooled to 0 °C. To the flask was added EDCI (2.59 g, 13.5 mmol), followed by DMAP (220.0 mg, 1.80 mmol, 0.2 eq). The reaction was stirred for 16 h, warming to r.t.. The mixture was washed with water and dried (Na2SO4). Purification by flash chromatography afforded title compound (1.35 g, 5.74 mmol, 63%). LCMS tR = 1.62 min, m/z = 237.2 [M+H]+.</p><!><p>Methyl 2-hydroxy-5- (trifluoromethoxy)benzoate (1.36 g, 5.74 mmol) was reacted following General Procedure C. Purification by flash chromatography afforded title compound (1.61 g, quant.). LCMS tR = 1.51 min, m/z = 252.1 [M+H]+.</p><!><p>Methyl 2- hydroxy-3-nitro-5-(trifluoromethoxy)benzoate (1.61 g, 5.74 mmol) was reacted following General Procedure B. The crude product was used for the next step without further purification (1.40 g, 5.59 mmol, 97% over 2 steps). LCMS tR = 1.42 min, m/z = 251.2 [M+H]+.</p><!><p>Methyl 3-amino-2-hydroxy-5-(trifluoromethoxy)benzoate (1.26 g, 5.0 mmol) and 18a were reacted following General Procedure A to afford a pale-brown solid (1.02 g, 2.2 mmol, 43%). 1H NMR (400 MHz, CDCl3) δH 11.10 (s, 1H), 8.68 (s, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.64 – 7.60 (m, 1H), 7.51 – 7.46 (m, 3H), 6.88 (d, J = 8.9 Hz, 1H), 3.96 (s, 3H); 19F NMR (376 MHz, CDCl3) δF −58.6; LCMS tR = 1.20 min, m/z = 485.8, 487.8 [M+H]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoate (1.02 g, 2.09 mmol) was reacted following General Procedure E to afford a colorless solid (575 mg, 1.22 mmol, 58%). 1H NMR (400 MHz, MeOH-d4) δH 7.82 (d, J = 2.5 Hz, 1H), 7.58 – 7.55 (m, 1H), 7.54 (dd, J = 8.8, 2.5 Hz, 1H), 7.45 – 7.41 (m, 1H), 6.86 (d, J = 8.8 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF −60.3; LCMS tR = 1.57 min, m/z = 471.8, 473.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-2-hydroxy-5-(trifluoromethoxy)benzoate (53.4 mg, 0.21 mmol) was reacted with 18b following General Procedure A to afford title compound as a solid (26.4 mg, 31%). LCMS tR = 1.80 min, m/z = 521.7 [M+H]+.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoate (10.5 mg, 0.02 mmol) was reacted following General Procedure E to afford a colorless solid (7.0 mg, 68%). 1H NMR (400 MHz, MeOH-d4) δ 7.74 (q, J = 2.4 Hz, 2H), 7.54 (d, J = 2.3 Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δ −60.3; LCMS tR = 1.66 min, m/z = 507.6 [M+H]+; Purity (AUC) ≥95%.</p><!><p>3-Bromo-5-(pentafluorosulfanyl) benzoic acid (1.05 g, 3.21 mmol) was dissolved in THF (16 mL) and MeOH (3.2 mL) and cooled to 0 °C. TMS diazomethane [2.0 M in Et2O] (1.77 mL, 3.53 mmol) was added dropwise, then the mixture stirred for 16 h. The mixture was concentrated and purified by flash chromatography to afford a colorless solid (1.02 mg, 2.97 mmol, 93%). 1H NMR (400 MHz, CDCl3) δH 8.36 (dd, J = 2.1, 1.3 Hz, 1H), 8.33 (t, J = 1.6 Hz, 1H), 8.07 (t, J = 2.0 Hz, 1H), 3.98 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 81.67 (d, J = 151 Hz, 1F), 62.94 (d, J = 151 Hz, 4F).</p><!><p>Methyl 3-bromo-5-(pentafluorosulfanyl)benzoate (890 mg, 2.61 mmol), tert-butyl carbamate (367 mg, 3.13 mmol), Pd2dba3 (48 mg, 0.05 mmol), Xantphos (91 mg, 0.16 mmol) and Cs2CO3 (2.55 g, 7.83 mmol) were combined in Toluene (10 mL) in a sealed tube and stirred at 90 °C for 16 h. The mixture was filtered through celite, concentrated and the residue was redissolved in DCM (25 mL). TFA (5 mL) was added and the mixture stirred at r.t. for 2 h. The mixture was washed with sat. aq. Na2CO3, concentrated and purified by flash chromatography to afford a brown solid (585 mg, 2.11 mmol, 81%). 1H NMR (400 MHz, CDCl3) δH 7.82 – 7.77 (m, 1H), 7.49 – 7.47 (m, 1H), 7.22 (t, J = 2.1 Hz, 1H), 3.95 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 83.9 (p, J = 150 Hz, 1F), 62.47 (d, J = 150 Hz, 4F).</p><!><p>Methyl 3-amino-5-(pentafluorosulfanyl)benzoate (139 mg, 0.50 mmol) and 18a were reacted following General Procedure A to afford a colorless solid (184 mg, 0.36 mmol, 72%). 1H NMR (400 MHz, CDCl3) δH 8.69 (d, J = 5.0 Hz, 2H), 8.26 (t, J = 1.7 Hz, 1H), 7.92 (br s, 1H), 7.74 – 7.69 (m, 2H), 7.56 (dd, J = 8.8, 2.4 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.98 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 81.6 (p, J = 151 Hz, 1F), 62.7 (d, J = 151 Hz); LCMS tR = 1.14 min, m/z = 528.8, 530.9 [M+NH4]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoate (68 mg, 0.13 mmol) was reacted following General Procedure E to afford a colorless solid (8 mg, 0.016 mmol, 12%). 1H NMR (600 MHz, MeOH-d4) δH 8.02 – 7.98 (m, 2H), 7.87 – 7.83 (m, 2H), 7.51 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF 81.4 (p, J = 149 Hz, 1F), 60.9 (d, J = 149 Hz, 4F); LCMS tR = 1.40 min, m/z = 519.7, 521.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-5-(pentafluorosulfanyl)benzoate (139 mg, 0.50 mmol) and 18b were reacted following General Procedure A to afford title compound as a colorless solid (131 mg, 0.24 mmol, 48%). LCMS tR = 1.67 min, m/z = 569.5 [M+NH4]+.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoate (72 mg, 0.13 mmol) was reacted following General Procedure E to afford a colorless solid (18 mg, 0.034 mmol, 26%). 1H NMR (400 MHz, MeOH-d4) δH 8.08 – 8.04 (t, J = 1.9 Hz, 1H), 8.02 (t, J = 1.9 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.84 (t, J = 1.9 Hz, 1H), 7.75 (d, J = 2.4 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF 81.4 (p, J = 149 Hz, 1F), 60.98 (d, J = 149 Hz, 4F); LCMS tR = 1.10 min, m/z = 533.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>(3-Bromo-4-methoxyphenyl)pentafluorosulfane, 34 (939 mg, 3.0 mmol), Pd(OAc)2 (34 mg, 0.15 mmol), P(tBu)3.HBF4 (174 mg, 0.60 mmol) and phenol (282 mg, 3.0 mmol) were taken in MeCN (13.7 mL) in a 40 mL reaction vial and phenyl formate (500 μL) and Et3N (1.25 mL, 9.0 mmol) were added. The reaction was run in 5-parallel reactions. The sealed vials were heated to 90 °C for 18 h, then combined after filtration through celite, concentrated, redissolved in EtOAc (100 mL) and washed with H2O (100 mL). Purification by flash chromatography affords a pale brown liquid (4.36 g, 12.3 mmol, 82%).</p><p>1H NMR (400 MHz, CDCl3) δH 8.41 (d, J = 2.9 Hz, 1H), 7.93 (dd, J = 9.2, 2.9 Hz, 1H), 7.48 – 7.40 (m, 2H), 7.31 – 7.26 (m, 2H), 7.07 (d, J = 9.2 Hz, 1H), 6.93 (tt, J = 7.3, 1.1 Hz, 1H), 4.00 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 84.52 (d, J = 150 Hz, 1F), 64.07 (d, J = 150 Hz, 4F); MS (ESI) m/z = 355.2.</p><!><p>Phenyl 2-methoxy-5-(pentafluorosulfanyl)benzoate (2.80 g) was reacted following General Procedure D, to afford a colorless solid (1.81 g, 5.31 mmol, 67%). 1H NMR (400 MHz, CDCl3) δH 10.88 (s, 1H), 8.50 (d, J = 2.8 Hz, 1H), 7.92 (dd, J = 9.2, 2.8 Hz, 1H), 7.53 – 7.45 (m, 2H), 7.40 – 7.31 (m, 1H), 7.29 – 7.20 (m, 3H), 7.11 (d, J = 9.2 Hz, 1H); 19F NMR (376 MHz, CDCl3) δ 84.58 (d, J = 151 Hz, 1F), 63.90 (d, J = 151 Hz, 4F); MS (ESI) m/z = 341.3.</p><!><p>Phenyl 2-hydroxy-5-(pentafluorosulfanyl)benzoate, 35 (1.81 g, 5.32 mmol) was reacted following General Procedure E, to afford title compound (1.15 g, 4.35 mmol, 82%). 1H NMR (400 MHz, MeOHd4) δH 8.27 (d, J = 2.9 Hz, 1H), 7.91 (dd, J = 9.2, 2.9 Hz, 1H), 7.07 (d, J = 9.2 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF 83.68 (d, J = 149 Hz, 1F), 62.35 (d, J = 149 Hz, 4F).</p><!><p>2-Hydroxy-5-(pentafluorosulfanyl)benzoic acid (300 mg, 1.14 mmol) was reacted following General Procedure C, to afford title compound (159 mg, 0.51 mmol, 45%). 1H NMR (400 MHz, MeOH-d4) δH 8.60 (d, J = 2.8 Hz, 4H), 8.53 (d, J = 2.8 Hz, 4H). 19F NMR (376 MHz, MeOH-d4) δF 80.9 (p, J = 149 Hz), 62.4 (d, J = 149 Hz).</p><!><p>To a mixture containing 2-hydroxy-3-nitro-5-(pentafluorosulfanyl)benzoic acid (60 mg, 0.19 mmol) in MeOH (1 mL), two drops of H2SO4 were added. The reaction mixture was heated to reflux and stirred overnight. The cooled mixture was concentrated and the residue dissolved in DCM and washed with water. The organic phase was concentrated to afford crude title compound which was used without purification (60 mg, 0.19 mmol, 96%). 1H NMR (400 MHz, CDCl3) δH 8.55 (d, J = 2.9 Hz, 1H), 8.51 (d, J = 2.9 Hz, 1H), 4.09 (s, 3H).</p><!><p>Methyl 2-hydroxy-3-nitro-5-(pentafluorosulfanyl)benzoate (223 mg, 0.69 mmol) was reacted following General Procedure B, to afford title compound (170 mg, 0.58 mmol, 84%). 1H NMR (400 MHz, Chloroform-d) δ 11.26 (s, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.20 (d, J = 2.6 Hz, 1H), 3.98 (br s, H); 19F NMR (376 MHz, Chloroform-d) δ 85.67 (p, J = 151 Hz, 1F), 63.71 (d, J = 150 Hz, 4F); LCMS tR = 1.06 min, m/z = 294.2 [M+H]+.</p><!><p>36 (50 mg, 0.17 mmol) and 18a were reacted following General Procedure A to afford title compound as a colorless solid (54 mg, 0.10 mmol, 60%). 1H NMR (400 MHz, CDCl3) δH 8.06 (d, J = 2.7 Hz, 1H), 8.03 (d, J = 2.7 Hz, 1H), 7.70 (d, J = 2.5 Hz, 1H), 7.48 (dd, J = 8.8, 2.5 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 3.99 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 83.5 (p, J = 151 Hz, 1F), 63.7 (d, J = 151 Hz, 4F). LCMS: tR = 1.24 min, m/z = 527.9, 529.9 [M+H]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfaneyl)benzoate (27 mg, 0.046 mmol) was reacted following General Procedure E, to afford a colorless solid (6 mg, 0.12 mmol, 26%). 1H NMR (400 MHz, MeOH-d4) δH 8.02 (d, J = 2.7 Hz, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.81 (d, J = 2.5 Hz, 1H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF 83.0 (p, J = 149 Hz, 1F), 62.2 (d, J = 149 Hz, 4F); LCMS tR = 1.07 min, m/z = 537.3 [M+Na]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-2-hydroxy-5-(pentafluorosulfanyl)benzoate (50 mg, 0.17 mmol) and 18b were reacted following General Procedure A, to afford a colorless solid (21 mg, 0.04 mmol, 22%). 1H NMR (400 MHz, CDCl3) δH 8.72 – 8.67 (m, 1H), 8.11 (d, J = 2.6 Hz, 1H), 8.02 (d, J = 2.6 Hz, 1H), 7.74 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 3.99 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 83.3 (p, J= 151 Hz, 1F), 63.7 (d, J = 151 Hz, 4F). LCMS: tR = 1.19 min, m/z = 562.1 [M+H]+.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoate (20 mg, 0.04 mmol) was reacted following General Procedure E, to afford title compound as a colorless solid (8 mg, 0.015 mmol, 41%). 1H NMR (400 MHz, Acetone-d6) δH 8.12 (d, J = 2.7 Hz, 1H), 8.09 (d, J = 2.7 Hz, 1H), 7.86 (d, J = 2.4 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δ 82.94 (p, J = 149 Hz), 62.21 (d, J = 149 Hz); LCMS tR = 1.111 min, m/z = 548.1, 550.1 [M+H]+, Purity (AUC) ≥95%.</p><!><p>To a solution of methyl 3-bromo-5-methylbenzoate, 52 (10.0 g, 44 mmol) in CHCl3 (200 mL) was added NBS (8.6 g, 48 mmol) followed by AIBN (730 mg, 4.4 mmol) and the mixture heated to reflux overnight. Upon cooling the mixture was filtered through celite and concentrated under reduced pressure. To the crude intermediate (6 g, 19.5 mmol) in THF (100 mL) was added TMSCN (1.93 g 19.5 mmol) and stirred for 30 min, then TBAF (1.0 M in THF, 19.5 mL) added. The mixture was stirred at r.t. for 16 h, then concentrated and purified by flash chromatography to afford title compound (2.0 g, 7.9 mmol, 18%). 1H NMR (400 MHz, CDCl3) δΗ 8.17 (s, 1H), 7.96 (s, 1H), 7.73 (s, 1H), 3.97 (s, 3H), 3.82 (s, 2H).</p><!><p>To a solution of methyl 3-bromo-5-(cyanomethyl)benzoate (500 mg, 2 mmol) and 1,3-diiodopropane (1.18 g, 4 mmol) in THF (30 mL) was added t-BuOK (448 mg, 4 mmol) and the mixture stirred at r.t. for 16 h. The mixture was diluted with EtOAc and washed with sat. aq. NH4Cl, concentrated, and purified by flash chromatography to afford title compound (353 mg, 1.2 mmol, 60%). 1H NMR (400 MHz, CDCl3) δΗ 8.13 (t, J = 1.7 Hz, 1H), 8.01 (t, J = 1.7 Hz, 1H), 7.74 (t, J = 1.7 Hz, 1H), 3.95 (s, 3H), 2.82 – 2.91 (m, 2H), 2.64 (td, J = 9.4, 12.0 Hz, 2H), 2.40 – 2.55 (m, 1H), 2.12 (td, J = 4.6, 7.0 Hz, 1H).</p><!><p>Methyl 3-bromo-5-(1-cyanocyclobutyl)benzoate (1.1 g, 3.7 mmol), tert-butylcarbamate (500 mg, 4.3 mmol), Cs2CO3 (2.33 g, 7.2 mmol) in DMF (20 mL) was added Pd2dba3 (171 mg, 0.19 mmol), Xantphos (220 mg, 0.38 mmol) and the mixture heated to 150 °C for 16 h. The cooled mixture was filtered through celite and concentrated. The residue was dissolved in DCM (20 mL) and TFA (5 mL) added, the mixture stirred for 1 h, then washed with sat. aq. NaHCO3 and concentrated. Purification by flash chromatography affords title compound (300 mg, 1.3 mmol, 35%). 1H NMR (400 MHz, CDCl3) δΗ 7.45 (s, 1H), 7.26 – 7.35 (m, 1H), 6.89 (t, J = 1.9 Hz, 1H), 3.91 (s, 3H), 2.74 – 2.93 (m, 2H), 2.55 – 2.69 (m, 2H), 2.42 (td, J = 8.8, 11.5 Hz, 1H), 1.94 – 2.13 (m, 1H); MS (ESI) m/z = 231.1 [M+H]+.</p><!><p>Methyl 3-amino-5-(1-cyanocyclobutyl)benzoate, 53 (230 mg, 1.0 mmol) and 18a were reacted according to General Procedure A. Purification by flash chromatography afforded a colorless solid (435 mg, 0.94 mmol, 94%). MS (ESI) m/z = 484.0 [M+NH4]+.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)benzoate (435 mg, 0.94 mmol) was reacted following General Procedure E (at r.t.) to afford a colorless solid (297 mg, 0.66 mmol, 70%). 1H NMR (400 MHz, MeOH-d4) δH 7.85 (d, J = 2.5 Hz, 1H), 7.79 (t, J = 1.8 Hz, 1H), 7.76 (d, J = 1.8 Hz, 1H), 7.50 (t, J = 1.8 Hz, 1H), 7.47 (dd, J = 8.8, 2.5 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 2.77 (ddt, J = 12.4, 8.8, 3.3 Hz, 2H), 2.61 – 2.49 (m, 2H), 2.38 (dp, J = 11.7, 8.8 Hz, 1H), 2.07 (dtt, J = 11.7, 9.2, 4.5 Hz, 1H); LCMS tR = 1.44 min, m/z = 467.8, 469.8 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>2-(3-Bromo-4-methoxyphenyl)acetonitrile (4.52 g, 20 mmol) and 1,3-dibromopropane (2.23 mL, 22 mmol) were dissolved in DMSO (100 mL), to this mixture was added NaH (60% dispersion in mineral oil, 2.0 g, 50 mmol) portion-wise. The solution was stirred for 16 h at r.t., then diluted with EtOAc:Et2O (200 mL, 1:1) and washed with water (3 × 400 mL). The combined aqueous layers were back-extracted with further EtOAc (200 mL). The combined organics were washed with sat. aq. NaCl, concentrated and purified by ISCO flash column chromatography (120 g, 0–25% EtOAc in hexanes) to afford a colorless oil (3.65 g, 13.7 mmol, 68%). 1H NMR (400 MHz, CDCl3) δH 7.61 (d, J = 2.4 Hz, 1H), 7.34 (dd, J = 8.6, 2.4 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 3.93 (s, 3H), 2.88 – 2.78 (m, 2H), 2.66 – 2.54 (m, 2H), 2.44 (dt, J = 11.6, 8.7 Hz, 1H), 2.16 – 2.04 (m, 1H); LCMS tR = 1.10 min, no mass observed.</p><!><p>1-(3-Bromo-4-methoxyphenyl)cyclobutane-1-carbonitrile (531 μL, 3.43 mmol), Pd(OAc)2 (39 mg, 0.17 mmol), P(tBu4).HBF4 (199 mg, 0.69 mmol) and phenol (323 mg, 3.43 mmol) were taken up in anhydrous MeCN (13.7 mL) in a sealed tube. Phenyl formate (687 μL, 6.86 mmol) and NEt3 (1.43 mL, 10.3 mmol) were added and the mixture heated to 90 °C for 18 h. This reaction was performed in quadruplicate. Upon cooling the four reactions were combined, filtered through celite, and concentrated. The residue was dissolved in EtOAc and washed with water. Purification by flash chromatography afforded a colorless solid (3.72 g, 12.1 mmol, 88%).1H NMR (CDCl3) δH 8.04 (d, J = 2.6 Hz, 1H), 7.61 (dd, J = 8.7, 2.6 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.33 – 7.23 (m, 3H), 7.09 (d, J = 8.7 Hz, 1H), 3.98 (s, 3H), 2.93 – 2.83 (m, 2H), 2.66 (dt, J = 11.8, 9.0 Hz, 2H), 2.55 – 2.41 (m, 1H), 2.19 – 2.06 (m, 1H); LCMS tR = 1.71 min, m/z = 308.1 [M+H]+.</p><!><p>Phenyl 5-(1-cyanocyclobutyl)-2-methoxybenzoate (3.72 g, 12.1 mmol) was reacted following General Procedure D to afford a colorless oil (3.47 g, 11.84 mmol, 98%). 1H NMR (400 MHz, CDCl3) δH 10.55 (s, 1H), 8.09 (d, J = 2.5 Hz, 1H), 7.59 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 – 7.43 (m, 2H), 7.34 (d, J = 7.4 Hz, 1H), 7.25 – 7.20 (m, 2H), 7.09 (d, J = 8.7 Hz, 1H), 2.91 – 2.81 (m, 2H), 2.72 – 2.59 (m, 2H), 2.53 – 2.38 (m, 1H), 2.16 – 2.05 (m, 1H) (Method A) LCMS: tR = 1.20 min, m/z = 294.1 [M+H]+.</p><!><p>Phenyl 5-(1-cyanocyclobutyl)-2-hydroxybenzoate (2.93 g, 10 mmol) was dissolved in DCE/H2O (1:1, 20 mL) and cooled to 0 °C in an ice/water bath. Tetrabutylammonium bromide (161 mg, 0.50 mmol) was added, followed by HNO3 (conc., 1.3 mL, 20 mmol). The mixture was stirred vigorously at 60 °C for 16 h, then cooled, diluted with CH2Cl2, and washed with water. Purification by flash chromatography afforded a pale-yellow solid (2.71 g, 8.0 mmol, 80%). 1H NMR (400 MHz, CDCl3) δH 8.42 (d, J = 2.5 Hz, 1H), 8.31 (d, J = 2.6 Hz, 1H), 7.55 – 7.48 (m, 2H), 7.43 – 7.35 (m, 1H), 7.32 – 7.24 (m, 3H), 3.00 – 2.89 (m, 2H), 2.76 – 2.63 (m, 2H), 2.61 – 2.49 (m, 1H), 2.25 – 2.11 (m, 1H); LCMS tR = 1.18 min, m/z = 339.0 [M+H]+.</p><!><p>Phenyl 5-(1-cyanocyclobutyl)-2-hydroxy-3-nitrobenzoate (2.71 g, 8.0 mmol) was reacted according to General Procedure B. Purification by flash chromatography afforded a cream solid (2.15 g, 7.0 mmol, 87%). 1H NMR (400 MHz, CDCl3) δH 10.70 (d, J = 0.7 Hz, 1H), 7.52 – 7.46 (m, 2H), 7.48 (d, J = 2.3 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.27 – 7.22 (m, 2H), 6.98 (dd, J = 2.3, 0.7 Hz, 1H), 2.90 – 2.77 (m, 2H), 2.71 – 2.58 (m, 2H), 2.54 – 2.39 (m, 1H), 2.16 – 2.07 (m, 1H); LCMS (Method B) tR = 1.08 min, m/z = 309.2 [M+H]+.</p><!><p>Phenyl 3-amino-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (308 mg, 1.0 mmol) and 18a were reacted according to General Procedure A. Purification by flash chromatography afforded a colorless solid (393 mg, 0.72 mmol, 72%). 1H NMR (400 MHz, CDCl3) δH 8.63 (dt, J = 4.4, 1.7 Hz, 2H), 7.90 (d, J = 2.3 Hz, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.49 – 7.45 (m, 2H), 7.38 – 7.35 (m, 2H), 7.23 – 7.17 (m, 2H), 6.88 (d, J = 8.8 Hz, 1H), 2.94 – 2.84 (m, 3H), 2.68 – 2.56 (m, 2H), 2.55 – 2.43 (m, 1H), 2.21 – 2.09 (m, 1H); LCMS tR = 1.86 min, m/z = 559.8, 561.7 [M+NH4]+.</p><!><p>Phenyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (108 mg, 0.20 mmol) was reacted following General Procedure E (at r.t. instead of 65 °C) to afford a colorless solid (61 mg, 0.16 mmol, 80%). 1H NMR (400 MHz, CDCl3) δH 7.85 (d, J = 2.5 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 8.8, 2.5 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 2.82 – 2.72 (m, 2H), 2.60 – 2.49 (m, 2H), 2.46 – 2.32 (m, 1H), 2.16 – 2.03 (m, 1H); LCMS tR = 1.44 min, m/z = 466.8, 468.7 [M+H]+; Purity (AUC) ≥95%.; HRMS (ESI/TOF) [M+Na]+ calculated for C18H15BrN2NaO6S 488.9726, found 488.9746.</p><!><p>Phenyl 3-amino-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (154 mg, 0.50 mmol) and 18b were reacted following General Procedure A to afford a brown solid (208 mg, 72%). 1H NMR (400 MHz, MeOH-d4) δH 7.79 (d, J = 2.5 Hz, 1H), 7.78 (d, J = 2.5 Hz, 1H), 7.75 (d, J = 2.4 Hz, 1H), 7.72 (d, J = 2.5 Hz, 1H), 2.05 (dd, J = 8.3, 3.5 Hz, 2H), 1.99 – 1.87 (m, 2H), 1.89 – 1.78 (m, 1H), 1.80 – 1.69 (m, 3H), 1.46 – 1.31 (m, 1H); LCMS tR = 1.97 min, m/z = 595.7, 597.7 [M+H]+.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (58 mg, 0.10 mmol) was reacted following General Procedure E at room temperature to afford a colorless solid (12 mg, 48%). 1H NMR (400 MHz, MeOH-d4) δH 7.76 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 2.82 – 2.72 (m, 2H), 2.62 – 2.51 (m, 2H), 2.46 – 2.32 (m, 1H), 2.14 – 2.03 (m, 1H); LCMS tR = 1.56 min, m/z = 500.7, 502.8 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+Na]+ calculated for C18H14BrClN2NaO6S 522.9337, found 522.9358.</p><!><p>2-(3-Bromo-4-methoxyphenyl)acetonitrile (4.52 g, 20 mmol) and 1,5-dibromopentane (3.0 mL, 22 mmol) were dissolved in DMSO (100 mL), to this mixture was added NaH (60% dispersion in mineral oil, 2.0 g, 50 mmol) portion-wise. The solution was stirred for 16 h at r.t., then diluted with EtOAc:Et2O (200 mL, 1:1) and washed with water (3 × 400 mL). The combined aqueous layers were back-extracted with further EtOAc (200 mL). The combined organics were washed with sat. aq. NaCl, concentrated, and purified by ISCO flash column chromatography (120 g, 0–25% EtOAc in hexanes) to afford a colorless solid (4.35 g, 14.8 mmol, 74%). 1H NMR (400 MHz, CDCl3) δH 7.62 (d, J = 2.5 Hz, 1H), 7.42 (dd, J = 8.6, 2.5 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 3.90 (s, 3H), 2.18 – 2.10 (m, 2H), 1.91 – 1.79 (m, 6H), 1.78 – 1.65 (m, 2H); LCMS (Method B) tR = 1.10 min, no mass observed.</p><!><p>1-(3-Bromo-4-methoxyphenyl)cyclohexane-1-carbonitrile (883 mg, 3.0 mmol), Pd(OAc)2 (39 mg, 0.17 mmol), P(tBu4).HBF4 (199 mg, 0.69 mmol) and phenol (323 mg, 3.43 mmol) were taken up in anhydrous MeCN (13.7 mL) in a sealed tube. Phenyl formate (687 μL, 6.86 mmol) and NEt3 (1.43 mL, 10.3 mmol) were added and the mixture heated to 90 °C for 18 h. This reaction was performed in quadruplicate. Upon cooling the four reactions were combined, filtered through celite, and concentrated. The residue was dissolved in EtOAc and washed with water. Purification by flash chromatography afforded a colorless solid (3.53 g, 10.5 mmol, 88%). 1H NMR (CDCl3) δH 8.07 (d, J = 2.7 Hz, 1H), 7.73 (dd, J = 8.8, 2.7 Hz, 1H), 7.49 – 7.40 (m, 2H), 7.33 – 7.22 (m, 3H), 7.08 (d, J = 8.8 Hz, 1H), 3.97 (s, 3H), 2.22 (d, J = 11.6 Hz, 2H), 1.98 – 1.74 (m, 8H); LCMS (Method A): tR = 1.86 min, m/z = 336.1 [M+H]+.</p><!><p>Phenyl 5-(1-cyanocyclohexyl)-2-methoxybenzoate (3.52 g, 10.5 mmol) was reacted following General Procedure D to afford a colorless oil (3.03 g, 9.49 mmol, 90%). 1H NMR (CDCl3) δH 10.55 (s, 1H), 8.19 (d, J = 2.6 Hz, 1H), 7.70 (dd, J = 8.8, 2.6 Hz, 1H), 7.54 – 7.45 (m, 2H), 7.40 – 7.33 (m, 1H), 7.28 – 7.22 (m, 2H), 7.10 (d, J = 8.9 Hz, 1H), 2.23 (d, J = 12.2 Hz, 2H), 2.03 – 1.72 (m, 8H); LCMS (Method A): tR = 1.75 min, m/z = 322.0 [M+H]+.</p><!><p>Phenyl 5-(1-cyanocyclohexyl)-2-hydroxybenzoate (3.03 g, 9.5 mmol) was dissolved in DCE/H2O (1:1, 20 mL) and cooled to 0 °C in an ice/water bath. Tetrabutylammonium bromide (153 mg, 0.47 mmol) was added, followed by HNO3 (conc., 1.2 mL, 19.0 mmol). The reaction was stirred vigorously at 60 °C for 18 h, then cooled, diluted with CH2Cl2, and washed with water. The crude material was carried forward without purification for hydrogenation, following General Procedure B. Upon purification a colorless oil was obtained (1.65 g, 4.9 mmol, 52%). 1H NMR (CDCl3) δH 10.55 (s, 1H), 8.19 (d, J = 2.6 Hz, 1H), 7.70 (dd, J = 8.8, 2.6 Hz, 1H), 7.52 – 7.46 (m, 2H), 7.39 – 7.32 (m, 1H), 7.28 – 7.22 (m, 2H), 7.10 (d, J = 8.8 Hz, 1H), 2.23 (d, J = 12.2 Hz, 3H), 1.99 – 1.72 (m, 8H); LCMS (Method B) tR = 1.28 min, m/z = 337.1 [M+H]+.</p><!><p>Phenyl 3-amino-5-(1-cyanocyclohexyl)-2-hydroxybenzoate (308 mg, 0.91 mmol) and 18a (313 mg, 1.1 mmol) were reacted according to General Procedure A. Purification by flash chromatography afforded a colorless solid (443 mg, 0.78 mmol, 85%).</p><p>1H NMR (400 MHz, MeOH-d4) δH 7.85 (d, J = 2.5 Hz, 1H), 7.73 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 2.08 – 1.87 (m, 4H), 1.88 – 1.77 (m, 1H), 1.74 (td, J = 12.9, 10.5, 6.3 Hz, 4H), 1.46 – 1.31 (m, 1H); LCMS tR = 1.29 min, m/z = 588.3, 590.3 [M+NH4]+.</p><!><p>Phenyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoate (29 mg, 0.05 mmol) was reacted following General Procedure E, to afford a colorless solid (17 mg, 69%). 1H NMR (400 MHz, MeOH-d4) δH 7.85 (d, J = 2.5 Hz, 1H), 7.73 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 2.08 – 1.87 (m, 4H), 1.88 – 1.77 (m, 1H), 1.74 (td, J = 12.9, 10.5, 6.3 Hz, 4H), 1.46 – 1.31 (m, 1H); LCMS tR = 1.65 min, m/z = 494.8, 496.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Phenyl 3-amino-5-(1-cyanocyclohexyl)-2-hydroxybenzoate (308 mg, 0.91 mmol) and 18b (336 mg, 1.1 mmol) were reacted according to General Procedure A. Purification by flash chromatography afforded a colorless solid (455 mg, 0.75 mmol, 82%). LCMS (Method B) tR = 1.39 min, m/z = 622.3 [M+NH4]+.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoate (30 mg, 0.05 mmol) was reacted following General Procedure E, to afford a colorless solid (17 mg, 64%). 1H NMR (400 MHz, MeOH-d4) δH 7.79 (d, J = 2.5 Hz, 1H), 7.78 (d, J = 2.5 Hz, 1H), 7.75 (d, J = 2.4 Hz, 1H), 7.72 (d, J = 2.5 Hz, 1H), 2.05 (dd, J = 8.3, 3.5 Hz, 2H), 1.99 – 1.87 (m, 2H), 1.89 – 1.78 (m, 1H), 1.80 – 1.69 (m, 3H), 1.46 – 1.31 (m, 1H); LCMS tR = 1.74 min, m/z = 530.7 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+Na]+ calculated for C20H18BrClN2NaO6S 550.9650, found 550.9681.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoate (109 mg, 0.25 mmol) was reacted with methylamine following General Procedure F, to afford a colorless solid (89 mg, 82%). 1H NMR (400 MHz, DMSO-d6) δH 7.80 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 8.8, 2.5 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 2.88 (s, 3H); LCMS tR = 1.57 min, m/z = 434.8, 436.8 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+Na]+ calculated for C14H12BrClN2NaO5S 456.9231, found 456.9247.</p><!><p>3-((5-Bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-chloro-2-hydroxybenzoic acid (23 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (5 mg, 21 %). 1H NMR (400 MHz, CDCl3) δH 10.94 (br s, 1H), 7.72 (dd, J = 9.2, 2.4 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 2.4 Hz, 1H), 7.63 (dd, J = 2.4, 1.2 Hz, 2H), 7.47 (s, 1H), 7.12 (d, J = 2.4 Hz, 1H), 6.27 (br s, 1H), 3.00 (d, J = 4.9 Hz, 3H); LCMS tR = 1.69 min, m/z = 470.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoate (23 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (21 mg, 91%). 1H NMR (400 MHz, DMSO-d6) δH 11.27 (s, 1H), 8.88 (s, 1H), 8.44 – 8.35 (m, 1H), 7.60 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 2.79 (d, J = 4.2 Hz, 3H), 1.94 – 1.79 (m, 1H), 0.95 – 0.81 (m, 2H), 0.45 – 0.34 (m, 2H); 19F NMR (376 MHz, DMSO-d6) δF −120.4; LCMS tR = 1.69 min, m/z = 458.8, 460.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-cyclopropyl-6-fluoro-2-hydroxybenzoate (49 mg, 0.1 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (36 mg, 73%). 1H NMR (400 MHz, MeOH-d4) δH 7.73 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 2.93 (s, 3H), 2.21 – 1.86 (m, 1H), 1.05 – 0.87 (m, 2H), 0.70 – 0.51 (m, 2H); 19F NMR (376 MHz, MeOH-d4) δF −121.7; LCMS tR = 1.83 min, m/z = 492.7, 494.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-5-(pentafluorosulfanyl)benzoic acid, 6l (25 mg, 0.05 mmol), was dissolved in DCM (0.5 mL), DIPEA (26 μL, 0.15 mmol) added and the mixture cooled to 0 °C before the addition of HATU (29 mg, 0.075 mmol). The mixture was stirred for 30 mins, then methylamine (2.0 M in THF, 125 μL, 0.25 mmol) was added and stirred for 16 h. The mixture was diluted with DCM, washed with water and purified by prep-HPLC to afford a colorless solid (6 mg, 0.01, 23%). 1H NMR (400 MHz, MeOH-d4) δH 7.99 (dt, J = 5.8, 1.5 Hz, 2H), 7.85 (dd, J = 2.4, 1.3 Hz, 2H), 7.50 (dd, J = 8.8, 2.5 Hz, 1H), 6.83 (d, J = 8.7 Hz, 1H), 3.92 (s, 3H); 19F NMR (376 MHz, MeOH-d4) δF 81.2 (p, J = 149 Hz, 1F), 61.0 (d, J = 149 Hz, 4F); LCMS tR = 1.33 min, m/z = 510.7, 512.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfaneyl)benzoate (26 mg, 0.05 mmol) was reacted following General Procedure F, to afford a colorless solid (7 mg, 0.013 mmol, 26%). 1H NMR (400 MHz, CDCl3) δH 8.34 (s, 1H), 8.05 (s, 2H), 7.68 (d, J = 2.4 Hz, 1H), 7.50 (dd, J = 8.8, 2.4 Hz, 1H), 7.28 (s, 1H), 6.87 (d, J = 8.8 Hz, 1H), 4.00 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 83.3 (d, J = 150 Hz), 63.7 (d, J = 150 Hz); LCMS tR = 1.89 min, m/z = 527.9, 529.9 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoate (18 mg, 0.03 mmol) was reacted following General Procedure F, to afford title compound as a colorless solid (3 mg, 0.005 mmol, 17%). 1H NMR (400 MHz, CDCl3) δH 8.04 (d, J = 2.4 Hz, 1H), 7.72 (d, J = 2.4 Hz, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.53 (d, J = 2.3 Hz, 1H), 6.42 (br s, 1H), 3.04 (d, J = 4.9 Hz, 3H); 19F NMR (376 MHz, CDCl3) δF 84.0 (p, J = 150 Hz), 64.1 (d, J = 150 Hz); LCMS tR = 1.16 min, m/z = 561.1, 563.1 [M+H]+; Purity (AUC) ≥95%.</p><!><p>53 (230 mg, 1.0 mmol) was taken in a solution of methylamine ([2.0 M in THF), 2 mL) and heated to 65 °C for 3 h. The mixture was concentrated to afford title compound (202 mg, 0.88 mmol, 88%) which was used without purification. MS (ESI) m/z = 230.1.</p><!><p>3-Amino-5-(1-cyanocyclobutyl)-N-methylbenzamide (23 mg, 0.1 mmol) and 18a (33 mg, 0.12 mmol) were reacted according to General Procedure A. Purification by reverse phase HPLC afforded a colorless solid (12 mg, 0.026 mmol, 26%). 1H NMR (400 MHz, DMSO-d6) δH 8.47 (s, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.60 – 7.42 (m, 3H), 7.28 (s, 1H), 6.88 (d, J = 8.8 Hz, 1H), 3.18 (s, 3H), 2.76 (d, J = 4.5 Hz, 3H), 2.74 – 2.65 (m, 2H), 2.38 – 2.21 (m, 2H), 2.04 – 1.92 (m, 2H); LCMS tR = 1.41 min, m/z = 463.8, 465.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>3-Amino-5-(1-cyanocyclobutyl)-N-methylbenzamide (23 mg, 0.1 mmol) and 18b (37 mg, 0.12 mmol) were reacted according to General Procedure A. Purification by reverse phase HPLC afforded a colorless solid (17 mg, 0.034 mmol, 34%). 1H NMR (400 MHz, DMSO-d6) δH 8.50 (d, J = 4.9 Hz, 1H), 7.91 (d, J = 2.5 Hz, 1H), 7.79 (d, J = 2.5 Hz, 1H), 7.55 (t, J = 1.8 Hz, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.28 (t, J = 1.9 Hz, 1H), 2.76 (d, J = 4.5 Hz, 3H), 2.77 – 2.65 (m, 2H), 2.37 – 2.22 (m, 1H), 1.99 (dtt, J = 9.1, 7.0, 4.5 Hz, 1H); LCMS tR = 1.50 min, m/z = 497.8, 499.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (31 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (18 mg, 75%). 1H NMR (400 MHz, MeOH-d4) δH 7.82 (d, J = 2.5 Hz, 1H), 7.56 – 7.47 (m, 3H), 6.84 (d, J = 8.8 Hz, 1H), 2.91 (s, 3H), 2.81 – 2.71 (m, 2H), 2.65 – 2.51 (m, 2H), 2.38 (dt, J = 11.5, 8.6 Hz, 1H), 2.12 – 2.02 (m, 1H); LCMS tR = 1.54 min, m/z = 479.8, 481.8 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+H]+ calculated for C19H19BrN3O5S 480.0223, found 480.0239.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (29 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (13 mg, 54%). 1H NMR (400 MHz, DMSO-d6) δH 11.07 (br s, 1H), 9.39 (br s, 2H), 9.12 (d, J = 4.6 Hz, 1H), 7.93 (d, J = 2.5 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.71 (d, J = 2.5 Hz, 1H), 7.35 (d, J = 2.1 Hz, 1H), 2.83 (d, J = 4.6 Hz, 3H), 2.75 – 2.65 (m, 2H), 2.58 – 2.46 (m, 2H), 2.25 (dt, J = 11.3, 8.6 Hz, 1H), 2.05 – 1.88 (m, 1H); LCMS tR = 1.66 min, m/z = 513.7, 515.8 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+H]+ calculated for C19H19BrClN3O5S 513.9834, found 513.9848.</p><!><p>Phenyl 3-((5-bromo-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoate (29 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (16 mg, 63%). 1H NMR (400 MHz, DMSO-d6) δH 11.30 (s, 1H), 9.09 (s, 1H), 8.88 (s, 1H), 7.71 (d, J = 2.6 Hz, 1H), 7.65 (d, J = 2.1 Hz, 1H), 7.58 (dd, J = 8.7, 2.1 Hz, 1H), 7.50 – 7.45 (m, 3H), 6.91 (d, J = 8.7 Hz, 1H), 2.81 (d, J = 4.3 Hz, 3H), 1.99 (d, J = 11.7 Hz, 2H), 1.90 – 1.51 (m, 8H); LCMS tR = 1.72 min, m/z = 507.8, 509.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclohexyl)-2-hydroxybenzoate (30 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (21 mg, 77%). 1H NMR (400 MHz, DMSO-d6) δH 9.09 (d, J = 4.4 Hz, 1H), 7.92 (d, J = 2.3 Hz, 1H), 7.72 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 2.82 (d, J = 4.4 Hz, 3H), 2.02 (d, J = 12.7 Hz, 2H), 1.88 – 1.51 (m, 7H), 1.26 (d, J = 12.7 Hz, 1H); LCMS tR = 1.84 min, m/z = 543.7 [M+H]+; Purity (AUC) ≥95%; HRMS (ESI/TOF) [M+H]+ calculated for C21H22BrClN3O5S 542.0147, found 542.0161.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (27 mg, 0.05 mmol), 3-amino-1,2,4-triazole (21 mg) and NEt3 (35 μL, 0.25 mmol) were heated to 90 °C in dioxane (1 mL) for 16 h. The mixture was concentrated and purified by prep-HPLC to afford a colorless solid (15 mg, 56%) after neutralization with sat. aq. K2CO3. LCMS tR = 1.41 min, m/z = 532.8, 534.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Phenyl 3-((5-bromo-3-chloro-2-hydroxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (27 mg, 0.05 mmol), (2-methoxyethyl)ethylamine (22 μL, 0.25 mmol) and NEt3 (35 μL, 0.25 mmol) were heated to 90 °C in dioxane (1 mL) for 16 h. The mixture was concentrated and purified by prep-HPLC to afford a colorless solid (16 mg, 61%). 1H NMR (400 MHz, MeOH-d4) δH 7.80 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.50 (dd, J = 8.7, 2.5 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 3.55 (d, J = 1.4 Hz, 4H), 3.36 (s, 3H), 2.81 – 2.70 (m, 2H), 2.62 – 2.51 (m, 2H), 2.44 – 2.30 (m, 1H), 2.11 – 1.97 (m, 1H); LCMS tR = 1.57 min, m/z = 523.8, 525.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>1-chloro-3-iodo-5-nitrobenzene (400 mg, 1.41 mmol), sodium methanesulfinate (174 mg, 1.7 mmol), CuI (27 mg, 0.14 mmol), L-proline (32 mg, 0.28 mmol) and NaOH (11 mg, 0.28 mmol) were taken in DMSO (3 mL) under an inert atmosphere and heated to 85 °C for 16 h. Upon cooling, the mixture was diluted with H2O and extracted with DCM. The organic phase was dried and concentrated to afford a colorless solid, which without purification was reacted following General Procedure B, to afford a yellow solid (108 mg, 0.53 mmol, 55% yield). 1H NMR (400 MHz, CDCl3) δH 7.23 (s, 1H), 7.08 (s, 1H), 6.86 (s, 1H), 4.11 (s, 2H), 3.04 (s, 3H).</p><!><p>3-Chloro-5-(methylsulfonyl)aniline (20 mg, 0.10 mmol) was reacted with 18a following General Procedure A, to afford a colorless solid (20 mg 0.046 mmol, 48%). 1H NMR (400 MHz, MeOH-d4) δH 7.88 (d, J = 2.5, 1H), 7.65 (t, J = 1.7 Hz, 1H), 7.58 (t, J = 1.7 Hz, 1H), 7.52 (dd, 8.8, 2.5 Hz, 1H), 7.47 (t, 1.7 Hz, 1H), 6.85 (d, J= 8.8 Hz, 1H), 3.06 (s, 3H); LCMS tR = 1.63 min, m/z = 458.7 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>3-Chloro-5-(methylsulfonyl)aniline (50 mg, 0.24 mmol) was reacted with 18b following General Procedure A, to afford a colorless solid (38 mg 0.085 mmol, 35%). 1H NMR (400 MHz, CDCl3) δH 7.79 (t, J = 1.9 Hz, 1H), 7.70 (d, J = 2.2 Hz, 2H), 7.52 (d, J = 1.9 Hz, 2H), 3.05 (s, 3H); LCMS: tR = 1.07 min, m/z = 492.8 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>To a solution of 2-bromo-4-chloro-6-nitrophenol (1000 mg, 3.96 mmol) in pyridine (10 mL) was added Cu (500 mg, 7.9 mmol) and dimethyl disulfide (713 μL, 7.9 mmol). The mixture was heated to 110 °C for 16 h. The cooled mixture was filtered through celite, suspended in 3 M HCl and extracted with DCM and concentrated. Purification by flash chromatography affords title compound (630 mg, 2.87 mmol, 72%). MS (ESI) m/z = 221.2 [M+H]+.</p><!><p>4-Chloro-2-(methylthio)-6-nitrophenol (1024 mg, 4.66 mmol) was dissolved in EtOH:H2O (1:1, 15 mL) and oxone (2.86 g, 9.32 mmol) added. The mixture was stirred at r.t. for 24 h. The mixture was diluted with water and extracted with DCM, and dried (MgSO4) to afford a yellow solid (1155 mg, 4.58 eq, 98%), which was used without further purification. 1H NMR (400 MHz, CDCl3) δH 11.40 (s, 1H), 8.39 (d, J = 2.7 Hz, 1H), 8.31 (d, J = 2.7 Hz, 1H), 7.26 (s, 1H), 3.34 (s, 3H).</p><!><p>4-chloro-2-(methylsulfonyl)-6-nitrophenol (68 mg, 0.20 mmol) was reacted following General Procedure B to afford title compound (60 mg, 0.2 mmol, 98%) which was taken forward without purification. 1H NMR (400 MHz, CDCl3) δH 7.00 (d, J = 2.3 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 3.12 (s, 3H). MS (ESI) m/z =</p><!><p>Amino-4-chloro-6-(methylsulfonyl)phenol (21 mg, 0.09 mmol) and 5-bromo-2-hydroxy-benzenesulfonyl chloride were reacted following General Procedure A, to afford a colorless solid (11 mg, 0.024 mmol, 26%). 1H NMR (400 MHz, CDCl3) δH 9.13 (s, 1H), 8.33 (s, 1H), 7.72 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.54 (dd, J = 8.9, 2.4 Hz, 1H), 7.48 (d, J = 2.5 Hz, 1H), 7.18 (s, 1H), 6.91 (d, J = 8.9 Hz, 1H), 3.08 (s, 3H); LCMS: tR = 1.71 min, m/z = 475.1 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>Amino-4-chloro-6-(methylsulfonyl)phenol (21 mg, 0.09 mmol) and 5-bromo-3-chloro-2-hydroxy-benzenesulfonyl chloride were reacted following General Procedure A, affording a colorless solid (12.2 mg, 0.024 mmol, 28%). 1H NMR (400 MHz, CDCl3) δH 7.76 (d, J = 2.5 Hz, 1H), 7.74 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 3.11 (s, 3H); LCMS: tR = 1.06 min, m/z = 509.1, 511.1 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-bromo-5-(pentafluorosulfanyl)benzoate (600 mg, 1.91 mmol), sodium methanesulfinate (146 mg, 1.4 mmol), CuI (23 mg, 0.12 mmol), L-proline (27 mg, 0.24 mmol) and NaOH (10 mg, 0.24 mmol) were taken in DMSO (2.5 mL) under an inert atmosphere and heated to 85 °C for 16 h. Upon cooling, the mixture was diluted with H2O and extracted with DCM. The organic phase was dried and concentrated to afford a colorless solid (164.1 mg, 0.48 mmol, 25% yield), which was taken forward without purification. 1H NMR (400 MHz, CDCl3-d) δH 8.74 (d, J = 1.7 Hz, 1H), 8.68 (t, J = 1.7 Hz, 1H), 8.50 (t, J = 1.7 Hz, 1H), 4.02 (s, 3H), 3.15 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 80.4 (t, J = 151 Hz), 63.0 (d, J = 151 Hz). LCMS tR = 0.98 min, m/z = 341.1 [M+H]+.</p><!><p>Methyl 3-(methylsulfonyl)-5-(pentafluorosulfanyl)benzoate (162 mg, 0.54 mmol) was reacted following General procedure E, to afford a colorless solid (117.7 mg, 0.36 mmol, 67%). 1H NMR (400 MHz, CDCl3) δH 8.79 (s, 1H), 8.73 (s, 1H), 8.56 (s, 1H), 3.17 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 80.2 (d, J = 151 Hz, 1F), 63.0 (d, J = 151 Hz, 4F).</p><!><p>3-(Methylsulfonyl)-5-(pentafluorosulfanyl)benzoic acid (117 mg, 0.36 mmol), diphenylphosphoryl azide (119 mg, 0.43 mmol) and tert-butanol (172 μL, 1.8 mmol) were taken in DME (1.8 mL) and heated to 100 °C for 16 h. The cooled mixture was diluted with EtOAc and washed with sat. aq. NH4Cl, sat. aq. NaHCO3, water, then concentrated. The residue was dissolved in DCM (5 mL), TFA (1 mL) added and the mixture stirred for 1 h at r.t. The mixture was concentrated and purified by flash chromatography to afford a colorless solid (51.2 mg, 0.17 mmol, 48%). 1H NMR (400 MHz, CDCl3) δH 7.61 (t, J = 1.8 Hz, 1H), 7.32 (t, J = 1.8 Hz, 1H), 7.25 (t, J = 1.8 Hz, 1H), 4.29 (s, 2H), 3.07 (s, 3H);19F NMR (376 MHz, CDCl3) δ 79.4 (p, J = 151 Hz, 1F), 59.53 (d, J = 150.5 Hz, 4F).</p><!><p>3-(Methylsulfonyl)-5-(pentafluorosulfanyl)aniline (20 mg, 0.07 mmol) and 18a were reacted following General Procedure A, to afford a colorless solid (8.4 mg, 0.016 mmol, 23%). 1H NMR (400 MHz, CDCl3) δH 8.08 (s, 1H), 7.82 (s, 1H), 7.77 (s, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.65 (s, 1H), 7.54 (dd, J = 8.8, 2.5 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 3.08 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 80.5 (d, J = 151 Hz), 62.9 (d, J = 151 Hz); LCMS: tR = 1.128 min, m/z = 548.8 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>3-(Methylsulfonyl)-5-(pentafluorosulfanyl)aniline (20 mg, 0.07 mmol) and 18b were reacted following General Procedure A, to afford a colorless solid (7 mg, 0.013 mmol, 19%). 1H NMR (400 MHz, CDCl3) δH 8.08 (s, 1H), 7.82 (s, 1H), 7.77 (s, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.65 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H), 3.08 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 80.5 (d, J = 151 Hz, 1F), 62.9 (d, J = 151 Hz, 4F); LCMS: tR = 1.201 min, m/z = 584.7 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>To 4-(pentafluorosulfaneyl)phenol (800 mg, 3.63 mmol) in acetic acid (4 mL) was added FeCl3 (59 mg, 0.36 mmol). The mixture was cooled to 0 °C and then Br2 (812 mg, 5.45 mmol) was added. The mixture was allowed to reach room temperature and stirred for 2 h. Then, the solvent was removed under reduced Stpressure and the crude material was purified by flash chromatography to afford title compound (439 mg, 1.5 mmol, 41%). 1H NMR (400 MHz, CDCl3) δH 7.90 (d, J = 2.6 Hz, 1H), 7.63 (dd, J = 9.0, 2.6 Hz, 1H), 7.06 (d, J = 9.0 Hz, 1H).</p><!><p>To 2-bromo-4-(pentafluorosulfanyl)phenol (439 mg, 1.47 mmol) in pyridine (4 mL) was added Cu (500 mg, 7.9 mmol) and dimethyl disulfide (713 μL, 7.9 mmol). The mixture was heated to 110 °C for 16 h. The cooled mixture was filtered through celite, suspended in 3 M HCl and extracted with DCM and concentrated. Purification by flash chromatography affords title compound (84 mg, 0.32 mmol, 32%). 1H NMR (400 MHz, CDCl3) δH 7.89 (d, J = 2.7 Hz, 1H), 7.63 (dd, J = 9.0, 2.7 Hz, 1H), 7.05 – 6.97 (m, 2H), 2.37 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 85.5 (d, J = 151 Hz), 64.3 (d, J = 151 Hz); MS (ESI) m/z = 266.0 [M+H]+.</p><!><p>2-(methylthio)-4-(pentafluorosulfanyl)phenol (84 mg, 0.32 mmol) was dissolved in EtOH:H2O (1:1, 1 mL) and oxone (197 g, 9.32 mmol) added. The mixture was stirred at r.t. for 24 h. The mixture was diluted with water and extracted with DCM, and dried (MgSO4) to afford title compound (44 mg, 0.25 mmol, 78%). 1H NMR (400 MHz, CDCl3) δH 9.25 (s, 1H), 8.11 (d, J = 2.7 Hz, 1H), 7.91 (dd, J = 9.2, 2.7 Hz, 1H), 7.13 (d, J = 9.2 Hz, 1H), 3.18 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 83.2 (d, J = 151 Hz), 64.0 (d, J = 151 Hz).</p><!><p>2-(Methylsulfonyl)-4-(pentafluorosulfanyl)phenol (74 mg, 0.25 mmol) was reacted following General Procedure C to afford a yellow solid (68 mg, 0.20 mmol, 80%).1H NMR (400 MHz, CDCl3) δH 8.81 (s, 1H), 8.70 (s, 1H), 3.38 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 80.0 (p, J = 152 Hz), 63.9 (d, J = 152 Hz).</p><!><p>2-(Methylsulfonyl)-6-nitro-4-(pentafluorosulfanyl)phenol (235 mg, 0.68 mmol) was reacted following General Procedure B to afford title compound as a pale brown solid (214 mg, 0.68 mmol, quant.), which was taken forward without purification. MS (ESI) m/z = 314.1 [M+H]+.</p><!><p>2-Amino-6-(methylsulfonyl)-4-(pentafluorosulfanyl)phenol (40 mg, 0.13 mmol) and 5-bromo-2-hydroxy-benzenesulfonyl chloride were reacted following General Procedure A, to afford the title compound (24 mg, 0.044 mmol, 34%). 1H NMR (400 MHz, CDCl3) δH 8.03 (d, J = 2.8 Hz, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 2.8 Hz, 1H), 7.62 (dd, J = 8.8, 2.5 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 3.31 (s, 3H); 19F NMR (376 MHz, MeOH-d4) δF 79.1 (p, J = 149 Hz), 59.4 (d, J = 149 Hz); LCMS tR = 1.01 min, m/z = 547.9, 549.9 [M+H]+; Purity (AUC) ≥95%.</p><!><p>2-Amino-6-(methylsulfonyl)-4-(pentafluorosulfanyl)phenol (21 mg, 0.09 mmol) and 5-bromo-3-chloro-2-hydroxy-benzenesulfonyl chloride was reacted following General Procedure A, to afford title compound (32 mg, 0.055 mmol, 42%). 1H NMR (400 MHz, CDCl3) δH 8.16 (d, J = 2.5 Hz, 1H), 7.85 (d, J = 2.4 Hz, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.71 (d, J = 2.5 Hz, 1H), 3.17 (s, 3H); 19F NMR (376 MHz, CDCl3) δF 79.0 (p, J = 151 Hz, 1F), 60.7 (d, J = 151 Hz, 4F); LCMS tR = 1.01 min, m/z = 566.3, 569.9 [M+H]+; Purity (AUC) ≥95%.</p><!><p>3-Bromo-5-methylaniline (2.0 g, 10.75 mmol), sodium methanesulfinate (1.32 g, 12.9 mmol), CuI (205 mg, 1.08 mmol), L-proline (248 mg, 2.15 mmol) and NaOH (86 mg, 0.24 mmol) were taken in DMSO (22 mL) under an inert atmosphere and heated to 85 °C for 16 h. Upon cooling, the mixture was diluted with H2O and extracted with DCM. The organic phase was dried and concentrated to afford title compound (1.2 g, 6.46 mmol, 60% yield). 1H NMR (400 MHz, CDCl3) δH 7.10 (d, J = 1.6 Hz, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.71 (dd, J = 2.1, 1.6 Hz, 1H), 3.90 (s, 2H), 3.01 (s, 3H), 2.36 – 2.31 (m, 3H); MS (ESI) m/z = 371.4 [2M+H]+.</p><!><p>A mixture containing 3-methyl-5-(methylsulfonyl)aniline (1196 mg, 6.46 mmol) and di-tert-butyl carbonate (1671 mg, 7.66 mmol) in MeOH (22 mL) was allowed to stir at r.t. for 16 h. The solvent was removed under reduced pressure and the crude material was purified by flash chromatography to afford title compound (826 mg, 2.89 mmol, 45%) 1H NMR (400 MHz, CDCl3) δH 7.71 (t, J = 1.9 Hz, 1H), 7.56 (s, 1H), 7.39 (d, J = 1.9 Hz, 1H), 6.86 (s, 1H), 3.03 (s, 3H), 2.39 (s, 3H), 1.51 (s, 9H). MS (ESI) m/z = 303.2 [M+NH4]+.</p><!><p>tert-Butyl (3-methyl-5-(methylsulfonyl)phenyl) carbamate (826 mg, 2.89 mmol) and NBS (618 mg, 3.47 mmol) were taken in CCl4 (25 mL) and the solution was degassed with Ar for 10 min. To this mixture was added AIBN (119 mg, 724 mmol) and the reaction mixture was heated to 80 °C for 16 h. The cooled reaction mixture was filtered. The filtrate was washed first with HCl [1 M] and then with a saturated solution of NaHCO3. The organic phase was dried using a phase separator and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography to give (961 mg, 2.64 mmol, 91%). 1H NMR (400 MHz, Chloroform-d) δ 7.84 (d, J = 1.8 Hz, 1H), 7.60 (s, 1H), 6.88 (s, 1H), 4.47 (s, 2H), 3.06 (s, 3H), 1.52 (s, 9H). MS (ESI) m/z = 381.3, 383.2 [M+NH4]+.</p><!><p>tert-Butyl (3-(bromomethyl-5-(methylsulfonyl)phenyl) carbamate (961 mg, 2.64 mmol) was dissolved in MeCN (5.0 mL) and cooled to 0 °C. K2CO3 (438 mg, 3.17 mmol) and TMSCN (0.420 mL, 3.17 mmol) were added and heated to 50 °C for 16 h. The cooled mixture was diluted with NaOH [3 M] and extracted with EtOAc. The crude material was purified by flash chromatography to title compound (441 mg, 1.42 mmol, 49%). 1H NMR (400 MHz, CDCl3) δH 7.89 (t, J = 1.8 Hz, 1H), 7.77 (s, 1H), 7.55 (t, J = 1.8 Hz, 1H), 6.82 (s, 1H), 3.81 (s, 2H), 3.07 (s, 3H), 1.53 (s, 9H). MS (ESI) m/z = 328.3 [M+NH4]+.</p><!><p>tert-Butyl (3-(cyanomethyl-5-(methylsulfonyl)phenyl) carbamate (421 mg, 1.36 mmol) and 1,3-dibromopropane (151 μL, 1.49 mmol) were dissolved DMSO (7 mL) was NaH (60% dispersion in mineral oil, 140 mg, 3.39mmol) was added with caution. The solution was stirred for 16 h at r.t., then diluted with EtOAc:Et2O (100 mL, 1:1) and washed with water (3 × 100 mL). The combined aqueous layers were back-extracted with further EtOAc (100 mL). The combined organics were washed with sat. aq. NaCl, concentrated and purified by flash column chromatography to afford a colorless oil (311 mg, 0.887 mmol, 65%). 1H NMR (400 MHz, CDCl3) δH 7.94 (s, 1H), 7.80 (s, 1H), 7.56 (s, 1H), 7.30 (s, 1H), 3.06 (s, 3H), 2.87 – 2.76 (m, 2H), 2.69 – 2.56 (m, 2H), 2.43 (dt, J = 11.9, 8.8 Hz, 1H), 2.13 – 2.04 (m, 1H), 1.50 (s, 9H); LCMS tR = 0.99 min, m/z = 368.3 [M+NH4]+.</p><!><p>tert-Butyl (3-(1-cyanocyclobutyl)-5-(methylsulfonyl)phenyl) carbamate (311 mg, 0.887 mmol) was dissolved in a solution of HCl (4 M in dioxane, 5 mL) and heated to 50 °C for 1 h. The solvent was removed under reduced pressure to afford title compound (253 mg, 0.88 mmol, quant) which was taken forward without purification. 1H NMR (400 MHz, CDCl3) δH 8.14 (s, 1H), 7.96 (s, 1H), 7.82 (s, 1H), 3.09 (s, 3H), 2.79 (s, 2H), 2.61 (d, J = 11.4 Hz, 2H), 2.44 – 2.34 (m, 1H), 2.07 (s, 1H); LCMS tR = 0.59 min, m/z = 251.0 [M+H]+.</p><!><p>1-(3-Amino-5-(methylsulfonyl)phenyl)cyclobutene-1-carbonitrile hydrochloride (83 mg, 0.29 mmol) and 5-bromo-3-chloro-2-hydroxy-benzenesulfonyl chloride were reacted following General Procedure A, to afford the title compound (55 mg, 0.11 mmol, 38%). 1H NMR (400 MHz, CDCl3) δH 7.90 (br s, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 7.62 (d, J = 1.8 Hz, 1H), 7.53 (t, J = 1.8 Hz, 1H), 3.08 (s, 3H), 2.92 – 2.81 (m, 2H), 2.64 – 2.52 (m, 2H), 2.52 – 2.42 (m, 1H), 2.16 – 2.07 (m, 1H); LCMS tR = 1.81 min, m/z = 518.9, 521.9 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>1-(3-Bromo-4-methoxyphenyl)cyclobutane-1-carbonitrile (200 mg, 0.75 mmol), sodium methanethiolate (55 mg, 0.79 mmol), Pd2dba3 (17 mg, 0.02 mmol), Xantphos (22 mg 0.04 mmol) were combined in a vial purged with argon. THF (3.75 mL) and Et3N (0.13 mL, 0.94 mmol) were added and the mixture heated to reflux for 18 h. The cooled mixture was filtered, concentrated and purified by flash chromatography to afford a yellow oil (174 mg, 0.746 mmol, 99% yield). 1H NMR (400 MHz, CDCl3) δH 7.21 – 7.13 (m, 2H), 6.87 – 6.80 (m, 1H), 3.90 (s, 3H), 2.88 – 2.75 (m, 2H), 2.59 (dt, J = 9.8, 8.0 Hz, 2H), 2.45 (s, 3H), 2.43 – 2.36 (m, 1H), 2.12 – 2.00 (m, 1H). MS (ESI) m/z = 234.2 [M+H]+.</p><!><p>1-(4-methoxy-3-(methylthio)phenyl)cyclobutane-1-carbonitrile (174 mg, 0.671 mmol) was dissolved in EtOH:H2O (1:1, 1.5 mL) and oxone (413 mg, 1.34 mmol) added. The mixture was stirred at r.t. for 24 h. The mixture was diluted with water and extracted with DCM, and dried (MgSO4) to afford a pale-yellow solid (188 mg, 0.58 mmol, 87% yield) that was taken forward without purification. 1H NMR (400 MHz, CDCl3) δH 8.03 (d, J = 2.5 Hz, 1H), 7.67 (dd, J = 8.6, 2.5 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 2.91 – 2.81 (m, 1H), 2.68 – 2.58 (m, 2H), 2.53 – 2.40 (m, 1H), 2.17 – 2.07 (m, 1H).</p><!><p>1-(4-Methoxy-3-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile (71 mg, 0.27 mmol) was reacted following General Procedure D, to afford an off-white solid (68 mg, 0.27 mmol, quant.). 1H NMR (400 MHz, CDCl3) δH 8.86 (s, 1H), 7.68 (d, J = 2.5 Hz, 1H), 7.58 (dd, J = 8.7, 2.5 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H), 3.15 (s, 3H), 2.91 – 2.77 (m, 2H), 2.69 – 2.54 (m, 2H), 2.54 – 2.39 (m, 1H), 2.17 – 2.02 (m, 1H).</p><!><p>1-(4-Hydroxy-3-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile (170 mg, 0.68 mmol) was reacted following General Procedure C to afford title compound (145 mg, 0.49 mmol, 84%). %). 1H NMR (400 MHz, Chloroform-d) δ 8.41 (d, J = 2.5 Hz, 1H), 8.33 (dt, J = 2.2, 1.0 Hz, 1H), 3.34 (s, 3H), 2.95 – 2.84 (m, 2H), 2.69 – 2.57 (m, 2H), 2.57 – 2.43 (m, 1H), 2.19 – 2.06 (m, 1H); MS (ESI) m/z = 314.3 [M+NH4]+.</p><!><p>1-(4-Hydroxy-3-(methylsulfonyl)-5-nitrophenyl)cyclobutane-1-carbonitrile (145 mg, 0.489 mmol) was reacted following General Procedure B to afford title compound (122 mg, 0.458 mmol, 94% yield1H NMR (400 MHz, Chloroform-d) δ 7.01 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H), 3.13 (s, 3H), 2.83 – 2.72 (m, 2H), 2.62 – 2.50 (m, 2H), 2.41 (dt, J = 11.5, 8.8 Hz, 1H), 2.11 – 1.98 (m, 1H); MS (ESI) m/z = 267.2 [M+H]+.</p><!><p>1-(3-Amino-4-hydroxy-5-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile (11 mg, 0.04 mmol) and 5-bromo-2-hydroxy-benzenesulfonyl chloride were reacted following General Procedure A, to afford the title compound (3 mg, 0.006 mmol, 15%). 1H NMR (400 MHz, CDCl3) δH 7.76 (d, J = 2.5 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 8.8, 2.5 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 3.27 (s, 3H), 2.80 – 2.65 (m, 2H), 2.56 – 2.24 (m, 3H), 2.15 – 1.94 (m, 1H); LCMS tR = 1.44 min, m/z = 501.0, 503.0 [M+H]+; Purity (AUC) ≥95%.</p><!><p>1-(3-Amino-4-hydroxy-5-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile (15 mg, 0.056 mmol) and 5-bromo-3-chloro-2-hydroxy-benzenesulfonyl chloride were reacted following General Procedure A, affording the title compound (8 mg, 0.015 mmol, 27% yield). 1H NMR (400 MHz, Acetonitrile-d3) δH 7.81 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 3.16 (s, 3H), 2.77 – 2.70 (m, 2H), 2.53 – 2.45 (m, 2H), 2.34 (dq, J = 11.5, 8.6 Hz, 1H), 2.06 – 1.97 (m, 1H); LCMS tR = 1.02 min, m/z = 552.2, 553.2 [M+NH4]+; Purity (AUC) ≥95%.</p><!><p>Amino-4-chloro-6-(methylsulfonyl)phenol (50 mg, 0.23 mmol) and 16a were reacted following General Procedure A to afford the title compound (3 mg, 0.006 mmol, 3%). 1H NMR (400 MHz, CDCl3) δH 9.24 (s, 1H), 8.00 (d, J = 2.5 Hz, 1H), 7.82 (d, J = 2.5 Hz, 1H), 7.68 (s, 1H), 7.63 (dd, J = 8.8, 2.4 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 3.96 (s, 3H), 3.11 (s, 3H). LCMS tR = 1.055 min, m/z = 489.2 [M+NH4]+; Purity (AUC) ≥ 95%.</p><!><p>Phenyl 3-((5-bromo-2-methoxyphenyl)sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (28 mg, 0.05 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (14 mg, 57%). 1H NMR (400 MHz, MeOH-d4) δH 7.88 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.9, 2.5 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H), 7.06 (d, J = 8.9 Hz, 1H), 3.90 (s, 3H), 2.89 (s, 3H), 2.81 – 2.70 (m, 2H), 2.62 – 2.50 (m, 2H), 2.38 (dq, J = 11.5, 8.5 Hz, 1H), 2.12 – 1.98 (m, 1H); LCMS tR = 1.74 min, m/z = 493.8, 495.8 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Amino-4-chloro-6-(methylsulfonyl)phenol (10 mg, 0.45 mmol) and 6-bromoquinoline-8-sulfonyl chloride were reacted following General Procedure A, affording the title compound (4 mg, 0.008 mmol, 18%). 1H NMR (400 MHz, CDCl3) δH 9.10 (dd, J = 4.3, 1.7 Hz, 1H), 8.48 (d, J = 2.2 Hz, 1H), 8.23 (d, J = 2.2 Hz, 1H), 8.20 (dd, J = 8.4, 1.7 Hz, 1H), 7.93 (d, J = 2.5 Hz, 1H), 7.62 (dd, J = 8.4, 4.3 Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 3.05 (s, 3H); LCMS tR = 1.038 min, m/z = 491.2, 493.2 [M+H]+; Purity (AUC) ≥95%.</p><!><p>1-(3-Amino-4-hydroxy-5-(methylsulfonyl)phenyl)cyclobutane-1-carbonitrile (21 mg, 0.079 mmol) and 6-bromoquinoline-8-sulfonyl chloride were reacted following General Procedure A, to afford the title compound (16 mg, 0.030 mmol, 38%). 1H NMR (400 MHz, CDCl3) δH 9.11 (dd, J = 4.4, 1.6 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.25 – 8.17 (m, 2H), 7.93 (d, J = 2.3 Hz, 1H), 7.62 (dd, J = 8.4, 4.3 Hz, 1H), 7.37 (d, J = 2.3 Hz, 1H), 3.06 (s, 3H), 2.82 (ddd, J = 11.9, 8.5, 4.1 Hz, 2H), 2.59 – 2.51 (m, 2H), 2.44 (dq, J = 11.5, 8.7, 8.1 Hz, 1H), 2.14 – 2.04 (m, 1H); LCMS tR = 1.02 min, m/z = 552.2, 553.2 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Phenyl 3-amino-5-(1-cyanocyclobutyl)-2-hydroxybenzoate 31 mg, 0.10 mmol) and 6-bromoquinoline-8- sulfonyl chloride were reacted following General Procedure D to afford a colorless solid (22 mg, 0.04 mmol, 38%). LCMS tR = 1.18 min, m/z = 578.3, 580.3 [M+H]+.</p><!><p>Phenyl 3-((6-bromoquinoline)-8-sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (71 mg, 0.12 mmol) was reacted following General Procedure E, to afford title compound (40 mg, 0.08 mmol, 65%). 1H NMR (400 MHz, MeOH-d4) δH 9.07 (dd, J = 4.3, 1.7 Hz, 1H), 8.41 – 8.38 (m, 2H), 8.34 (dd, J = 8.4, 1.7 Hz, 1H), 7.79 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 8.4, 4.3 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 2.79 – 2.69 (m, 2H), 2.59 – 2.48 (m, 2H), 2.44 – 2.30 (m, 1H), 2.06 (ddt, J = 9.0, 6.9, 4.5 Hz, 1H); LCMS tR = 1.04 min, m/z = 502.3, 504.2 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Phenyl 3-((6-bromoquinoline)-8-sulfonamido)-5-(1-cyanocyclobutyl)-2-hydroxybenzoate (22 mg, 0.04 mmol) was reacted with methylamine following General Procedure F to afford a colorless solid (7 mg, 36%). 1H NMR (400 MHz, MeOH-d4) δH 9.09 (dd, J = 4.3, 1.7 Hz, 1H), 8.42 (d, J = 2.2 Hz, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.37 (dd, J = 8.4, 1.8 Hz, 1H), 7.70 – 7.66 (m, 1H), 7.68 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 2.82 (s, 3H), 2.79 – 2.71 (m, 2H), 2.63 – 2.52 (m, 1H), 2.43 – 2.32 (m, 1H), 2.11 – 2.00 (m, 1H); LCMS tR = 1.19 min, m/z = 514.8, 516.7 [M+H]+; Purity (AUC) ≥95%.</p><!><p>Methyl 3-amino-2-hydroxy-5-(pentafluorosulfanyl)benzoate (35 mg, 0.12 mmol) and 6-bromoquinoline-8-sulfonyl chloride were reacted following General Procedure A, to afford title compound as a colorless solid (28 mg, 0.05 mmol, 42%). MS (ESI) m/z = 563.2 [M+H]+.</p><!><p>Methyl 3-((6-bromoquinoline)-8-sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoate (14 mg, 0.022 mmol) was reacted following General Procedure E to afford title compound (8 mg, 0.015 mmol, 66%). 1H NMR (400 MHz, MeOH-d4) δH 9.04 (dd, J = 4.3, 1.7 Hz, 1H), 8.45 – 8.39 (m, 2H), 8.37 (dd, J = 8.4, 1.7 Hz, 1H), 8.15 (d, J = 2.8 Hz, 1H), 7.92 (d, J = 2.7 Hz, 1H), 7.67 (dd, J = 8.4, 4.3 Hz, 1H); 19F NMR (376 MHz, MeOH-d4) δF 83.0 (p, J = 150 Hz, 1F), 62.1 (d, J = 150 Hz, 4F); LCMS tR = 1.13 min, m/z = 549.1, 551.2 [M+H]+, Purity (AUC) ≥95%.</p><!><p>Phenyl 3-((6-bromoquinoline)-8-sulfonamido)-2-hydroxy-5-(pentafluorosulfanyl)benzoate (14 mg, 0.022 mmol) was reacted following General Procedure F to afford title compound (9 mg, 0.015 mmol, 69%). 1H NMR (400 MHz, CDCl3) δH 9.11 (dd, J = 4.4, 1.7 Hz, 1H), 8.43 (d, J = 2.2 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 8.20 – 8.12 (m, 2H), 7.58 (dd, J = 8.4, 4.3 Hz, 1H), 7.41 (d, J = 2.5 Hz, 1H), 6.41 – 6.36 (m, 1H), 2.94 (d, J = 4.8 Hz, 3H); 19F NMR (376 MHz, CDCl3) δF 84.6 (p, J = 150 Hz, 1F), 64.1 (d, J = 150 Hz, 4F); LCMS tR = 1.19 min, m/z = 563.2 [M+H]+; Purity (AUC) ≥95%.</p>

PubMed Author Manuscript

Temperature-dependent activity of kinesins is regulable

Cytoskeletal transport in cells is driven by enzymes whose activity shows sensitive, typically Arrhenius, dependence on temperature. Often, the duration and outcome of cargo transport is determined by the relative success of kinesin vs. dynein motors, which can simultaneously bind to individual cargos and move in opposite direction on microtubules. The question of how kinesin and dynein activity remain coupled over the large temperature ranges experienced by some cells is one of clear biological relevance. We report a break in the Arrhenius behavior of both kinesin-1 and kinesin-3 enzymatic activity at 4.7\xc2\xb0C and 10.5\xc2\xb0C, respectively. Further, we report that this transition temperature significantly changes as a function of chemical background: addition of 200 mM TMAO increases transition temperatures by ~6\xc2\xb0C in all cases. Our results show that Arrhenius trend breaks are common to all cytoskeletal motors and open a broad question of how such activity transitions are regulated in vivo.

temperature-dependent_activity_of_kinesins_is_regulable

INTRODUCTION<!>Results<!>Discussion<!>Motility Assays:<!>Motor purification:

<p>Mechanochemical enzymes of the kinesin and dynein families enable active transport of cargos along microtubules (MTs) which is essential for eukaryotic cell function (1). Cargo motility is often driven by motor ensembles, which may result in saltatory, biased bidirectional or unidirectional motility on a single microtubule, or a variety of cargo navigation phenotypes at MT intersections. The balance of ensemble motor activity, which is critical to the cargo navigation outcome, received significant experimental and theoretical attention but almost always assuming fixed environmental conditions (2). However, temperature changes have clear biological relevance even for mammalian thermoregulated organisms, e.g. extreme variations during hibernation (3). Crucially, for a single cargo, driven by a motor ensemble, a temperature-driven change in an underlying parameter such as motor velocity can result in a qualitative change in the character of motility (4). Therefore, quantitatively understanding collective motor activity across temperatures is an issue of rising importance in cytoskeletal biophysics.</p><p>The simplest temperature-dependence of enzymatic rate is an Arrhenius trend, corresponding to a chemical reaction proceeding forward by crossing an energy barrier along a one-dimensional reaction pathway. The Arrhenius trend has a characteristic value (activation energy) which corresponds to the height of this energy barrier, and is also a readout of the rate-limiting step of the enzymatic cycle. However, piecewise Arrhenius trend is extremely common (5, 6), and is reflective of a temperature-dependent change in the enzymatic cycle. Arrhenius breaks (transitions between distinct Arrhenius trends) have been previously observed for two of the three major families of molecular motors: in conventional myosin at 5°C, and in various dyneins at 15°C and 17°C (6, 7). Previous studies of kinesin-1 were conducted above 5°C and have not observed a break (4, 8). However, prior report of Arrhenius break for myosin encouraged our search for the same in kinesins due to high homology of motor architecture (9). Here, by extending experiments into a lower temperature range, we show that kinesins do exhibit Arrhenius breaks and we further show that the temperature at which the break occurs is dependent on environmental/chemical conditions.</p><!><p>Prior reports show no Arrhenius break for kinesin-1 above ~5°C (4, 10, 11). Here we extended motility measurements down to 0°C. Motility was steady and distributions of motor velocities were approximately Gaussian in all measured conditions (Fig. 1). The data across all temperatures (Fig. 2A, black) was best fit to a piecewise-Arrhenius trend, with a break at ~4.7°C. The activation energies were significantly different above and below the break.</p><p>We next aimed to perturb the enzymatic cycle of kinesin by using saturating amounts of GTP to drive motor activity. This reduced average velocity at room temperature by approximately threefold relative to ATP background, consistent with a previous report (12), but still allowed for accurate velocity measurements at low temperatures. We again observed an Arrhenius break at ~3.7°C (Fig 2A, red) – not significantly different from ATP results. In the GTP assays, the activation energies were significantly different above and below the break, and greater than in the ATP background, consistent with ATP being the preferred nucleotide for driving mechanochemical activity (12).</p><p>We then investigated how chemical factors may impact the Arrhenius breaks. We focused on tri-methyl amine oxide (TMAO), which is a crowding mimic at near-molar concentrations (13) and stabilizes kinesin activity at elevated temperatures (14). Under saturating ATP conditions in 200 mM TMAO background the Arrhenius break was found at ~11°C (Fig. 2B). Activation energies were significantly different above and below the break.</p><p>Finally, we sought to determine whether this break was specific to kinesin-1 or a more general feature of kinesin motors. We repeated our experiments with dimeric hKIF1A, a kinesin-3 family motor known to be involved in fast axonal transport (15). Previous studies of fungal kinesin-3 did not observe an Arrhenius break down to ~5°C (10), but the temperature dependence of human KIF1A has not been previously studied. We did identify an Arrhenius break at ~10.5°C for hKIF1A with significantly different activation energies above and below the break (Fig. 3, red). In 200 mM TMAO background the Arrhenius break temperature was found at ~16°C, with significantly different activation energies above and below the break (Fig 3, black).</p><!><p>Here, we report an Arrhenius break in both the kinesin-1 and kinesin-3 families. It is tempting to speculate that this feature is a general property of kinesins, and likely of all cytoskeletal motors (4, 6). Prior work on kinesin's randomness parameter has established that there are at least two steps in the enzymatic cycle of kinesin-1 which are either rate-limiting or close (16). If the durations of these steps have differing temperature dependence, then the nature of the rate-limiting step could change abruptly as a function of temperature as is the case for e.g. myosin (6). Further work is necessary to provide definitive insight into the atomic-scale mechanism of the observed behavior and likely shed further light on the nature of rate-limiting steps in kinesin's enzymatic cycle.</p><p>The observed significant shift in the transition temperature in the presence of 200 mM TMAO shows that chemical/environmental regulation of Arrhenius breaks is possible for cytoskeletal motors. Notably, shifting the Arrhenius break in Kinesin-1 from 4.7°C to 11°C brings its temperature-dependent trend much closer to that of mammalian cytoplasmic dynein (7) hinting that kinesin and dynein motors in cells may be better matched across a wider temperature range than previously appreciated. Our TMAO work also raises the question of how other families of motors (particularly cytoplasmic dyneins) would perform under the same environmental conditions. Overall, our findings open a new set of hypotheses bearing on how transport regulation depends on temperature.</p><!><p>Assays were performed similar to (11). Upon MT attachment, surfaces were passivated with 1% Ficoll solution. Motors were adsorbed to ∅1 μm beads via streptactin conjugation (30 min streptactin then 15 min. kinesin incubations).</p><!><p>KIF1A motors were purified as previously described (17). Amino acid residues 1-556 of human KIF5A (top) were cloned into the pET28a vector followed by an mScarlet-strepII tag using isothermal assembly. The construct was verified by sequencing. To express the protein, BL21 (DE3) RIPL cells were transformed and grown in LB medium to an OD of ~0.4. Cells were induced with 0.1mM IPTG overnight at 18°C. Cells were harvested and resuspended in lysis buffer: 50mM Tris pH 8.0, 150mM KAce, 2mM MgSO4, 1mM EGTA, 10% glycerol along with 1mM PMSF and 1mM DTT. Cells were disrupted via high-pressure homogenization using an Emulsiflex C3 (Avestin). The lysate was centrifuged at 28,000 x g for 20 min and the supernatant was pumped over a column of Streptactin XT resin (IBA) for ~1 hour at 4°C. The column was then washed with excess lysis buffer to remove unbound material and the motors were eluted in lysis buffer containing 50 mM biotin. Eluted protein was further purified via anion exchange chromatography using a TSKgel SuperQ-5PW (Tosoh bioscience) 7.5 mm ID × 7.5 cm. column equilibrated in HB buffer (35 mM PIPES-KOH pH 7.2, 1 mM MgSO4, 0.2 mM EGTA, 0.1 mM EDTA). Bound proteins were eluted with a 45mL linear gradient of X mLs from 0-1M KCL in HB buffer. Fractions containing the motor were combined and concentrated on amicon spin filters with a 50 KDa cutoff after addition of 0.1 mM ATP and 10% glycerol. Concentrated motors were frozen in LiN2 and stored at −80°C.</p>

PubMed Author Manuscript

Novel action and mechanism of auranofin in inhibition of vascular endothelial growth factor receptor-3-dependent lymphangiogenesis

Auranofin is a gold compound initially developed for the treatment of rheumatoid arthritis. Recent data suggest that auranofin has promise in the treatment of other inflammatory and proliferative diseases. However, the mechanisms of action of auranofin have not been well defined. In the present study, we identify vascular endothelial growth factor receptor-3 (VEGFR3), an endothelial cell (EC) surface receptor essential for angiogiogenesis and lymphangiogenesis, as a novel target of auranofin. In both primary EC and EC cell lines, auranofin induces downregulation of VEGFR3 in a dose-dependent manner. Auranofin at high doses (\xe2\x89\xa51 \xce\xbcM) decreases cellular survival protein thioredoxin reductase (TrxR2), TrxR2-dependent Trx2 and transcription factor NF-\xce\xbaB whereas increases stress signaling p38MAPK, leading to EC apoptosis. However, auranofin at low doses (\xe2\x89\xa40.5 \xce\xbcM) specifically induces downregulation of VEGFR3 and VEGFR3-mediated EC proliferation and migration, two critical steps required for in vivo lymphangiogenesis. Mechanistically, we show that auranofin-induced VEGFR3 downregulation is blocked by antioxidant N-acetyl-L-cysteine (NAC) and lysosome inhibitor chloroquine, but was promoted by proteasomal inhibitor MG132. These results suggest that auranofin induces VEGFR3 degradation through a lysosome-dependent pathway. Auranofin may be a potent therapeutic agent for the treatment of lymphangiogenesis-dependent diseases such as lymphedema and cancer metastasis.

novel_action_and_mechanism_of_auranofin_in_inhibition_of_vascular_endothelial_growth_factor_receptor

INTRODUCTION<!>Reagents<!>Cell culture<!>Cell growth inhibition assay<!>Assay for Apoptosis<!>Cell migration assay<!>Gene expression<!>BrdU incorporation assay in vitro<!>Western Blot Analysis<!>Immunofluorescence microscopy (IF)<!>Statistical Analysis<!>Auranofin inhibits cell growth and induces cell apoptosis in a dose-dependent manner<!>Auranofin at a high dose inhibits EC survival and induces EC apoptosis<!>Auranofin specifically reduces VEGFR3 at a low dose<!>Auranofin induces VEGFR3 degradation via a lysosome-dependent pathway<!>Auranofin at a low dose inhibits VEGFR3-dependent EC proliferation and migration<!>DISCUSSION

<p>The gold compound Auranofin is a metal phosphine complex that was developed for a treatment of rheumatoid arthritis as gold-based drugs 1. Recent data suggest that auranofin has promise in the treatment of other inflammatory and proliferative diseases 2. However, the mechanisms of action of auranofin have not been well defined.</p><p>Lymphatics play a substantial role in conditions such as chronic airway inflammation, tumor growth and metastasis, rheumatoid arthritis, inflammatory bowel disease, renal and corneal transplant rejection, and atopic dermatitis and psoriasis 3. Lymphatic vessels express distinct cellular markers such as Prox-1, podoplanin 4, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) 5 as well as surface receptors VEGFR2 and VEGFR3. VEGFR3 is expressed in the blood endothelium during development, but is restricted to the lymphatics in the adult with the exception of few blood capillaries in some organs 6 as well as the microvasculature of tumors and wounds 7. Therefore, VEGFR3 is critical for lymphangiogenesis and lymphangiogenesis-associated diseases 8.</p><p>To determine whether auranofin regulates lymphangiogenesis, we investigated the effects of auranofin on endothelial cell (EC) survival, proliferation and migration in the mouse lymphatic endothelial cell line (SVEC) and in primary human lymphatic EC (HLEC). Our data demonstrate that auranofin at high doses induces EC apoptosis while at low dose specifically inhibits VEGFR3 expression and VEGFR3-dependent EC proliferation and migration. To the best of our knowledge, this is the first report to demonstrate that auranofin inhibits VEGFR3 and lymphangiogenesis.</p><!><p>Auranofin, SB203580, NAC, and chloroquine were purchased from Sigma-Aldrich (St. Louis, MO). MG132 was from Millipore (Billerica, MA). VEGFR3 antibody for Western blotting was from eBioscience (San Diego, CA, USA) and for immunostaining was from R&D Systems (Minneapolis, MN). TrxR2 and Trx2 antibodies were from ABCAM (Cambridge, MA). Other primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-linked antibodies were from GE Healthcare Bio-Sciences Corp (Piscataway, NJ, USA). Alexa Fluor 488- or 594-conjugated secondary antibodies were from Life Technologies (Grand Island, NY, USA).</p><!><p>Human dermal lymphatic endothelial cells (HLEC) (HMVEC-dLyAd) were purchased from Lonza, and were cultured in EGM-1 MV media on cell culture dishes coated with fibronectin (10 μg/ml) as we described previously 9. For characterization, cells were stained with Prox-1 and podoplanin, nuclear and transmembrane markers for HLEC, respectively. Earlier passage cells were >95% positive for LEC markers. All experiments were performed in early passages (passages 3–6) and cells remained Prox-1 positive. SVEC cell line (ATCC® CRL-2181™) is a mouse endothelial cell line derived by SV40 (strain 4A) transformation of endothelial cells from axillary lymph node vessels. SVEC cells were cultured in Dulbecco's modified Eagle's medium with 4mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum, 100 units/ml Penicillin, and 100 μg/ml Streptomycin, at 37°C in a humidified incubator with 5% CO2 atmosphere.</p><!><p>[Berridge MV, Herst PM, and Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, 11: 127–152 (2005)]. The SVEC cells (1×104/well) were seeded into 96-well culture plates and cultured at 37°C in a 5% CO2/air environment for 12 h. After cells were treated with different dose of auranofin for 4–24 h, 10 μL solution of MTT (0.5mg/mL in PBS) was added to each well. After incubation 4h, the MTT agent was removed and the formazan particles were solubilized with 150 μl DMSO. The absorbance was read at 570 nm with a microplate reader X-wave BioTek Synergy (BioTek Instruments). Cell viability was expressed as a percentage of control.</p><!><p>SVEC (10×104/well) were seeded 12-well culture plates and cultured at 37°C in a humidified incubator with 5% CO2 for 12 h. After cells were treated with 1 μM and 2 μM auranofin for 4h, Incubate the cells in 0.5mL Annexin-binding buffer 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4 with 5uL Annexin V conjugated Alexa-Fluor® 488 Life Technologies, A13201 and 1 uL Hoechst3342 ( Life Technologies) at room temperature for 15 min. Apoptosis of cells were observed using a Zeiss Axiovert 200 fluorescence microscope (Carl ZeissMicroImaging; Thornwood, NY), and images were captured using Openlab3 software (Improvision, Lexington, MA).</p><!><p>For monolayer migration, SVEC were seeded in 12-well tissue culture plates and grown to confluence. Cells were serum starved for 12 h and were then subjected to "wound injury" assay with a 200 μl plastic pipette tip. Fresh media containing 3% serum alone or serum with VEGF-C (50 ng/m) in the absence or presence of 0.25 μM auranofin. Cell were cultured for 12 h. The SVEC migration in culture was determined by measuring wound areas in cell monolayers. Wound images were captured by a digital camera under a Zeiss Axiovert microscope (10X). Wound healing (% wound closure) was measured and analyzed by NIH Image 1.60 9.</p><!><p>Total RNAs were isolated from cells by using the RNeasy kit with DNase I digestion (Qiagen, Valencia, CA). Reverse transcription (RT) was done by standard procedure (iScript™ cDNA Synthesis Kit) using 1μg total RNA. Quantitative real-time polymerase chain reaction (PCR) was performed by using iQ™ SYBR® Green Supermix on iCycler real time detection system iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Inc, Hercules, CA). RT-PCR with specific primers were described previously 9.</p><!><p>BrdU incorporation assay was performed as we described previously 10. Briefly, SVEC were grown on 0.1% gelatin-coated glass chamber slides for 12 h. Cells were treated with 0.25 μM auranofin for 12h, and then incubated with 10 μM BrdU for 4 h prior to fixation with 4% PFA for 30 min at room temperature. Once fixed, cells were acid denatured with 2M HCl in 0.1% PBS-Tween for 30 min and then washed three times in PBS. Cells were then permeabilized and blocked nonspecific epitopes by 1%BSA/10% normal horse serum in 0.1% PBS-Tween for 1 h following by primary anti-BrdU antibody and secondary antibody incubation.</p><!><p>All Immunoblotting were performed as previously described10. In brief, SVEC were treated by auranofin, cells were washed two times by cold PBS and lysed in cold 1×SDS buffer. Then, protein samples were boiled for 10 min, resolved in 8~10% polyacrylamide gels and transferred to polyvinylidene difluoride membrane and blocked with 5% milk diluted in PBS containing 0.05 % Tween20 (PBST). Membranes were then immunoblotted with the specified antibodies (1:1000 dilution in PBST) using horseradish peroxide-conjugated secondary antibodies (1:2,000 dilution; GE Healthcare Life Sciences/Amersham Biosciences) and enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Amersham Biosciences).</p><!><p>SVEC were grown on fibronectin-coated glass chamber slides for 12h. Cells were then treated by auranofin as indicated. Cells were fixed with 4% PFA in PBS for 15 min at room temperature, permeabilized with 0.1% triton-X buffer, blocked in 1% horse serum diluted in PBS for 1 h and stained 2 h at room temperature or 4 °C overnight using specified antibodies, followed by Alexa Fluor 488- or 594-conjugated secondary antibodies (donkey anti-goat, donkey anti-rabbit or donkey anti-mouse or a combination for double IF diluted at 1:1000 in PBST). Slides were observed using a Zeiss Axiovert 200 fluorescence microscope (Carl ZeissMicroImaging; Thornwood, NY), and images were captured using Openlab3 software (Improvision, Lexington, MA).</p><!><p>Experiments were carried out at least in triplicate and results were expressed as mean± SEM. Statistical analysis was performed using SPSS statistical package (SPSS 16.0 for Windows; SPSS, Inc. Chicago, IL). Difference between two groups was analyzed by two-tailed Student's t test, and that between two or more groups was analyzed by one-way ANOVA multiple comparisons followed by Bonferroni's post-hoc test. Difference with P<0.05 (*) or P<0.01 (**) was considered statistically significant.</p><!><p>Studies in tumor cells have suggested that auranofin, a gold complex (Fig. 1A for structure), induces mitochondrial dysfunction and cell death 1d, 11. However, the responses of endothelial cells (EC) to auranofin have not been investigated. To this end, we examined the effects of auranofin on cell viability in primary EC and several EC lines by MTT assay. Auranofin treatment of SVEC (an SV40 T antigen-immortalized lymphatic EC line expressing both VEGFR2 and VEGFR3) resulted in decrease of EC viability in dose- and time-dependent manner. Treatment of auranofin for 12 h caused significant reduction of cell survival at ≥ 1 μM but not at lower doses (≤0.5) (Fig. 1B). Annexin-V and Hoechst-3342 double staining revealed that auranofin at ≥ 1 μM induced EC apoptosis (Fig. 1C with quantification in D). These data suggest that auranofin induces mitochondrial dysfunction and EC apoptosis at high concentrations.</p><!><p>Auranofin has been shown as an inhibitor of thioredoxin reductase (TrxR; both cytosolic TrxR1 and mitochondrial TrxR2) and induces apoptosis in several cancer cell lines where auranofin at high doses activates ROS generation, p38 mitogen-activated protein kinase (p38MAPK) with reduction of the survival transcription factor NF-κB 1d, 11–12. To elucidate the intracellular mechanisms of EC mitochondrial dysfunction and apoptosis induced by auranofin, we measured expression of TrxR2, Trx2, generation of ROS, activation of p38MAPK, and reduction NF-κB activity. Correlating with the effects of auranofin on EC survival, auranofin at high doses (≥1 μM) induced significant reductions in expression of TrxR2 and Trx2 (Fig. 2A), the critical anti-oxidant system in the mitochondria 13. Therefore, auranofin induced mitochondrial ROS generation in a concentration-dependent manner as measured by mitochondrial ROS probe Dihydrorhodamine 123, which can be blocked by anti-oxidant N-acetyl-cysteine (NAC) (Fig. 2B). Auranofin at high doses also induced phosphorylation of p38MAPK with a reduction in phosphorylations of NF-κB as previously reported in other cell types. Interestingly, we observed significant reduction in expression of the EC-specific survival receptor VEGFR3 at various concentrations (Fig. 2A). As we reported recently 9, protein analysis of VEGFR-3 by Western blot revealed 3 bands (Fig. 1E). While the band at 175 kDa is considered to be the intracellular and unglycosylated precursor, the band with molecular weight 125 kDa represents the mature form of VEGFR-3 14. SVEC is immortalized EC cell line where another important survival receptor VEGFR2 is expressed at a very low level. We therefore examined effects of auranofin in primary human lymphatic EC (HLEC) which express both VEGFR3 and VEGFR2 8a. Auranofin induced activation of p38MAPK (Fig. 2C) and ROS generation (Fig. 2D) in HLEC as observed in SVEC. In these primary EC auranofin also induced a significant downregulation of VEGFR3 even at a low dose (≤0.5 μM), but only weakly reduced the expression of VEGFR2 (Fig. 2C).</p><!><p>We then investigate the mechanism by which auranofin at a low dose (≤0.5 μM) specifically downregulates VEGFR3 in SVEC. Cells were treated with a low dose of auranofin (0.25 μM) for various times (0–24 h), and VEGFR3 and protein were determined. Results indicated that auranofin induced downregulation of VEGFR3 protein without significant alterations on VEGFR3 mRNA (Fig. 3A). LYVE-1, a specific marker for lymphatic EC, was not affected by auranofin at its mRNA or its protein level. Auranofin at a low dose had no effect on the activation of p38MAPK (Fig. 3B). The downregulation of VEGFR3 in SVEC was further confirmed by indirect immunofluorescence staining. Consistent with the immunoblotting results, auranofin significantly reduced VEGFR3 immunostaining with no effects on LYVE-1 (Fig. 3C).</p><!><p>Recently we have demonstrated that VEGFR3 stability is regulated in EC 10. Since VEGFR3 mRNA was not significantly altered by auranofin, we examined how VEGFR3 protein is regulated by auranofin. We first examined if auranofin induced ROS generation is required for VEGFR3 downregulation. SVEC were incubated with auranofin in the absence or presence of anti-oxidant NAC or p38MAPK inhibitor SB203580 compound. Consistent with that auranofin at 0.25 μM induces a low level of ROS but not activation of p38MAPK, auranofin-induced VEGFR3 downregulation was blocked by NAC but not by p38MAPK inhibitor (Fig. 4A). Of note, p38MAPK inhibitor (which blocks p38MAPK kinase activity towards its substrates) stabilized phosphor-p38MAPK.</p><p>To determine which degradation pathway is involved in VEGFR3 degradation, SVEC were treated with auranofin in the presence of proteasomal inhibitor MG132 or lysosomal inhibitor chloroquine (CHQ) 15. Results showed that CHQ, but not MG312, significantly blocked auranofin-induced VEGFR3 degradation in SVEC (Fig. 4B). Surprisingly, MG132 alone strongly induced degradation of VEGFR3. These data indicate that auranofin induces VEGFR3 downregulation through a lysosome-dependent degradation pathway.</p><!><p>We have recently shown that VEGFR3 is critical for VEGF-C-induced EC proliferation and migration, two critical steps for lymphangiogenesis in vivo 9–10. We finally examined the effects of auranofin on VEGF-C-induced SVEC proliferation and migration. The effect of auranofin on SVEC proliferation was detected by BrdU incorporation assay in vitro. Under normal culture media with growth factors containing VEGF-C, treatment of auranofin at 0.25 μM for 12h significantly reduced BrdU+ SVEC cells (Fig. 5A with quantification in 5B), suggesting that a low dose of auranofin reduced SVEC proliferation.</p><p>To determined effects of auranofin on EC migration, SVEC migration was stimulated with VEGF-C in the presence or absence of 0.25 μM of auranofin, and cell migration was assessed by a scratch wound assay. Auranofin significantly reduced both basal and VEGF-C-induced wound closure with more profound effects on VEGF-C-induced EC migration (Fig. 5C with quantifications in 5D by comparing ARF effects on basal and VEGF-C-induced migration). In other words, VEGF-C had stronger responses in promoting LEC migration in the absence of auranofin (Fig. 5E by comparing VEGF-C responses in the absence and presence of ARF). In contrast, High doses (≥ 1 μM) of auranofin induced LEC apoptosis and completely blocked both basal and VEGF-C-induced LEC migration (not shown). These data indicate that auranofin at a low dose specifically inhibits VEGFR3-dependent lymphangiogenesis.</p><!><p>In the present study, the molecular mechanisms underlying the auranofin-induced inhibition of lymphangiogenesis in vitro were investigated. The results of this study are summarized in a schematic representation (Fig. 6). Auranofin at a high dose causes cell apoptosis by downregulating survival factors TrxR2, Trx2 and NF-κB, while activating p38MAPK, leading to EC apoptosis (see Fig. 1) and complete blockade of EC migration. Auranofin at a low dose inhibits VEGFR3 expression, VEGFR3-dependent EC proliferation and migration. Therefore, we have identified VEGFR3 as a novel target of auranofin. Moreover, we show that auranofin-induced VEGFR3 downregulation is blocked by antioxidant NAC and lysosome inhibitor chloroquine, but promoted by proteasomal inhibitor MG132. Our data reveal a novel mechanism by which auranofin specifically regulates VEGFR3 degradation through a lysosome-dependent pathway in lymphatic EC.</p><p>Auranofin has been widely used as a Trx reductase inhibitor. Therefore, despite the decline in its clinical applications in rheumatoid arthritis, auranofin shows promise in the treatment of several different diseases, including leukemia, carcinoma, and parasite, bacterial and viral infections. This is because the Trx reductase system and its thiol redox activity are important for cell survival and proliferation 13a, 16. TrxR2 converts Trx2 from an oxidized inactive form of Trx2 to a reduced active. Our results show that auronafin treatment causes reduction of Trx2 protein, suggesting that TrxR2 may stabilize Trx2 in EC and oxidized Trx2 is more accessible for degradation. Auronafin induces ROS generation and ROS-activated p38MAPK in EC, likely resulting from the reduction of the TrxR2-Trx2 proteins 13. In addition, Trx reductase is also known to maintain NF-κB p65 protein stability 1d, 11–12, 16–17. Consistent with this notion, we have observed that auranofin, by inhibiting Trx reductase activity, induces downregulation of NF-κB p65.</p><p>Auranofin, as Trx reductase inhibitor, has also been used as a tool to define cellular thiol-dependent signaling pathway and their functions. For example, Auranofin was used by Dr. Dean Jones group to specifically inhibit Trx reductase without oxidation of the GSH/GSSG while buthionine sulfoximine was used to deplete GSH without detectable oxidation of Trx1. Based on this specificity of auranofin, they have shown that auranofin can modify proteins associated with cellular glycolysis, cytoskeleton remodeling, protein translation and cell adhesion 18. Consistent with their findings, we have observed that auranofin at a high dose (2 μM) strongly disrupts cytoskeleton and EC-junctions as visualized by phalloidin and anti-ZO-1, respectively (Supplemental Fig. S1).</p><p>The most novel finding in our work, by examining effects of auranofin at a low dose on lymphatic EC, is the identification of VEGFR3 as a specific target of auranofin. Auranofin at a low does not induce EC apoptosis, correlating with its lack of effect on protein expression of TrxR2, Trx2, NF-κB p65 or p38MAPK activation. However, auranofin at a low dose specifically reduces VEGFR3 protein in both immortalized lymphatic EC lines and primary lymphatic EC with no effect on lymphatic EC marker LYVE-1. Since auranofin at a low dose reduces VEGFR3 protein but not VEGFR3 mRNA levels, we have explored potential mechanisms by which auranofin induces VEGFR3 degradation. Our surprising finding is that a lysosomal inhibitor chloroquine, but not a proteasomal inhibitor MG132, blocks auranofin-induced VEGFR3 degradation; MG132 in fact induced VEGFR3 degradation. Our results clearly support that auranofin induces VEGFR3 downregulation through a lysosome-dependent degradation pathway. The exact mechanism by which auranofin induces a lysosome-dependent degradation is unclear. Recent studies have suggested that VEGFR3 surface and protein stability are regulated by endocytosis, mediated by AIP1, Dab2, the transmembrane protein Ephrin B2 and the cell polarity regulator Par-3 10, 19. It is plausible that auranofin induces endocytosis of VEGFR3 in lymphatic EC by modulating the AIP1-Dab2-Ephrin-Par3 complex. Meanwhile, auranofin may also prevent VEGFR-3 recycling or/and promote VEGFR-3 trafficking to lysosome. We are currently investigating this possibility. Consistent with that auranofin at a low dose induces a low level of ROS but not activation of p38MAPK, auranofin-induced VEGFR3 downregulation was blocked by NAC but not by p38MAPK inhibitor SB203580. The role of ROS in promoting VEGFR3 endocytosis needs to be further determined.</p><p>The generation of new lymphatic vessels through lymphangiogenesis and the remodelling of existing lymphatics are thought to be important steps in cancer metastasis 8b. The VEGF-C/VEGFR3 axis is considered to be a major driver of tumour lymphangiogenesis 6–7 Our study demonstrate that auranofin specifically inhibits the VEGF-C/VEGFR3-dependent lymphangiogensis. Our study suggests auranofin as therapeutic agent for the treatment of lymphangiogenesis-mediated disease such as such as lympheda and cancer metastasis 8.</p>

PubMed Author Manuscript

Role of Branching of Hydrophilic Domain on Physicochemical Properties of Amphiphilic Macromolecules

A novel series of amphiphilic macromolecules (AMs) composed of a sugar backbone, aliphatic chains, and branched, hydrophilic poly(oligoethylene glycol) methyl ether methacrylate (POEGMA)were developed for drug delivery applications. The branched, hydrophilic domains (POEGMA homopolymers with one hydroxyl group) were prepared via atom transfer radical polymerization (ATRP) of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) monomers using 2-hydroxyethyl-2-bromoisobutyrate (HEBiB) as an initiator and copper bromide/bipyridine (CuBr/Bpy) as the catalyst system. To form the amphiphilic structures, the branched POEGMAs were coupled to hydrophobic domains that were formed via acylation of a sugar backbone. The impact of branching in the hydrophilic domain was investigated by comparing the AMs\xe2\x80\x99 solution and thermal properties with those of the linear counterparts. Although these highly branched AMs showed similar critical micelle concentration (CMC) values as compared to linear analogues, they possessed quite low glass transition (Tg) temperatures. Consequently, these novel AMs with branched hydrophilic domain combine the desirable thermal properties of POEGMA with favorable solution properties of amphiphilic architectures, which make them suitable for injectable drug delivery systems.

role_of_branching_of_hydrophilic_domain_on_physicochemical_properties_of_amphiphilic_macromolecules

Introduction<!>Results and Discussion<!>Synthesis of POEGMA Homopolymers<!>Synthesis of Amphiphilic Macromolecules<!>Thermal Properties<!>Hydrodynamic Size<!>Critical Micelle Concentration (CMC) values<!>Materials<!>Measurements<!>Synthesis of POEGMA<!>Synthesis of branched amphiphilic macromolecules<!>Conclusions

<p>Amphiphilic molecules have been used as nanoscale drug delivery vehicles for hydrophobic drugs.1-16 Above a critical micelle concentration (CMC), these molecules aggregate spontaneously in aqueous solution to form micelles, with hydrophobic cores and hydrophilic shells. This aggregation is thermodynamically driven and the micelle stability is reflected in the CMC value.9 Within micelles, hydrophobic drug molecules can be solubilized in the hydrophobic cores and are protected by the hydrophilic barrier. We previously reported the successful development of amphiphilic macromolecules (AMs) comprised of sugar backbones, aliphatic chains, and poly(ethylene glycol) (PEG) for drug delivery applications.17-22 This design allows for the precise tuning of AM chemical structures and has generated a range of physicochemical properties, including different colloidal stability, aggregation sizes, and thermal properties. Our studies have shown that altering the hydrophobic region by increasing the length and number of aliphatic chains attached to the sugar backbone, greatly improves hydrophobic interactions, micelle formation, and hence micelle stability.18 Additionally, we reported that replacing the single linear PEG chain with two shorter PEG chains (pseudo-branched analogues)22 yielded AMs that formed micelles of smaller size and higher water solubility, thus higher micelle stability, as compared to analogues containing a single, longer PEG chain.22 This enhancement of micelle stability is highly favorable for effective drug delivery systems to achieve sustained release and targeted site accumulation.23</p><p>Thermo-sensitive hydrogels have also attracted attention as drug delivery systems. 24, 25 These liquid hydrogels can solidify upon exposure to physiological temperatures into a drug depot which provides controlled release of encapsulated drugs. Poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA) is a thermo-sensitive polymer; its low critical solution temperature (LCST) can be controlled by the oligo(ethylene glycol) (OEG) chain length.26, 27 POEGMA consists of biocompatible components: methacrylate and pendant OEG. This composition imparts water-solubility and hydrophilicity comparable to PEG, as it provides numerous sites for hydrogen bonding, and has increased chain flexibility. We envisioned that the introduction of a thermo-responsive polymer as the hydrophilic domain within the previously established AMs can maintain the AMs' micelle stability, provide beneficial thermo-sensitivity, and make them suitable for formulation into hydrogels. Therefore we investigated the effect of structurally modifying the AMs' hydrophilic domain on AMs' solution and thermal properties.</p><p>Herein, we report the synthesis and physicochemical characterization of AMs with branched, hydrophilic domains shown schematically in Figure 1. Synthesis of the branched, PEG-containing chain was accomplished by atom transfer radical polymerization (ATRP). This method has emerged as one of the most powerful and widely used synthetic techniques in polymer science,28-30 as it enables polymer synthesis with predetermined molecular weights and narrow molecular weight distributions.28-30 In this study, linear palm-tree-like POEGMA homopolymers were successfully obtained (Figure 1) via ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) monomers using 2-hydroxyethyl-2-bromoisobutyrate (HEBiB) as an initiator and copper bromide/bipyridine (CuBr/Bpy) as the catalyst system (Figure 2).</p><p>Previously published hydrophobic domains, consisting of a sugar (mucic acid or tartaric acid) acylated with lauroyl chloride to introduce four or two twelve carbon chains, respectively, were used.17 These hydrophobic domains were then coupled to hydrophilic POEGMA of various molecular weights to generate the branched AMs. The adapted nomenclature system for these molecules consisted of either M12P5 or T12P5 for the PEG-containing macromolecules: where M stands for mucic acid, T stands for tartaric acid, 12 refers to the number of carbon atoms of each aliphatic chain, and P5 indicates 5 KDa PEG. With POEGMA, however, the system consists of M12(Pn) or T12(Pn) where Pn refers to POEGMA with the specified number (n) of OEGMA repeats. The chemical structures of these novel polymers were confirmed by proton nuclear magnetic resonance (1H NMR) spectroscopy, infrared (IR) spectroscopy, and gel permeation chromatography (GPC). Additionally, aggregation size was quantitatively assessed via dynamic light scattering and micelle stability was determined by measuring CMCs using fluorescence spectroscopy. Lastly, thermal properties were determined using differential scanning calorimetry (DSC). These branched AMs were compared with linear AM analogues as shown in Figure 1, with the expectation that the branched, flexible hydrophilic domain would provide new AMs with similar solution stability but different thermal properties.</p><!><p>Amphiphiles can self-assemble to form micelles which show potential as colloidal drug delivery systems, especially for hydrophobic drug entities.1-16 Upon micellization, these molecules can solubilize hydrophobic drug molecules within their core11-16 and are capable of enhancing the bioavailability, increasing the blood circulation time, and minimizing the toxicity of the solubilized bioactives.1-16 A limitation for micelles' in vivo applications, however, has been their solution stability, namely the ability to withstand drastic dilution.31 To overcome this limitation, we previously developed a series of AMs containing branched hydrophobic domains and linear PEG, a biocompatible and non-immunogenic hydrophilic domain. Our AMs possessed remarkably low CMC values (10−6 to 10−7 M) as compared to established surfactants (e.g., sodium dodecyl sulfate CMC = 8.6 × 10−3 M).32 This low CMC was attributed to the strong hydrophobic interactions between the hydrophobic domains, which facilitate micelle formation. The hydrophilic PEG domain then provides a hydrophilic barrier which prevents unwanted protein adsorption and cell adhesion.11 Therefore, we hypothesized that the introduction of branching in the hydrophilic domain within the previously established AMs would enhance the AMs' in vivo stability. To investigate the relationship between the branched, hydrophilic domains and solution stability, we prepared several AMs with varying degrees of branching in the hydrophilic domain. Specifically, we investigated the importance of branching by synthesizing AM analogues with equivalent molecular weights yet with varying degrees of branching in the hydrophilic domain. POEGMA, a hydrophilic polymer consisting of repeat units of OEGMA was chosen as a branched equivalent of PEG. Inclusion of the thermo-sensitive POEGMA was also expected to decrease the melting point and provide AMs with potential for application as hydrogels.</p><!><p>POEGMA homopolymers with one hydroxyl end group were prepared via ATRP of OEGMA using HEBiB as an initiator with a CuBr/Bpy catalyst system as shown in Figure 2. The initial conditions and results of ATRP of OEGMA are listed in Table 1. These conditions allowed for the successful synthesis of polymers with predetermined molecular weights, high conversion (100%), and narrow polydispersities (PDI< 1.5).The new POEGMA polymers were characterized by both 1H NMR and GPC. The experimental degree of polymerization (Dp) of POEGMA was calculated according to 1H NMR by comparing signal at δ 3.3-3.4 ppm from OEGMA with signal at δ 4.0-4.2 ppm from both OEGMA and HEBiB, respectively. Good correlation was found between target Dp based on the [Monomer]0/[Initiaor]0 and experimental Dp, which indicated our good control over the polymerization under this reaction condition. Mn,GPC were determined from GPC using PMMA as standards while Mn,NMR were calculated from Dp determined by 1H NMR. These results are consistent with literature, considering that conventional GPC measurements are not necessarily suitable for branched polymers like POEGMA and NMR is a better measurement.33-35</p><p>We also successfully practiced the ATRP of P50 and P100 (data not shown). It was observed that increasing the molecular weight resulted in qualitative increase in viscosity; therefore, properties can thus be tuned through careful choice of the OEG repeat number, which gives rise to the desired molecular weight. P10 and P25 were used to determine the impact of a branched, hydrophilic domain on AM physical properties (including thermal and solution properties).</p><!><p>POEGMA was conjugated onto the previously synthesized hydrophobic domains (M12 or T12) to form the final AM structures. The coupling of M12 or T12 onto the mono-hydroxyl substituted POEGMA was accomplished with 1,3-dicyclohexylcarbodiimide (DCC) as a coupling agent and DPTS as a catalyst to yield the AMs (Figure 2). 1H NMR data confirmed successful coupling of POEGMA to the acylated sugars. Although the spectra were dominated by the POEGMA peaks, the peaks corresponding to the terminal methyl and methine groups of the aliphatic chains were prominent. In IR spectra, disappearance of the hydroxyl group signal was the further evidence for successful reaction.</p><!><p>Thermal characterization of polymers was performed using DSC methods to evaluate the thermal stability, which may correlate to their in vivo stability. POEGMA homopolymers were viscous liquids which exhibited low glass transition temperatures between −56 and −59 °C. The low glass transition temperatures of POEGMA homopolymers is attributed to a plasticizer effect exerted by the OEG side chains.26, 27 Similarly, the branched AMs were thick liquids exhibiting glass transition temperatures at slightly higher temperatures of −50 and −53 °C (Table 2). AMs with linear PEG chains; however, were solids which possessed a melting temperature of 56 and 58 °C and did not exhibit glass transition temperatures. This result implies that the glass transition temperature is mainly determined by the more flexible OEG side chains of POEGMA. Overall, the chemical structure of the hydrophilic OEG domain dictates thermal properties of the new molecules.</p><!><p>Formation of nanoscale aggregates in aqueous solutions is highly desirable for drug carriers. The aggregation sizes of the macromolecules were determined by using dynamic light scattering. All hydrodynamic sizes of the POEGMA homopolymers were in the low nanoscale range (5-18 nm) (Table 2). The size of PEG-containing AMs ranged from 7 to 20 nm, whereas the POEGMA-containing AMs were relatively larger (45-140 nm). This size difference can be attributed to efficiency of packing of these molecules into aggregates. By comparing the sizes of linear PEG-based AMs with branched POEGMA-based AMs which have same hydrophobic domain and molecular weight of hydrophilic component (e.g., M12P5 and M12(P25)), it's interesting to find the sizes of branch POEGMA-based AMs were about seven times of their linear counterparts. By comparing the sizes of mucic acid-based AMs with tartaric acid-based AMs, we note that the increase in branching in the hydrophobic domain increases the overall hydrodynamic size - regardless of the hydrophilic domain. This effect is more pronounced in the POEGMA-containing AMs. These results indicate that aggregation sizes are dependent on the interactions of both hydrophobic and hydrophilic domains within each macromolecule. Despite this difference most aggregation sizes were within the nanoscale range36 required for drug delivery purposes.</p><!><p>CMC values provide relevant information regarding solution stability of the micelles in aqueous media; lower CMC values indicate higher stability upon drastic dilutions, as is experienced under physiological conditions.37 Therefore, preventing such micelle dissociation increases the stability of micelle-encapsulated hydrophobic drugs.23 Assessment of the CMC values was performed by fluorescence spectroscopy using a pyrene probe.23, 38 Figure.3 shows the change of intensity ratio (I334.5nm/332nm) of pyrene as a function of M12(P10) concentration. As indicated in Table 2, all AMs had very low CMC values (ranging from 10−5 to 10−7) as compared to commercially available surfactants.39 These values indicate their ability to form stable micelles and withstand dilution that would occur upon injection, for example. Comparing AMs with mucic acid versus those with tartaric acid reveals that mucic acid derivatives display lower CMC values. However, similar CMC values were obtained for branched and linear polymers, indicating that the hydrophobic domain is the key factor influencing the overall CMC. These observations directly correlate with the calculated hydrophiliclipophilic balance (HLB) values, which reflect the degree of hydrophilicity.40 As indicated in Table 2, although higher HLB values were obtained for branched hydrophilic chains, owing to their more hydrophilic nature, their CMC values were similar to those of their linear counterparts. This confirms that CMC is dictated by the stronger hydrophobic interactions of the hydrophobic domain alkyl chains. The low micromolar values indicate the ability of these novel AMs to withstand dilution and show their potential for drug delivery applications.</p><!><p>Oligo(ethylene glycol) methyl ether methacrylate (OEGMA) with an average molecular weight of 300 g/mol was obtained from Sigma-Aldrich and purified by passing through a neutral alumina column prior to use. HEBiB, CuBr and Bpy, mucic acid, tartaric acid, lauroyl chloride, zinc chloride, dimethyl amino pyridine (DMAP), p-toluene sulphonic acid, and DCC were purchased from Aldrich and used as received. Dimethyl amino pyridine ptoluene sulphonate (DPTS), M12 (acylated mucic acid derivative with four twelve carbon aliphatic chains (Figure 2)), and T12 (acylated tartaric acid derivative with two twelve carbon aliphatic chains (Figure 2)) were prepared as previously published.17</p><!><p>1H NMR spectra were recorded on Varian VNMRS 400 MHz spectrometer. Samples were dissolved in CDCl3 with tetramethylsilane (TMS) as an internal reference. IR spectra were recorded on a Thermo Nicolet/Avatar 360 spectrophotometer by solvent-casting onto a NaCl plate. Each spectrum was an average of 32 scans. Molecular weights were determined by gel permeation chromatography using a PerkinElmer series 200 LC system equipped with a PL-Gel column. Tetrahydrofuran (THF) was used as eluent for analysis and solvent for sample preparation. The sample (10 mg/mL) was dissolved into THF and filtered using a 0.45 μm PTFE syringe filter (Whatman, Clifton, NJ) before injection into the column at a flow rate of 0.5 mL/min. The average molecular weight of the sample was calibrated against narrow molecular weight poly(methyl methacrylate) (PMMA) (Poly[Anayltik]n, Ontario, CA). Thermal properties (melting temperature, Tm and glass transition temperature, Tg) were determined by a TA instrument Q200. Data was analyzed by TA Instruments Universal Analysis 2000 software version 4.5 A. Samples (4-8 mg) were heated under dry nitrogen gas from −70 °C to 100 °C, and data were collected at heating and cooling rates of 10 °C/min with a three-cycle minimum. CMC measurements were carried out by fluorescence studies on a Spex fluoro Max spectrofluorometer at 25 °C. A stock solution of 5 × 10−7 M pyrene in water was prepared and used as the probe molecule. Samples were dissolved in water, diluted to specific concentrations, then added to the stock pyrene solution,. Excitation was performed from 300 to 360 nm, with 390 nm as the emission wavelength. Upon micelle formation, the pyrene maximum absorption shifted from 332 to 334.5 nm. The ratio of absorption of pyrene with polymer (334.5 nm) to pyrene only (332 nm) was plotted as the logarithm of polymer concentrations and the inflection point was taken as the CMC value. Hydrodynamic diameter and zeta potential analyses were performed using a NanoZS90 instrument (Malvern Instruments, UK). Samples (10 mg/mL were prepared using HPLC grade water and filtered through 0.45 µm PTFE syringe filters before measurement. Each sample was run at room temperature three separate times with 20 measurements per run.</p><!><p>ATRP of OEGMA was carried out using HEBiB as an initiator and CuBr/Bpy as the catalyst system in isopropanol at 30 °C. The general procedures of the polymerization were as follows: OEGMA, CuBr, BPy and solvent (isopropanol) were transferred into a dry round-bottomed flask equipped with a magnetic stirring bar, placed in an ice-bath. The flask was sealed and the solution degassed by purging with argon for 15 min. The initiator (HEBiB) was mixed separately with a small amount of solvent and the resulting solution degassed with argon for 15 min. To start the polymerization, the initiator solution was added to the monomer solution, and placed in an oil-bath at 30 °C for 24 h. Aliquots were withdrawn from the flask at timed intervals to monitor the polymerization progress. Conversion of the OEGMA monomer was determined by comparison of the intensity of the 1H NMR signals corresponding to vinylic protons (5.6 and 6.1 ppm) with aliphatic protons of CH3O (~3.4 ppm) or -OCOCH2CH2O- (~4.1 ppm). Using this method, 100% conversion was reached by 24 hr.</p><p>The catalyst was removed by passing the polymer solution through a silica column. Subsequently, the polymers were precipitated from THF into cold n-hexane, filtered and lyophilized. 1H NMR spectrum of POEGMA homopolymer reveals the characteristic signals of POEGMA at (δ): 4.1 (br s, 2H, CO2CH2CH2O-), 3.65 (br s, 14H,OCH2CH2-), 3.55 (s, 2H, CH2OCH3), 3.38 (s, 3H, OCH3), 1.74-2.1(m, 2H, -CH2C(CH3)-) and 0.8-1.02 (m, 3H, -CH2C(CH3)-). IR spectroscopy of POEGMA homopolymers with one hydroxyl end group confirmed the successful ATRP of OEGMA. IR (NaCl, cm−1): 3513 (OH), 2874 (C-H), 1728 (C=O) and 1109 (C-O-C). The molecular weight results are listed in Table 1.</p><!><p>The preparation of M12(P25) is presented as an example. (P25) refers to POEGMA homopolymers with 25 OEG repeat units, (P10) refers to POEGMA homopolymers with 10 OEG repeat units POEGMA (Mn = 7711) (1.1 g, 0.14 mmol) was dehydrated by azeotropic distillation from toluene (50 mL) under vacuum. M12 (0.40 g, 0.42 mmol) and DPTS (36 mg, 0.12 mmol) were dissolved in THF (3.0 mL) and methylene chloride (10 mL), and the solution then added at room temperature to POEGMA. After 10 min under nitrogen, 0.50 mL of DCC solution (1.0 M in methylene chloride) was added dropwise over 15 min. After 24 h, the DCC side product (dicyclohexylurea) was removed by vacuum filtration. The filtrate was washed with 0.1 N HCl, then twice with brine. The organic layer was dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was purified by precipitation into diethyl ether (100 mL). Product was obtained as colorless oil (360 mg, 30% yield).The ethylene oxide of POEGMA comprises a significant component of branched AMs and dominates the 1H NMR and IR spectra. Thermal and properties are listed in Table 2.</p><p>M12(P25): Product was obtained as colorless oil (360 mg, 30% yield). 1H NMR (CDCl3) (δ): 4.08 (br s, 52H, CH), 3.65 (br s, 350H, CH2), 3.55 (s, 52H, CH2), 3.38 (s, 75H, CH3), 1.89 (m, 50H, CH2), 1.25 (m, 64H, CH2), 1.02 (m, 81H, CH3), 0.88 (t, 12H, CH3). IR (NaCl, cm−1): 2921 (C-H), 1728 (C=O) and 1109 (C-O-C).</p><p>T12(P25): Product was obtained as brown oil (460 mg, 40% yield).1H NMR (CDCl3) (δ): 4.08 (br s, 52H, CH), 3.65 (br s, 350H, CH2), 3.57 (s, 52H, CH2), 3.38 (s, 75H, CH3), 1.89 (m, 50H, CH2), 1.25 (m, 32H, CH2), 1.02 (m, 81H, CH3), 0.88 (t, 12H, CH3). IR (NaCl, cm−1): 2874 (C-H), 1728 (C=O) and 1111 (C-O-C).</p><p>M12(P10): Product was obtained as colorless oil (170 mg, 30% yield). 1H NMR (CDCl3) (δ): 4.08 (br s, 22H, CH), 3.65 (br s, 140H, CH2), 3.56 (s, 22H, CH2), 3.38 (s, 30H, CH3), 1.88 (m, 20H, CH2), 1.26 (m, 64H, CH2), 1.02 (m, 36H, CH3), 0.88 (m, 12H, CH3). IR (NaCl, cm−1): 2874 (C-H), 1728 (C=O) and 1107 (C-O-C).</p><p>T12(P10): Product was obtained as brown oil (210 mg, 40% yield).1H NMR (CDCl3) (δ): 4.08 (br s, 22H, CH), 3.65 (br s, 140H, CH2), 3.55 (s, 22H, CH2), 3.38 (s, 30H, CH3), 1.89 (m, 20H, CH2), 1.25 (m, 32H, CH2), 1.02 (m, 36H, CH3), 0.88 (t, 12H, CH3). IR (NaCl, cm−1): 2873 (C-H), 1728 (C=O) and 1108 (C-O-C).</p><!><p>A series of new AMs were synthesized to study the impact of branched versus linear hydrophilic domains on solution and thermal properties. Solution properties of branched and linear AMs, including hydrodynamic size, and CMC, were similar. This result indicated that solution properties are dictated by the interactions of the hydrophobic domain. Adversely, a significant difference in thermal properties, on the other hand was observed. These studies show that the hydrophilic domain contributes significantly to thermal properties and affects the AMs' overall physical characteristics. Together the low CMC and glass transition temperature make these new AMs envisioned to be appropriate for drug delivery of hydrophobic drug entities in the form of hydrogels or injectables. Further studies are underway to determine their behavior under simulated physiological conditions.</p>

PubMed Author Manuscript

Homochiral nanotubes from heterochiral lipid mixtures: a shorter alkyl chain dominated chiral self-assembly

It is an important topic to achieve homochirality both at a molecular and supramolecular level. While it has long been regarded that "majority rule" guides the homochiral self-assembly from an enantiomer mixture, it still remains a big challenge to manipulate the global homochirality in a complex system containing chiral species that are not enantiomers. Here, we demonstrate a new example wherein homochiral nanotubes self-assembled from a mixture of heterochiral lipids that deviated from the "majority rule". We have found that when two heterochiral lipids with mirror headgroups but a 2-methylene discrepancy in alkyl chain length are mixed, homochiral nanotubes are always formed regardless of their mixing ratio. Remarkably, the helicity of the nanotube is exclusively controlled by the molecular chirality of the lipids with shorter alkyl chains, i.e., the chiral self-assembly was dominated by the lipid with the shorter alkyl chain. MD simulation reveals that the match of both the alkyl chain length and hydrogen-bonding between two kinds of lipids plays an important role in the assembly. This work provides a new insight into the supramolecular chirality of complex systems containing multi chiral species.

homochiral_nanotubes_from_heterochiral_lipid_mixtures:_a_shorter_alkyl_chain_dominated_chiral_self-a

Introduction<!>Results and discussion<!>Self-assembly of heterochiral lipid mixtures<!>Supramolecular chirality of the composite nanotubes from heterochiral lipid mixtures<!>Conclusions<!>Conflicts of interest

<p>Homochirality in living organisms, i.e. almost all of the amino acids and sugars are L-and D-enantiomers, respectively, is one of the most mysterious phenomena and has attracted long-term interest in biology, chemistry, physics and material science. [1][2][3][4][5][6][7][8][9][10][11][12] Such molecular homochirality in the biological system requires the design of drug molecules as a single enantiomer, 13 which is suggested to be related to the different interactions between proteins and enantiomers of drug molecules. 14,15 Thus, the homochirality issue [16][17][18][19][20][21] is extended to a supramolecular level such that the stereochemical communication or chiral-chiral interaction between various chiral species becomes vitally important. [22][23][24][25] So far, two important rules on stereochemical communication, the "majority rule" [26][27][28][29][30][31][32][33][34][35][36] and "sergeant-andsoldiers rule", 27,31,[37][38][39][40][41][42] have been well-established with respect to covalent and non-covalent bonding of chiral polymers or supramolecular assemblies. Generally, the "majority rule" is related to two chiral molecules with mirrored conguration and states that the global chirality of the system is always determined by the chirality of the excess enantiomeric species. The "sergeants-and-soldiers rule" deals with the interaction between chiral sergeants and achiral soldiers and states that the chirality of the whole system follows the chirality of the sergeant. However, there is still a big challenge to manipulate the interaction or communication between different chiral species in complex systems, 3,[43][44][45][46][47][48] such as chiral lipids with different chain lengths in a biological membrane, [49][50][51] where the chiral species are not necessarily in exact mirror congurations. [52][53][54] Here, we designed a series of enantiomeric glutamide lipids with various alkyl chain lengths and investigated their selfassembly behaviours (Fig. 1). Absolutely mirrored heterochiral lipid mixtures are found to follow the "majority rule", i.e. the majority enantiomers control the global chirality of the system and the racemate is oen achiral. However, when two heterochiral lipids with mirror headgroups but a 2-methylene discrepancy in alkyl chain length were mixed, a homochiral composite nanotube was always obtained. Remarkably, the helical sense was not determined by the majority component but by the lipids with the shorter alkyl chain no matter how small the amount of that lipid. This phenomenon deviates from the reported stereochemical communication rules and has never been reported before. It demonstrates that a small variation in molecular structure also plays an important role in stereochemical communication apart from intrinsic molecular chirality. By combining various experimental characterization methods and theoretical molecular dynamics (MD) simulation, the mechanism of this unprecedented phenomenon is disclosed.</p><!><p>Lipid molecule design and synthesis N,N 0 -bis(alkyl)-D/L-glutamic diamide lipids, with enantiomerically pure glutamic acid as the polar headgroup and double hydrophobic nonpolar alkyl tails, were designed to mimic natural amphiphilic chiral lipids with different chain lengths (Fig. 1). The lipid molecules were synthesized by two simple steps, as previously reported: 55 the tert-butoxycarbonyl (Boc)protected D/L-glutamic acid was rstly connected to two equimolar alkyl amines, then the Boc group was eliminated to free the polar amine headgroup.</p><!><p>The self-assembly of the lipids was all performed in ethanol medium through a heat-and-cooling gelation process. Briey, lipids or their mixtures were dispersed into ethanol at room temperature and then heated to a transparent solution. Aer the solution was cooled down to room temperature, the gel was formed. All the lipids as well as their mixtures could form white opaque gels and self-assembled into well-dened nanostructures upon gelation (see Experimental section for details).</p><p>Fig. 1 Self-assembly of chiral lipids. (a) Enantiomerically pure D-and L-lipids form M-and P-helices, respectively. (b) Mixing of racemates follows the "majority-rule". However, mixing of two heterochiral lipids with mirror chiral head groups but a 2-methylene discrepancy in alkyl chain length leads to the homochiral composite nanotube, whose helical sense is exclusively determined by the molecular chirality of the lipid with the shorter alkyl chain regardless of their mixing ratios. Characterization of the self-assembled nanostructures from heterochiral lipid mixtures Fig. 2 shows the representative morphologies of the nanostructures, and three important features can be found.</p><p>First, all the pure enantiomers formed chiral nanotubes with helicity following their molecular chirality, regardless of their chain length, i.e., D-and L-lipids produced le-and righthanded nanotubes, respectively (Fig. 2a and b).</p><p>Second, when two opposite enantiomeric lipids with absolute mirror-conguration (n ¼ m) such as 20D/20L, 18D/18L, and 16D/16L were mixed, they obeyed the "majority rule", i.e., the helicity was determined by the excess enantiomeric lipid. In particular, a planar nanosheet without any chirality was formed for an equimolar mixture (Fig. 2a and b).</p><p>Third, when two pseudo-enantiomeric heterochiral lipids, i.e., with opposite chiral head groups and a 2-methylene discrepancy in chain lengths, such as the combinations of 20L/18D, 20D/18L, 18D/16L, 18L/16D, 16L/14D, and 16D/14L, were mixed, helical nanotubes were exclusively formed at various mixing ratios, even for equimolar mixtures (Fig. 2c-e, S1 and S2 †). In this case (n-m ¼ 2 system), the "majority rule" is no longer operative.</p><p>In order to elucidate these new observations, various characterization methods, such as XRD, FTIR spectroscopy, CD spectroscopy and DSC thermal analysis, were carried out. Hereaer, the self-assembly of the 18D/18L and 18D/16L systems will be studied as an example.</p><p>FTIR spectra are powerful in discriminating molecular interactions. As shown in the FT-IR spectra (Fig. S3 †), all the nanostructures showed obvious H-bonded vibrations from N-H, amide I and amide II. However, their precise vibrations are different for the different lipid mixtures. The N-H, amide I and amide II bands at 3326, 1636, and 1531 cm À1 for the 18D (18L) nanotube shied to 3302, 1633, and 1544 cm À1 for the 18D/18L nanosheet, indicating that the 18D/18L nanosheet has stronger hydrogen bonding interactions than that of either the 18D or 18L nanotube 55 (Fig. 3a and S3, Table S1 †). This was further conrmed by DSC thermogram analysis of the 18D/18L nanostructures (Fig. 3b and S4 †), where the phase transition temperature (T m ) is ca. 121 C regardless of the mixing ratio, indicating the miscible nature of the 18D and 18L lipids. 56 This means that the nanoscale chirality is counterbalanced at a molecular level. 55 Consequently, the helical torsion force in the racemate bilayer is decreased, which is evidenced by the dspacing expansion of the racemate bilayer (4.85 nm, equimolar 18D/18L) compared to the enantiomerically pure bilayers (4.23 nm, 18D) (Fig. 3c). Therefore, achiral planar nanosheets are produced for 18D/18L at an equimolar ratio.</p><p>In contrast, the FTIR spectra showed scarcely any change of the hydrogen bonding interaction in all 18D/16L combinations compared to the 18D or 16L nanotubes (Fig. 3a and S3, Table S2 †). It seems that the helical torsion force in the heterochiral bilayer of 18D/16L is unaffected. Therefore, the nanotube rather than the planar sheet formed for all heterochiral 18D/16L combinations. However, only one T m peak was found in the DSC thermograms of the 18D/16L nanotubes (Fig. S4 †) and the plot of T m value to mixing ratio is a U-shaped curve (Fig. 2b), with a T m value of 115 C for equimolar 18D/16L lower than those of either 18D (121 C) or 16L (119 C), suggesting the mutual diluent effect and co-self-assembly 30 of 18D and 16L. 56 Moreover, the XRD patterns (Fig. 3c) showed a single bilayer (4.08 nm) just between that of 18D (4.23 nm) and 16L (3.92 nm), further suggesting the co-assembly of all 18D/16L combinations. It should be noted that if two respectively self-assembled nanotubes were mixed, we can observe two sets of peaks (Fig. 3c). Therefore, we can conclude that when 18D and 16L were mixed, they tended to co-assemble rather than self-sort.</p><!><p>Given that two opposite chiral lipids are involved in the n-m ¼ 2 system, the supramolecular and nanoscale chirality of the composite nanotubes is alluring. High-resolution SEM images (Fig. 4a-f) show that the composite nanotube is chiral at the nanoscale. Moreover, the chirality is exclusively one handed, which is always consistent with that of the nanotubes formed from the shorter lipids alone. Specically, the composite 18D/ 16L nanotubes are always right-handed (Fig. 4b-f) like the 16L nanotube and the 18L/16D nanotubes are le-handed (Fig. 4a and S5 †) like the 16D nanotube. Obviously, the helicity of the composite nanotubes from heterochiral lipids is basically determined by the molecular chirality of the shorter lipids.</p><p>The helicity of the nanotubes was further investigated by CD spectroscopy. Since these lipid molecules do not possess any chromophore, an achiral dye, meso-tetra(4-sulfonatophenyl) porphyrin (TPPS), was used as a probe [57][58][59] to reect the helicity of the nanotube through aggregation on the surface of the nanotubes (Fig. 4g, h and S6 †). UV/Vis spectra displayed two strong bands at 493 and 708 nm, indicating induced Jaggregation of TPPS at the surface of all nanotubes. 57 The CD spectra of the D-lipids displayed two strong Cotton effects at 495(À) and 486(+) with a crossover at 490 nm, and 430(À) and 415 (+) with a crossover at 422 nm, while the L-lipids showed mirrored Cotton effects to those of the D-lipids, which reected the chiral packing manner of the lipids at the surface of the nanotubes, i.e. an M-helix for D-lipids and P-helix for L-lipids. Both 18D/18L and 16D/16L were CD silent, indicating achiral packing at the surface of the planar nanosheets. On the other hand, 18L/16D and 18D/16L displayed strong negative and positive Cotton effects, respectively. Once 16L was involved in the system, 18D/16L exclusively showed positive CD signals regardless of the molar ratios of 16L to 18D (Fig. 4h). The CD results are well consistent with the SEM observations, indicating that the heterochiral lipid nanotubes are globally homochiral and that the helicity is essentially determined by the lipids with the shorter alkyl chain.</p><p>Theoretical analysis and molecular dynamics (MD) simulation [60][61][62][63] To further disclose the unprecedented phenomenon and deeply understand the chiral self-assembly process, theoretical analysis was carried out via MD simulation. According to the previous theoretical studies, 64,65 the handedness of aggregates is dependent on the molecular orientation, which is actually the orientation of amide groups in the lipid molecules here. Besides, the alkyl chain should be matched to maintain the bilayer. Therefore, we mainly focus on the alkyl chain length match and the orientation of amide groups to analyze the handedness of the heterochiral lipid bilayer. There are two amide groups in both 16L and 18D. The a-amide and amino groups can form an intramolecular hydrogen bond, which induces the a-amide to produce an orientation, while the direction of the g-amide is uncertain. The interaction of the oppositely chiral headgroups leads to a ca. 90 difference in the directors (d) of the a-amide groups in 16L and 18D (Fig. 5a, b and S8 †). The theoretical studies by Selinger et al. showed that rotating the tilt direction by 90 should change the curvature direction by 90 , giving a handedness reversal. 64 Therefore, the different chirality of 16L and 18D bilayers can be easily understood.</p><p>Planar 16L (18D) bilayer aggregation has two different stacking manners (Fig. S9 †). However, the aggregation with C2 symmetry (essentially the orientations of a-amides on two sides) where the rotation axis lies along the bilayer aggregation direction will lead to damage of the bilayer structure aer MD simulation. Only the pre-assembly aggregation with C2 symmetry where the rotation axis lies perpendicular to the bilayer plane can result in chiral bilayer structures. For obtaining the chiral structures of the pure 16L and 18D systems, we built planar bilayer aggregates containing two layers and a total of 120 molecules with a 3.6 Å d-space for MD simulations. Aer the equilibriums were reached, we sampled one snapshot per 1 ps and extracted the average congurations during 5.5-6 ns for the pure 16L and 18D systems. It was found that the 16L molecules form a P-helix bilayer structure (Fig. 5a), while the 18D molecules form an M-helix (Fig. 5b). For the 16L/16D mixture with a 1 : 1 ratio, the lengths of alkyl tails perfectly match with each other and the intermolecular hydrogen bonds can form between a-amide groups. Moreover, the a-amide orientations of 16L and 16D are perpendicular to each other, nally resulting in the achiral nanosheet structure.</p><p>However, in the 16L/18D mixture, a conformation rearrangement on the molecular structure of 18D happened due to the existence of 16L. As presented in Fig. 5c, when the a-amide in 16L was connected to the g-amide in 18D, and the g-amide in 16L was connected to the a-amide in 18D, the length of the alkyl chains between the two molecules could be perfectly matched. In this situation, the orientation of the a-amide in 18D was lost, while the orientation of the g-amide in 18D was induced and it pointed in the same direction as that of the a-amide in 16L. Hence, for further study on the 16L/18D aggregate by MD simulation, we built a pre-assembly bilayer with a planar structure containing two layers and a total of 120 molecules (16L/18D ¼ 1/1). As with the pure systems, the orientations of the amides on both sides of 16L/18D should keep C2 symmetry where the rotation axis is perpendicular to the bilayer plane. Aer the equilibrium was reached, we also sampled one snapshot per 1 ps and extracted the average conguration during 5.5-6 ns for the 16L/18D system. It was found that a P-helix was achieved for the 16L/18D aggregate. The MD simulation is consistent with the experimental results, and well explains the unprecedented phenomenon.</p><!><p>In summary, the self-assembly behaviors of two heterochiral lipids and their mixtures were systematically investigated (Fig. 6). For individual chiral lipid self-assembly, the intramolecular hydrogen bond between the a-amide and amino groups induces the a-amide to produce an orientation, and the oppositely chiral headgroups cause a ca. 90 difference in the directors of the a-amide groups. Consequently, L-lipids always form P-helical nanotubes and D-lipids form M-helical nanotubes.</p><p>For the absolutely mirrored heterochiral lipid mixtures (n ¼ m system), the lengths of the alkyl tails can perfectly match with each other and intermolecular hydrogen bonds can form between a-amide groups. In the composite bilayer, the a-amide orientations of the L-lipids and D-lipids are perpendicular to each other, nally resulting in the achiral nanosheet structure.</p><p>For the two heterochiral lipids with mirror headgroups but a 2-methylene discrepancy in alkyl chain length (n-m ¼ 2 system), under alkyl chain communication, the conformation of the longer lipids is rearranged in order to match the shorter lipids (Fig. 6). Consequently, the a-amide of the short lipids was connected to the g-amide of the longer lipids, and the g-amide of the shorter lipids was connected to the a-amide of the longer lipids. In this situation, the alkyl chain length between the two lipids could be perfectly matched. Moreover, the orientation of the g-amide of the longer lipids was induced and pointed in the same direction as that of the a-amide of the shorter lipids, while the orientation of the a-amide of the longer lipids was lost. Finally, the alkyl chain packing, hydrogen-bonding connection and orientation of the two lipids were perfectly matched. Thus, globally homochiral nanotubes are produced and the helicity of the heterochiral lipid nanotube is exclusively determined by the As for 18D/16L (c), the shorter lipid 16L induced a conformation rearrangement of the longer lipid 18D, leading to the disappearance of the orientation of the a-amide and an induced orientation of the g-amide in 18D, which is the same as that of the aamide in 16L. In this case, the alkyl chains between the two lipids are also perfectly matched. Therefore, a P-helical bilayer was achieved for the 18D/16L heterochiral lipid mixture like that of pure 16L. Fig. 6 "Induced conformation rearrangement" mechanism of homochiral nanotube from heterochiral lipids. Alkyl chain communication between heterochiral lipids induced the conformation of the longer lipids to rearrange in the presence of the shorter lipids. Consequently, the orientation of the g-amide of the longer lipids was induced and pointed in the same direction as that of the a-amide of the shorter lipids, while the orientation of the a-amide was lost. Finally, the alkyl chain packing, hydrogen-bonding connection and orientation of the two lipids were perfectly matched. Thus, globally homochiral nanotubes were produced and the helicity was exclusively determined by the molecular chirality of the shorter lipids.</p><p>This journal is © The Royal Society of Chemistry 2019 Chem. Sci., 2019, 10, 3873-3880 | 3877 molecular chirality of the shorter lipids. The "induced conformation rearrangement" mechanism well interpreted the formation of the homochiral nanotube from heterochiral lipid mixtures regardless of the mixing ratio.</p><p>The present contribution sheds new light on the understanding of homochirality at a supramolecular and nanoscale level in complex lipid systems and provides new guidance in exploring homochiral materials in complex supramolecular systems. 3,[43][44][45] Experimental Self-assembly procedure For the self-assembly of pure lipids: the lipid solids (3 Â 10 À5 mol) were put into a seal-capped vial with 1 mL of ethanol added (0.03 M). Then, the sample vial was heated up to 75 C for a while to make a clear solution and subsequently allowed to cool down to room temperature naturally (25 C, cooling rate was about 10 C min À1 ). White gels were obtained, which were fully aged for 12 hours under ambient conditions before being measured. For the self-assembly of mixed lipids: the required amount of D-and L-lipids was mixed at a specic proportion in one sample vial and 1 mL of ethanol was added (the total concentration was kept at 0.03 M). Then, the sample was treated using the above procedure.</p><p>Characterization SEM and TEM. The fully aged gel was transferred from a sample vial to single-crystal silica wafers with a thin lm of Pt coating for SEM observation and to carbon-coated Cu grids stained with 2% uranyl acetate (wt%, aqueous, about 2 min) for TEM observation; images were taken using a Hitachi S-4300 or S-4800 FE-SEM (15 kV) and a JEM-2010 (200 kV), respectively.</p><p>XRD. The quartz-plate-sustained xerogel lms of selfassembled lipids or lipid mixtures were used for XRD measurements on a Rigaku D/Max-2500 X-ray diffractometer (Japan) with Cu/Ka radiation (l ¼ 1.5406 Å, 40 kV, 200 mA). For t18D/t16L, two respectively pre-self-assembled nanotubes of 18D lipid and 16L lipid were vacuum-dried, and the solids were mixed and ground with an agate mortar and pestle for fully mixing.</p><p>FTIR spectroscopy. KBr pellets of vacuum-dried xerogels were prepared for Fourier-transform infrared (FTIR) spectral measurements on a Bruker Tensor 27 FTIR spectrometer (resolution: 4 cm À1 ).</p><p>DSC. The vacuum-dried self-assembled solids (3-5 mg) of pure and mixed lipids were recorded on a METTLER TOLEDO DSC882e to obtain DSC thermograms in a nitrogen atmosphere at a heating rate of 5 C min À1 from 35 to 135 C. For thermal analysis of the mechanical mixture of 18D and 16L nanotubes (18D ¼ 16L), the dried solids of respectively pre-self-assembled 18D nanotubes (2.17 mg) and 16L nanotubes (1.98 mg) were directly added into the sample pan and measurements were performed under the same conditions as described above.</p><p>UV/Vis and CD. A 10 À3 M aqueous solution of TPPS (tetrakis(4-sulfonatonphenyl)porphine, Dojindo Laboratories) was prepared and divided into several aliquots in which the asprepared lipid gels were added. The mixtures were gently shaken for a while and settled overnight under ambient conditions for full absorption of TPPS on the surface of the nanostructures. The excess TPPS in the aqueous solutions was removed using a centrifuge (Anke TGL-16C, Shanghai) at 6000 rpm for 5 min. The green sediments were dispersed in water and re-centrifuged several times until the supernatant liquid was colourless. Aer that, the sediments were dispersed into 3 mL of aqueous hydrochloric acid (0.1 M) and then centrifuged to remove residual acid. Finally, the sediments were re-dispersed into methanol for UV/Vis and CD spectral measurement on a JASCO UV-550 and J-815 CD spectrophotometer, respectively.</p><p>MD simulation. [60][61][62][63] The pre-assembly aggregates of bilayers were solvated in H 2 O boxes with sufficient capacity by the PACKMOL program. Then, MD of solution systems was performed within the NPT ensemble (constant number of atoms, pressure, and temperature) in GROMACS-4.6.7. A Berendsen thermostat with a time-step of 1 fs was employed to regulate the temperature at 298 K. All simulations were carried out for 6 ns to achieve a fully relaxed conguration by using the General Amber Force-Field (GAFF).</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Allosteric Activation of Trypanosomatid Deoxyhypusine Synthase by a Catalytically Dead Paralog*♦

Background: Deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent modification of translation factor eIF5A.Results: Trypanosomatid DHS activity is increased 3000-fold by heterotetramer formation with a catalytically dead paralog, and both gene products are essential for parasite growth.Conclusion: Trypanosomatid DHS is a complex between catalytically impaired and inactive DHS subunits.Significance: This activation mechanism uniquely evolved for two independent enzymes within the trypanosomatid polyamine pathway.

allosteric_activation_of_trypanosomatid_deoxyhypusine_synthase_by_a_catalytically_dead_paralog*♦

Introduction<!><!>Introduction<!>Ethics Statement<!>Anti-DHS Antibody Production<!>Multiple Sequence Alignment<!>Cloning of TbDHSc, TbDHSp, and TbeIF5A<!><!>In Vitro Growth and Transfection of T. brucei<!>Generation of Tet-inducible TbDHSc and TbDHSp T. brucei Expression Constructs<!>Generation of T. brucei Gene Knock-out Constructs and Cell Lines<!>T. brucei Mouse Infection Model<!>Recombinant Expression of DHS and eIF5A<!>Expression and Purification of Yeast SUMO Protease, Ulp1<!>DHS Enzyme Activity Assay<!>Sedimentation Velocity<!>Protein Quantitation<!>RNA and DNA Purification<!>Quantification of RNA Levels by qPCR<!>Western Blot Analysis<!>Immunoprecipitation<!>Phylogenetic Analysis of the DHS Genes in the Kinetoplastids<!><!>TbDHSc and TbDHSp Genes Are Essential for T. brucei Growth<!><!>DHS Is Essential for Infectivity of T. brucei in Mice<!>TbDHSc and TbDHSp Form a Functional Complex<!><!>TbDHSc and TbDHSp Form a Functional Complex<!><!>TbDHSc and TbDHSp Form a Functional Complex<!><!>TbDHSc and TbDHSp Form a Functional Complex<!>DISCUSSION<!>

<p>Trypanosomatid parasites cause several fatal vector-borne human diseases, including the following: human African trypanosomiasis (HAT),3 American trypanosomiasis (Chagas disease), and leishmaniasis (1). Together, these parasites infect more than 20 million people primarily in tropical and subtropical regions. In particular, Trypanosoma brucei gambiense and T. brucei rhodesiense, the causative agents of HAT, are endemic in 36 countries in sub-Saharan Africa and are responsible for a debilitating neurological disease that invariably leads to death if untreated.</p><p>Eflornithine (difluoromethylornithine) is a suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) (Fig. 1A), which in combination with nifurtimox, is a front line treatment for HAT, demonstrating the importance of polyamine function for parasite growth (2). The cationic polyamines (putrescine and spermidine) are essential for growth of most eukaryotic cells and have been explored as potential targets for the treatment of both infectious disease and cancer (2, 3). Spermidine has been implicated in the regulation of translation and transcription, modulation of chromatin structure, and ion channel function (4, 5). In addition, in trypanosomatids spermidine is used in the synthesis of trypanothione (N1,N8-bis(glutathionyl)spermidine), required to maintain intracellular thiol-redox balance (6, 7).</p><p>Biosynthesis and metabolism of polyamines are tightly controlled; in mammalian cells regulation is orchestrated by a complex array of transcriptional, translational, and post-translational mechanisms (3, 4) that are generally lacking in trypanosomatids. Instead, these parasites have evolved a novel mechanism to control activity and expression of a key enzyme required for spermidine biosynthesis, S-adenosylmethionine decarboxylase (AdoMetDC) (2). Previously, we found that the functional trypanosomatid AdoMetDC was a heterodimer between a catalytically impaired subunit and a catalytically dead paralog, both of which were essential for cell growth (8, 9). We defined the term prozyme to describe activating subunits that arose via gene duplication of their partner enzyme. Heterodimer formation between AdoMetDC and the AdoMetDC prozyme led to a 1200-fold activation of AdoMetDC activity. Furthermore, the AdoMetDC prozyme protein levels appear to be translationally regulated, suggesting T. brucei modulates prozyme expression to control AdoMetDC activity and flux through the polyamine pathway (9).</p><p>A specialized yet essential function of the polyamine spermidine in eukaryotic cells is to serve as a precursor for the hypusine modification of eukaryotic initiation factor 5A (eIF5A) (10). Hypusine-modified IF5A is present in both eukaryotes and archaea; although its functions are poorly understood, eIF5A is essential in yeast and mammalian cells (11). In bacteria, the eIF5A homolog elongation factor P, which is lysinylated instead of hypusinated, was shown to relieve ribosome stalling in the presence of polyproline tracks (12, 13). In yeast, eIF5A associates with translating ribosomes in a hypusine-dependent manner and is required for translation elongation (14, 15). Synthesis of hypusine requires two enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. DHS catalyzes the modification of eIF5A to eIF5A-deoxyhypusine in a four-step NAD+-dependent reaction that proceeds through two imine intermediates (Fig. 1A and Scheme 1) (16). The reaction is highly specific and unique to eIF5A. The x-ray structure of human DHS (HsDHS) shows the protein is a homotetramer formed from a dimer of dimers with each containing two active sites at the interface between monomers (17).</p><!><p>Reaction mechanism of DHS.</p><!><p>Genomes of kinetoplastids such as T. brucei and Leishmania species encode two homologs of HsDHS, one of which appears to be catalytically dead. In Leishmania donovani, one of these homologs was shown to be essential and to encode a functional DHS, although it was significantly less active than the mammalian enzyme (18). The functional role of the second DHS homolog was not established. Here, we examine the roles of both homologs in T. brucei and demonstrate that both are required for optimal enzyme activity. Similar to AdoMetDC, we show that the two T. brucei DHS genes encode one catalytically active DHS subunit and one catalytically dead subunit that associate as a heterotetramer to form the active enzyme commensurate with a 3000-fold increase in catalytic activity. We also show that both genes are essential for parasite growth and infectivity in vivo and that the functional form of DHS in the parasite is the heterotetramer. These data demonstrate that the trypanosomatids have independently evolved an analogous strategy to activate two key enzymes involved in polyamine synthesis through oligomerization with a catalytically dead paralog. Trypanosomatids represent the only known species where this strategy is used to generate the catalytically active species of both DHS and AdoMetDC.</p><!><p>Animal experiments were approved by the Ethical Review Committee at the University of Dundee and performed under the Animals (Scientific Procedures) Act of 1986 (UK Home Office Project License PPL 60/4039) in accordance with the European Communities Council Directive (86/609/EEC). To minimize animal suffering, mice with a terminal parasitemia (>108 cells ml−1) were humanely killed.</p><!><p>Antibodies were raised in rabbits by Covance Inc., Denver, PA, against recombinant TbDHSc and TbDHSp purified from Escherichia coli (see below). Generation of rabbit polyclonal antibodies to T. brucei dihydroorotate dehydrogenase (TbDHODH) was described previously (19).</p><!><p>DHS sequences were obtained using NCBI BLASTP searches of the kinetoplastid protein database with HsDHS (P49366) as the search query. Sequences were aligned with Clustal Omega (version 1.1.0). Phylogenetic trees were constructed with Mega5 software using the Neighbor-Joining algorithm with Kimura-2 parameters. DHS sequence accession numbers are listed in Fig. 1 and supplemental Fig. S1.</p><!><p>Genes (TriTrypDB accession numbers are as follows: TbDHSp, Tb927.1.870; TbDHSc, Tb927.10.2750; and TbEIF5A, Tb11.03.0410) were PCR-amplified from T. brucei single marker genomic DNA, cloned into pCR®8/GW/TOPO® (Invitrogen), and sequenced (Applied Biosystems Big Dye Terminator 3.1 chemistry and capillary instrumentation) to confirm that no mutations were introduced (see Table 1 for primers). No nucleotide polymorphisms were identified compared with the published genomic sequence of T. brucei gambiense. In addition, the 5′UTRs from both TbDHSc and TbDHSp genes were cloned by PCR from total RNA using the splice leader sequence as a forward primer and gene-specific reverse primers and sequenced in their entirety (Table 2).</p><!><p>Cloning primers</p><p>Restriction sites are shown in boldface type.</p><p>UTR sequence of TbDHSc and TbDHSp</p><p>5′UTRs for TbDHSc (Tb927.10.2750) and TbDHSp (Tb927.01.870) were cloned by amplification from Tb427 cDNA using the splice leader sequence as a primer with a reverse primer in the ORF. Sequences of the cloned fragments are displayed. The spliced leader sequence is not shown.</p><!><p>Mammalian bloodstream forms (BSF) of T. brucei were cultured at 37 °C with 5% CO2 in HMI-11 media supplemented with 10% heat-inactivated tetracycline (Tet)-free fetal bovine serum (Atlanta Bio) as described (20). BSF single marker cells expressing T7 RNA polymerase and Tet repressor were used for genetic experiments (21). Transfection was performed with an Amaxa Nucleofector II as described (22) using NotI-linearized DNA (5 μg) followed by selection with the appropriate antibiotic. Antibiotic concentrations were as follows: 2 μg ml−1 G418 (Sigma), 2.5 μg ml−1 phleomycin (InvivoGen), 2 μg ml−1 blasticidin S (Sigma), 2 μg ml−1 hygromycin B (Sigma), and 1 μg ml−1 Tet (RPI). Cell growth was monitored with a hemocytometer; cell number was defined as cell density × culture volume × dilution factor. For GC7 EC50 determination, cell density was monitored using PrestoBlue (20 μl, Invitrogen) after 72 h of incubation with GC7 (0.01–1000 μm). Chicken serum was used for these studies to avoid polyamine oxidase-mediated toxicity of GC7. Fluorescence (560 nm excitation/590 nm emission) was measured using a SynergyTM H1 Hybrid Multi-Mode Microplate Reader (BioTek), normalized to untreated cells, and data were fitted to Equation 1 using GraphPad Prism.</p><!><p>Constructs were generated with and without N-terminal tags to allow expression of either native TbDHSc and TbDHSp, AU1-tagged TbDHSc, or FLAG-tagged TbDHSp. Forward PCR primers contained the DNA sequence of the desired tag as follows: AU1 tag (amino acids MDTYRYI) or FLAG tag (amino acid sequence MDYKDDDDK). Untagged genes and AU1-tagged TbDHSc were then subcloned using HindIII and BamHI into pLew100v5 (phleomycin resistance (21)), whereas FLAG-tagged TbDHSp was cloned into pLew300 (blasticidin resistance (9)). Both vectors allow integration of the plasmid into the rRNA locus and support Tet-inducible expression of the tagged gene in T. brucei.</p><!><p>PCR fragments containing 5′- and 3′-flanking regions for the TbDHSc and TbDHSp genes on either side of the blasticidin or hygromycin resistance genes were generated by fusion PCR from T. brucei single marker genomic DNA. The 5′-flanking region of the TbDHSc (374 bp) or the TbDHSp genes (437 bp) were amplified with gene-specific primers (Table 1, flank primer sets). The reverse primer included an overhang complementing the blasticidin resistance gene (19 bases) or hygromycin resistance gene (21 bases). Similarly, the 3′-flanking regions of the TbDHSc (384 bp) or TbDHSp genes (499 bp) were amplified starting directly after the annotated stop codon, and the forward primer included an overhang to complement the resistance gene. The hygromycin resistance gene was amplified from the pLew90 vector (21) and the blasticidin resistance gene from the pLew300 vector (9). Amplified fragments were gel-purified and used in a second PCR with the TbDHSc or TbDHSp nesting primers (Table 1) and the amplified blasticidin or hygromycin resistance gene. PCR mixtures (50 μl) contained the following: 1× Phusion HF buffer, 200 μm dNTPs, 0.5 μm nesting primers, 20 ng of flanking fragment, 50 ng of resistance gene, and 1 unit of Phusion polymerase (New England Biolabs). PCR cycling conditions were as follows: 94 °C for 15 s, 65 °C for 30 s, and 72 °C for 2 min for 30 cycles. This reaction led to the joining of the TbDHSc or TbDHSp flanking regions in a cassette containing the resistance marker for replacement of the allele. The PCR product was gel-purified using the High Pure PCR product purification kit (Roche Applied Science) and transfected into T. brucei parasites directly.</p><p>For transfections, the first allele of the TbDHSc or TbDHSp gene was replaced with the hygromycin resistance gene, and clonal lines were obtained by limited dilution. The resultant single knock-out (SKO) cells were then transfected with the tagless TbDHSc- or TbDHSp-regulatable gene expression constructs and selected using phleomycin. Confirmation of ectopic expression upon Tet induction was obtained by quantitative PCR (qPCR) using a primer in the ectopic UTR and a primer within the gene (Table 1) and by Western blot using rabbit antiserum to recombinant T. brucei DHS. Expression levels from the Tet-induced ectopic copy were considerably higher than from the genomic DHS alleles. SKO cell lines that were confirmed to show good expression from the ectopic gene copy were then used to create conditional double knock-out (cDKO) cell lines by replacement of the second allele with the blasticidin resistance gene. Knock-outs were confirmed by PCR. Tet-regulatable cDKO cell lines were propagated in media containing Tet to maintain expression of TbDHSc or TbDHSp and with G418, hygromycin B, phleomycin, and blasticidin to maintain selection. To study the effects of DHS knockdown, TbDHSc and TbDHSp cDKO cells were washed three times with Tet-free media prior to plating in fresh media. Growth curves were analyzed for n = 3 biological replicates.</p><!><p>A second independent set of cDKO clones was generated at University of Dundee and used for in vivo studies. Methods were essentially as described (23) using flanking primers and PCR (Table 1) to generate gene replacement constructs for TbDHSc and TbDHSp containing the hygromycin or puromycin resistance genes. These cell lines showed similar in vitro behavior to those characterized in Fig. 2, A and C. Wild-type and cDKO BSF T. brucei parasites were cultured in the absence of selectable drugs for 24 h with cDKO cells grown ±Tet. Cells were used to infect n = 3 mice per group (dosed ± doxycycline (Dox)) by a single intraperitoneal injection of 104 parasites as described (24).</p><!><p>TbDHSc, TbDHSp, and TbEIF5A genes were cloned into pE-SUMO Kan (Life Sensors) for expression as N-terminal His6-SUMO-tagged fusion proteins. PCR fragments generated with primers shown in Table 1 were digested with BsaI/XbaI and cloned directly into BsaI-linearized pE-SUMO. Untagged TbDHSp was also cloned into the HindIII-KpnI site of the pT7-FLAGTM-MAT-Tag®-2 vector (Sigma) where the C-terminal His tag was removed by insertion of a stop codon. HsDHS (P49366.1) and human eIF5A (HseIF5A) (P63241.2) sequences were codon-optimized for E. coli, synthesized by GenScript, and cloned into pE-SUMO Kan as above. Genes were expressed in T1 phage-resistant E. coli BL21(DE3) cells selected with kanamycin (50 μg ml−1) for single gene expression or kanamycin (50 μg ml−1) and ampicillin (100 μg ml−1) for co-expression of SUMO-TbDHSc and TbDHSp. Protein expression was induced at A600 of 0.5 with isopropyl β-d-1-thiogalactopyranoside (0.25 mm) for 16 h at 16 °C. Cells were harvested by centrifugation (1000 × g for 0.5 h), resuspended in Buffer A (50 mm Hepes, pH 8.0, 300 mm NaCl, 50 mm imidazole, 2 mm β-mercaptoethanol, 2 mm phenylmethylsulfonyl fluoride (PMSF)), and lysed by high pressure disruption (EmulsiFlex-C5, Avestin). Lysate was clarified (15,000 × g for 0.5 h), and protein was purified from the soluble fraction by Ni2+-affinity chromatography (HiTrap Chelating HP column, GE Healthcare) using a linear gradient from 50 to 320 mm imidazole in Buffer A for elution. SUMO tag was removed by treatment with Ulp1 (5 μg/ml final) (purified as described below) for 16 h at 4 °C. Sample was then diluted 20-fold in Buffer A, and the now tagless DHS was separated from the His6-SUMO by Ni2+-affinity chromatography. DHS-containing fractions (flow-through) were combined and dialyzed against DHS buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm DTT). The TbDHSc-TbDHSp complex was further purified by gel filtration chromatography on a Superdex 200 Prep Grade (GE Healthcare) using DHS buffer. Purified protein concentrations were calculated using the following A280 extinction coefficients: TbDHSc, 46.4 cm−1 mm−1; TbDHSp, 25.9 cm−1 mm−1; TbDHSc-TbDHSp, 72.3 cm−1 mm−1; TbeIF5A, 4.1 cm−1 mm−1; HsDHS, 39.9 cm−1 mm−1, and HseIF5A, 4.5 cm−1 mm−1 (computed using ProtParam, ExPASy, Swiss Institute of Bioinformatics).</p><!><p>The pET28b-Ulp1 expression construct was a gift from Dr. Kim Orth (University of Texas Southwestern). The protein was expressed with an N-terminal His6 tag in E. coli BL21 (DE3). Protein expression was induced as above except 1 mm isopropyl β-d-1-thiogalactopyranoside was used, and induction was for 2 h at 37 °C. Cells lysates were prepared as above and protein-purified by Ni2+-affinity chromatography as above except protein was eluted with Buffer A plus 250 mm imidazole in a single step.</p><!><p>Activity was measured at 37 °C in 1-h reactions using a nitrocellulose filter binding assay to detect the incorporation of [3H]spermidine into eIF5A as described (25, 26). Reaction mixture (20 μl) contained recombinant DHS (4–40 nm HsDHS, 10–40 μm TbDHSc, or 5–20 nm TbDHSc-TbDHSp), eIF5A (0.1–100 μm), and [3H]spermidine (7.5 μm hot or a hot-cold mixture ranging in concentration from 0.2 to 150 μm), NAD+ (0.5–1000 μm), DTT (1 mm), and glycine-NaOH buffer (0.2 m, pH 9.3). Initial rates of velocity data were fitted to the Michaelis-Menten equation using GraphPad Prism. The catalytic rate constant, kcat, was calculated based on active monomer concentration. For GC7 IC50 determination, velocity data were fitted to Equation 1 using GraphPad Prism. Data were collected in triplicate, and error represents the mean ± S.D.</p><!><p>TbDHSc-TbDHSp complex (0.5 ml) was prepared at a range of concentrations (A280 0.7, 0.2, and 0.05) in assay buffer (50 mm Hepes, 150 mm NaCl, pH 8.0) and then loaded into ultracentrifuge cells assembled with sapphire windows after overnight incubation at 4 °C. A280 and interference data were collected at 20 °C in an An-50 Ti rotor monitored continually for 16 h at 40,000 rpm in an Optima XL-I ultracentrifuge (Beckman-Coulter). Complete sedimentation was observed by 5 h. Interference data were analyzed using SEDFIT (27) to calculate sedimentation-coefficient distributions (c(s)) and estimate molecular weight. c(s) plots were generated in GUSSI.</p><!><p>Protein concentration was quantitated using a protein assay (Bio-Rad) and a bovine serum albumin (BSA) standard curve, unless otherwise stated.</p><!><p>RNA was isolated from T. brucei single marker BSF cells (2 × 108 cells), washed in phosphate-buffered saline (PBS), pH 7.4, and then lysed using TRIzol reagent (Invitrogen) followed by purification with the RNeasy mini kit as recommended by the manufacturer (Qiagen). DNA was isolated from T. brucei single marker BSF cells (107 cells) that were harvested and washed twice with PBS, pH 7.4, before being resuspended in DNA lysis buffer (1 m Tris-HCl, pH 8.0, 0.5 m EDTA, 5 m NaCl, 20% SDS, 0.1 mg of proteinase K) and incubated for 6 h at 55 °C. RNA was digested using RNase A at 37 °C for 30 min. An equivalent volume of chloroform was added, and the DNA was extracted in the aqueous layer, ethanol-precipitated, and resuspended in 50 mm Tris-HCl, pH 8.0.</p><!><p>cDNA was synthesized from isolated RNA using the SuperScript®III first-strand synthesis system (Invitrogen). The reaction mixture (20 μl) contained the following: 2 μg of total RNA, 50 ng of random hexamers, 250 μm dNTPs, 1× RT buffer, 5 mm MgCl2, 10 μm DTT, 40 units of RNaseOUTTM, and 200 units of SuperScriptTMIII RT. RNA and primers were initially denatured at 65 °C for 5 min before the remaining components were added. The subsequent reaction conditions were as follows: annealing at 25 °C for 10 min, synthesis at 50 °C for 50 min, termination at 85 °C for 5 min, and removal of RNA with RNase H (2 units) at 37 °C for 20 min. The synthesized cDNA was used directly for qPCR without further purification. Relative gene abundance was quantified using the iQTM SYBR® Green Supermix and CFX 96-Real Time System by Bio-Rad. The reaction mix (20 μl) contained 100–150 ng of cDNA, 1× iQTM SYBR® Green Supermix, and 100 nm primers (Table 1). PCR cycling conditions included a one-time initial denaturation at 95 °C for 3 min followed by 40 cycles of the denaturation at 95 °C for 15 s and annealing/extension at 56 °C for 60 s. After completion of the cycles, melt curve analysis was done from 55 to 95 °C in 0.5 °C increments. Relative gene abundance was calculated by ΔΔCt using telomerase reverse transcriptase (TERT) as the reference gene (28).</p><!><p>Cells (typically 2 × 108) were harvested by centrifugation (2000 × g, 10 min); pellets were washed twice with PBS, pH 7.4 (1 ml), resuspended in Tryp Lysis Buffer (50 mm Hepes, pH 8.0, 100 mm NaCl, 5 mm β-mercaptoethanol, 2 mm PMSF, 1 μg ml−1 leupeptin, 2 μg ml−1 antipain, 10 μg ml−1 benzamidine, 1 μg ml−1 pepstatin, 1 μg ml−1 chymostatin), and lysed with three freeze/thaw cycles. The lysate was clarified by centrifugation (13,000 × g, 10 min, 4 °C), and supernatant (30 μg of total protein) was separated by SDS-PAGE and transferred to a PVDF membrane (iBlot®, Invitrogen). The membrane was blocked with 5% milk in Tris-buffered saline (TBS) (20 mm Tris-HCl, pH 7.6, 137 mm NaCl) and incubated with primary antibody. Primary antibodies anti-TbDHSc (rabbit polyclonal), anti-TbDHSp (rabbit polyclonal), anti-FLAG/M2 (mouse monoclonal, Sigma), or anti-AU1 (mouse monoclonal, Covance) were used at a 1:1000 dilution, and rabbit anti-TbDHODH was used at a 1:2500 dilution. Blots were washed with TBS + 0.1% Tween 20 and incubated with the appropriate secondary antibody at 1:10,000, goat anti-rabbit antibody or goat anti-mouse antibody conjugated to alkaline phosphatase (Sigma). Protein was detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). For a loading control, membranes were stripped and reprobed with antibody to TbDHODH as described previously (19). Membranes were stripped with Restore Western blot stripping buffer (Thermo Scientific, Rockford, IL) for 20 min at RT and rinsed with TBS before blocking again with 5% milk in TBS.</p><!><p>BSF T. brucei cells (108 cells) co-transfected with the AU1-TbDHSc and FLAG-TbDHSp expression plasmids were induced with Tet for 24 h before harvesting by centrifugation (2000 × g, 10 min). Cell pellet was washed twice with PBS, pH 7.4 (1 ml), resuspended in hypotonic buffer (10 mm Tris, pH 7.5, 2 mm PMSF, 1 μg ml−1 leupeptin, 2 μg ml−1 antipain, 10 μg ml−1 benzamidine, 1 μg ml−1 pepstatin, 1 μg ml−1 chymostatin), and incubated on ice for 1 h followed by three freeze/thaw cycles resulting in lysis and adjusted with salt buffer (10 mm Tris, pH 7.5, 400 mm NaCl) to 80 mm NaCl. Cell lysate was clarified by centrifugation (10,000 × g, 10 min, 4 °C). Total soluble protein (50 μg) was incubated alone or with either mouse monoclonal anti-AU1 antibody (Covance) or mouse monoclonal M2 anti-FLAG antibody (Sigma) (1:150 dilution for both antibodies) for 12 h at 4 °C. Dynabeads® protein A (50 μl, Invitrogen) was added, and the antibody-antigen complex was captured with a magnetic stand. The beads were washed three times with TBS, pH 7.6, and the antibody-antigen complex was eluted with 40 μl of citrate buffer, pH 3. Eluent was neutralized with 0.1 m NaOH (5 μl) before separation by SDS-PAGE and Western blot analysis as described above.</p><!><p>Two distinct clades of trypanosomatid DHS proteins were identified by BLASTP analysis of the translated trypanosomatid genome using HsDHS as the search model (Fig. 1 and supplemental Fig. S1). Multiple sequence alignment and comparison of key residues show that one group consists of a protein that contains the catalytic Lys, shown to form the key imine intermediate with substrate (Scheme 1) (10, 17), while the other DHS group lacks the catalytic Lys despite containing many putative substrate-binding residues. The genes encoding these proteins are present on different chromosomes. We refer to the T. brucei gene products as TbDHSc (Tb927.10.2750) reflecting the presence of the catalytic (c) Lys, and TbDHSp (Tb927.1.870) where prozyme (p) designates an activating function. TbDHSc exhibits 28% amino acid sequence identity with human HsDHS but is 92 amino acids larger due to internal expansions. TbDHSp shares 40% identity with HsDHS but only 30% identity with TbDHSc. Trypanosoma cruzi has two DHSc gene homologs (TcDHS(B) and TcDHS(C)) that are closely related and group together on the tree and one copy of the more diverged DHSp gene (TcDHS(A)), whereas Leishmania species and T. brucei contain only a single copy of each gene. BLASTP analysis of eukaryotic DHS proteins showed that Entamoeba species also contain two significantly diverged paralogs of DHS, one with the catalytic Lys that groups with TbDHSc and one without the Lys that groups with TbDHSp. It is not clear if a single gene duplication event led to the generation of both the trypanosomatid DHSp and Entamoeba DHSp homologs or if they arose from independent events. All other eukaryotes appear to contain either only a single DHS gene or closely related gene duplicates that retain the catalytic Lys and are thus likely to be functionally equivalent and catalytically competent.</p><!><p>Phylogenetic analysis of DHS genes in trypanosomatids. A, spermidine and hypusine metabolic pathway in T. brucei. B, partial sequence alignment of DHS from select eukaryotes chosen to include a representative of each of the major eukaryotic lineages in the analysis: Opisthokonta (humans, Trichoplax, and Saccharomyces); Excavata (trypanosomatids, Giardia, and Naegleria); Amoebozoa (Entamoeba and Acanthamoeba); Archaeplastida (Arabidopsis and Chlamydomonas), and Alveolata (Perkinsus). Highlighted in yellow is the catalytic lysine residue. For organisms that contain more than one DHS homolog, duplicates are indicated using consecutive letters (A–C, etc.), except for those where function has been demonstrated in this paper (e.g. T. brucei DHSc and DHSp). Gene IDs are as follows: Homo sapiens (P49366); Trichoplax adherens (EDV28024.1); Chlamydomonas reinhardtii (A, EDP09680.1; B, EDP01029.1); Acanthamoeba castellanii (ELR12881.1); Naegleria gruberi (EFC43118.1); Saccharomyces cerevisiae (P38791); Giardia lamblia (EFO61259.1); Arabidopsis thaliana (A, AED90939.1; B, AAG53621.2; C, AED90940.1); Perkinsus marinus (A, EER15074.1; B, EER03596.1); T. brucei (TbDHSp, Tb927.1.870; TbDHSc, Tb927.10.2750); T. cruzi (A, Tc00.1047053511421.60; B, Tc00.1047053504119.29; C, Tc00.1047053506195.300); Leishmania major (A, LmjF.20.0250; B, LmjF.34.0330), and Entamoeba dispar (A, EDR24093.1; B, EDR21721.1). The full sequence alignment is shown in supplemental Fig. S1. C, Neighbor-Joining tree constructed with Mega5.</p><!><p>We generated cDKO of the TbDHSc and TbDHSp genes in the T. brucei bloodstream form cells to determine whether one or both of the DHS genes were essential for cell growth. T. brucei is a diploid organism, so for both genes one endogenous locus was replaced with the hygromycin resistance antibiotic selection marker generating the SKO cell lines; a Tet-regulated copy of the respective DHS gene was inserted into the rRNA locus to serve as a rescue plasmid, and the second locus was then replaced with a blasticidin or puromycin resistance antibiotic selection marker generating the final cDKO cell lines. Independent cDKO lines were generated in each laboratory for each gene, and representative data are shown (Fig. 2). TbDHSc and TbDHSp cDKO lines were initially evaluated for growth defects in vitro. For TbDHSc cDKO cells, removal of Tet led to a >90% reduction in TbDHSc RNA and protein within 24 h, to a slowed growth by day 4, and to complete parasite clearing by day 6 (Fig. 2, A and B). Likewise, for the TbDHSp cDKO parasites, no detectable TbDHSp RNA or protein was observed 24 h after Tet withdrawal, and cell death occurred by day 8 (Fig. 2, D and E). Cultures were monitored by microscopy for an additional 4 days after cell death, and no live parasites were observed. These data demonstrate that both TbDHSc and TbDHSp are essential for survival of BSF T. brucei in vitro. Interestingly, in both cDKO cell lines, knock-out of one DHS gene (either TbDHSc or TbDHSp) led to the simultaneous loss of both TbDHSc and TbDHSp proteins (Fig. 2, A and D) despite the finding that the RNA as expected was only depleted for the gene targeted for knockdown (Fig. 2, B and E). These data suggested that TbDHSc and TbDHSp form a complex in the cell and that the individual proteins were not stable when the complex was disrupted.</p><!><p>Effects of DHS knockdown on T. brucei growth and survival. Panel 1, effects of TbDHSc knockdown; panel 2, effects of TbDHSp knockdown. A and D, cell growth curve of log(cell number × dilution factor) over time. Data represent an average mean ± S.E. for multiple independent biological replicates. A, TbDHSc cDKO cells (n = 6); D, TbDHSp cDKO cells (n = 3); blue circle, + Tet (0.5 μg/ml); green square, −Tet. Panel inset, representative Western analysis performed with rabbit polyclonal antibodies to the indicated protein (30 μg of total protein); TbDHODH was detected as a loading control. B and E, qPCR analysis of mRNA levels for TbDHSc cDKO cells (B) and TbDHSp cDKO cells (E). The symbol < indicates RNA levels were below the limit of detection. Error bars represent the mean ± S.D. for n = 3 replicates. C and F, Kaplan-Meier survival curves of infected mice (n = 3 per group). C, TbDHSc cDKO; F, TbDHSp cDKO cells; SM (single marker); T. brucei wild-type cells (purple), and cDKO-infected mice treated with (blue) or without (green) Dox.</p><!><p>Mice were infected with TbDHSc and TbDHSp cDKO lines. One set of animals received Dox in their drinking water to maintain expression of the respective DHS proteins, and for the other set Tet was removed 24 h prior to inoculation, and mice were not administered Dox. Mice infected with TbDHSc or TbDHSp cDKO lines that received Dox in their water succumbed to parasitemia by day 6 after inoculation and showed an identical time course to mice infected with the control parental cell line (Fig. 2, C and F). In contrast, in the absence of Dox, mice infected with the cDKO of TbDHSc survived to the end of the experiment (day 30), at which time they remained parasite free and were assumed to be cured (Fig. 2C). Mice infected with cDKO of TbDHSp showed a prolonged survival time, but they eventually succumbed to parasitemia on day 24 after infection (Fig. 2F). The relapse of parasitemia in the TbDHSp cDKO infection suggests that a small number of parasites survived most likely through mutation in the Tet promoter, allowing re-expression of the TbDHSp protein, as documented previously for other proteins with this system (21). These data demonstrate that TbDHSc and TbDHSp are essential to sustain an in vivo infection of T. brucei in mice.</p><!><p>To determine whether TbDHSc and TbDHSp form a complex, we generated a stable T. brucei BSF cell line that co-expressed N-terminally tagged AU1-TbDHSc and FLAG-TbDHSp. Immunoprecipitation of AU1-TbDHSc from soluble T. brucei lysates using monoclonal antibody to AU1 was performed followed by Western blot analysis with anti-AU1 and anti-FLAG antibody. Both AU1-TbDHSc and FLAG-TbDHSp were found in the immunoprecipitate (Fig. 3A). Likewise, if a monoclonal antibody to FLAG was used for immunoprecipitation, both AU1-TbDHSc and FLAG-TbDHSp were detected (Fig. 3A). Thus, we can conclude that TbDHSc and TbDHSp form a protein complex in T. brucei.</p><!><p>Biochemical characterization of T. brucei DHS. A, co-immunoprecipitation of AU1-TbDHSc and FLAG-TbDHSp from BSF T. brucei. Protein was immunoprecipitated with anti-AU1 or anti-FLAG antibody followed by Western blot analysis. B, SDS-PAGE analysis of TbDHSc (50 kDa) and TbDHSp (37 kDa) co-purified by Ni2+-affinity chromatography and gel filtration column chromatography. C, sedimentation velocity analysis of purified TbDHSc-TbDHSp complex. The observed c(s), signal population is shown as a function of S. D, NaBH3CN trapping of DHS reaction intermediates for TbDHSc-TbDHSp (0.1 μm) and TbeIF5A (10 μm). Protein was separated by SDS-PAGE. [3H]Spermidine-labeled proteins were visualized by autoradiography.</p><!><p>To assess the activity of TbDHSc and TbDHSp, the open reading frames (ORFs) for these proteins and the substrate T. brucei eIF5A (TbeIF5A) (Tb11.03.0410) were cloned into vectors for expression in E. coli, which is not capable of carrying out modification of eukaryotic eIF5A. HsDHS and HseIF5A expression vectors were also generated to serve as controls. The proteins were expressed and purified as described under "Materials and Methods." Unlike what was observed in T. brucei, both TbDHSc and TbDHSp could be expressed as stable proteins in E. coli. The ability of purified recombinant DHS to catalyze hypusine modification of eIF5A was measured with either T. brucei or human eIF5A as substrate using [3H]spermidine and a previously described filter binding assay (25, 26). The specific activity of purified TbDHSc using TbeIF5A as substrate was ∼103-fold lower than the activity of HsDHS on HseIF5A (Table 3), the latter being in agreement with previous reports (29, 30). The low observed activity of TbDHSc was similar to that reported for the Leishmania enzyme (18). No activity was detectable for TbDHSc with HseIF5A as the substrate. Recombinant TbDHSp showed no activity within the limit of detection with either eIF5A substrate (Table 3).</p><!><p>Comparison of specific activity between DHS homotetramers and heterotetramers</p><p>Data were collected at fixed substrate concentrations (1 mm NAD+, 7.5 μm [3H]spermidine, and 10 μm eIF5A). Error represents the mean ± S.D. for six replicates.</p><!><p>To assess the activity of the TbDHSc-TbDHSp protein complex, tagless TbDHSp was co-expressed with His6-SUMO-TbDHSc in E. coli. Following purification by Ni2+-affinity chromatography, the SUMO tag was removed using Ulp1 protease and the tag-free protein complex further purified by size exclusion chromatography. TbDHSc and TbDHSp were present in approximately equimolar amounts in the peak fraction from this column confirming that the two paralogous gene products form a stable complex (Fig. 3B). Velocity sedimentation and analytical ultracentrifugation (Fig. 3C) revealed a single species of 175 kDa consistent with a 2:2 TbDHSc-TbDHSp heterotetramer. The specific activity of the heterotetramer was ∼3000-fold higher than for the TbDHSc homotetramer, and it was functional on both T. brucei and human eIF5A substrates (Table 3). Substrate titrations were performed using the heterotetrameric TbDHS (Table 4) and showed that the kcat and the Kmapp values for TbeIF5A were similar to what has been reported for HsDHS/HseIF5A, although the Kmapp values for NAD+ and spermidine were ∼10-fold higher than reported for HsDHS (30). These data demonstrate that the heterotetrameric TbDHS complex is the functional enzyme in T. brucei.</p><!><p>Steady-state kinetic parameters for T. brucei heterotetrameric DHS</p><p>Variable concentrations of the substrate under determination were used with fixed concentrations (1 mm NAD+, 100 μm TbeIF5A, and 100 μm spermidine) of the other substrates. Error represents the standard deviation for three independent experiments.</p><!><p>To further characterize TbDHS activity, sodium cyanoborohydride was used to trap the imine reaction intermediates (Scheme 1). Reaction mixtures containing [3H]spermidine were treated with sodium cyanoborohydride, TCA-precipitated, separated by SDS-PAGE, and analyzed by autoradiography (Fig. 3D). In reactions containing TbDHSc-TbDHSp and TbeIF5A, two bands were detected as follows: a strong band corresponding in size to TbeIF5A, and a weaker band corresponding to TbDHSc (Fig. 3D). These data show that as expected DHS is transiently labeled during the reaction and that the labeled substrate is transferred to TbeIF5A resulting in deoxyhypusine modification (Fig. 1A and Scheme 1). No labeling of either TbeIF5A or TbDHSc was detected for reactions containing only TbDHSc as the catalyst, again showing that on its own TbDHSc is highly impaired in catalytic function and that only in complex with TbDHSp is it fully functional.</p><p>GC7 is a structural analog of spermidine and a known inhibitor of HsDHS (31). GC7 inhibited the activity of TbDHSc-TbDBHSp and the growth of BSF cells at similar concentrations (IC50 = 1.5 ± 0.15 μm and EC50 = 8.0 ± 1.5 μm, respectively). When AU1-TbDHSc or FLAG-TbDHSp was overexpressed in BSF cells independently, there was not a significant shift in the EC50 value for GC7 (EC50 = 5–6 μm). However, overexpression of AU1-TbDHSc and FLAG-TbDHSp together reduced sensitivity to GC7 (EC50 = 26 ± 3.0 μm), while TbDHSc and TbDHSp SKO lines were somewhat more sensitive (EC50 = 3.8 ± 0.4 and 5.5 ± 0.84, respectively). These data suggest that the mechanism of action of cell killing by GC7 is mediated by DHS inhibition, providing further evidence that TbDHSc-TbDHSp is the functional DHS species in T. brucei.</p><!><p>Regulation and control of gene expression and modulation of enzyme activity are critical aspects of cellular function. Although diverse mechanisms for regulating enzyme activities are well known, we report here a new paradigm for potential enzyme regulation in the trypanosomatids based on activators that are catalytically dead enzyme paralogs termed prozymes. Remarkably, these parasitic protozoa have independently evolved this mechanism in two different steps in the same essential biochemical pathway, the biosynthesis of spermidine and the subsequent hypusine modification of a critical lysine in the translation factor eIF5A. Gene duplication of both trypanosomatid AdoMetDC and DHS led to the evolution of an enzyme activation mechanism in which one paralog retained limited catalytic function and the other lost key catalytic residues, but retained the ability to oligomerize with the catalytic subunit to greatly enhance catalytic activity. A pivotal feature of this model is that observed activation by the prozyme component is dramatically large (1000–3000-fold) and is thus likely to result from cooperative structural changes.</p><p>The functional significance of prozyme activation of DHS has been clearly demonstrated by our studies. We show that both TbDHSc and TbDHSp are essential for the growth of mammalian blood form T. brucei and for infection of a mammalian host. Additionally, GC7, a known inhibitor of DHS, was found to have anti-trypanosomal activity. We demonstrate that the functional species of DHS in T. brucei is a heterotetrameric complex between TbDHSc and TbDHSp and that complex formation is required not only for full activity but also for stability of the proteins in the parasite. These data genetically and chemically validate T. brucei DHS as a potential drug target and demonstrate the importance of the functional heterotetrameric DHS complex.</p><p>Despite the similarities to the AdoMetDC example, significant differences in mechanism are also present. The two DHS subunits are not stable in T. brucei unless in complex with each other. In contrast AdoMetDC prozyme and AdoMetDC levels are independent, and one can exist stably in excess over the other, which is a significant factor in AdoMetDC regulation in T. brucei (8, 9). T. brucei up-regulates AdoMetDC prozyme protein levels in response to inhibition or knockdown of AdoMetDC. It remains to be determined if T. brucei also regulates levels of DHSp to control deoxyhypusine formation in the parasite. Finally, although the AdoMetDC prozyme mechanism appears novel to the trypanosomatids, BLASTP analysis identified potential DHSp homologs in Entamoeba species in addition to the trypanosomatids. Functional analysis is needed to determine whether the Entamoeba DHSp paralog is also required to activate DHSc in this genus.</p><p>Our discovery that T. brucei DHS is activated by a prozyme mechanism adds to the list of unusual and novel mechanisms that cells have evolved to regulate polyamine metabolism or modulate enzyme activity. Polyamine metabolism is tightly regulated in mammals, plants, and yeast, although interestingly, no regulation of DHS has been described (3–5). Regulation occurs through common mechanisms such as transcriptional control but also through novel pathway-specific mechanisms. The intracellular turnover rate of ODC is controlled by expression of a protein inhibitor termed antizyme that targets ODC for degradation by the 26 S proteasome. Antizyme expression is in turn initiated by translational frame-shifting of antizyme mRNA when spermidine levels are high (32), and it is further regulated by the antizyme inhibitor, which is itself an inactive paralog of ODC (33). AdoMetDC expression is controlled by a small ribosome-stalling upstream open reading frame (uORFs) that is also sensitive to spermidine levels (34). Trypanosomatids not only lack these mechanisms but unlike other eukaryotes are also unable to regulate RNA polymerase II transcription (35–37). The protein coding genes typically lack introns (38) and are transcribed as large polycistronic clusters, which undergo 5′-leader splicing of the pre-mRNA (39). Regulation instead occurs during mRNA processing, mRNA degradation, translation, protein processing, and protein turnover (36). Furthermore, as a consequence of the mRNA trans-splicing reaction, 5′-UTRs are short, and translational control by uORFs has not been observed. Thus, the driving force to evolve novel mechanisms to regulate the polyamine pathway in trypanosomatids may have been the paucity of other potential mechanisms. Given the large investment made by cells to control and regulate polyamine levels, together with the number of novel mechanisms that have been uncovered, it is clear that regulation of this pathway is a key cellular function.</p><p>Inactive paralogs have been identified in a wide variety of gene families in metazoan species, although they are most prevalent in the kinase, protease, sulfotransferase, and RAS-like protein families (40–43). Inactive paralogs are perfectly poised to play regulatory roles, retaining the ability to bind both ligands and regulatory molecules. It has been shown that when duplicate genes evolve complementary mutations, the ability of cells to maintain both duplicates is enhanced, allowing novel function to evolve (44), thus providing a platform for the evolution of a regulatory function. With the exception of pseudo-kinases, there is still limited functional data on the roles of inactive paralogs. Examples of regulation by both inhibitory and activating mechanisms have been described, although most of the examples involve inhibition or dominant negative effects. The sheer magnitude of the activation observed for T. brucei DHS and AdoMetDC is unprecedented, and the observation that this occurs at two points in the same metabolic pathway is new.</p><p>In conclusion, the ability to regulate enzyme activity with a catalytically dead paralog provides cells with another tool for post-transcriptional regulation. Trypanosomatids represent the only known species where this regulatory strategy is available to potentially control the activity of DHS and AdoMetDC. Evolution of the prozyme mechanism in the trypanosomatids may have been driven by the need to control polyamine synthesis and function in an organism that lacks transcriptional control of gene expression and the frame-shifting and uORF-based mechanisms employed by many other eukaryotes. The discovery of this novel enzyme activation mechanism first for AdoMetDC and now for DHS powerfully confirms the importance of polyamines in the parasite, first exemplified by the discovery of trypanothione (6). Our data suggest that the paradigm of enzyme activation by a catalytically dead paralog may be more widespread than currently known. Indeed, many additional examples of this mechanism for the regulation of enzyme function in eukaryotes are likely still undiscovered.</p><!><p>This work was supported, in whole or in part, by National Institutes of Health Grant 2R37 AI034432 (to M. A. P.). This work was also supported by Welch Foundation Grant I-1257 (to M. A. P.).</p><p>This article was selected as a Paper of the Week.</p><p>This article contains supplemental Fig. 1.</p><p>human African trypanosomiasis</p><p>S-adenosylmethionine decarboxylase</p><p>ornithine decarboxylase</p><p>deoxyhypusine synthase</p><p>T. brucei deoxyhypusine synthase (catalytic subunit)</p><p>T. brucei deoxyhypusine synthase prozyme (activating) subunit</p><p>human DHS</p><p>eukaryotic initiation factor 5A prior to hypusine modification</p><p>eIF5A with hypusine modification</p><p>bloodstream form T. brucei</p><p>N1-guanyl-N7-diaminoheptane</p><p>tetracycline</p><p>doxycycline</p><p>single knockout cell line</p><p>Tet-regulated conditional double knockout cell line</p><p>quantitative PCR.</p>

PubMed Open Access

The Interfacial Properties of Apolipoprotein B292-593 (B6.4-13) and B611-782 (B13-17). Insights into the Structure of the Lipovitellin Homology Region in ApoB

The N-terminal sequence of apolipoprotein B (apoB) is critical in triacylglycerol-rich lipoprotein assembly. The first 17% of apoB (B17) is thought to consist of three domains: B5.9, a \xce\xb2-barrel, B6.4-13, a series of 17 \xce\xb1-helices and B13-17, a putative \xce\xb2-sheet. B5.9 does not bind to lipid while B6.4-13 and B13-17 contain hydrophobic interfaces that can interact with lipids. To understand how B6.4-13 and B13-17 might interact with triacylglycerol during lipoprotein assembly, the interfacial properties of both peptides were studied at the triolein/water interface. Both B6.4-13 and B13-17 are surface active. Once bound the peptides can neither be exchanged or pushed off the interface. Some residues of the peptides can be ejected from the interface upon compression but re-adsorb on expansion. B13-17 binds to the interface more strongly. The maximum pressure the peptide can withstand without being partially ejected (\xce\xa0MAX) is 19.2mN/m for B13-17 compared to 16.7mN/m for B6.4-13. B13-17 is purely elastic at the interface while B6.4-13 forms a viscous-elastic film. When spread at an air/water interface, the limiting area and the collapse pressures are 16.6\xc3\x852/aa and 31mN/m for B6.4-13, 17.8 \xc3\x852/aa and 35mN/m for B13-17, respectively. The \xce\xb1-helical B6.4-13 contains some hydrophobic helices that stay bound and prevent the peptide from leaving the surface. The \xce\xb2-sheets of B13-17 bind irreversibly to the surface. We suggest that during lipoprotein assembly, the N-terminal apoB starts recruiting lipid as early as B6.4 but additional sequences are essential to form a lipid pocket that can stabilize lipoprotein emulsion particles for secretion.

the_interfacial_properties_of_apolipoprotein_b292-593_(b6.4-13)_and_b611-782_(b13-17)._insights_into

<!>Materials<!>Interfacial tension (\xce\xb3) measurements<!>Instantaneous compression and expansion of the interface<!>Determination of the \xce\xa0MAX values<!>Oscillation of the interface and the elasticity analysis<!>Buffer exchange procedure<!>Langmuir balance studies at the Air/Water (A/W) interface<!>Both B6.4-13 and B13-17 are surface active and bind to TO/W interface<!>B6.4-13 only partially desorbs from the TO/W interface on compression and cannot be washed off the interface<!>B13-17 only partially desorbs from the TO/W interface on compression and cannot be washed off the interface<!>\xce\xa0MAX values for both B6.4-13 and B13-17<!>B13-17 is almost purely elastic while B6.4-13 has viscous component on the TO/W interface<!>Monolayers of B6.4-13 and B13-17 at the A/W interface<!>Discussion

<p>Apolipoprotein B (apoB) -containing lipoproteins including chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL) are the major carriers of triacylglycerol (TAG) and cholesterol in human plasma (1, 2). ApoB is the major non-exchangeable component of these apoB-containing lipoproteins. It exists in two forms in human, the full length of apoB (B100) and the truncated N-terminal 48% of apoB (B48). B100 in liver and B48 in intestine recruit and assemble phospholipids, TAGs and cholesterol to form nascent TAG-rich emulsion particles which are secreted into plasma in the form of VLDL and chylomicrons, respectively. In plasma these lipoproteins are remodeled by the action of lipid exchange proteins and various lipases. Chylomicrons are converted to chylomicron remnants and VLDL is converted to IDL and then LDL. These lipoproteins are taken up mainly in the liver via receptor regulated endocytotic pathways. Elevated levels of apoB-containing lipoprotein particles are a major risk factor for atherosclerosis (1, 2). However, adequate apoB is required for transporting TAG and cholesterol in the blood stream to peripheral tissues for energy usage, fat storage, cell membrane synthesis and maintenance, and steroid hormone production. ApoB binds irreversibly to lipid and has an extensive conformational flexibility to fulfill the need to cover and stabilize the lipoprotein particles of varied sizes and different lipid and apolipoprotein compositions (3-7).</p><p>B100 is a huge glycoprotein consisting of 4536 amino acid residues with a molecular mass of 550 kDa. Electron Micrographs of ApoB solubilized in sodium deoxycholate micelles shows a 650Å-long flexible beaded ribbon of varied width (8). In Electron Microscopy reconstructions LDL particles appear to be quasi spherical particles approximately 220-240 Å in diameter with a high density protein ring surrounding a low density lipid core (9). The N-terminal appears to form a domain projecting from the surface. It is generally accepted that apoB can be divided into five super domains: NH2-βα1-β1-α2-β2-α3-COOH (10-12). The α2, α3 and the β1, β2 domains comprise predominantly amphipathic α helix (AαH) and amphipathic β strand (AβS) structures, respectively, and are believed to be the major lipid associating motifs of apoB (10-15). On the other hand, the βα1 superdomain encompassing the N-terminal up to B22 (residues 1-1000), contains both α-helix and β-sheet structure and is predicted to have a globular, multidomain structure (10). The assembly of nascent lipoprotein particles happens cotranslationally in the ER, i.e. when the N-terminal of apoB is folded with lipids to form a precursor lipoprotein, the C-terminal is still being synthesized by the ribosome (16-18). Moreover, microsomal triglyceride transfer protein (MTP), an essential ER-localized cofactor is required in lipoprotein assembly (19, 20). Two MTP binding sites are present in the N-terminal βα1 domain, residues 1-300 and 430-570 (21, 22). Further evidence shows that loss of the disulfide bonds in the N-terminal abolishes secretion out of the cell (23-25). Thus the N-terminal sequence is responsible for initiating lipid recruitment and proper folding of the ba1 domain is critical in lipoprotein assembly and secretion.</p><p>Dashti et al. showed (26) that in McA-RH7777 cells B22 (the first 1000 amino acids) are necessary for the formation of a nascent lipoprotein particle containing both TAG and phospholipids. Alternatively, Shellness et al. used apoB/MTP transfected COS cells (27) to show that B19.5 (the first 884 amino acids) is capable of forming some lipoproteins. B17 (the first 782 amino acids) secreted from MTP-deficient C127 cells contains a very small amount of surface lipid (28). However B17 binds to dimyristoylphosphatidylcholine (DMPC) (27, 28), phospholipid-TAG emulsions (29) and TAG droplets (30) in vitro. B19 and B20.1 also bind to TAG droplets (31). Thus the N-terminal 22% of apoB contains lipid associating domains and has the required elements to form a primordial lipoprotein particle, and under certain conditions shorter constructs are also adequate (27).</p><p>Sequence alignment and analysis show that the βα1 domain is homologous in sequence and amphipathic motifs with lipovitellin (LV) (21, 32-36). LV is an ancient lipid transport and storage protein that delivers lipids into oocytes and appears to be a putative ancestor protein of apoB (36, 37). The crystal structure of Lamprey LV has been solved at 2.8Å resolution (32). It is comprised of a globular β-barrel domain (also called the N-sheet domain), an α-helical domain and a lipid binding pocket which is lined up by two anti-parallel β-sheet domains (the C-sheet and α-sheet). Based on the structure of LV, homologous models for the N-terminal domain of apoB have been built. Mann et al. (21) modeled the first 587 amino acid of apoB and showed that the homologous β-barrel domain and the central helical domain are conserved. Segrest et al. (33) modeled the whole βα1 domain and showed that it contains domains homologous to the β-barrel, α-helix and two β-sheet domains and proposed a lipid pocket model for the initiation of the lipoprotein particle assembly.</p><p>Limited proteolysis results show that the β-barrel, α-helix and the C-sheet domains are three independently folded domains in apoB containing secondary structures consistent with the B20.5 model (Fig. 1) (35, 38). Lipid binding experiments show that B5.9 does not bind phospholipids, while B6.4-13 and B13-17 have hydrophobic interfaces which interact with DMPC to form discoidal particles (38). Other fragments of apoB such as B6.4-9, B13-15, B6.4-15 and B6.4-17 bind phospholipids as well. Thus, B17 contains lipid binding structures that could bind lipids during, or immediately after translocation to initiate the lipoprotein assembly.</p><p>Oil-drop tensiometry has been used to study the lipid binding of apolipoproteins and consensus peptides at a TAG/water interface (13, 30, 31, 39-44). It measures the surface behaviors in terms of surface adsorption, desorption, re-adsorption and elastic properties to yield information about lipid-protein interactions. In a previous study, Mitsche, et. al. (44) studied the surface behavior of B6.4-17 which contains both the α-helical (B6.4-13) and the β-sheet domains (B13-17) of apoB. B6.4-17 binds to a TAG/water interface and cannot be completely ejected once bound. However, part of the peptide is pushed off the surface if compressed above 16.7mN/m (ΠMAX) and very rapidly snaps back on upon re-expansion. The authors suggested that the N-terminal 8 or 9 helices (B6.4-9) are the structures being pushed off above 16.7mN/m while B13-17 remains bound even at high pressures. Thus, this peptide exhibits properties of both α and β structure and retains some globular structure at the hydrophobic interface. In the present work, we used a similar approach to study the helical (B6.4-13) and sheet (B13-17) domains individually to test the lipid associating ability of these domains and determine the mechanism of lipid-protein interaction.</p><!><p>Two apoB fragments encompassing residues 292-593 (B6.4-13), and 611-782 (B13-17) were expressed from pET24a vectors (Novagen) in BL21 DE3 E. coli cells, and purified as described previously (35). Both peptides contain a 6-His tag at the carboxyl terminus. Before interfacial tension studies, B6.4-13 was desalted to 10 mM sodium phosphate/100 mM sodium chloride buffer at pH 7.4 using a PD-10 desalting column (GE biosciences, Pittsburgh, PA) and B13-17 was extensively dialyzed against 5 mM sodium phosphate buffer at pH 7.4. Freshly made B6.4-13 and B13-17 were divided into small aliquots, rapidly frozen in liquid nitrogen and stored at −80 °C. Each peptide aliquot was thawed right before the experiment and discarded afterwards. The concentrations of peptide stocks were determined by Lowry protein assay (45).</p><p>Triolein (>99% pure) was purchased from NU-CHEK PREP, INC. (Elysian, MN) and its interfacial tension against buffer is 32 mN/m. All other reagents are of analytical grade. KCl was heated to 600°C for 6 h to remove all organic contaminants before use (41).</p><!><p>The interfacial tension of the triolein/water (TO/W) interface in the presence of different amounts of B6.4-13 or B13-17 in the aqueous phase was measured with an I. T. CONCEPT Tracker oil-drop tensiometer (Longessaigne, France) (46). Peptide stocks were added to the aqueous phase (2 mM pH 7.4 phosphate buffer) to obtain different peptide concentrations from 1.9×10−8M to 2×10−7M for B6.4-13 and from 7.5×10−8M to 3×10−7M for B13-17. A 16 μL triolein drop was formed in the aqueous phase and the interfacial tension (γ) was recorded continuously until it approached an equilibrium level. The surface pressure, Π, is the γ of the interface without peptide (γTO=32 mN/m) minus γ of the interface with peptide (γpep), i.e. Π = γTO − γpep. All experiments were carried out at 25 ± 0.1°C in a thermostated system.</p><!><p>To study the desorption and re-adsorption behavior of bound peptides at the interface, near-instant compression and expansion experiments were carried out. Once γ approached an equilibrium level, the oil drop (16 μL) was compressed by suddenly decreasing the volume at different ratios (6% to 75%). The compressed volume was held for several minutes and then was increased back to the original volume and held for several minutes until γ re-equilibrated. The sudden decrease in drop volume (V) instantaneously decreases the drop surface area (A) and results in a sudden compression causing γ to drop abruptly to a certain level, γ0. The surface pressure generated is: Π0 = γTO − γ0, where γTO is the surface tension of pure triolein (32 mN/m). If bound peptide or regions of the peptide, desorbs from the surface γ will rise towards an equilibrium value (desorption curve). If the peptide does not desorb, then, the γ will remain essentially constant at the same low level. On re-expansion, free peptide molecules in the aqueous phase or regions of the peptide being ejected from the surface can re-adsorb onto the newly formed surface and γ will fall back to the equilibrium (re-adsorption curve). If the re-adsorption curve (dγ/dt) is the same as the original adsorption curve then we conclude that the entire peptide is ejected from the surface. If however, dγ/dt is much faster, then only part of the peptide is pushed off at compression and snaps back very rapidly when the area is re-expanded (40).</p><!><p>To estimate the maximum pressure (ΠMAX) that a peptide can withstand without being fully or partly ejected from the interface, a series of instantaneous compression and re-expansion experiments using different changes in surface area and over a wide range of peptide concentrations were carried out. The tension change, Δγ, over the compression period was plotted against the instant pressure generated right after the compression, Π0. Then the data was fitted to a straight-line. The intercept at Δγ=0 gives the Π at which the peptide molecules show no net adsorption or desorption, this is ΠMAX (40, 41).</p><!><p>Oscillations were carried out after the equilibrium γ (γe) was reached. The drop volume (16 μL) was sinusoidally oscillated at varied amplitudes (6%-50% change in volume) and periods (8 to 128 sec). The changes in area (A) and γ were followed as the volume (V) oscillated. In the elasticity analysis, the interfacial elasticity modulus, ε (ε = dγ/d ln A), the phase angle, φ, between the compression and the expansion, the elasticity real part, ε′, and the elasticity imaginary part, ε″, were obtained (ε′ =|ε|cosφ, ε″ =|ε|sinφ) (47, 48).</p><!><p>With the surface tension at equilibrium (γe), the aqueous phase buffer (6 mL) containing the peptide was exchanged for buffer without peptide using the protocol described previously (39, 44). The aqueous phase was continuously removed from the aqueous surface and the new buffer was continuously infused near the bottom of the stirred cuvette. At least ~150 mL buffer was exchanged. If peptide desorbs into the aqueous phase during or after buffer exchange, the surface concentration of peptide will fall and γ will rise.</p><p>The instant compression and expansion experiments and the oscillation experiments were also carried out after buffer exchange and compared with that before buffer exchange. Three basically different behaviors are possible. First, if bound peptide is pushed off the surface during compression, then on re-expansion γ will rise to a higher level and stay there since there is virtually no peptide available in the aqueous phase to adsorb back onto the newly formed surface. Second, if only part of the peptide molecule is pushed off the surface on compression, then on re-expansion γ will fall rapidly back to the same equilibrium level since the ejected part of peptide re-adsorbs back on the surface. Finally, compression may cause a conformational change in the peptide which sequesters part of it from the surface and the sequestered part only very slowly re-binds to the surface after re-expansion to lower γ back to the equilibrium level (39).</p><!><p>A solution of each peptide in 30% w/v isopropanol/2 mM phosphate buffer (pH 7.4) was spread slowly (~50 μL/min) on a clean surface of a 3.5 M KCl, 10 mM pH 7.4 phosphate buffer on a KSV 5000 mini trough of a Langmuir/Pockles surface balance (Helsinki, Finland) at 25°C, according to the techniques of Phillips and Krebs (49). Then the surface was compressed at a rate of 5 mN/m/min and the Π-A curves for the peptide monolayer were obtained. To check the reversibility of the Π/A isotherms the peptide monolayer was compressed to a certain Π then re-expanded to a Π lower than 1 mN/m at a rate of 5 mN/m/min, and the compression and expansion curves were compared. The state of the peptide monolayer (liquid, condensed viscous or solid phase) was detected by putting talc powder on the surface, directing a fine jet of air and directly observing the motion of talc particles (50, 51). In the liquid state the talc particles move rapidly and freely, in the condensed viscous state they move slowly and in the solid state they are nearly stationary.</p><!><p>The equilibrium interfacial tension of TO/W interface was measured with different amount of peptides present in the aqueous phase. Fig.2A and Fig. 2B are examples of interfacial tension-time curves of B6.4-13 and B13-17. Both peptides are surface active and lower the surface tension (γ) of the TO/W interface (32mN/m) to reach an equilibrium level. The equilibrium γ is dependent on the peptide concentration in the aqueous phase. In general, the higher the peptide concentration the lower the equilibrium γ, and the less time it takes to reach the equilibrium. The γ of the TO/W interface (32mN/m) is lowered to 14.1mN/m with 1.5×10−7M B6.4-13 in the aqueous phase, and 12.6mN/m with B13-17 at the same concentration. Thus, at similar concentrations, B13-17 decreases γ 1.5mN/m more than B6.4-13 indicating that B13-17 has a higher affinity for the TO/W interface.</p><!><p>Fig. 3A shows an example of tension and area changes during the sudden compression and expansion of the interface for B6.4-13 before and after buffer exchange. The concentration of B6.4-13 in the aqueous phase is 1.9×10−7M before buffer exchange, and the original volume of the triolein drop is 16μl. As the figure shows, after γ approaches an equilibrium level (~13.3mN/m), the drop is compressed by decreasing the volume by 1μL, 2μL, 4μL, 8μL, 10μL and 12μL, respectively. The actual volume changes varied from 5% to 66% and the corresponding area changes varied from 3% to 51%. Every instant compression makes γ fall but then γ rapidly rises towards an equilibrium value while the compressed volume is held for several minutes. The drop is then re-expanded back to 16μl after each compression. Each expansion makes the γ increase above γe initially. However, γ returnes back to the equilibrium value (13.4±0.1mN/m) over time. Smaller compressions induces a smaller fall in γ while larger compression induces a greater fall in γ. For example, at 1μL compression, γ droppes from 13.3 to 12.1mN/m and then rises back to 12.8mN/m; at the following expansion, γ rises to 14.1mN/m and then relaxes back to the equilibrium (13.3mN/m). While at 12μL compression, the tension droppes from 13.6mN/m to 3.1mN/m and then rises back to 9.0mN/m; at the following expansion, γ rises to 17.5mN/m and then relaxes back to the equilibrium (13.5mN/m). After every compression γ decreases first and then rises to a new equilibrium level indicating that some material desorbes from the surface on instant compression.</p><p>The instant compression and expansion protocol is repeated after buffer exchange (Fig. 3A, right) to find out whether the whole molecule or only some part of the bound B6.4-13 desorbes from the TO/W interface during compression. The buffer exchange procedure is started at 7300 sec when γ is at the equilibrium level and stops at 10300 sec (shown by the bar in Fig. 3A). About 150 mL buffer is exchanged and the concentration of B6.4-13 in the aqueous phase reduces to virtually 0. During the buffer exchange γ remaines the same equilibrium value (13.5±0.1mN/m) which indicates that bound B6.4-13 does not desorb from the interface. Then the instant compression and expansion procedure is applied to the triolein drop again (Fig. 3A, right) like that before the buffer exchange (Fig. 3A, left). The changes in γ upon compression/expansion are very similar to that before the buffer exchange. The γ falls to the similar level on compression, rises to the similar level and then relaxes back to the similar equilibrium level after re-expansion. These data clearly show that only some part of the peptide is pushed off the interface by compression. If the whole peptide molecule is ejected from the interface on compression then γ will remain high after re-expansion because there are virtually no peptide molecules in the aqueous phase available to re-adsorb onto the surface.</p><p>Furthermore, the re-adsorption curves at each corresponding expansion, before and after the buffer exchange, are very similar indicating that the re-adsorption is not dependent on the peptide concentration in the aqueous phase. The ejected part of the bound peptide re-adsorbed covering the newly generated surface so quickly that free peptide cannot adsorb onto the surface from the aqueous phase. This data further confirms that only part of the B6.4-13 can be pushed off the surface. However, before or after the buffer exchange, upon compression, the γ always equilibrate to a level lower than the value before compression indicating that the remaining part of the peptide can stand compression and stays compressed to some extent. The fact that the area can be reduced 51% and on re-expansion the γ returns to equilibrium suggests that about ½ of the peptide is pushed off the surface in a form that can rapidly rebind.</p><!><p>Fig. 3B shows an example of tension and area changes during the instant compression and expansion of the interface for B13-17 before (2.1×10−7M in the aqueous phase) and after the buffer exchange. γ changes in a very different way from that of B6.4-13. There are three kinds of changes in γ during the compression and re-expansion of the oil droplet at different ratios before the buffer exchange (Fig. 3B, left), shown as 1, 2 and 3 in Fig. 3B. (1) At smaller compressions, (e.g. 1μL volume decrease), γ drops from the equilibrium value 12.2mN/m to 11.6mN/m. γ remains constant when the compressed volume is held. Upon re-expansion, γ rises to 12.4mN/m and quickly equilibrate back to the original value of 12.2mN/m. No desorption is observed. This indicates that no B13-17 is pushed off the surface and bound B13-17 is simply compressed at the surface. (2) At intermediate compression, (e.g. 2, 4 or 8 μL volume decrease), γ falls much further and quickly rises (0.5-1.5mN/m) when the compressed volumes are held. At each following re-expansion, γ rises to a level higher than the original equilibrium value of 12.2mN/m and then relaxes back towards the equilibrium. Thus, at these intermediate compressions, part of the peptide (about 25%) is pushed off the interface but snaps back very quickly upon re-expansion. (3) At bigger compressions, (e.g. 10 and 12μL volume decrease), γ falls deeper but rises back very little. The desorption is slow compared to those in the intermediate compressions. At the following re-expansion, γ rises high and falls back rapidly to levels higher than the equilibrium. We suggest that at these bigger compressions, part of the peptide is pushed off the surface causing conformational changes in a fraction of the peptide which can't re-spread rapidly. After re-expansion, the ejected region of the peptide needs a much longer time to re-arrange its conformation to re-adsorb onto the surface. This results in a very slow change (hours) in γ towards the equilibrium level. Similar behavior is observed with the AβS domain B37-41 (39).</p><p>In contrast with B6.4-13, the desorption curves for B13-17 upon compressions bigger than 1 μL has only a slightly smaller increase in γ while the compressed volume is held. For instance at the 8μL compression (about 30% change in the area), the γ falls from the equilibrium to 4.9mN/m and only rises back to 6.2mN/m, that is a 1.3mN/m tension increase and is very small compare to the tension increase at 8μL expansion of B6.4-13 which is 5.0mN/m (dropped to 5.0mN/m and rises back to 10mN/m). This indicates that there is only a little net desorption of B13-17 at bigger compression, most part of the peptide stayes compressed on the surface.</p><p>A buffer exchange is run (shown as the bar in Fig. 3B) to remove B13-17 in the aqueous phase. Similar to that of B6.4-13, γ remaines the same during buffer exchange indicating that no bound peptide desorbes from the interface. Instant compression and expansion measurements are carried out (Fig. 3B, right) using the same protocol as before the buffer exchange (Fig. 3A, left). The same there kinds of changes in the desorption and re-adsorption curves are observed as before the buffer exchange. Therefore only a small part of B13-17 is pushed off the surface upon compression and the majority of B13-17 remains on the surface. If highly compressed the ejected region forms a conformation which is slow to re-spread.</p><!><p>Instant compression followed by re-expansion measurements were carried out at varied peptide concentrations to estimate the ΠMAX values for both peptides. Fig. 4 shows that ΠMAX of B6.4-13 is 16.7mN/m and ΠMAX of B13-17 is 19.2mN/m. The data points shown are a mixture of points taken from the compression and expansion experiments before or after buffer exchange. For both peptides, the whole peptide molecule is not pushed off the surface at our study pressures, so the ΠMAX values shown here are the pressures at which parts of the peptides are ejected. A higher Π is needed to push off the ejected region of B13-17 from the interface indicating that the ejected region of B13-17 binds to the TO/W interface more tightly than the ejected region of B6-13. In addition, △γ is always much bigger for B6.4-13 than B13-17 at the same pressure indicating that more structure of B6.4-13 tends to desorb from the interface while more structure of B13-17 tends to stay. As a matter of fact, both B6.4-13 and B13-17 can stand a very high surface pressure, e.g. 29mN/m (Fig. 3A and Fig. 3B) at a large compression, e.g. 12 μL (~50% decrease in area), indicating that both peptides have structures that bind strongly to the TO/W interface.</p><!><p>Equilibrium oscillations of B6.4-13 and B13-17 at different amplitudes and periods were carried out before and after buffer exchange. These compressions differ from the instantaneous compressions in that they are slow, steady, sinusoidal compressions that are performed at set of rates. Two sets of the surface pressure-area (Π-A) curves for B6.4-13 and B13-17 (all at 1.5×10−7M in the aqueous phase) derived from the oscillations before the buffer exchange are shown in Fig. 5A and Fig. 5B. The Π-A curves for B6.4-13 show significant hysteresis between compression and expansion. The corresponding phase angles, φ, are relatively larger, up to 30° (data not shown) indicating a visco-elastic surface. On the other hand, the Π-A curves for B13-17 show little hysteresis and the phase angles are relatively small, less than 10° (data not shown) indicating a pure elastic surface. There is very little desorption and re-adsorption of the B13-17 during these oscillations. This is further evidence that only a small region of B13-17 desorbs and re-adsorbs. We did series study on the elasticity for both peptides over a wide range of peptide concentration, oscillation amplitude, and oscillation period (data not shown). The φ values average over all experimental conditions for B6.4-13 is 16.6±7.7° and for B13-17 is 4.5±3.8°, indicating that B6.4-13 forms a visco-elastic surface on TO/W interface while B13-17 is mainly elastic. Between the two peptides, B13-17 has a higher modulus (38±7.3mN/m) than B6.4-13 (25.6±6.5mN/m). The elasticity modulus is the increase in the surface tension for a small increase in area of a surface element (ε = dγǀ d ln A), is the mathematical description of the surface's tendency to be deformed elastically (i.e., non-permanently) when a force is applied to it. A higher elasticity modulus indicates a more rigid molecule. This is consistent with the larger phase angles for B6.4-13 relative to B13-17. In other words, B13-17 is more rigid and less compressible than B6.4-13.</p><p>Examples for the elasticity modulus, ε, from the oscillations before and after buffer exchange are compared for both peptides in Fig. 6. Similar results are present either before or after the buffer exchange at the same peptide, same amplitude and same period. ONE-WAY ANOVA analysis at 0.05 level showes that the ε, φ and ε' of corresponding oscillations are not significantly different before and after buffer exchange (data not shown). This indicates that there is no whole molecule desorption from the interface or re-adsorption onto the interface.</p><!><p>Fig. 7 shows the pressure/area (Π-A) isotherms for B6.4-13 and B13-17. The limiting area for B6.4-13 is 16.6 Å2/aa and the B6.4-13 monolayer collapses at 31 mN/m at the area of 12.2 Å2/aa. The limiting area for B13-17 is 17.8 Å2/aa and the B13-17 monolayer collapses at 35 mN/m at the area of 13.3 Å2/aa. Both B6.4-13 and B13-17 can be reversibly compressed and expanded at pressures below the collapse pressure (31mN/m and 35mN/m), respectively (data not shown). By observing the movement of the Talc powder on the surface, we found that B13-17 monolayer starts solidify right after the lift up (Π>2mN/m) which is typical for a β sheet structure (39). In contrast, the B6.4-13 monolayer does not solidify until Π is over 10mN/m and becomes solid when Π reaches 20 mN/m. The average area per residue for proteins adsorbed flat on a surface is 15-25 Å2/aa, so both peptides are likely flat at the A/W interface.</p><!><p>When proteins or peptides come to hydrophobic interfaces like oil/water or air/water (A/W) interfaces, different structures behave differently. When some water soluble globular proteins adsorbs to the hydrophobic interface, since they hide most of their hydrophobic residues in the core of the molecule, they undergo major conformational changes after binding and denature to gradually decrease the interfacial tension (47, 52). On the contrary, apolipoproteins are specifically evolved for lipid binding with the characteristic secondary structures of AαH and AβS (10-14). When approaching a hydrophobic interface, they don't denature, instead they rapidly adsorb and retain the secondary structures.</p><p>AαH and AβS structures show characteristic interfacial properties at TO/W interface in terms of the adsorption, the desorption and re-adsorption, the elasticity and the compressibility. Both AαH and AβS structures are surface active, they adsorb onto the surface and lower the surface tension rapidly (13, 15, 40, 43). AβS structures show strong irreversible binding to TO/W interface (13, 39). They can be compressed to a very high surface pressure (about 28-29 mN/m) and stay compressed without leaving the surface. Bound AβS structures display purely elastic properties with very small phase angles at the surface when the surface area changes less than 15% (13). When applied to an A/W interface on a monolayer trough, AβS structures quickly interact with each other when compressed to a low pressure and form solid monolayer suggesting a large extended β sheet structure at very low pressure (1 to 2 mN/m) (39). This large β sheet can be compressed and expanded reversibly up to its collapse pressure (usually around or higher than 35mN/m) (39 and unpublished data). AαH structures show reversible binding to TO/W surface. In general they desorb from the surface when the surface is compressed above 13-19mN/m (15, 40, 42) and re-adsorb quickly when the surface is re-expanded. This reversible on and off behavior of AαH was proposed in the review by Segrest, et al. (10). Some AαH structures with lower surface affinity, like the N-terminal domain of apoA-I (residue 1-44), can partially desorb from the surface without compression during exchanging the peptide out of the aqueous phase (43 and unpublished data). AαH show purely elastic properties only at smaller changes in the surface area and at fast rates, otherwise they are visco-elastic at the surface (15, 40, 42). When applied to an A/W interface, AαH structure does not become a solid monolayer until the pressure reaches well above 10 mN/m. As observed for AβS, AαH monolayer can also be reversibly compressed and expanded as long as the pressure remains below the collapse pressure.</p><p>According to the LV homology model of B17, B6.4-13 and B13-17 are distinct domains containing α and β structures with amphipathic features (35, 38, and Fig. 8). Our study show that B6.4-13 binds to TO/W interface (Fig. 2A) but unlike most AαH, cannot be completely pushed off the TO/W interface (Fig. 3A). B6.4-13 does show typical partial desorption and re-adsorption curves of AαH structures, i.e. fast desorption at compression and fast re-adsorption at re-expansion towards the equilibrium. This indicates that the majority of the secondary structure of this domain is AαH. This is consistent with the model prediction and the CD spectra that show nearly 60% its secondary structure is α helix in B6.4-13 (35). Up to 50% of B6.4-13 desorbs from the TO/W interface at pressures above 16.7 mN/m (Fig. 4). This is within the ΠMAX range for the AαH structures in exchangeable apolipoproteins (13-19 mN/m) and is consistent with the ΠMAX value of B6.4-17 (16.7 mN/m) (44). Thus, when B6.4-17 is compressed at the interface, regions in the B6.4-13 domain are pushed off at lower pressures than B13-17. The B13-17 domain has a higher lipid binding affinity with a ΠMAX of 19.2 mN/m (Fig. 4), but most of the peptide remains bound, is elastic and cannot desorb. B6.4-13 has visco-elastic properties at the TO/W interface like other AαH structures in the exchangeable apolipoproteins as well. At an A/W interface, the limiting area for B6.4-13 monolayer is 16.6 Å2 per residue and the collapse pressure is 31 mN/m indicating that the peptide is lying flat on the surface (Fig. 7).</p><p>That B6.4-13 cannot be pushed off the interface is a little surprising because all other AαH structures (apoA-I, apoA-I fragments and the consensus peptides) we have studied can be pushed off the interface by compression (15, 40, 42). We note that some of the α helix structures in B6.4-13 domain are not those classic type A or type Y AαH found in exchangeable apolipoproteins or in the α2 or α3 domains of apoB (Fig. 8). Some of the α-helixes are very hydrophobic without a clear hydrophilic face, e.g. helices 5, 6 and 8. These helices are more similar to transmembrane α-helices, but shorter. We speculate that they may fully insert into the hydrophobic lipid and then cannot be pushed off by compression. Helices 5, 6 and 8 face the lipid pocket in the B17 model (Fig. 8), especially helix 6 and 8 that bind to the lipid directly in the LV structure. Some helices in the C-terminal of B6.4-13 are typical for globular proteins and have a lot of charged residues. They do not show strong lipid binding affinity for DMPC (38). In the LV homology model, those charged residues interact with the charged side of the β sheet formed by B13-17 domain. Mitsche, et. al. (44) have studied the interfacial properties of B6.4-17 which encompasses both the α-helical domain (B6.4-13) and the β-sheet domain (B13-17) at TO/W and A/W interfaces. Only about 60% of the expected area of B6.4-17 binds to the A/W interface. The authors suggest that the C-terminal part of the helical domain (B9-13) retains protein-protein interactions with the hydrophilic face of the β sheet domain and thus does not contact the surface. In our study, lacking protein-protein interactions with B13-17, those C-terminal helices of B6.4-13 with charged residues might partially denature like a globular structure and irreversibly bind to the hydrophobic interface. That is another possible reason for B6.4-13 not being fully pushed off by compression.</p><p>B13-17 showes strong binding to the TO/W interface (Fig. 2B). It has a very high ΠMAX value of 19.2 mN/m (Fig. 4) and is almost purely elastic (Fig. 5B). B13-17 cannot be washed off or pushed off the interface (Fig. 3B). Only a small region of the sequence can be pushed off the interface by compression as shown by the very small increase in the surface tension after compression (Fig. 3B). This is the typical desorption behavior of AβS structures (13, 39), and is consistent with the model which predicts that B13-17 contains 6 anti-parallel β strands forming a β sheet (Fig. 8). The re-adsorption curves of B13-17 at intermediate compression shows a very rapid fall back to the equilibrium level (Fig. 3B), and at larger compression, the re-adsorption curves return to near the equilibrium values, and there is no significant difference after the buffer exchange. Together, this suggests that only a small region of B13-17 is pushed off the surface and re-adsorbed rapidly.</p><p>We notice that at larger compression, the re-adsorption curves do not fall back to the equilibrium level (different by 1-2mN/m). We have shown similar phenomena in our study of B37-41, a large AβS structure (39). When B37-41 is compressed by 36% and then re-expanded γ rises to 23.9 mN/m then relaxes back in 40 min to 22.7 mN/m (6.5 mN/m higher than the equilibrium, e.g. 16.2 mN/m). Then after ~12 hours, the tension gradually fell back to 19.7 mN/m. We suggest that larger compression causes some conformational changes of the ejected region that requires more time to return to the equilibrium. In the LV homology model of B20.5 (Fig. 1), a large loop of amino acids (656-729) between strands 4 and 5 of the β-sheet in B13-17 is missing because this part is not present in the LV structure. This region has been predicted to contain an AαH structure (33). CD study shows 30.7% of a content in B13-17 (35). Those helical structures might be the region that leaves the surface upon compression and re-adsorbs at expansion.</p><p>Thus, both B6.4-13 and B13-17 display strong lipid binding to TO/W interface. Both of them contain structures irreversibly bind to lipid. B13-17 has a higher affinity and is more elastic than B6.4-13. They show typical surface behaviors of AαH and AβS structures suggesting that the main secondary structures of the two domains are α-helix and β-sheet respectively, which are consistent with the homologous B17 model and with the surface study of B6.4-17 (Table 1) (35, 38, 44).</p><p>There are two competing models for the initiation of apoB lipoprotein assembly. A lipid pocket model is proposed that the N-terminal 1000-residues βα1 domain of apoB forms a lipid pocket homologous to that of lamprey lipovitellin during translation, which contains two β sheet connected by a central α-helical domain and a helix-turn-helix motif close the pocket (53). This pocket is gradually filled with phospholipids and converted to a nascent TAG-core emulsion particle as the translation continuous. While the βα1 domain contains the required sequence and structural elements for initiating assembly, the amino acid residues between 931 and 1000 maybe critical for the formation of a stable, bulk-lipid containing nascent lipoprotein particle (53). In an alternative "intercalation/desorption model", the two sides of the lipovitellin-like cavity form solvent-accessible surfaces with strong interfacial binding properties interact with the ER membrane to sequester neutral lipids to nucleate the oil droplet formation and ultimately desorbs from the membrane as a small neutral lipid core-containing precursor particle (27). The suggestion that B19.5 is secreted in a small particle with a neutral lipid core (27) and that B17, B19 and B20.1 bind to a TAG/W interface (30, 31) are used in support of this model. Both models agree that the N-terminal domain contains the structure and required elements for lipid binding. But they differ on the mechanism and what sequence is involved in the lipid binding.</p><p>Our study shows that as early as B6.4 in the N-terminal domain of apoB, the structure possesses the ability to bind to neutral lipids. Both B6.4-13 and B13-17 contain the elements that strongly bind lipids. This is consistent with the study showing that B6.4-17, B17, B19 and B20.1 all bind to TAG interface in vitro (30, 31, 44). In vivo studies show that truncated apoB of different size is secreted with different lipid composition. B17 is secreted with only a small amount of phospholipids (28). B22 and B19.5 are claimed to be the minimum size required for lipoprotein particle assembly and secretion in different cell lines (26, 27). We think that the lipid recruitment may start early but only sequences containing a more complete lipid pocket (i.e. B19.5 or B22) can stabilize the emulsion particle and be secreted. B32.5, B37 and B41 are secreted as stable lipoprotein particles containing increasing core lipids (54). So, during apoB translocation, the lipid recruiting may start as early as B6.4 but future sequences as translation continues are required to produce a stable emulsion particle which can be secreted.</p><p>In summary, our interfacial studies on the two distinct domains (B6.4-13 and B13-17) in the LV homologous N-terminal domain of apoB show that both domains contain lipid associating structures. The majority secondary structure of the two domains is AαH and AβS respectively, which is consistent with the B20.5 model. The two domains show different surface behaviors at TO/W interface but they work together to form a hydrophobic face to bind to TAG surface.</p>

PubMed Author Manuscript

Metabolic kinases moonlighting as protein kinases

Protein kinases regulate every aspect of cellular activity, whereas metabolic enzymes are responsible for energy production and catabolic and anabolic processes. Emerging evidence demonstrates that some metabolic enzymes, such as pyruvate kinase M2, phosphoglycerate kinase 1, ketohexokinase isoform A, hexokinase (HK), and nucleoside diphosphate kinase 1 and 2 (NME1/2), that phosphorylate soluble metabolites can also function as protein kinases and phosphorylate a variety of protein substrates to regulate the Warburg effect, gene expression, cell cycle progression and proliferation, apoptosis, autophagy, exosome secretion, T-cell activation, iron transport, ion channel opening, and many other fundamental cellular functions. The elevated protein kinase functions of these moonlighting metabolic enzymes in tumor development make them promising therapeutic targets for cancer.

metabolic_kinases_moonlighting_as_protein_kinases

<!>PKM2<!>PGK1<!>KHK-A<!>Hexokinase<!>Histidine Kinases<!>How can one establish unequivocally that a metabolic kinase acts as a protein kinase?<!>Prospective

<p>Protein kinases are important regulators of intracellular signal transduction pathways that mediate the development and regulation of both unicellular and multicellular organisms. They play critical roles in cell growth, division, differentiation, adhesion, motility, and death (Brognard and Hunter, 2011; Lu and Hunter, 2009). These protein kinases, more than 500 of which exist in the human genome, can be primarily subdivided into tyrosine (Y)- and serine (S)/threonine (T)-specific kinases based on their catalytic specificity (Manning et al., 2002). Disruption of normal protein kinase functions by mutations, altered expression, or dysregulation can cause many human diseases, including cancer and diabetes (Lu and Hunter, 2009).</p><p>Cell metabolism comprises the life-sustaining chemical reactions ultimately responsible for all cellular processes; production of ATP and building blocks for proteins, lipids, nucleic acids, and some carbohydrates; and elimination of nitrogenous wastes. Metabolic enzymes catalyze these reactions, facilitating cell growth and proliferation and the response to all intracellular and extracellular signaling and stimuli. Each metabolic enzyme is known to catalyze a unidirectional and/or bidirectional reaction in a specific metabolic pathway. However, recent studies have demonstrated that several metabolic enzymes can also moonlight as protein kinases and phosphorylate multiple protein substrates. These phosphorylations are critical for key cellular functions, including the Warburg effect, a feature of which is a high rate of glycolysis and lactic acid fermentation in the cytosol of cancer cells regardless of oxygen level (Li et al., 2016b; Li et al., 2016d; Yang and Lu, 2015). This review highlights the roles of the unexpected protein kinase activity of the metabolic enzymes pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase isoform A (KHK-A, or fructokinase A), hexokinase (HK) and the NME1/2 histidine (H) kinases in regulation of a variety of cellular functions and the impact of this regulation on tumorigenesis.</p><!><p>Pyruvate kinase regulates the final rate-limiting step of glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to produce pyruvate and adenosine triphosphate (ATP) (Yang and Lu, 2015). This kinase has four isoforms: PKL, PKR, PKM1, and PKM2. PKL and PKR, which are encoded by the PKLR gene, are expressed in the liver and erythrocytes, respectively, whereas PKM1 and PKM2, which are encoded by the PKM gene, are expressed in different types of cells and tissues. The heterogeneous nuclear ribonucleoproteins A1 (hnRNPH1) and A2 (hnRNPH2) and polypyrimidine-tract (PPT) binding protein splicing factors regulate alternative splicing of PKM pre-mRNA and generate PKM2 via the inclusion of the PKM2-specific exon 10 and exclusion of the PKM1-specific exon 9 (David et al., 2010; Noguchi et al., 1987). An isoform switch from PKM1 to PKM2 and enhanced PKM2 expression have been found in many cancer types (Bluemlein et al., 2011; Desai et al., 2014; Yang et al., 2012a).</p><p>Genetic replacement of PKM2 with PKM1 inhibits aerobic glycolysis and tumor growth in mice, although PKM1 has higher glycolytic activity than PKM2 does (Christofk et al., 2008; Guminska et al., 1988; Mellati et al., 1992). One of the fundamental functional differences between PKM1 and PKM2 is that the latter has unique nuclear functions. Specifically, PKM2 contains a nuclear localization signal (NLS) encoded by exon 10, whereas PKM1 lacks an NLS. Activated extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinases bind to an ERK docking groove encoded by PKM2 exon 10 thus allowing ERK1/2 to phosphorylate PKM2 but not PKM1 at Ser37. Once phosphorylated, the pSer37.Pro38 bond in PKM2 is subject to cis-trans isomerization by the phospho-specific peptidyl-proline isomerase protein interacting with never in mitosis A-1 (PIN1), causing a conformational change in PKM2, conversion of PKM2 from a tetramer to a monomer, and exposure of the PKM2 NLS for importin α5 binding and subsequent nuclear translocation (Yang and Lu, 2013; Yang et al., 2012c). Interaction of PKM2 with JMJD5, a Jumonji C domain-containing dioxygenase, hinders PKM2 tetrameric assembly and facilitates PKM2's nuclear translocation (Wang et al., 2014a). In addition, enhanced nuclear translocation of PKM2 is induced by sumoylation of PKM2 mediated by the SUMO-E3 ligase PIAS3 and by acetylation of PKM2 at Lys433 mediated by the p300 acetyltransferase that prevents the binding of fructose-1,6-bisphosphate to PKM2 and formation of a PKM2 tetramer (Lv et al., 2013; Spoden et al., 2009). This evidence supports the possibility that PKM2 translocates into the nucleus as a monomer.</p><p>In the nucleus, PKM2 phosphorylated at Ser37 in the cytosol in response to growth factor receptor (EGFR) activation, is dephosphorylated by the dual-specificity Cdc25A phosphatase, thereby forming a complex with β-catenin that has been phosphorylated by c-Src – at Tyr333, which then binds to β-catenin–regulated target promoter regions of genes, such as MYC and CCND1 (Liang et al., 2016b; Yang et al., 2011). Using PEP as a phosphate donor, nuclear PKM2 phosphorylates histone H3 at Thr11 in nucleosomes associated with gene promoter regions. This phosphorylation is required for the dissociation of histone deacetylase 3 (HDAC3) from the gene promoter regions and the subsequent acetylation of histone H3 at Lys9. PKM2-dependent histone H3 phosphorylation is essential for EGFR-promoted cell proliferation and tumorigenesis as well as expression of specific genes, including c-Myc–regulated genes such as GLUT1 and lactate dehydrogenase A (LDHA), and polypyrimidine-tract binding (PTB) protein, which increases PKM2 mRNA expression levels via alternative splicing of PKM pre-mRNA. Thus, nuclear PKM2 enhances the Warburg effect by upregulating expression of glycolytic genes, including itself (Yang et al., 2012b; Yang et al., 2012c). PKM2 also binds to other transcription factors, such as hypoxia-inducible factor (HIF) 1α, signal transducer and activator of transcription (STAT)3, and Oct4, and enhances their target gene expression (Gao et al., 2012; Luo et al., 2011; Yang and Lu, 2015). Notably, PKM2 phosphorylates STAT3 at Tyr705 and promotes STAT3 transcriptional activity (Gao et al., 2012). Although one study of the ability of recombinant PKM2 to phosphorylate proteins in PKM2-deficient cell lysates failed to detect PEP-dependent protein kinase activity, possibly due to insufficient levels of key target substrate proteins in the cell lysates and the low concentration of 32P-PEP used in the reactions (Hosios et al., 2015), a later study using similar phosphoproteomic analyses of the proteome of renal cell carcinoma cells demonstrated that recombinant PKM2 in the presence of PEP phosphorylated 974 protein substrates (He et al., 2016). In addition, multiple research groups have demonstrated and validated PKM2's protein kinase activity using both yeast and mammalian cells (Keller et al., 2014; Li et al., 2015). Succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5′-phosphate (SAICA) binds to PKM2 and enhances PKM2-mediated histone H3 phosphorylation at Thr11, as well as phosphorylation of ERK1/2 and more than 100 other proteins (Keller et al., 2014). Another report demonstrated that the yeast PKM2 homolog PYK1 directly phosphorylates histone H3 at Thr11 and regulates the cross-talk between H3 Thr11 phosphorylation and H3 Lys4 methylation in autoregulation of PYK1 expression (Li et al., 2015). Transgenic mouse studies demonstrated that PKM2 is not required for breast cancer formation promoted by BRCA1 tumor suppressor deficiency but is essential for BCR-ABL – or MLL-AF9–induced leukemia development (Israelsen et al., 2013; Wang et al., 2014b), suggesting that the roles of PKM2 in tumor suppressor- and oncogene-induced tumorigenesis can differ.</p><p>Besides regulating gene expression, PKM2's protein kinase activity is instrumental for many critical cellular activities. PKM2 binds to the spindle checkpoint protein Bub3 during mitosis and phosphorylates Bub3 at Tyr207 (Jiang et al., 2014a). This phosphorylation leads to recruitment of the Bub3-Bub1 complex to the outer kinetochore protein Blinkin and governs kinetochore-spindle attachment and the mitotic checkpoint, thereby promoting accurate chromosome segregation and proliferation of tumor cells (Jiang et al., 2014a). PKM2 is also involved in cytokinesis. When PKM2 Thr45 is phosphorylated by Aurora B, PKM2 interacts with myosin light chain 2 (MLC2) in the contractile ring regions of mitotic cells and phosphorylates it at Tyr118, and MLC2 Tyr118 phosphorylation is greatly enhanced by EGFR variant III, K-Ras G12V, and B-Raf V600E oncogenic mutant expression (Jiang et al., 2014a). This phosphorylation primes MLC2 phosphorylation at Ser15 mediated by Rho-associated protein kinase 2 (ROCK2) and is required for contraction of the actomyosin complex at the cleavage furrow, completion of cytokinesis, and tumor cell proliferation (Jiang et al., 2014b). In addition, PKM2, whose expression is transcriptionally enhanced by EGFR-dependent activation of nuclear factor (NF)-κB, promotes hormonal and nutrient signal-independent activation of mammalian target of rapamycin complex 1 (mTORC1) via phosphorylation of mTORC1 inhibitor AKT1 substrate 1 (AKT1S1) at Ser202/203 and its release from Raptor (He et al., 2016; Yang et al., 2012a). In renal cell carcinomas and breast cancers, PKM2 overexpression has been correlated with elevated AKT1S1 phosphorylation at Ser202/203, activated mTORC1, and reduced autophagy (He et al., 2016).</p><p>The protein kinase activity of PKM2 is also involved in cell apoptosis. In response to oxidative stress, PKM2 translocates to the outer membrane of mitochondria, where heat shock protein (HSP)90α1-mediates a conformational change in PKM2, which then interacts with and phosphorylates Bcl-2 at Thr69. This phosphorylation prevents the binding of a Cul3-based BCR (BTB-CUL3-RBX1) E3 ligase to Bcl-2, thereby stabilizing Bcl-2 and enhancing the resistance of tumor cells to oxidative stress (Liang et al., 2016a). PKM2 also participates in the remodeling of tumor microenvironments by regulating tumor cell exosome secretion. PKM2 acts as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23) at Ser95, which in turn enables the formation of the SNARE complex to facilitate exosome release (Wei et al., 2017).</p><p>In summary, in addition to its originally characterized metabolic function in the glycolytic pathway, it has now been established that PKM2 also acts as a dual-specificity protein kinase that can phosphorylate protein substrates at both serine/threonine and tyrosine residues. PKM2 possesses nonmetabolic functions, acting as a protein kinase and phosphorylating a variety of protein substrates to regulate the Warburg effect, gene expression, mitosis and cytokinesis progression, cell proliferation, apoptosis, and exosome secretion (Fig. 1).</p><!><p>Pyruvate kinase and PGK1 are the only two ATP-generating enzymes in the glycolysis pathway. PGK1 is the first ATP-generating enzyme in this pathway and is highly expressed in many types of cancer (Li et al., 2016d). It catalyzes the reversible conversion of 1,3-diphosphoglycerate and ADP to 3-phosphoglycerate and ATP, respectively (Li et al., 2016d). Nuclear PKM2 enhances the Warburg effect by upregulating the expression of glycolytic enzymes, including PKM2 itself, to increase glucose uptake and lactate production (Yang et al., 2011), but how this enhanced aerobic glycolysis coordinates with suppression of mitochondrial pyruvate metabolism is a central question in understanding the Warburg effect. Our recent work demonstrated that EGFR activation, oncogenic K-Ras G12V and B-Raf V600E mutant expression, and hypoxic stress all result in ERK activation-dependent mitochondrial translocation of a small portion of cytosolic PGK1 (Li et al., 2016a). Activated ERK1/2 phosphorylates PGK1 at Ser203. This phosphorylation recruits the PIN1 prolyl isomerase, leading to isomerization of the Ser203.Pro204 bond and subsequent exposure of the presequence of PGK1 (38-QRIKAA-43) on its surface. The exposed PGK1 presequence is then recognized by the mitochondrial translocase of the outer membrane (TOM) complex, leading to translocation of PGK1 into the mitochondria. In the mitochondria, PGK1, acting as a protein kinase, interacts with and directly phosphorylates pyruvate dehydrogenase kinase isozyme 1 (PDHK1) at Thr338 using ATP as a phosphate donor. This phosphorylation activates PDHK1 and enhances PDHK1-mediated pyruvate dehydrogenase E1α phosphorylation at Ser293, which inactivates the pyruvate dehydrogenase complex preventing conversion of pyruvate and coenzyme A (CoA) to acetyl-CoA and CO2 in the mitochondria. This PGK1-mediated phosphorylation event suppresses mitochondrial oxidative phosphorylation and thereby increases extracellular acidification and lactate production by shunting pyruvate from the mitochondria into the cytosol, thereby promoting tumorigenesis (Li et al., 2016a). Thus, tumor cells promote the Warburg effect via nuclear PKM2-dependent upregulation of glycolytic gene expression and through mitochondrial PKG1-mediated PDHK1 activation and subsequent inhibition of mitochondrial pyruvate metabolism. Thus, the protein kinase activities of both PKM2 and PGK1 play critical roles in this regulation.</p><p>Rapid tumor growth results in outgrowth of the existing vasculature and ischemia, and the tumor inevitably encounters metabolic stress, which induces autophagy to maintain cellular homeostasis (Lin et al., 2012). During the initiation of autophagy, autophagosome nucleation requires functional Beclin1, which recruits the class III phosphatidylinositol (PI) 3-kinase VPS34 into a complex with VPS15 and ATG14L to generate PI 3-phosphate (PI(3)P) (Funderburk et al., 2010). PI(3)P recruits proteins with PI(3)P–binding domains to modulate intracellular trafficking and autophagosome formation (Kim et al., 2013). Recent studies demonstrated that PGK1 plays a critical role in initiation of autophagy. In response to glutamine deprivation and hypoxia, mTOR-mediated phosphorylation of acetyltransferase ARD1 at Ser228 is inhibited, leading to an association of ARD1 with PGK1 and subsequent PGK1 acetylation at Lys388. Acetylated PGK1 interacts with Beclin1 and phosphorylates it at Ser30. This phosphorylation alters VPS34 conformation and dramatically enhances its ability to bind to PI, thereby significantly increasing VPS34 activity and PI(3)P production. As a consequence, PGK1-mediated Beclin1 phosphorylation at Ser30 is required for initiation of autophagy, which is instrumental for tumor development. In addition, Beclin1 phosphorylation at Ser30 positively correlates with poor prognosis for glioblastoma (Qian et al., 2017).</p><p>These studies revealed that PGK1 can function as a protein kinase to regulate mitochondrial function and cellular stress-induced autophagy initiation and that integrated regulation of glycolysis, mitochondrial metabolism, and autophagy by PGK1 is instrumental to promotion of tumor cell proliferation and maintenance of cell homeostasis (Fig. 1).</p><!><p>Like glycolysis, fructose metabolism is a critical component of cell metabolism, with aberrant fructose metabolism leading to development of liver diseases (Ishimoto et al., 2012). In the fructose metabolic pathway, KHK, also known as fructokinase, the first rate-limiting metabolic enzyme, catalyzes the conversion of fructose and ATP to fructose 1-phosphate (F1P) and ADP, respectively. F1P is metabolized into dihydroxyacetone phosphate and glyceraldehyde by aldolase and subsequently converges on the glycolysis pathway (Li et al., 2016b). Mutually exclusive splicing of adjacent exons 3A and 3C in the KHK precursor RNA results in expression of KHK isoform A (KHK-A) or isoform C (KHK-C). KHK-A and KHK-C have low and high fructose phosphorylation activity, respectively, because only KHK-C has high binding affinity for fructose, whereas KHK-A has low fructose-binding affinity and a high Km for phosphorylation of fructose (~7 mM) (Asipu et al., 2003; Bonthron et al., 1994).</p><p>KHK-C is predominantly highly expressed in the liver, kidneys, and pancreas. In contrast, KHK-A is ubiquitously expressed at low levels (Ishimoto et al., 2012). In hepatocellular carcinoma (HCC) cells, KHK-C expression switches to KHK-A expression as a result of c-Myc–induced high level expression of hnRNPH1 and hnRNPH2, which bind to a motif located in the intron close to the 3′ end of exon 3C resulting in alternative splicing of the KHK pre-mRNA and expression of KHK-A. KHK-A expression results in much lower fructose catabolism rates, ATP consumption, and reactive oxygen species production in HCC cells than in normal hepatocytes (Li et al., 2016c). Importantly, instead of binding fructose, KHK-A interacts with the rate-limiting enzyme phosphoribosyl pyrophosphate synthetase 1 (PRPS1) in the de novo nucleic acid synthesis pathway. This allows KHK-A to function as a protein kinase and directly phosphorylate PRPS1 at Thr225 (Km, ~0.2 μM), which lies in the binding region for PRPS1's allosteric inhibitor ADP. This phosphorylation abrogates the feedback inhibition of PRPS1 by blocking the binding of ADP to PRPS1, leading to elevated de novo nucleic acid synthesis via constitutive activation of PRPS1 in HCC cells, and thereby promotes HCC proliferation and growth in the livers of mice. Immunohistochemical staining of human HCC specimens demonstrated that KHK-A and PRPS1 pThr225 were positively correlated with each other and that the levels of KHK-A and PRPS1 pThr225 staining were inversely correlated with survival duration, supporting a pivotal role for KHK-A–dependent PRPS1 phosphorylation in HCC progression (Li et al., 2016b; Li et al., 2016c).</p><p>Thus, in addition to the identified protein kinase activity of PKM2 and PGK1 in the glycolytic pathway, KHK-A in the fructose metabolic pathway acts as a protein kinase that plays an instrumental role in de novo nucleic acid synthesis and promotion of HCC development (Fig. 1).</p><!><p>Hexokinase (HK) catalyzes the phosphorylation of glucose to produce glucose 6-phosphate, using ATP as a phosphate donor. Four isoforms of hexokinase (HK 1–4) exist in mammalian tissues [1]. Incubation of HK-PII, an isoenzyme from Saccharomyces cerevisiae, with γ-32P ATP shows that HK-PII can autophosphorylate itself (Fernandez et al., 1988). Phosphoamino acid analyses reveal that HK1 purified from rat brain can autophosphorylate at serine, threonine, and tyrosine residues (Adams et al., 1994). In addition, in vitro phosphorylation assays show that HK1 can phosphorylate purified histone H2A, and that this phosphorylation and the autophosphorylation activity of HK1 is inhibited in the presence of its normal substrate glucose (Adams et al., 1991). Although HK1 exhibits protein kinase activity in vitro, whether HK1 as well as other HK isoforms can act as protein kinases in vivo and the physiological role of such phosphorylation activity in the regulation of HK and cellular activities remain to be determined.</p><!><p>In prokaryotes and lower eukaryotes (yeast, fungi, and plants), protein-histidine kinase activity of metabolic enzymes was identified in two-component and multicomponent signaling systems and this activity plays an important role in sugar phosphorylation (Hess et al., 1988; Kennelly and Potts, 1996; Swanson et al., 1994). In the PEP/sugar phosphotransferase system, enzyme I, which converts phosphoenolpyruvate to pyruvate, is autophosphorylated at His189, with PEP being used as the phosphate donor. The phosphate group in enzyme I is then transferred sequentially to a histidine in the HPr enzyme and then to a histidine in enzyme IIA before being transferred to a cysteine residue in enzyme IIB, which, in complex with enzyme IIC, phosphorylates intracellular glucose (Attwood and Wieland, 2015). Thus, the histidine kinase activity of enzyme I plays a critical role in glucose metabolism.</p><p>PEP can be used as a phosphate donor to phosphorylate proteins by enzyme I in prokaryotes and lower eukaryotes and by PKM2 in eukaryotes. The low sequence homology between enzyme I and PKM2 and their different structures suggest that they will have different substrates for phosphorylation. Moreover, enzyme I is a histidine kinase that autophosphorylates itself and then transfers this phosphate onto its protein substrates, whereas PKM2 does not use a phosphoenzyme intermediate, and transfers phosphate from PEP onto both ADP and protein substrates in a concerted reaction. In addition, although PKM2 can phosphorylate several different proteins, these phosphorylations are regulated by posttranslational modification, oligomerization state or alteration of PKM2's subcellular compartmental localization (Jiang et al., 2014a; Yang et al., 2012b; Yang et al., 2012c).</p><p>In mammals, nucleoside diphosphate kinase (NDPK, or NDK) is a ubiquitous enzyme that catalyzes the conversion of nucleoside diphosphates (NDPs) into nucleoside triphosphates (NTPs) by transferring terminal γ-phosphate groups from 5′-triphosphate-nucleotides to 5′-diphosphate-nucleotides. NDPKs are encoded by the NME gene family, which is comprised of 10 family members (Boissan et al., 2009). Besides their NDPK activity, NDPK-A (also known as NME1) and NDPK-B (also known as NME2) function as protein-histidine kinases (Attwood and Wieland, 2015). Similar to the phosphoenolpyruvate/sugar phosphotransferase system in lower organisms, the NDPK catalytic mechanism involves NTP (usually ATP)-dependent autophosphorylation of a highly conserved histidine residue in its active site and this phosphate group is then transferred from the phosphohistidine either to an NDP molecule or to the histidine in substrate proteins (Attwood and Wieland, 2015).</p><p>Several proteins have been identified as substrates of NDPK-A and NDPK-B protein kinase activity. Specifically, NDPK-A phosphorylates the histidine at the catalytic site of ATP citrate lyase (ACLY), a cytosolic enzyme that converts citrate to acetyl-CoA and is critical for fatty acid synthesis (Attwood and Wieland, 2015). Autophosphorylated NDPK-A transfers the phosphate group from NDPK-A to ACLY. Citrate then binds to ACLY, and the phosphate is transferred to citrate to generate citryl-phosphate, an intermediate product of the reaction that forms acetyl-CoA (Wells, 1991). Like NDPK-A, NDPK-B can also act as a histidine kinase, forming a complex with G protein βγ dimers and phosphorylating His266 in the Gβ subunit. The phosphate on His266 is highly energetic and can be transferred onto guanosine diphosphate (GDP), leading to formation of guanosine triphosphate (GTP) and contributing to G-protein activation (Cuello et al., 2003). NDPK-B also binds directly to and activates the Ca2+-activated K+ channel KCa3.1 by phosphorylating H358 in the cytoplasmic C-terminal tail of KCa3.1, thus relieving copper-dependent inhibition of KCa3.1 channel function, and promoting subsequent activation of CD4+ T cells (Srivastava et al., 2006; Srivastava et al., 2016). In addition, NDPK-B phosphorylates His711 in the C-terminal tail of transient receptor potential-vanilloid-5 (TRPV5), which regulates urinary Ca2+ excretion by mediating active Ca2+ reabsorption in the distal convoluted tubule of the kidney. His711 phosphorylation activates TRPV5 channel activity (Cai et al., 2014).</p><p>Histidine phosphorylation, like serine, threonine, and tyrosine phosphorylation, is reversible. Using monoclonal antibodies that specifically recognize phosphorylated histidine in proteins (Fuhs et al., 2015), phosphoglycerate mutase family 5 (PGAM5) was identified as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic pHis118 on NDPK-B and negatively regulates CD4+ T cell activity by inhibiting NDPK-B–mediated histidine phosphorylation and activation of the K+ channel KCa3.1, which is required for T-cell receptor (TCR)-stimulated Ca2+ influx and cytokine production (Panda et al., 2016). On the other hand, NDPK-phosphorylated ACLY, Gβ, and KCa3.1 are substrates for a phosphohistidine-specific phosphatase, PHPT1, which dephosphorylates the phosphorylated active site histidine and reverses the functional consequences of NDPK-dependent phosphorylation (Klumpp et al., 2003; Maurer et al., 2005; Srivastava et al., 2008). In addition, phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), a conserved phosphatase that hydrolyzes P-N bonds in synthetic substrates in vitro, is potentially a third phosphohistidine protein phosphatase, but LHPP is otherwise poorly characterized.</p><p>Thus, in addition to regulating nucleoside triphosphate production, NDPK-A and NDPK-B function as histidine kinases and regulate a spectrum of cellular activities in a cooperative manner with phosphohistidine phosphatases.</p><!><p>The recent finding that many metabolic kinases can also act as protein kinases is unexpected and raises a number of mechanistic questions. The most obvious is how the active site of an enzyme designed to recognize ATP/ADP and a second small metabolite molecule for phosphate transfer is also able to recognize a specific Ser, Thr or Tyr residue in a selected protein substrate and phosphorylates it. This in turn raises the issue of whether the protein substrate occupies the same binding site as the small molecule substrate that is normally phosphorylated. One way to answer this is to show that the small molecule substrate can compete with the protein substrate for phosphorylation in vitro, which has only been demonstrated in a few cases. The ultimate way to establish how a protein substrate binds will be to solve a structure of the protein bound to the metabolic kinase as an enzyme intermediate complex. So far this has not been achieved for any metabolic kinase and its proposed protein substrate. Detailed structural analyses will also elucidate how posttranslational modifications of metabolic enzymes, such as Aurora B-mediated PKM2 Thr45 phosphorylation and ARD1-mediated PGK1 Lys388 acetylation (Jiang et al., 2014a; Qian et al., 2017), enable these metabolic enzymes to bind to and phosphorylate their protein substrates.</p><p>Another concern is that the observed protein kinase activity could be due to contamination with an authentic protein kinase, particularly if the metabolic kinase is isolated from a eukaryotic cell. To address this issue, recombinant forms of the metabolic kinase and its substrates should be used, and ideally a kinetic analysis of protein substrate phosphorylation should be carried out to demonstrate that the protein kinase activity has properties consistent with this activity being of physiological relevance in cells.</p><p>To establish unequivocally that the protein kinase activity of a metabolic kinase is an intrinsic and physiologically activity, we propose a number of criteria that should be met. Table 1 lists these criteria and indicates which of them have been satisfied for the different metabolic kinases with reported protein kinase activities that we have reviewed.</p><!><p>The original characterization of the human kinome identified more than 500 protein kinases (Manning et al., 2002). Characterization and demonstration of the protein kinase activities of metabolic enzymes expands the kinome. Identification of additional metabolic enzymes, which are able to transfer phosphate groups during their metabolic reactions, as protein kinases is a reasonable expectation. Demonstration of multiple roles of metabolic enzymes, such as PKM2, PGK1, KHK-A, HK1, and NDPK-A/B, in plural cellular functions provides new insights into integrated regulation of cell metabolism and many other important cellular activities, such as cell growth, proliferation, survival, autophagy, and apoptosis.</p><p>The existence of non-canonical functions for PKM2, PGK1, and KHK-A in tumor development make these enzymes promising targets for new therapeutic interventions for human cancer. Structural elucidation of the binding and phosphorylation of protein substrates of these metabolic enzymes will facilitate identification of specific interventions that can selectively inhibit their protein kinase activity rather than their metabolic enzymatic activity. Disruption of processes involved in their subcellular localization will inhibit their important subcellular compartment-specific protein kinase activity that is critical for tumor development. In addition, cancer type-specific expression of some of these metabolic enzymes, such as HCC-specific splicing of the KHK gene and expression of KHK-A (Li et al., 2016c), afford the possibility of cancer type-specific treatment by targeting the protein kinase activity of these metabolic enzymes. Advances in understanding the protein kinase activity of metabolic enzymes could lead to the development of new and specific therapeutic cancer interventions.</p>

PubMed Author Manuscript

Gas Adsorption Selectivity in Topologically Disordered Metal-Organic Frameworks

Disordered metal-organic frameworks are emerging as an attractive class of functional materials, however their applications in gas storage and separation have yet to be fully explored. Here, we investigate gas adsorption in the topologically disordered Fe-BTC framework and its crystalline counterpart, MIL-100. Despite their similar chemistry and local structure, they exhibit very different sorption behaviour towards a range of industrial gases, noble gases and hydrocarbons. Virial analysis reveals that Fe-BTC has enhanced interaction strength with guest molecules compared to MIL-100. Most notably, we observe striking discrimination between the adsorption of C3H6 and C3H8 in Fe-BTC, with over a twofold increase in the amount of C3H6 being adsorbed than C3H8. Thermodynamic selectivity towards a range of industrially relevant binary mixtures is probed using ideal adsorbed solution theory (IAST). Together, this suggests the disordered material may possess powerful separation capabilities that are rare even amongst crystalline frameworks.

gas_adsorption_selectivity_in_topologically_disordered_metal-organic_frameworks

Introduction<!>Materials<!>MIL-100<!>Fe-BTC<!>Powder X-ray Diffraction<!>Gas Sorption<!>Structural Characterisation and Nitrogen Adsorption<!>& S1, & Table<!>Hydrogen and Carbon Dioxide<!>Noble Gases<!>Hydrocarbons<!>Analyte<!>Thermodynamic Gas Selectivity<!>Conclusions<!>Author Contributions

<p>Metal-organic frameworks (MOFs) are hybrid materials known for their chemical and structural diversity. 1 With surface areas reported in excess of 7,000 m 2 g −1 , they are emerging as promising candidates for gas storage. 2,3 Furthermore, their pore architectures, tuneable between 3 and 100 Å in diameter, are well suited for various separation processes, such as industrial gases and hydrocarbons. [4][5][6][7][8][9][10] MOFs are capable of gas separation through thermodynamic, kinetic, molecular sieving, and even quantum effects. 11 Separation depends on many properties of the framework; surface area, pore size, surface chemistry, the presence of functional groups, unsaturated metal sites, extra-framework ions and water molecules. 5 The kinetic diameter, polarizability and permanent polarity of the guest also play an important role, complicating the prediction of porous material's separation capabilities prior to measuring adsorption isotherms.</p><p>The separation of industrial gases, specifically the capture of CO2 from CO2/N2 or CO2/CH4 mixtures, is increasing in importance given the environmental impact of the rising global greenhouse gas emissions. 12 Another key area is the petrochemical industry, which relies on the separation of hydrocarbons for use as fuels and chemical feedstocks for the production of polymers. 7 Separation of hydrocarbons of the same chain length is not easily achieved given their similar physical properties. For example, the boiling points of C2H4 and C2H6 differ by 15 K, while for C3H6 and C3H8 the difference is only 5 K. 13 Coupled with a molecular size difference of less than 0.4 Å, C3H6/C3H8 separation is a very challenging and extremely energy-intensive process. 7 Very few crystalline MOFs have been found to successfully discriminate between the two gases. Developing MOFs for these applications requires consideration of not only the selectivity itself but also the chemical stability, recyclability, handling stability and capacity of the material, which are all important for practical use within an industrial setting. 14,15 MOFs are particularly attractive candidates due to the intricate ways in which their pore architecture and surface chemistry can be modified to tune the interactions with guest molecules. 16 MIL-100 is a crystalline MOF, comprising Fe(III) centres and 1,3,5-benzenetricarboxylate linkers, arranged into oxo-centred trimer motifs that further assemble into hybrid tetrahedra [Figs. 1a & b]. 17 Its hierarchical structure has a specific surface area in excess of 2,000 m 2 g −1 and contains two types of mesopore with internal diameters of 25 and 29 Å, accessible through 5.5 and 8.6 Å apertures, respectively [Fig. 1c]. The crystallographic unit cell has a cell volume in excess of 390,000 Å 3 [Fig. 1d].</p><p>Activation of MIL-100 at temperatures below 150 °C generates coordinatively unsaturated Fe(III) sites, while temperatures above 150 °C induce partial reduction to form unsaturated Fe(II) sites, which may lead to enhanced interactions with certain adsorbates. 18 These unsaturated metal sites are effective in aiding the separation of light hydrocarbons, with interaction strengths following the order C2H2 > C2H4 > C2H6 > CH4. 19 A similar increase in interaction strength was observed between MIL-100 and C3H6 compared to C3H8 upon generation of unsaturated Fe(II) sites through activation, suggesting the potential for C3H6/C3H8 separation using materials activated at temperatures above 150 °C. 20 MIL-100 has also found applications in water harvesting and as a Friedel-Crafts reaction catalyst. 21 Amorphous MOFs share the local structure of their crystalline counterparts but lack any long-range order. 22,23 Typically, they are obtained via the collapse of a crystalline material through the application of heat or pressure. Structural disorder within MOFs has been demonstrated to improve conductivity, mechanical response, and mass transfer capacity compared to crystalline frameworks. 24 Notable examples of amorphous MOFs are found within the zeolitic imidazolate framework (ZIF) family, such as the hybrid glass agZIF-62 [Zn(Im)2(bIm)2-x;</p><p>Im -imidazolate, bIm -benzimidazolate; ag denoting the glass state], which is obtained through the meltquenching of crystalline ZIF-62. 25 Recently, mechanical amorphisation of ZIF-8 [Zn(mIm)2; mIm -2-methylimidazolate], to form amZIF-8 (am denoting amorphisation by ball milling), was found to enhance the adsorption selectivity of C3H6 over C3H8. 26,27 Despite this, investigations into disordered MOFs for applications in gas storage and separation are of comparatively low prevalence with respect to their crystalline counterparts, particularly in the area of C3H6/C3H8 separation.</p><p>Fe-BTC, known commercially as Basolite® F300, has the same chemical composition as MIL-100 and is topologically disordered, lacking long-range order [Fig. 1e]. 28,29 X-ray absorption near edge structure analysis of Fe-BTC revealed the presence of octahedrally coordinated Fe(III) ions at ambient temperature. 30 Furthermore, analysis of the extended X-ray absorption fine structure region revealed Fe-BTC possessed the same trimer unit structure present in MIL-100. Synchrotron X-ray pair distribution function analysis revealed the presence of mixed hierarchical local structure within Fe-BTC, confirming the trimer unit's existence and the presence of tetrahedral assemblies as observed in MIL-100. 29 This was used to produce the first atomic-scale model of Fe-BTC.</p><p>Upon activation at 120 °C, removal of water molecules coordinated to the Fe(III) ions in the trimer unit leads to the formation of coordinatively unsaturated metal sites. 30 This causes a lowering of the octahedral symmetry but does not induce the structural rearrangement of the framework. Upon activation at the slightly higher temperature of 150 °C, a small proportion of unsaturated Fe(II) sites were detected using NO-probed infrared spectroscopy in Fe-BTC and MIL-100. 31 At 250 °C the proportion of Fe(II) sites was greater in MIL-100 than Fe-BTC; beyond this temperature, Fe-BTC began to show signs of decomposition. Fe-BTC has been studied for its catalytic ability and outperforms MIL-100 in Lewis acid catalysis. 32 This has been attributed to its unsaturated metal sites and additional Brønsted acid sites, which are likely to influence its interactions with guest molecules. 31,33 The morphological and porous nature of Fe-BTC is highly synthesis dependent. Often obtained via a sol-gel route, the Fe-BTC gel can subsequently be dried (i) through exchange with supercritical CO2 (sCO2) to obtain hierarchically porous aerogels, (ii) at room temperature for several days to afford xerogels, or (iii) at higher temperatures overnight to produce powdered samples. 28 The use of sCO2 exchange avoids destructive capillary forces, which cause the collapse of the hierarchically porous architecture. Aerogel samples of Fe-BTC possessed a total pore volume of 5.62 cm 3 g −1 and a single point Brunauer-Emmett-Teller (BET) surface area of 1,618 m 2 g −1 . Xerogel samples had a BET surface area of around 800 m 2 g −1 that could be increased to 1,182 m 2 g −1 with a total pore volume of 0.71 cm 3 g −1 through the ageing of the gel before drying. The powdered sample of Fe-BTC was least porous but was easiest to prepare. Quenched solid density functional theory analysis of the N2 adsorption isotherm revealed that the xerogel had a broad pore size distribution in both the micro-and mesopore range up to 4.5 nm with maxima at 1.3 and 3.0 nm. The aerogel contained an even broader distribution in the whole range of micro-and mesopores with the same maximum at 1.3 nm. 28 More recent investigations, using the harsher drying conditions employed in this study, gave rise to powdered Fe-BTC samples that were essentially non-porous to nitrogen at 77 K, yet still retained the same local structure as MIL-100. 29 Basolite® F300, whose exact synthetic route remains undisclosed, has a BET surface area in the range 1,300 to 1,600 m 2 g −1 as reported by the manufacturers. Independent experimental measurements have reported the BET surface area to be around 685 to 840 m 2 g −1 , with a total pore volume of 0.29 cm 3 g −1 and pore size of 2.2 nm. 31,34 Computational modelling has suggested that the degree of tetrahedral assembly in Fe-BTC materials influences the porosity, with accessible surface area increasing with the proportion of tetrahedral assemblies. 29 One atomic-scale model of Fe-BTC, for example, contained appreciable internal porosity while remaining non-porous to a nitrogen-sized probe. This model demonstrated the successful percolation of a 3.19 Å diameter spherical probe through the structure and revealed a maximum spherical cavity size of 9.15 Å. This analysis was performed Please do not adjust margins Please do not adjust margins from a purely geometric perspective and did not consider dynamics within the real material. This suggests that Fe-BTC may have adsorption capacity for other gases given the dynamic nature of the material under realistic conditions. Given the existing promise of MIL-100 in gas sorption and separation, the absence of long-range order in Fe-BTC means it is well placed to explore how topological disorder affects sorption and selectivity in MOFs. The area of C3H6/C3H8 separation is particularly interesting, given the limited number of crystalline MOFs reported with this behaviour. Ultimately, we aim to demonstrate the importance of disorder as a tool to augment and enhance properties of MOFs in the field of gas sorption and separation.</p><!><p>All chemicals were obtained from commercial suppliers and used as received. Iron (III) nitrate nonahydrate (99.95%), 1,3,5-benzenetricarboxylic acid (95%), methanol (99.8%), ethanol (99.8%), ammonium fluoride (99.99%), sodium hydroxide pellets (98%) and iron (II) chloride tetrahydrate (99.99%) were all purchased from Sigma Aldrich. Ultrahigh purity gases were used as received from BOC Gases.</p><!><p>MIL-100, Fe3(OH)(H2O)2O[(C6H3)(CO2)3]2.nH2O, was synthesised following the procedure in Ref. 35. 1,3,5-benzenetricarboxylic acid (1.676 g), dissolved in 1 M aqueous sodium hydroxide (23.72 g), was added dropwise to a solution of iron (II) chloride tetrahydrate (2.260 g) dissolved separately in water (97.2 mL). The green suspension was left to stir at room temperature for 24 hours. The product was recovered by centrifugation, washed thoroughly with ethanol (3×20 mL), and dried overnight at 60 °C. The orange powder was purified following Ref. 36. Briefly, the powder was dispersed and heated for 3 hours in each water (700 mL at 70 °C), ethanol (700 mL at 65 °C) and 38 mM aqueous ammonium fluoride solution (700 mL at 70 °C). The powder was recovered between each stage by centrifugation. The final product was dried overnight at 60 °C.</p><!><p>Fe-BTC was synthesised following the procedure in Ref. 29. Both iron (III) nitrate nonahydrate (2.599 g) and 1,3,5benzentricarboxylic acid (1.177 g) were dissolved in methanol (20 mL each). The two solutions were combined at room temperature and left to stir for 24 hours, forming a viscous orange solution. This was washed with ethanol (3×20 mL) before drying overnight at 60 °C. The powder was then purified as described above and left to dry overnight at 60 °C.</p><!><p>Powder X-ray diffraction data were collected at room temperature using a Bruker D8 diffractometer using Cu Kα1 (λ = 1.5406 Å) radiation and a LynxEye position-sensitive detector with Bragg-Brentano parafocusing geometry. Samples of finely ground powder were dispersed onto low-background silicon substrates and loaded onto the rotating stage of the diffractometer. Data were collected over the angular range 2° < 2θ < 50°. Pawley refinements were carried out using TOPAS Academic (V6) software. 37 The unit cell parameters were refined against those previously reported for MIL-100. 17 A modified Thompson-Cox-Hasting pseudo-Voigt peak shape and simple axial divergence correction were employed.</p><!><p>Gas adsorption isotherms and kinetic profiles were measured using a Quantachrome iQ2 instrument. Prior to measurement, samples were degassed at 150 °C for 12 hours. Sample masses were measured using degassed samples after the sample tube was backfilled with N2. Sample temperatures were accurately equilibrated at 273 K and 293 K using a temperature-controlled water bath and at 77 K using a Dewar filled with liquid N2. Under these conditions, MIL-100 and Fe-BTC are expected to contain coordinatively unsaturated Fe(III) sites with little to no partial reduction to Fe(II) occurring. See Supplementary Methods for details on the surface area, non-local density functional theory, virial, and ideal adsorbed solution theory analyses.</p><!><p>Samples of MIL-100 and Fe-BTC were prepared following previously reported procedures (see Experimental Methods for details). Powder X-ray diffraction measurements confirmed the crystalline nature and phase purity of MIL-100 [Figs. S1]. The diffraction pattern for Fe-BTC did not contain sharp Bragg scattering. Instead, very broad regions of weak scattering were observed, consistent with its lack of Please do not adjust margins Please do not adjust margins long-range order yet possession of trimer-based local structure similar to that present in MIL-100. 29 The N2 adsorption isotherm for MIL-100 at 77 K displayed the expected intermediate Type I and IV behaviour indicative of the presence of both micro-and mesopores with a secondary uptake at approximately 0.12 P/P0, a signature of the dual-pore architecture [Fig 3]. 17 The BET surface area was 1,465 m 2 g −1 , with a maximal uptake of 479.4 cm 3 g −1 [Table S2]. Fe-BTC, however, exhibited an almost negligible maximal uptake of 42.1 cm 3 g −1 and hence the BET surface area (68 m 2 g −1 ) cannot readily be regarded as reliable.</p><!><p>Non-local density functional theory can be used to extract the pore size distribution from an adsorption isotherm [Figs. S2 & S3]. This approach can be informative, despite the limitations discussed in the Supplementary Methods. The pore size distribution for MIL-100 had maxima at 9.5 and 16.9 Å, while Fe-BTC possessed maxima at 9.1 and 17.3 Å [Figs. S4 & S5]. The pores in Fe-BTC were present in a significantly lower amount than in MIL-100, hence the lower porosity of Fe-BTC. Notably, the contribution of the larger pore cavity was appreciably less in Fe-BTC than in MIL-100, suggesting the porous interior of Fe-BTC is largely comprised of the smaller pore structure. This pore cavity has a smaller crystallographic window aperture of 5.5 Å (c.f. 8.6 Å aperture in the larger pore). It is the ordered network structure of MIL-100 that facilitates uptake and diffusion of guest molecules through the pores and connected apertures. In contrast, the topologically disordered nature of Fe-BTC disrupts the accessibility of the pores and results in a decrease in accessible surface area towards N2 at this temperature. This has also been observed in ZIFs and their melt-quenched glass counterparts. 38 These results suggest that the porous interior of Fe-BTC is inaccessible to N2 at 77 K. Measurement of the N2 isotherms at 273 K revealed that Fe-BTC is not a dense, non-porous material [Fig. S6]. At 77 K there is an 11-fold decrease in the maximal uptake of N2 in Fe-BTC compared to MIL-100. Whereas at 273 K this is greatly reduced to a factor of only 1.4. This is symptomatic of activated diffusion, where N2 molecules cannot successfully diffuse through Fe-BTC at 77 K but can overcome this energetic barrier at higher temperatures. 39 Motivated by this, we further investigated the porous nature of these two materials by measuring a series of gas sorption isotherms (H2, CO2, Xe, Ar, CH4, C2H4, C2H6, C3H6 and C3H8) at 77, 273 or 293 K up to 100 kPa [see Table S3 for analyte properties].</p><!><p>Pure isotherms of H2 were measured at 77 K for MIL-100 and Fe-BTC [Fig. 4]. The initial uptake was similar in both materials up to 20 kPa, exhibiting rapid adsorption kinetics. 40 The H2 isotherms deviate at higher pressure where the larger surface area of MIL-100 allows for greater uptake of H2. At the highest pressure, the maximal uptake of MIL-100 was 110.7 cm 3 g −1 , and 74.7 cm 3 g −1 for Fe-BTC, though neither isotherm reached saturation at the pressures studied here [see Table 1 for maximal uptakes]. Hence the extent of adsorption correlates with the guest molecule binding strength to the framework between 0 to 100 kPa.</p><p>Gas sorption of CO2, with its highly polar bonding, is a simple way to probe a MOF's textural properties [Fig. 4]. MIL-100 displayed a maximal uptake of 64.4 cm 3 g −1 at 273 K, while Fe-BTC adsorbed 35.4 cm 3 g −1 in comparison. This further demonstrates that Fe-BTC is indeed capable of adsorption and is not dense, nor does it possess a collapsed porous interior. They both exhibited mild hysteresis upon desorption, indicating the adsorption and desorption branches are not in equilibrium.</p><p>The BET surface areas derived from these isotherms were 127 and 77 m 2 g −1 for MIL-100 and Fe-BTC, respectively [Table S4]. 41 These values represent the lower limit of accessible surface area, however they are nonetheless useful, comparative values given the limited utility of the N2 isotherms at 77 K. These results are further diagnostic of activated diffusion occurring in Fe-BTC with the higher temperature of 273 K enabling diffusion of CO2 through the structure. Hence, the ratio of maximal uptakes for CO2 is more comparable to N2 at 273 K than 77 K. Please do not adjust margins Please do not adjust margins 2]. 18 This is consistent with stronger interactions occurring in Fe-BTC due to confinement of the CO2 molecules within the smaller pore cavity compared to MIL-100. Qst decreases at higher loadings of CO2 in both materials.</p><!><p>Pure isotherms for Xe and Ar were measured at 273 K for MIL-100 and Fe-BTC [Fig. 5]. The Xe isotherm for MIL-100 was almost linear in the pressure range studied, with a maximal uptake of 43.1 cm 3 g −1 , comparable to previous reports. 42 Fe-BTC had a maximal Xe uptake of 20.1 cm 3 g −1 . The Xe isotherm for Fe-BTC began to plateau at higher pressures, indicating it was approaching saturation. Such behaviour is consistent with reduced pore space available in Fe-BTC compared to MIL-100. Fe-BTC displayed large hysteresis upon desorption, suggesting restricted diffusion of Xe through the structure, compared to the more accessible pores of MIL-100. Both materials exhibited similar sorption of Ar, with nearlinear adsorption and no hysteresis upon desorption. The maximal adsorption to MIL-100 was 4.4 cm 3 g −1 whilst Fe-BTC adsorbed 4.0 cm 3 g −1 ; these values are comparable to that of ZIF-8 and HKUST-1. 43 MIL-100 and Fe-BTC's maximal Ar uptakes are considerably lower than for Xe, which exhibits a greater adsorption strength due to its higher boiling point and results in the hysteresis upon desorption for Fe-BTC. The smaller size of Ar enables easier diffusion through the structure leading to no hysteresis being observed.</p><!><p>Gas sorption isotherms of five short-chain hydrocarbons (CH4, C2H4, C2H6, C3H6 and C3H8) were collected at 273 K [Fig. 6 & 7]. In MIL-100, maximal uptakes at 100 kPa were primarily dictated by the hydrocarbon chain length (increasing C1 < C2 < C3) due to the higher boiling points of the longer chain molecules resulting in stronger adsorbate-adsorbent interactions. Conversely, Fe-BTC did not follow this trend, instead exhibiting some interesting adsorption behaviour.</p><p>In both MIL-100 and Fe-BTC, CH4 exhibited the lowest maximal uptakes, adsorbing 10.8 and 10.6 cm 3 g −1 , respectively [Fig. 6a & b]. Neither material exhibited hysteresis and both isotherms were near-linear, together indicating that the pores were far from saturation. An additional isotherm of CH4 was collected at 293 K for MIL-100 and Fe-BTC [Figs. S12 & S13]. In both materials the maximal uptake of CH4 was reduced at the higher temperature. Virial analysis revealed Qst values at nearzero coverage of 11. S6]. Qst values for CH4 remained almost constant as the adsorbate loading increased.</p><p>In MIL-100, C2H4 exhibited steeper initial uptake than C2H6 up to 50 kPa [Fig. 6a]. As pressures increased, the C2H4 and C2H6 isotherms intersect and at 100 kPa the maximal uptake of C2H6 (70.7 cm 3 g −1 ) was marginally higher than C2H4 (63.0 cm 3 g −1 ). Neither C2H4 nor C2H6 displayed hysteresis upon desorption in MIL-100. This inability to discriminate between C2H4 and C2H6 is very common due to the similar physical properties of the two gases. 7 In Fe-BTC, C2H4 also showed steeper initial uptake than C2H6 at low pressure [Fig. 6b]. The maximal uptakes of C2H4 and C2H6 were 32.5 and 28.7 cm 3 g −1 , respectively. Upon desorption, Fe-BTC exhibited hysteresis with C2H6 but not C2H4. The steeper</p><!><p>Temperature (K) MIL-100 (cm 3 g −1 )</p><p>Fe-BTC (cm Please do not adjust margins Please do not adjust margins initial adsorption of C2H4 in both materials occurs due to unsaturated metal sites that possess a greater affinity towards the unsaturated hydrocarbons at low pressure. C2H6 is only slightly larger than C2H4 (c.a. 0.2 Å), and both have similar polarizability. Together this results in very similar maximal uptakes of C2H4 and C2H6 in both materials. However, the slightly larger size of C2H6 results in diffusion limitations through the disordered Fe-BTC structure. Hence, appreciable hysteresis is observed upon desorption of C2H6 in Fe-BTC, but not in the more accessible MIL-100 pore network. The most interesting observations were made with the adsorption of C3H6 and C3H8. In MIL-100, C3H6 exhibited steeper initial uptake than C3H8 at low pressure [Fig. 7a]. At the highest pressure, the maximal uptakes of C3H6 (147.4 cm 3 g −1 ) and C3H8 (141.7 cm 3 g −1 ) were almost identical, and neither isotherm demonstrated hysteresis upon desorption, similar to previous reports. 44 Again, this inability to discriminate between C3H6 and C3H8 is very common amongst most MOFs. 7 Strikingly, Fe-BTC demonstrated very different sorption behaviour. C3H6 showed significantly steeper initial uptake than C3H8, suggesting very different interactions upon initial adsorption; the maximal uptakes of C3H6 and C3H8 were 44.0 and 18.8 cm 3 g −1 , respectively [Fig. 7b]. Unambiguous discrimination exists between the two gases, with over twice the amount of C3H6 adsorbed than C3H8 in Fe-BTC. This level of discrimination is uncommon in the crystalline MOF domain and remains even rarer amongst disordered materials. Both gases exhibited hysteresis upon desorption. Similar to C2H4, the steeper initial adsorption of C3H6 in both materials is due to the increased interaction between the unsaturated metal sites and the unsaturated molecules. The marginally larger size and very similar polarizability of C3H8 compared to C3H6 has little effect in MIL-100, with near-identical maximal uptakes of the two. However, the disordered structure of Fe-BTC, predominantly containing the smaller pore cavity with a window aperture of only 5.5 Å, results in a pronounced magnification of the different physical properties of C3H6 and C3H8. This enables a significantly higher maximal uptake of C3H6, which can more freely occupy the pore space of Fe-BTC, at 100 kPa. We hypothesize that the larger kinetic diameter of C3H8 is comparable to the window aperture size of the small pore, which leads to substantial diffusion restrictions and hysteresis upon desorption.</p><p>Motivated by these results, additional C3H6 isotherms were collected at 293 K [Figs. S7]. Further highlighting the high affinity of Fe-BTC for C3H6.</p><p>We measured the kinetics of the uptake of C3H6 by MIL-100 and Fe-BTC [Fig. 8]. In MIL-100, we observed rapid adsorption kinetics with saturation being obtained in under three minutes. This is due to the large pores and apertures enabling unhindered diffusion. Fe-BTC exhibited significantly slower uptake of C3H6 compared to MIL-100, reaching saturation over a time of around 75 minutes. The slower diffusion of C3H6 in Fe-BTC is consistent with our structural model of this material, which contains tortuous diffusion pathways with bottlenecks and apertures that are comparable to the kinetic diameter of C3H6. We speculate that there is potential for the kinetic discrimination of C3H6 and C3H8 by Fe-BTC based on the additional kinetic barriers that are encountered by the latter.</p><!><p>The topological disorder in Fe-BTC has a large impact on its adsorption properties. Given the technical challenges associated with measuring adsorption isotherms of gaseous mixtures, ideal adsorbed solution theory (IAST) was employed to quantify the thermodynamic gas selectivities in MIL-100 and Fe-BTC. 45,46 Specifically, the selectivities towards 50:50 binary mixtures of CO2/N2, CH4/N2 and CO2/CH4 were investigated at 273 K. These mixtures are industrially relevant for the processing of flue gases, biogas purification, and natural gas purification. 45 Initially, each pure-component adsorption isotherm was fitted to one of four models before being used to calculate the thermodynamic selectivities of MIL-100 and Fe-BTC [See Supplementary Methods & Fig. S22 & Table S8].</p><p>In MIL-100, the CO2/N2 selectivity was 55.7 at 1 kPa, which increased by a factor of 2.5 to 139.7 at 100 kPa, while Fe-BTC had an initial selectivity of 67.8 that increased by a factor of almost 2.6 to 174.8 at 100 kPa [Fig. 9a]. This favourable adsorption of CO2 over N2 relates to the activated diffusion phenomenon discussed previously and the reduced impact of the molecular sieving effect experienced by CO2. Our results for MIL-100 are approximately four times higher than that previously reported; however, this is likely due to the lower experimental temperatures used here. 45 The adsorption of CH4 and N2 is equally competitive, and the resulting selectivity was an order of magnitude smaller than for CO2/N2 [Fig. 9b]. The CH4/N2 selectivity for MIL-100 was 3.3 at 1 kPa and 2.5 at 100 kPa, while Fe-BTC was 7.2 at 1 kPa and 5.0 at 100 kPa. Neither material exhibited pressure dependence.</p><p>The CO2/CH4 selectivity for MIL-100 was 21.8 at 1 kPa and 17.4 at 100 kPa, consistent with previous reports [Fig. 9c]. 45 Fe-BTC had a CO2/CH4 selectivity of 12.8 at 1 kPa and 11.7 at 100 kPa. Again, neither material showed significant pressure dependence. The slightly enhanced selectivity in MIL-100 is consistent with the larger, relative Qst value derived for CO2 with respect to CH4 for MIL-100 compared to Fe-BTC, which results from the large quadrupole moment of CO2.</p><p>Due to the adsorption and desorption branches of Fe-BTC's C3H6 isotherm not being at equilibrium, we avoided performing IAST analysis on these data to examine its thermodynamic selectivity towards C3H6/C3H8 mixtures.</p><!><p>As perhaps anticipated, the giant pore architecture and high porosity of crystalline MIL-100 mean that it exhibits a higher maximal uptake for many gases compared to its disordered counterpart, Fe-BTC. However, the absence of long-range order in the disordered material effects higher affinity towards certain gases, such as CO2 and CH4. It also leads to the emergence of highly sought after C3H6/C3H8 discrimination capabilities and highlights the prospective utility of disordered MOFs in the field of gas sorption. Please do not adjust margins Please do not adjust margins Critically, our study has established that while the ordered nature of MIL-100 facilitates accessibility to the larger internal pore network, the presence of substantial structural disorder, as in Fe-BTC, may impart powerful separation abilities on the framework. The balance between order and disorder in MOFs as a route to augment their sorption properties is an appealing avenue for future investigation. Two potential routes to tune the interplay between selectivity and capacity in these materials include (i) the progressive incorporation of defects into MIL-100 to introduce disorder or (ii) adjusting the synthesis of Fe-BTC to retain a greater degree of porosity.</p><p>The number of disordered, functional MOF structures is rapidly growing; simultaneously, computational methods further accelerate the discovery of new disordered materials, 23 and databases set up to store the few characterised amorphous MOF configurations. 47 In the future, we anticipate the curation of disordered MOF structural databases, much like the crystalline CoRE MOF database, that can be screened for desirable properties. 48 Ultimately, this will enable us to survey the broad synthetic landscape of disordered MOFs in the search for high-performance materials in applications such as hydrocarbon separation.</p><p>While we have used the MIL-100 and Fe-BTC pairing here as an important demonstration that structural disorder can enhance the gas sorption properties of MOFs, we are certain that this behaviour extends beyond these two specific materials. The use of structural disorder within MOFs as a general tool to enhance gas storage and separation abilities remains widely underappreciated, which we believe hinders the realisation of the full potential of this fascinating class of materials.</p><!><p>A.F.S. and T.D.B. designed the project. A.F.S. synthetised the samples and collected the powder X-ray diffraction data. C.W.A., L.K.M. and S.J.L collected the gas sorption data. A.F.S. analysed the data with the help of S.G.T. A.F.S. wrote the manuscript with the input of all authors.</p>

ChemRxiv

Application of a Trifunctional Reactive Linker for the Construction of Antibody-Drug Hybrid Conjugates

A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin \xce\xb14\xce\xb21-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin \xce\xb14\xce\xb21. The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.

application_of_a_trifunctional_reactive_linker_for_the_construction_of_antibody-drug_hybrid_conjugat

<p>The development of antibody-drug conjugates has been undertaken for the treatment of cancer, in part because this approach may improve selectively and pharmacokinetics (PK).1,2 Tissue-specificity and increased serum half-life are typically governed by the antibody component, while the cytotoxic or radioactive drug cargo provides the therapeutic effect. Alternate immunoconjugates, termed "chemically programmed antibodies" (cpAbs), have also been described that employ cell-targeting by the drug cargo rather than by the antibody.3–5 An important advantage of cpAbs over traditional immunoconjuates is that a single antibody can be directed to multiple targets via conjugation to different antigen-specific peptides or small molecules. Such an approach expands the versatility of a given antibody while endowing the small molecule with the effector functions and PK characteristics of an antibody.</p><p>To broaden the scope of immunoconjugate-based chemotherapy, we recently reported a genre of cpAbs that does not require antibody-variable domains.6 Instead, while the antigen-specific small molecule provides target specificity, an IgG1-derived Fc fragment improves the PK properties of the small molecule and allows alternative routes of administration such as interaction with the neonatal Fc receptor (FcRn).6 In addition, an engineered C-terminal selenocysteine (Sec) residue on the Fc protein (Fc-Sec) insures site-specific attachment of a single drug molecule. To achieve this, we designed a flexible trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker that simultaneously allows cell targeting, regiospecific conjugation to the Fc protein and conjugate detection. For cell-targeting we employed LLP2A 1 (Figure 1) a recently developed peptidomimetic that binds with high affinity and specificity to the cell-surface protein integrin α4β1 (IC50 = 2 pM).7</p><p>Integrin α4β1 has been shown to promote metastasis and angiogenesis in a variety of cancers, and it plays a key role in the onset of drug-resistance that can lead to relapse following chemotherapy for acute myelogenous leukemia (AML).8–10 Although targeting integrin α4β1 is not without its risks,11 studies suggest that integrin α4β1 antagonists may be particularly valuable therapeutic agents for the treatment of hematologic malignancies, such as multiple myeloma and AML.10,12 We wondered whether conjugation of LLP2A to Fc-Sec could overcome undesirable PK characteristics of LLP2A while maintaining its potency and selectivity.6,13</p><p>In conjugating 1 to Fc-Sec the linking segment needed to be sufficiently long to allow 1 to bind to integrin α4β1 without steric interference from the relatively large Fc protein. For this reason PEG-SU was chosen because it can be extended in a modular fashion depending on the number of PEG-SU units employed. Additionally, the N-Fmoc and N-Boc protected forms of PEG-SU can be used in solid-phase syntheses.14 A PEG-SU dimer was utilized in the Fc-Sec-LLP2A conjugate, since it had been previously shown that 1 retains its affinity for integrin α4β1 when indirectly coupled to streptavidin through a biotin attached via this linker.7 The versatility of PEG-lysine-based linkers is also known.7,15 For our purposes, two lysine residues were introduced at the C-terminal end of the PEG-SU spacer to provide primary amines as attachment points for (1) auxiliary functionality and (2) an alkylating agent that could be used for conjugation to the Sec residue of the Fc protein. Inclusion of biotin as the auxiliary functionality yielded the prototype construct LLP2A-(PEG-SU)2-Lys(Nε-Biotin)-Lys(Nε-R)-amide (2), where R would be the nucleophile acceptor suitable for conjugation with the Sec residue of the Fc protein (Figure 1).6, 16</p><p>The solid-phase preparation of final products 2b – 2e (Figure 1) required the previously reported acids 3 – 5 (Figure 2).7 Coupling of Nε-Mmt-Nα-Fmoc-L-Lys to Rink amide MBHA resin followed by Nε-biotin-Nα-Fmoc-L-Lys using standard Fmoc protocols provided the intermediate 6 (Scheme 2). Two subsequent coupling cycles with N-Fmoc-PEG-SU (3) gave the resin-bound intermediate 7, onto which was constructed the LLP2A sequence.7 Treatment of the resulting resin 8 with 1% TFA in CH2Cl2 removed the acid-labile Nε-Mmt-Lys group without cleavage from the resin. Subsequent acylation of the resulting free amine followed by resin cleavage (95% TFA) yielded the peptides 2a – 2e. However, it was found that the N-haloacetamide acylation products proved difficult to obtain in pure form. Iodoacetamide-containing 2b could not be prepared using standard protocols,17,18 and although 2c could be prepared in low yield, it could not be obtained in pure form, even following preparative reverse-phase HPLC.</p><p>In contrast to 2b and 2c, peptide 2d was obtained with relatively few side products. One significant side product resulted from deletion of the Aad residue from 2d (approximately 30%) due to incomplete coupling of residues amino-proximal to the sterically hindered aminocyclohexanecarboxylic acid (Ac6c). However, pure 2d could be obtained through a two-stage protocol that involved the initial solid-phase synthesis of 2a followed by a final solution-phase coupling step to yield 2d. This two-stage route provided pure 2d in sufficient quantity for cell-based assays. A more efficient solid-phase route resulting in improved coupling of residues amino-proximal to the Ac6c, was achieved using 2-(1H-7-azabenzotriazol-1-yl)-tetramethyluronium hexafluorophosphate (HATU).19 Introducing a capping cycle (1-acetylimidazole) also facilitated the separation of deletion by-products (Scheme 2).</p><p>The chemoselective alkylation of Sec residues by 2d was examined using a variety of Fc constructs. Inclusion of a biotin handle within 2d allowed facile detection of Fc covalent adducts by avidin pull-down experiments followed by ELISA visualization. This showed that conjugation only occurred when the Sec residue was present in the Fc protein (Figure 3A).6,20 The Fc–Sec–2d conjugate was then incubated with integrin α4β1-expressing cells. The cells were washed and then treated with Cy5-labeled rabbit anti-human IgG. Under these conditions, cells would only fluoresce if both the Fc and LLP2A components of the hybrid construct were present. As shown in Figure 3B, cells treated with the Fc–Sec–2d conjugate were detected by flow cytometry while cells treated with Fc-Stop (Fc protein minus the Sec residue) or 2e alone, did not exhibit such dual specificity (Figure 3B).21</p><p>Competition experiments with an anti-integrin α4 mAb (purchased from Serotec) indicated that the Fc–Sec–2d hybrid and the mAb exhibit identical/overlapping epitopes as evidenced by the diminished binding of Fc–Sec–2d to lymphoma cells in the presence of the Serotec mAb (Figure 3C).22 In contrast, the binding of an anti-integrin α4 mAb from a different source (R&D Systems) could not be competed with the Serotec mAb, suggesting that the two mAbs recognize different epitopes on integrin α4β1. These results indicate that the binding of the Fc–Sec–2d hybrid to the cells examined is mediated by the peptidomimetic LLP2A (1) and not by the Fc protein portion, and that the targeting specificity of the parent peptidomimetic for integrin α4 is retained in the Fc–Sec–2d hybrid.</p><p>In conclusion, the trifunctional linker A–(PEG-SU)nLys(Nε–B)Lys(Nε–C), where A is a targeting moiety, B is auxillary functionality such as a fluorescent tag and C is a nucleophile acceptor, is well-suited for the preparation of immunoconjugates in which a small synthetic molecule governs cell-targeting.3,5 In the current study, this linker allowed the conjugation of a LLP2A-biotin construct to Fc–Sec in a chemoselective manner. Subsequent in vitro analysis revealed that all three components of the linker system—targeting agent (1), tag (biotin), and antibody fragment (Fc–Sec), were fully functional, with the affinities of the parent 1 and Fc protein for integrin α4β1 and Fc receptor respectively, being retained.6 The trifunctional PEG-SU-Lys-Lys-maleimide linker may have more general utility as an organic tether for the construction and evaluation of antibody-drug conjugates.</p>

PubMed Author Manuscript

Timescales of Coherent Dynamics in the Light Harvesting Complex 2 (LH2) of Rhodobacter sphaeroides

The initial dynamics of energy transfer in the light harvesting complex 2 from Rhodobacter sphaeroides were investigated with polarization controlled two-dimensional spectroscopy. This method allows only the coherent electronic motions to be observed revealing the timescale of dephasing among the excited states. We observe persistent coherence among all states and assign ensemble dephasing rates for the various coherences. A simple model is utilized to connect the spectroscopic transitions to the molecular structure, allowing us to distinguish coherences between the two rings of chromophores and coherences within the rings. We also compare dephasing rates between excited states to dephasing rates between the ground and excited states, revealing that the coherences between excited states dephase on a slower timescale than coherences between the ground and excited states.

timescales_of_coherent_dynamics_in_the_light_harvesting_complex_2_(lh2)_of_rhodobacter_sphaeroides

<p>Understanding the molecular mechanisms underpinning energy transfer events in photosynthetic complexes has been of long standing interest, for both technological and pedagogical purposes.1–3 The light harvesting complex 2 (LH2), a peripheral antenna complex from purple bacteria, has been heavily studied by a wide variety of experimental and theoretical techniques.4–6 The LH2 complex is composed of 27 bacteriochlorophyll a chromophores arranged circularly in two concentric rings, shown in Figure 1.7 The inner ring contains eighteen strongly interacting chromophores known as the B850 chromophores due to their prominent absorption at 850 nm. The outer ring of nine more weakly interacting chromophores comprises the B800 ring, which contribute to the absorption at 800 nm. A large variety of spectroscopic methods have successfully mapped out the energy landscape8, 9, the timescales of the relaxation dynamics following photoexcitation,10, 11 as well as characterizations of the electronic environment of the surrounding protein.12, 13 Detailed modeling of these various experiments has allowed for a detailed understanding of the dynamics, however the experimental energy transfer timescales can be reproduced by both modified Redfield theories as well as multichromophoric Förster resonance energy transfer (FRET).14–16 These two models provide strikingly different pictures of the underlying dynamics.</p><p>More recently the question of the presence of coherent electronic motion in this system has started to be addressed, which may help distinguish the more correct relaxation mechanism. A model that is consistent with a wide variety of spectroscopic observables revealed that the fast decay in pump probe anisotropy experiments conducted on the 800 nm region of the spectrum could be attributed to excitonic dephasing, as opposed to relaxation dynamics as previously assigned.14 This result indicates that energy transfer between the relatively weakly interacting chromophores in the B800 ring may not be adequately described by a FRET like mechanism as had been formerly thought. Two-dimensional electronic spectroscopy (2DES) experiments have observed coherent oscillations between regions of the two broad absorption bands, revealing a rich interplay between the dephasing dynamics and coherent electronic motion.17 The persistence of coherent electronic motion has now been observed in a wide variety of photosynthetic complexes,18–21 suggesting that it may be a common feature present in these systems.</p><p>In this letter, we extend the previous results through a coherence specific variant of two-dimensional spectroscopy. By controlling the polarization of the incident laser pulses, we selectively detect signals which evolve as a coherence (superposition) between two states with differing dipole moment directions.22–24 This particular method is well suited to studying coherent evolution which is difficult to distinguish with other methodologies such as pump-probe spectroscopy. Two color photon echo techniques19, 25 as well as pump-probe anisotropy methods14, 26 and two-dimensional experiments18 are also strong probes of coherent dynamics. The coherence-specific polarization scheme has been implemented previously to study the molecular structure of a small dipeptide in solution22 as well as coherent dynamics in the LHCII complex of photosystems II24 and the reaction center of purple bacteria.27 The coherence specific polarization sequence is given by π/4, − π/4, π/2, and 0 radians for pulses one, two, three, and the local oscillator, respectively.22 Coherences involving states with parallel or anti-parallel transition dipoles cannot be observed with this methodology. Coherent vibrational motion could also contribute to this signal, provided that the transition dipole direction is dependent on nuclear coordinates. Such non-Condon effects have been observed in photosynthetic antenna complexes containing phycocyanobilin pigments, where high frequency out-of-plane vibrational modes were measured in transient absorption anisotropy experiments.28</p><p>To acquire the coherence-specific data, we utilize a single-shot variant of 2DES called GRadient Assisted Photon Echo (GRAPE) spectroscopy.29, 30 In this experiment, we create a temporal gradient between the first two pulses across a homogeneous sample. An imaging spectrometer captures and resolves the temporal gradient thereby acquiring an entire two-dimensional map in parallel. GRAPE reduces the need for long term phase stability and power stability by reducing the acquisition time by two orders of magnitude. The inclusion of three half-wave plates in the paths of beams 1–3 allows complete control of the polarization pulse sequence. We generate broadband optical pulses by focusing the output of a regenerative amplifier into Argon at ~1.3 atm generating spectrally broadened pulses centered at 800 nm with ~ 100 nm of bandwidth. This spectrally broadened pulse was compressed with an SLM based pulse shaper (Biophotonics Solutions) to 15 fs (FWHM). The waiting time was sampled from −100 to 500 fs in 10 fs steps. The sample was isolated and prepared as previously described.31 The peak absorbance at 850 nm was ~0.2 OD in a 200 µm length cell. All experiments were conducted at room temperature. Due to uncertainties in the relative phases of our excitation pulses, we report only the absolute value of our signal. The GRAPE spectrometer acquires only the rephasing portion of the third order optical response.</p><p>The coherence-specific 2D spectra are shown in Figure 2. At early waiting times, the presence of signal simply reveals the presence of a superposition state. The identity of the coherence can be determined by examining the location of the signal in the 2D map, and the dephasing of that particular coherence is resolved by examining the signal with increasing waiting time. Two diagonal features are resolved, corresponding to the two bands centered at 850 nm and 800 nm in the linear absorption spectrum. The spectra show intense features on the diagonal even though theory predicts the rephasing portion of the coherence signal to be located off the diagonal. We attribute this shift to dispersive contributions to the signal, which broaden the peaks in the absolute value of the signal. Cross-peaks between the two main features are also clearly resolved. As the waiting time is increased, the system relaxes due to interactions between the chromophores and the surrounding protein matrix, spurring dephasing. The signal rapidly decays with increasing waiting time, most predominantly in the lower energy band and cross peaks between the two bands. Unlike the conventional all parallel polarization scheme, contributions from populations are highly suppressed in the coherence-specific scheme allowing us to directly Fourier transform the full complex data. In this fashion, we can distinguish positive from negative frequencies. Traces of the Fourier transforms of the cross peak between the features at 800 nm and 834 nm reveal frequencies centered at the difference between the axes and rotating in opposite directions. This effect is not predicted by the theory of a purely vibrational coherence, leading us to conclude that the coherence is of an electronic origin.32, 33 The magnitudes of the traces are roughly equal and are consistent with theoretical predictions. Integration over the λτ and λt domains reveals the average timescales of dephasing present in the system.24 The total signal undergoes a fast initial decay followed by a slower decay, with the signal nearly vanishing by 500 fs. The signal is well modeled by the sum of a Gaussian, exponential, and constant offset. The parameters of the fit are listed in table 1.</p><p>We have utilized a Frenkel-excitonic framework to model the absorption spectrum of the complex and to connect the spectroscopic signatures to the molecular structure. Material parameters for the site energies, couplings, and spectral densities were taken from previous studies of the complex.12, 13, 15, 34 Complete details of the model are provided in the supplemental information section. Our model provides a reasonable fit to both the absorption spectrum of the native complex as well as a modified complex which lacks the B800 chromophores. We then classify states based on their probability amplitude and group states into three categories. The first two categories are states that are > 90% localized on either the B850 or B800 chromophores, and the third category is comprised of states that are delocalized across both rings. Calculations of the absorption spectrum of these states then reveals that the B850 pigments largely absorb at 850 nm along with a lower wavelength tail that extends past the 800 nm feature. The 800 nm region of the spectrum is predominantly comprised of excitations that are localized on the B800 pigments or mixed states involving both rings of chromophores, in agreement with previous studies.14</p><p>Two-dimensional slices through different values of λτ are shown in Figure 3. We see that coherent oscillations occur throughout the entire spectral region and closely follow the energy difference between the axes. A slice through λτ = 774 nm reveals that coherence is maintained between the highest energy region of the 800 nm band and the 850 nm band. This coherence can be definitively assigned to coherence between lower and higher lying electronic excitations on the inner B850 ring. Lower frequencies within the 850 nm region are also observed, revealing that coherence within the B850 chromophores is maintained at short times. Because the contribution to the absorbance at 800 nm from the B850 chromophores is minor, we preliminarily attribute this feature to the B800 chromophores. This assignment is strengthened by the significantly smaller intensity of the signal in the lower wavelength region which we can unambiguously attribute to the B850 ring. Our data reveals that the coherent signal is more intense within the individual absorption bands, but a weaker signal is still present connecting the two bands. The timescales that we extract are in reasonable agreement with previous studies conducted at 77 K. For the 800 nm region, the 177 fs decay that we observe at room temperature is faster than the 300 – 500 fs timescale extracted from anisotropy experiments which were conducted at 77 K.14 Similarly, the initial decay of anisotropy within the 850 nm region at 77 K was found to be significantly faster with a timescale of roughly 60 fs compared with our Gaussian decay time of 15 fs from our experiment. The increased temperature, yielding amplified thermal motion, most likely explains the increased dephasing rates.</p><p>A complete simulation of the dephasing dynamics would require consideration of the full nonlinear response, which is non-trivial. Instead, we present a minimal theoretical framework to provide a qualitative understanding of the measured dynamics. Within the secular approximation, which decouples populations and coherences, we can approximate the evolution of the coherence between excited states i and j with the following equation:35–37 (1)ρji(t)∝〈exp[−iωjit−gjj(t)−gii(t)+2Re(gij(t))−(kj+ki)t]〉. Here ωij is the difference in energy between the states i and j divided by the reduced Planck's constant, gjj(t) is the lineshape function which describes the dephasing induced by interactions with the bath for state j, gij(t) is the lineshape function which describes the extent to which the fluctuations induced by the bath between the states i and j are correlated, and ki is the rate of population loss of the state i. The angular brackets indicate an average over disorder in the system, reflecting the ensemble nature of the measurement. The dynamics of this coherence between excited states differs from the observed dynamics of the coherence between the ground state and excited state j, which can be approximated by:35–37 (2)ρjg∝〈exp[−iωjg−gjj(t)−kjt]〉. Here, ωjg is the transition energy between the ground state and the jth excited state. Comparing these two equations reveals that the interactions governing the dephasing of coherences between excited states and coherence between the ground and excited states are largely shared, differing only to the extent that fluctuations (either static disorder or dynamic disorder) are correlated. Correlated fluctuations can arise from excitonic mixing between states as well as from correlated environmental motions.19 Due to the strong interactions between chromophores in the LH2 complex, the dominant contribution to the correlated fluctuations most likely arises from simply excitonic mixing. The electronic states involving the B850 chromophores are highly delocalized across ~4–5 chromophores at room temperature.4 Recent calculations utilizing the hierarchical equations of motion found the presence of coherent electronic motion within the B850 chromophores persisting on a 150 fs timescale at room temperature for a single realization of the Hamiltonian (i.e. neglecting disorder in the system).38 This result suggests that the strong coupling which yields delocalized excitations is the physical mechanism that yields persistent coherent motion on the excited state and confirms that coherence persists longer in a single system when compared to ensemble measurements. Similarly, the 77 K pump-probe anisotropy measurements could also be reproduced without assuming correlated spectral motion.14 We can experimentally measure the dephasing of the ground and excited state coherences through separately acquired all parallel polarization 2DES. Traces through coherence time τ at different emission wavelengths λt then reveal the dephasing dynamics of the ground excited state coherences. This signal is equivalent to a wavelength resolved three pulse photon echo peak shift experiment.39 We compare the dephasing dynamics between these two classes of coherences in figure 4.</p><p>The all parallel 2DES signal oscillates at an optical frequency and fully decays by 150 fs. This dephasing time is rather long when compared to most small molecules in solution at room temperature, which typically dephase by 50 fs. A defining characteristic of protein environments seems to be reduced coupling to the environment.40 The maximum of the signal is shifted from a coherence time of zero due to inhomogeneity present and the rephasing process of a photon echo. The recovered shift of 31 fs is in good agreement with previous measurements.39 The coherence-specific signal shows a similar short 31 fs timescale, as well as a longer lived 177 fs exponential decay, shown by both the integrated intensity and individual traces. The short timescale is associated with coherence between the states at 800 and 850 nm as well as states within the 850 nm band. The longer lived coherence is primarily located within the 800 nm feature, though weaker long-lived features are also present within the 850 nm feature. The lifetime extracted in the coherence-specific experiment is shorter than the previously reported timescale extracted from an all parallel polarization 2DES experiment due to our increased temporal resolution.17</p><p>It is somewhat counter intuitive that coherence persists longer among the more weakly bound chromophores rather than the more strongly interacting chromophores. We interpret this paradox as arising from the ensemble nature of our experiment, meaning the ensemble measurement likely differs from measurements of individual members of the ensemble. Previous photon echo peak shift measurements of the 800 nm and 850 nm spectral regions have revealed that the protein environments are distinct between the two absorption bands.12, 13 The 800 nm spectral region has a reduced overall coupling strength to the bath, on both ultrafast timescales and slower timescales. The slower timescales, present in most proteins, lead to strong wavelength dependence of the beating observed in Figure 3. This ensemble inhomogeneity leads to an artificial dephasing in our measurement that would not be present if decoherence in a single protein complex was measured.41–43 Because the 850 nm spectral region is more inhomogeneous, these slower timescales will most likely lead to an increased dephasing rate. Thus coherence persists longer than the timescale measured in this experiment, and furthermore, the dephasing rate in the 850 nm region is artificially shortened to a greater degree. Although we cannot recover the true decoherence timescale with the current technique without making assumptions to the magnitude of shared fluctuations in equation (1), we do know that our measurement represents a lower bound for the coherence lifetime. We can then compare this lifetime to the coherence lifetime between ground and excited states. We measure the coherence lifetime between ground and excited states using the rephasing process of photon echo signals, which represents an accurate measure of the decoherence time because the echo signal and is largely free from inhomogeneous artifacts. We conclude that coherence among excited states persists on a longer timescale than coherence between the ground and excited states, indicating a strong correlation between the electronic states.</p><p>Utilizing the coherence-specific polarization sequence, we directly observe coherences among all excited states. The persistence of coherence between all states indicates that a purely FRET based model is not consistent with describing the energy transfer dynamics in this system. Due to the inhomogeneous nature of the protein and the rephasing capability of the photon echo signals, we conclude that excited state coherences persist longer than coherences between the ground and excited states. The background-free detection of excited state coherence clarifies the observation of lower frequency oscillations as well as the distinction between positive and negative frequencies, revealing results consistent with excitonic theory.</p>

PubMed Author Manuscript

Downregulation of miR‐33b promotes non‐small cell lung cancer cell growth through reprogramming glucose metabolism miR‐33b regulates non‐small cell lung cancer cell growth

AbstractGlucose metabolism is a common target for cancer regulation and microRNAs (miRNAs) are important regulators of this process. Here we aim to investigate a tumor‐suppressing miRNA, miR‐33b, in regulating the glucose metabolism of non‐small cell lung cancer (NSCLC). In our study, quantitative real‐time polymerase chain reaction (qRT‐PCR) showed that miR‐33b was downregulated in NSCLC tissues and cell lines, which was correlated with increased cell proliferation and colony formation. Overexpression of miR‐33b through miR‐33b mimics transfection suppressed NSCLC proliferation, colony formation, and induced cell‐cycle arrest and apoptosis. Meanwhile, miR‐33b overexpression inhibited glucose metabolism in NSCLC cells. Luciferase reporter assay confirmed that miR‐33b directly binds to the 3′‐untranslated region of lactate dehydrogenase A (LDHA). qRT‐PCR and Western blot analysis showed that miR‐33b downregulated the expression of LDHA. Moreover, introducing LDHA mRNA into cells over‐expressing miR‐33b attenuated the inhibitory effect of miR‐33b on the growth and glucose metabolism in NSCLC cells. Taken together, these results confirm that miR‐33b is an anti‐oncogenic miRNA, which inhibits NSCLC cell growth by targeting LDHA through reprogramming glucose metabolism.

downregulation_of_mir‐33b_promotes_non‐small_cell_lung_cancer_cell_growth_through_reprogramming_gluc

<!>INTRODUCTION<!>Tissue samples and cell lines<!>Quantitative real‐time polymerase chain reaction<!>Cell transfection<!>Cell proliferation assays<!>Colony formation assay<!>Cell cycle analysis<!>Detection of lactate production, glucose consumption, and ATP levels<!>Luciferase reporter assay<!>Western blot<!>Statistical analysis<!>miR‐33b is significantly downregulated in NSCLC tissues and cell lines<!><!>miR‐33b inhibits the growth of NSCLC cells<!><!>miR‐33b regulates glucose metabolism in NSCLC cells<!><!>LDHA is a direct target of miR‐33b<!><!>LDHA expression attenuates the growth inhibitory effect of miR‐33b on NSCLC cells<!><!>LDHA expression attenuates the inhibitory effect of miR‐33b on glucose metabolism in NSCLC cells<!><!>DISCUSSIONS<!>CONCLUSIONS<!>CONFLICTS OF INTEREST<!>AVAILABILITY OF DATA AND MATERIALS<!>ETHICS APPROVAL AND CONSENT TO PARTICIPATE<!>CONSENT FOR PUBLICATION

<p>Zhai S , Zhao L , Lin T , Wang W . Downregulation of miR‐33b promotes non‐small cell lung cancer cell growth through reprogramming glucose metabolism miR‐33b regulates non‐small cell lung cancer cell growth. J Cell Biochem. 2019;120:6651‐6660. 10.1002/jcb.27961 30368888</p><p>Shengping Zhai, Lingyan Zhao, and Tiantian Lin contributed equally to this study.</p><!><p>Lung cancer is the first leading cause of cancer‐related deaths in the US. Non‐small lung cancer (NSCLC) constitutes approximately 85% of the cases.1, 2 Moreover, one‐third of diagnosed patients are suffering from NSCLC of advanced stages (stage III or IV).3 NSCLC is the most lethal form of lung cancer and the majority of patients die within first 5 years of diagnosis. The five‐year survival of NSCLC is only 17%.4 Recurrence occurs in 30%‐60% of patients with NSCLC.5, 6 Current treatment options against NSCLC include surgery, adjuvant therapy, chemotherapy, radiotherapy, and immunotherapy. However, effective treatment of NSCLC is still lacking, particularly for advanced stage cancers.7 Therefore, it is imperative to develop new therapeutics for this cancer.</p><p>MicroRNAs (miRNAs) are small non‐coding RNA of 20 to 24 nucleotides. They bind to the 3′ untranslated region (UTR) of the target gene to induce mRNA degradation.8 Altered expression of miRNAs has been observed in different diseases, including cancer.9 Recent evidence indicate that miRNAs are important cancer regulatory molecules. One mechanism of this regulation is through the mediation of cancer metabolism. Cancer is known for its unique glucose metabolism to support its rapid growth even under extreme conditions, such as hypoxia. As a result, abnormal glucose metabolism promotes cancer invasiveness and metastasis.10, 11 It was shown that several miRNAs play a role in glucose metabolism of cancer. For example, miRNA‐195‐5p is known to directly regulate GLUT3 (Glucose Transporter regulator).12 miR‐32 have been documented to control the expression of the SLC45A3 protein, which functions as glucose transporter.13 miR‐223 is known to upregulate GLUT4 while miR‐133 is known to downregulate its expression.14, 15 Moreover, miR‐23 indirectly regulates translocation of GLUT4 by regulating SMAD416 and translocation of GLUT4 in adipocytes is regulated by miR‐21.17 Several other miRNAs have been reported to regulate the glycolysis. For example, the miR‐143 expression is inversely proportional to the expression of hexokinase 2 (HK2), which mediates aerobic glycolysis.18, 19 miR‐138 regulates HK1.18 One more important mediator of glycolysis in is aldolase A, which was found as a direct target of miR‐122 in liver cells.20</p><p>Here we study miR‐33b, which was shown as an important tumor suppressor in a variety of cancers, including breast cancer,21 osteosarcomas,22 colorectal cancer,23 and lung adenocarcinoma.24 MiR33‐b has been also demonstrated to regulate glucose metabolism.25 However, in lung cancer, the role of miR‐33b has not been studied. The aim of our study was to elucidate the role of miR‐33b in NCSLC and study the exact mechanism of this regulation. We found that miR‐33b inhibits the growth of NSCLC through targeting LDHA, thereby reprogramming glucose metabolism. These results could potentiate miR‐33b as a new therapeutic target in NCSLC.</p><!><p>Paired non‐small cell lung cancer (NSCLC) and adjacent non‐tumor lung tissues from 22 patients were acquired at Yaitai Yuhuangding Hospital affiliated to Qingdao University. This study was approved by the Ethics Committee of Yaitai Yuhuangding Hospital affiliated to Qingdao University and informed consent was obtained from each patient. Four NSCLC cell lines, A549, SPC‐A1, H1299, and H460, and a normal bronchial epithelial cell line (16HBE) were acquired from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA) in humidified incubator maintained at 37 ℃ with 5% CO2.</p><!><p>Total RNA was isolated from cells or tissues using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. cDNA synthesis and quantitative real‐time polymerase chain reaction (qRT‐PCR) reactions were performed using SYBR Green Assay and the ABI PRISM 7500 Sequence Detection System (ABI). The primers for miR‐33b and lactate dehydrogenase A (LDHA) were 5′‐ATTCTTTCGAACTGTCTTGG‐3′ (miR‐33b, forward), 5′‐TTCACCCTCGGCTGTCCTGACA‐3′ (miR‐33b, reverse) and 5′‐TTGGTCCAGCGTAACGTGAAC‐3′ (LDHA, forward), 5′‐CCAGGATGTGTAGCCTTTGAG‐3′ (LDHA, reverse). U6 snRNA was used as a house‐keeping gene for miR‐33b quantification. qRT‐PCR analyses for LDHA and the normalization control gene GAPDH were performed using SYBR Premix Ex Taq (TaKaRa, Dalian, China). Quantification was performed using the 2‐△△Ct method.</p><!><p>miR‐33b mimics (5′‐AGGAU CGGUU UGUGCACA‐3′), miR‐33b inhibitor (5′‐AUCGG AUGUG GUGCA CUA‐3′), negative control (NC) (5′‐AUUUGCCAGG UCGGA AUG‐3′) and inhibitor NC (5′‐AGGUC AAGCA GUUCG UUG‐3′) were designed and synthesized by GenePharma (Shanghai, China). Solutions were dissolved in DEPC at a concentration of 20 μM. Approximately 5 to 6 × 105 cells in logarithmic growth phase were seeded in a six‐well plate, followed by adding medium containing serum and double antibody. miR‐33b mimics, NC and serum‐free DMEM were added to cells to a final concentration of 50 nM. miR‐33b inhibitor, inhibitor NC and serum‐free medium were added to cells to a final concentration of 150 nM. For each group, 12 μL of HiPerFect transfection reagent was added to the samples. After incubation at room temperature for 5 to 10 minutes, the cells were added transfection mixture.</p><!><p>To monitor cell growth, transfected cells were seeded in 24‐well plates (4000 cells per well) in triplicate. At 24, 48, 72, or 96 hours after seeding, the cells were trypsinized and the cell number was counted by a hemocytometer to plot the cell growth curve. In Cell Counting Kit‐8 (CCK‐8, Beyotime) assay, cells were seeded and cultured in 96‐well plates overnight, followed by adding 10 μL of CCK‐8 reagent. After a 2‐hour incubation, the 96‐well plate was placed in a 37°C, 5% CO2 incubator, and the absorbance was measured at 450 nm using a microplate reader.</p><!><p>Cells were seeded in 12‐well plates at a density of 5900 cells per well. Fresh culture medium added every 3 days. After 7 days, crystal violet staining was performed and the numbers of colonies were counted.</p><!><p>Cells were collected by trypsinization and washed twice using cold PBS, followed by fixation in 70% ethanol overnight at 4°C. Cells were subsequently incubated with 20 μg/mL propidium iodide (Sigma‐Aldrich) for 20 minutes at room temperature. Cell cycle analysis was performed with FACS flow cytometry (BD Biosciences, Franklin Lakes, NJ).</p><!><p>Cells were cultured in DMEM without phenol red for 15 hours. The culture media were then harvested, and the lactate and glucose concentrations were measured using a Lactate Assay kit (BioVisionCA) and glucose assay kit (Sigma‐Aldrich), respectively. ATP levels were quantified using a CellTiter‐Glo® Luminescent Cell Viability Assay (Promega, Madison, WI). Protein concentration, measured using a bicinchoninic acid (BCA) protein assay, were used to normalize all lactate, glucose, and ATP measurements.</p><!><p>The 3′‐UTR sequence of wild‐type LDHA and that of a target‐site Mutant (MT) were amplified by PCR, cloned into a dual‐luciferase reporter plasmid (Promega), yielding pGL3‐LDHA–3′‐UTR‐wild‐type (WT) and pGL3‐LDHA‐3′‐UTR‐MT respectively. Cells were inoculated into 96‐well plates at the density of 1.5 × 104 cells per well. Cells were co‐transfected with the WT or MT vector and miR‐33b mimics, NC, miR‐33b inhibitor, or inhibitor NC using the Attractene Transfection Reagent (Qiagen). The ratio of firefly to Renilla luciferase activity was assessed at 48 hours after transfection.</p><!><p>Cells lysed in ice‐cold RIPA buffer (Beyotime, China) supplemented with 10 nM PMSF. The protein samples of equal amount were resolved on 10% SDS polyacrylamide gels and transferred to polyvinylidene fluoride membranes at 100 V for 2.5 hours. 5% fat‐free milk in TBST was used to block the membrane, followed by adding primary antibodies (Abcam, Cambridge) (anti‐LDHA, 1: 500) and incubation at 4°C overnight. Secondary antibodies (1:5000) were added and incubated for 2 hours at room temperature. The protein bands were visualized using the chemiluminescence method (Millipore, MA). Image J software (National Institutes of Health, Bethesda) was used to analyze the protein expression levels. GAPDH (1:1000) was used as the control.</p><!><p>Statistical analysis was performed using the SPSS 17.0 software. Data were expressed as mean ± SD. The independent‐samples t‐test was used for comparisons between two groups. The one‐way ANOVA test, followed by Bonferroni's post‐hoc test, was performed to analyze difference among more than two groups. P values less than 0.05 were considered significant.</p><!><p>The Expression of miR‐33b in 22 NSCLC and adjacent nontumoral normal tissue samples was measured by qRT‐PCR. It was shown that miR‐33b was significantly decreased in NSCLC tissues compared to that in the non‐tumor normal tissues (P < 0.01) (Figure 1A). Assessment of miR‐33b expression in four NSCLC cell lines (A549, SPC‐A1, H1299, and H460) and normal human bronchial epithelial cells (16‐HBE) by qRT‐PCR showed that NSCLC cells exhibited significantly lower miR‐33b expression than 16‐HBE cells (P < 0.05), with the lowest expression levels detected in SPC‐A1 and H1299 cells (Figure 1B). These two cell lines were therefore used for subsequent functional experiments.</p><!><p>miR‐33b is downregulated in human NSCLC tissues and cell lines. A, The expression levels of miR‐33b were analyzed by qRT‐PCR in 22 pairs of NSCLC tissues and adjacent normal tissues. B, MiR‐33b expression levels in four NSCLC cell lines were measured by qRT‐PCR with snRNA U6 levels as an internal control. *P < 0.05, **P < 0.01. miR, microRNA; NSCLC, non‐small cell lung cancer; qRT‐PCR, quantitative real‐time polymerase chain reaction</p><!><p>We then tested the effect of miR‐33b on SPC‐A1 and H1299 cell growth. Firstly, miR‐33b mimics, non‐coding RNA (NC), miR‐33b inhibitor, and inhibitor NC were transfected into SPC‐A1 and H1299 cells and miR‐33b level were assessed by qRT‐PCR. As shown in Figure 2A, the level of miR‐33b in the miR‐33b mimics group was significantly higher than that in the NC group in both SPC‐A1 and H1299 cells (P < 0.01). In addition, miR‐33b expression in the miR‐33b inhibitor group was significantly lower than that in the inhibitor NC group (P < 0.01). Moreover, as shown in Figure 2B and 2C, cells transfected with miR‐33b mimics had suppressed cell proliferation, whereas cells transfected with miR‐33b inhibitor showed increased cell proliferation (P < 0.05, P < 0.01). Consistently, CCK‐8 assay indicated that overexpressing miR‐33b significantly suppressed cell proliferation, and reduction of miR‐33b enhanced that in both SPC‐A1 and H1299 cells (P < 0.01) (Figure 2D). We also confirmed the effect of miR‐33b on the long‐term proliferative capacity of SPC‐A1 and H1299 cells, as evidenced in colony formation assay. As shown in Figure 2E, in both NSCLC cell lines, miR‐33b overexpression significantly inhibited colony formation, while reduction of miR‐33b promoted that (P < 0.01). Furthermore, flow cytometry was used to analyze the effect of miR‐33b on the cell cycle of NSCLC cell lines. It was shown that upregulation of miR‐33b in SPC‐A1 cells led to a significant increase in the cellular population in G0/G1 phase but a sharp decrease in the S phase, while downregulation of miR‐33b in H1299 cells noticeably induced the opposite effect (P < 0.01) (Figure 2E).</p><!><p>miR‐33b inhibits the growth of NSCLC cells. A, miR‐33b expression levels changed after transfection of SPC‐A1 and H1299 cell lines. B, C, The ectopic expression of miR‐33b significantly suppressed or promoted the cell proliferation of SPC‐A1 and H1299 cells in a time‐dependent manner. D, The results of CCK‐8 assay showed miR‐33b mimics suppressed cell proliferation, and inhibition of miR‐33b leads to enhanced cell proliferation. E, The long‐term proliferative capacity of SPC‐A1 and H1299 cells was detected by colony formation. F, The cell cycle analysis in SPC‐A1 and H1299 cell lines was performed using flow cytometry. *P < 0.05, **P < 0.01. miR, microRNA; NSCLC, non‐small cell lung cancer</p><!><p>To explore the role of miR‐33b in glycolysis in NSCLC, differences in metabolic parameters were detected in SPC‐A1 and H1299 cells after transfection. We show that upregulation of miR‐33b in SPC‐A1 cells efficiently reduced glucose consumption (Figure 3A), lactate production (Figure 3B) and ATP levels (Figure 3C), while downregulation of miR‐33b increased these metabolic parameters (P < 0.01). The similar results were also observed in another NSCLC cell line, H1299 cells (P < 0.01) (Figure 3A‐C).</p><!><p>miR‐33b regulates glucose metabolism in NSCLC cells. A‐C, Glucose consumption, lactate production, and ATP levels were detected after SPC‐A1 and H1299 cells were transfected. *P < 0.05, **P < 0.01. miR, microRNA; NSCLC, non‐small cell lung cancer; ATP, adenosine triphosphate</p><!><p>To elucidate the mechanism of miR‐33b regulation in NSCLC, we utilized bioinformatics analysis and identified that LDHA might be a putative target gene of miR‐33b (Figure 4A). To corroborate this, we explored whether miR‐33b had a functional role in regulating LDHA expression. LDHA WT or MT 3′‐UTR was subcloned into a luciferase reporter vector, followed by co‐transfection with miR‐33b mimic or miR‐33b inhibitor into SPC‐A1 and H1299 cells. It was shown that luciferase activity in miR‐33b mimic was significantly lower than that of cells transfected NC in the LDHA‐3′UTR‐WT group (P < 0.01), but that was no significant difference in the LDHA‐3′UTR‐MT group (P > 0.05), while luciferase activity was significantly higher in miR‐33b inhibitor than that for cells transfected with inhibitor NC in the LDHA‐3′UTR‐WT group (P < 0.01), and luciferase activity showed no significant difference in the LDHA‐3′UTR‐ MT group (P > 0.05) (Figure 4B). Furthermore, overexpression of miR‐33b significantly downregulated LDHA mRNA and protein levels, while reduction of miR‐33b significantly upregulated that in both NSCLC cell lines (P < 0.01) (Figure 4C, D).</p><!><p>LDHA is a direct target of miR‐33b. A, Predicted binding between miR‐33b and the seeds matched in the 3′‐UTRs of LDHA. B, Luciferase assay in SPC‐A1 and H1299 NSCLC cells. C, LDHA mRNA levels were analyzed after miR‐33b transfection by qRT‐PCR. D, miR‐33b transfection affects LDHA protein levels. **P < 0.01. miR, microRNA; NSCLC, non‐small cell lung cancer; LDHA, lactate dehydrogenase A; qRT‐PCR, quantitative real‐time polymerase chain reaction</p><!><p>To further determine the role of LDHA in miR‐33b‐regulated NSCLC cells, we designed an LDHA vector and co‐transfected SPC‐A1 and H1299 cells with this vector and miR‐33b mimics. It was found that the LDHA vector attenuated the inhibitory effect of miR‐33b mimics on LDHA protein in NSCLC cells (P < 0.01) (Figure 5A). The LDHA vector also attenuated the growth inhibitory effect of miR‐33b on NSCLC cells (P < 0.05, P < 0.01) (Figure 5B‐F).</p><!><p>LDHA expression attenuates the growth inhibitory effect of miR‐33b on NSCLC cells. A, LDHA vector attenuated the inhibitory effect of miR‐33b mimics on LDHA protein. B‐D, The results the of the growth curve and CCK‐8 assays showed LDHA vector attenuated the inhibitory effects of miR‐33b mimics on cell proliferation of SPC‐A1 and H1299 cells. E, The long‐term proliferative capacity of SPC‐A1 and H1299 cells was detected by colony formation. F, The cell cycle analysis in SPC‐A1 and H1299 cell lines was performed using flow cytometry. *P < 0.05, **P < 0.01. miR, microRNA; NSCLC, non‐small cell lung cancer; LDHA, lactate dehydrogenase A</p><!><p>Finally, we explored whether miR‐33b could inhibit the glycolysis of NSCLC by targeting LDHA. We show that glycolysis‐suppressing effects induced by miR‐33b could be reversed by LDHA overexpression, as evidenced by increased glucose consumption, lactate production and ATP levels after LDHA vector transfection (P < 0.05, P < 0.01) (Figure 6).</p><!><p>LDHA expression attenuates the inhibitory effect of miR‐33b on glucose metabolism in NSCLC cells. A‐C, Glucose consumption, lactate production, and ATP levels were detected after SPC‐A1 cells were transfected. D‐F, Glucose consumption, lactate production, and ATP levels were detected after H1299 cells were transfected. *P < 0.05, **P < 0.01. miR, microRNA; NSCLC, non‐small cell lung cancer; ATP, adenosine triphosphate</p><!><p>Treatment of NSCLC still remains a challenge. Currently, surgery is the primary treatment modality for NSCLC patients in stage I, II &III. Chemotherapy is normally given to the patients suffering from stage IV cancer. However, the therapeutic outcome of these treatments is still suboptimal. The use of miRNAs as regulatory molecules of lung cancer is emerging. Here we show that miR‐33b is downregulated in NSCLC tissue and cells, which is in line with previous reports that miR‐33b serves as a tumor suppressor.26, 27 Further, we showed that miR‐33b overexpression attenuated the proliferation, colony formation of NSCLC cells and promoted cell cycle arrest and apoptosis (Figure 2). These data implicated the use of miR‐33b for NSCLC therapy. Indeed, recent evidence identified a number of miRNAs as novel NSCLC suppressors, e.g miR‐513a‐3p, miR‐200b, miR‐100, let‐7c, miR‐101, miR‐186, miR‐34ac, miR‐24, and miR‐148a, or promoters, e.g miR‐21, miR‐135a, miR‐30c, and miR‐100.28 Overexpression or suppression of these molecules was shown to effectively attenuate NSCLC progression. For example, Zhang et al showed that miR‐143 targets epidermal growth factor receptor (EGFR) and suppresses the cell proliferation and invasion of NSCLC in vitro, suggesting miR‐143 as potential therapeutic target against NSCLC.29 Therefore, our study underscored the critical role of miRNAs in cancer and miRNAs other than miR‐33b may also be important regulators of NSCLC. It is also worth noting that our study is limited to in vitro analysis of the cell physiologies affected by miR‐33b, and it is imperative to validate the therapeutic potential of miR‐33b in vivo.</p><p>Despite recent advances in the understanding of miRNA regulation in cancer, investigation of the role of miRNAs in cancer metabolism, whereas, is rarely studied. Our study is preceded by evidence showing that miRNAs participate in the regulation of glucose and fatty acid metabolism in other diseases.30 Cancer are characteristic of elevated glucose metabolism, a mechanism that has been utilized in imaging detection of cancer, such as the 18F‐FDG PET.31 Here we for the first time showed that miR‐33b attenuates glucose metabolism in NSCLC. When the SPC‐A1 and H1299 NSCLC cells were transfected with miR‐33b, significantly reducing of the metabolic parameters, including consumption of glucose, ATP and lactate production was seen. In line with this, downregulation of miR‐33b by using its inhibitor results in increased glucose consumption, ATP levels and lactate production within cells.</p><p>Further, as a mechanistic study, we identified LDHA as a target of NSCLC. LDHA is an enzyme of the glycolytic pathway, which plays a role in the regulation of glycolysis in anaerobic conditions. LDHA has been used as a biomarker in various cancers.32 In our study, the interaction between LDHA and miR‐33b was verified by luciferase reporter assay. Moreover, overexpression of LDHA was shown to antagonize the tumor‐suppressing effects of miR‐33b. Several signaling pathways have been previously identified to regulate LDHA, including JMJD2A‐LDHA (Jumonji C domain 2A‐Lactate dehydrogenase A) and KLF4/LDHA (Krüppel‐like factor 4‐ Lactate dehydrogenase A) pathways, which are crucial to glycolysis regulation in cancer.33, 34 Zhao et al demonstrated the inhibitory role of miR‐33b in malignant melanoma by regulating LDHA.26 Notably, miR‐33b also regulates the metabolism of fatty acids,35 and other cancer‐related processes, such as angiogenesis, hypoxia, etc., which presumably all contribute to the tumor‐attenuation effects of miR‐33b.26, 27 Besides, as a broad‐spectrum regulatory molecules, miR‐33b has also been shown to regulate genes, such as NPC1 (Niemann Pick C), ABCA1 (ATP‐Binding Casette A1), ABCG1 (ATP‐Binding Casette G1), CPT1A (carnitine palmitoyltransferase 1A), CROT (carnitine O‐octanoyltransferase), and HADHB (hydroxyacyl‐CoA dehydrogenase/3‐ketoacy‐CoA thiolase/enoyl‐CoA hydratase β subunit).36 These factors should also be considered to maximize the therapeutic utility of miR‐33b.</p><!><p>In this study, it has been found that in NSCLC cells/tissue the miR‐33b is significantly downregulated. Moreover, miR‐33b overexpression not only inhibits the growth of NSCLC cells but also attenuates the glucose metabolism. The rate of glucose metabolism is inversely proportional to the level of miR‐33b. More miR‐33b expression results in a decreased rate of glucose metabolism. The study also found that inhibition of NSCLC cells growth by miR‐33b may be regulated through targeting LDHA. It has also been found that miR‐33b inhibits the glucose metabolism in NSCLC cells by targeting LDHA. In short, miR‐33b acts as an anti‐NSCLC molecule by reprogramming glucose metabolism through targeting LDHA.</p><!><p>The authors declare that they have no conflicts of interests.</p><!><p>The analyzed data sets generated during the study are available from the corresponding author on reasonable request.</p><!><p>The current study was approved by the Ethics Committee of the Yaitai Yuhuangding Hospital affiliated to Qingdao University. All patients and healthy volunteers provided written informed consent before their inclusion within the study.</p><!><p>All authors have read and approved the final manuscript.</p>

PubMed Open Access

Post-synthetic modification of covalent organic frameworks via in situ polymerization of aniline for enhanced capacitive energy storage

Covalent organic frameworks (COFs) with layered architecture with open nanochannels and high specific surface areas are promising candidates for energy storage. However, the low electrical conductivity of two-dimensional COFs often limits their scope in energy storage applications. The conductivity of COFs can be enhanced through post-synthetic modification with conducting polymers. Herein, we developed polyaniline (PANI) modified triazine-based COFs via in situ polymerization of aniline with the porous frameworks. The composite materials showed high conductivity of 1.4-1.9 x 10 -2 S cm -1 at room temperature with 10-fold enhancement in specific capacitance than the pristine frameworks. The fabricated supercapacitor exhibited a high energy density of 24.4 W h kg -1 and a power density of 200 W kg -1 at 0.5 A g -1 current density. Moreover, the device fabricated using the conducting polymer-triazine COF composite can light up a green lightemitting diode for 1 min after being charged for 10 s.

post-synthetic_modification_of_covalent_organic_frameworks_via_in_situ_polymerization_of_aniline_for

Introduction:<!>Results and discussion:<!>𝐶 𝑠 = ∫ 𝐼 . 𝑑𝐸 2𝜈𝑚𝐸<!>𝐶 𝑠 = 𝐼 . 𝑡 2𝐸<!>Conclusions<!>Conflicts of interest

<p>Porous organic materials owing to the low skeleton density, high specific surface area, tunable pore size, and robust hydrothermal stability due to covalent bonds have attracted a great deal of attention for task-specific applications ranging from gas adsorption, catalysis, molecular separation, water purification, sensing to electrical energy storage. [1][2][3][4] In the context of affordable and sustainable energy sources, supercapacitors have emerged as promising energy storage systems in the carbon-neutral economy due to the high-power density, fast charge/discharge capability, high coulombic efficiency, and long cycling life. 5,6 The mechanism for storing charges in a supercapacitor relies on two distinct processes: nonfaradaic double-layer formation via the electrostatic adsorption of ions on the electrolyte/electrode interface (electric double-layer capacitance, EDLC) and faradaic redox reactions at the surface of the electrode (pseudocapacitance, PC). 7 Carbon based amorphous polymeric porous materials have been studied extensively as supercapacitive electrode materials due to their large specific surface area and relatively high packing density. 8 However, the lack of control over the pore-size distribution and pore structure often leads to the sluggish ion-diffusion resulting in undesirable capacitance loss during the fast charging-discharging. 9 Owing to the controlled pore size distribution and special pore functionality, covalent organic frameworks (COFs), an emerging class of crystalline porous organic materials obtained through dynamic covalent chemistry, 10 have been explored for supercapacitor applications. 11 The layered architecture of 2D COFs possesses 1D channels that facilitate ion diffusion and mass transport. 12 However, the strong π-π stacking of COF layers leads to inaccessibility of redox-active sites deeply buried inside the pore channels, limiting the charge storage capacity. 13,14 On the other hand, the electron localization on the heteroatom linkages and defects in the framework arising due to insufficient reversible condensation interrupt the extended π-electron conjugation. 15 Consequently, it results in low electrical conductivity of COFs retarding practical usefulness in energy storage applications. 15 The electrical properties of COFs can be improved by doping conducting polymers into the porous frameworks. [16][17][18] Dichtel and co-workers first showed the strategy by introducing polyethylenedioxythiophene (PEDOT) via electropolymerization within a redox-active anthraquinonebased COF (DAAQ-TFP COF) to achieve high volumetric energy storage capacity with fast charging rates. 16 Awaga and coworkers adopted in situ solid-state polymerization of PEDOT inside the nanochannels of anthraquinone-based COF with the enhancement of electrical conductivity and high specific capacitance. 17 Later on, Mai and co-workers grew the COF on the surface of commercial polyaniline. 18 In this strategy, the coating of COF on conducting polyaniline hides the redox-active sites that diminish the pseudocapacitance contribution. The electronic charge density on heteroatoms in the frameworks with a high specific surface area facilitates the ion adsorption to enhance the double-layer capacitance. 19,20 Besides, the electroactive building units, like N-rich triazine cores enhance the relative permittivity of the electrode materials and also contribute to the pseudocapacitance. 21 Herein, we synthesized two triazine-based covalent organic frameworks via solvothermal Schiff-base polycondensation by combining C 2 and C 3 -symmetric (triazine) building units, TCOF-1, and connecting two C 3 -symmetric triazine units, TCOF-2 (Fig. 1a). We modified TCOFs via in situ polymerizations of aniline using a chemical oxidation method to improve the electrical conductivity resulting in polyaniline modified composites, PANI@TCOF-1, and PANI@TCOF-2. The composite materials showed high electrical conductivity, which further improved the specific capacitance with cyclic stability. We fabricated asymmetric supercapacitor devices derived from PANI@TCOF-2, and the lighting up of a green light-emitting diode (LED) was demonstrated.</p><!><p>Triazine-based covalent frameworks (TCOFs) are the class of porous organic networks that can be synthesized via ionothermal trimerization of aromatic polynitriles in the presence of molten ZnCl 2 . 22a Most of the CTFs obtained under the harsh ionothermal conditions are amorphous. 22b We employed triazine moiety within the monomeric building unit to get highly ordered crystalline frameworks with a large specific surface area. The triazine-based covalent organic frameworks, TCOF-1, and TCOF-2, were fabricated via Schiffbase polycondensation reactions following reported procedures (Fig. 1a). 23,24 TCOF-1 was synthesized by a solvothermal reaction of 2,4,6-tris(4-formylphenoxy)-1,3,5triazine (TFPOT, C 3 ) and p-phenylenediamine (C 2 ), resulting in a pale-yellow solid with 70% yield. Similarly, TCOF-2 was synthesized using TFPOT (C 3 ) and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT, C 3 ′ ) to afford a yellow solid with 75% yield.</p><p>The post-synthetic modification of TCOFs was carried out through in situ polymerization of aniline via chemical oxidation method within the porous frameworks (Fig. 1d). Fourier transform infrared (FTIR) spectroscopy was used to confirm the formation of TCOFs and PANI@TCOFs. The disappearance of the C=O (1704 cm -1 ) and N-H (3460-3210 cm -1 ) stretching bands indicates the consumption of aldehyde and amino groups of the monomers. Whereas, the appearance of the C=N stretching band at 1622-1626 cm -1 confirmed imine bond formation in TCOFs via condensation reaction. The FTIR spectra of polyaniline-TCOF composites (PANI@TCOFs) showed characteristic benzenoid-quinonoid nitrogen vibration at 1564 and 2,4,6-tris(4-formylphenoxy)-1,3,5triazine (TCOF-1) and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 2,4,6-tris(4-formylphenoxy)-1,3,5-triazine (TCOF-2). PXRD patterns of (b) TCOF-1 and (c) TCOF-2: comparison between the experimental pattern (blue), Pawley refined profile (red), the refinement differences (grey), and the simulated pattern (green) for eclipsed AA stacking mode of TCOF-1 and slipped AA stacking mode of TCOF-2; space-filling models representing the stacking along the c-axis with the layer distances are shown. (d) Schematic representation depicting in situ polymerization of aniline with TCOFs. (e) FT-IR spectra of TCOFs and PANI@TCOFs. (f) Pore size distribution plots of TCOFs and PANI@TCOFs obtained through the analysis of respective adsorption isotherms employing the non-local density functional theory (NLDFT) method.</p><p>cm -1 and aromatic amine peak at 1296 cm -1 (Fig. 1e). The blue shift of characteristic bands of polyaniline (8-12 cm -1 ) in composite materials ascertains the inclusion of polyaniline within the TCOFs. 25,26 The TCOFs were characterized by powder X-ray diffraction (PXRD) analysis. The PXRD pattern of TCOF-1 showed an intense diffraction peak at 2.68° attributed to the (100) plane (Fig. 1a). The other diffraction peaks appearing at 4.69°, 5.43°, 7.21°, and 9.46° correspond to the (110), ( 200), (210), and ( 220) facets, respectively. The PXRD profile of TCOF-2 exhibited a prominent peak at 3.98° and relatively weak peaks at 6.87°, 7.96°, and 10.56°, which were indexed to (100), ( 110), ( 200), and (210) facets, respectively (Fig. 1c). The experimental PXRD pattern accorded well with the simulated pattern based on the AA stacking for TCOF-1 and slipped AA stacking for TCOF-2 (Fig. 1b corresponding to (001) plane were observed at 2θ = 16.6° and 25.8° for TCOF-1 and TCOF-2, respectively (Fig. 1b, 1c). The PXRD data and the structural analysis of TCOF-1 and TCOF-2 are consistent with the earlier reports. 23,24 The suppression of the high intensity peaks at low 2θ in PXRD suggests a lowering of crystallinity in PANI@TCOFs. The surface area and porosity of TCOFs and PANI@TCOFs were estimated by the nitrogen adsorption−desorption isotherms at 77 K. The sorption curves of both TCOFs showed type-IV isotherms. The TCOF-1 sorption curves having the H1 hysteresis loop indicate the cylindrical-like pore channels. 27 Whereas, the H4 hysteresis loop of TCOF-2 suggests the narrow slit-shaped pores. 27a The Brunauer−Emmett−Teller (BET) specific surface area of TCOF-1 and TCOF-2 were found to be 1756 and 1110 m 2 g −1 , respectively. The pore volumes were 1.64 cm 3 g −1 for TCOF-1 and 0.85 cm 3 g −1 for TCOF-2, estimated from the nitrogen adsorption amount at P/P 0 = 0.95. The uniform pore size distribution (2.6 nm for TCOF-1; 1.4 and 3.9 nm for TCOF-2) was obtained based on the non-local density functional theory (NLDFT) method. The lowering of BET specific surface area and the changes in the pore size distribution of PANI@TCOFs infer the successful integration of PANI within the porous framework of TCOFs (Fig. 1f). The field emission scanning electron microscopy (FESEM) images depict the aggregated-particles like morphology for TCOF-1 and fused flake-like morphology for TCOF-2 (Fig. 2a, 2b). A distinctly different morphology was noticeable for PANI@TCOFs compared to the pristine TCOFs (Fig. 2c, 2d). PANI@TCOF-1 and PANI@TCOF-2 showed the well-distributed intricate network structure attributing to polyaniline loading with the porous framework. PANI@TCOF-2 showed whisker-like morphology of polyaniline that grew over the framework of TCOF-2 resulting in faster ion diffusion through the nanochannels. 25 The transmission electron microscopy (TEM) images of TCOFs and PANI@TCOFs further revealed the porous nature of the frameworks.</p><p>The heteroatom rich porous framework structure of TCOFs and PANI@TCOFs motivated us to explore the electrochemical performance of TCOFs and PANI@TCOFs as active electrode materials for supercapacitors. The electrochemical measurements were carried out using a typical three-electrode electrolytic cell with 1 M H 2 SO 4 as an aqueous electrolyte. TCOFs and PANI@TCOFs were coated on platinum-foil and used as the working electrode. A platinum wire and saturated calomel electrode (SCE) were employed as the counter and reference electrodes, respectively. The electrochemical performances of the electrodes were investigated by cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) measurements and electrochemical impedance spectroscopy (EIS). The cyclic voltammetry was carried out at different scan rates, and specific capacitance from the cyclic voltammograms (CVs) was calculated using the following equation.</p><!><p>(1) C s , I, ν, m, and E denote the specific capacitance, current, scan rate, mass of active material deposited on the electrodes, and the potential applied, respectively. Fig. 3a and 3c depict the cyclic voltammograms of PANI@TCOF-1 and PANI@TCOF-2, respectively, recorded at different scan rates from 10 to 100 mV s −1 in the potential window from 0 to 0.8 V. The CV curves of the composite materials show a rectangular shape featuring redox peaks. As revealed from CVs, the capacitive response is due to the combined effect of both electric double-layer capacitance and pseudocapacitance. Here, the pseudocapacitance contribution comes from the reversible redox activity of polyaniline. The voltammograms show two sets of distinct redox peaks. A redox couple between 0.1 and 0.35 V vs. SCE is associated with the conversion of the fully reduced leucoemeraldine base to the partially oxidized emeraldine (Fig. 3c). The redox peaks occurring between 0.35 and 0.6 V vs. SCE are due to the conversion of emeraldine to the oxidized pernigraniline form of PANI. 28 The highest specific capacitance of PANI@TCOF-1 and PANI@TCOF-2 were found to be 156 and 258 F g -1 at 1 mV s -1 scan rate, respectively. The galvanostatic charge-discharge (GCD) experiments were carried out at different current density for further investigations. The specific capacitance was calculated from the GCD plots using the following equation.</p><!><p>(2) I, t, and E represent the applied current density, time taken for the charge/discharge process, and the potential window, respectively. The charge-discharge curves of PANI@TCOF-1 and PANI@TCOF-2 at varying current densities exhibited typical triangular shapes (Fig. 3b and 3d). The asymmetric nature of the GCD curves also suggests the contribution from pseudocapacitance along with EDLC to the total specific capacitance value. 21c The specific capacitance of PANI@TCOF-1 and PANI@TCOF-2 from GCD curves were calculated to be 154 and 275 F g -1 at 0.5 A g -1 current density. PANI@TCOFs exhibited contrastingly higher specific capacitance compared to pristine TCOFs (10-20 F g -1 , Fig. 1e). Despite of high specific surface area and narrow pore size distribution, the low capacitance values of TCOFs could be due to poor electrical conductivity. 15 We measured the electrical conductivity by four-probe current-voltage (I-V) method. As anticipated, the electrical conductivity of TCOFs was very low, below the acceptable range of detection. The electrical conductivities of PANI@TCOF-1 and PANI@TCOF-2 were estimated as 1.4 x 10 -2 and 1.9 x 10 -2 S cm −1 , respectively, at room temperature and increased linearly with temperature (Fig. 1f). The significant enhancement of conductivity of PANI@TCOFs compared to pristine TCOFs is attributed to the integration of conducting polymer with porous frameworks. Besides, the whisker-like morphology of polyaniline reduces the diffusion length improving ion transport within the framework that results in considerably high conductivity and specific capacitance of PANI@TCOF-2. 25 The specific capacitance of PANI@TCOFs decreased with increasing the current density, and 43% retention was observed for PANI@TCOF-2, suggesting substantial rate capability of the composite materials (Fig. 3g). The cyclic stability of PANI@TCOFs coated electrodes at a 100 mV s -1 scan rate up to 1000 cycles was examined. The capacitance retention of 69% and 62% was observed for PANI@TCOF-1 and PANI@TCOF-2, respectively (Fig. 3h). Furthermore, the charge-discharge cycling employing PANI@TCOF-2 was performed for 500 cycles at 10 A/g current density. The high retention of coulombic Fig. 3 The electrochemical performance of composite materials in 1 M H 2 SO 4 . Cyclic voltammograms at different scan rates and galvanostatic charge-discharge curves at different current densities (a, b) for PANI@TCOF-1 and (c, d) PANI@TCOF-2, respectively. (e) Specific capacitances of TCOFs and PANI@TCOFs at 0.5 A g -1 current density. (f) Temperaturedependent conductivity profiles of PANI@TCOF-1 and PANI@TCOF-2 (symbols and solid lines denote the experimental and fitted data, respectively). (g) Specific capacitances of PANI@TCOFs obtained at different current densities. (h) Cyclic stability tests of PANI@TCOFs at a scan rate of 100 mV s -1 . (i) Nyquist plots of PANI@TCOFs at frequency range 100 mHz-100 kHz; symbols and solid lines denote the experimental and fitted data, respectively. The lower inset shows the magnified Nyquist plots in the high-frequency region. The upper inset shows the equivalent circuit model to fit the Nyquist plots; R S is the solution resistance or equivalent series resistance, R ct is charge transfer resistance at the electrode/electrolyte interface, Q dl is a constant phase element for double layer capacitance, W d is Warburg element, and Q ps is the pseudocapacitive charging. (j) Ragone plots of gravimetric energy density against power density for PANI@TCOFs-based supercapacitors. (k) Four individual solid-state asymmetric supercapacitors employing PANI@TCOF-2 are connected in a series; the device can power a green light-emitting diode (LED) glowing for ∼1 min.</p><p>efficiency of 93% suggests the efficient charge storage and durability of the composite materials. The electrochemical impedance spectroscopy (EIS) was performed further to get a detailed insight into the electrochemical behaviour of the materials. EIS measurements were carried out in a frequency range from 10 5 Hz to 0.1 Hz at open circuit potentials. The Nyquist plots for PANI@TCOF-1 and PANI@TCOF-2 feature a nearly vertical rise of impedance along the imaginary axis, indicating an excellent capacitive performance (Fig. 3i). 21c,29 The electrical equivalent circuit (EEC) based on EIS results was constructed (Fig. 3f, upper inset), and the different components of the electrochemical system were analyzed. R s is the solution resistance or the equivalent series resistance, and R ct is the charge transfer resistance at the electrode/electrolyte interface. The lower value of R ct for PANI@TCOFs compared to pristine TCOFs indicates the facile charge transfer among electrolyte ions and PANI@TCOFs at the electrode interface. 30 The double layer capacitance at the electrode/electrolyte interface because of the surface inhomogeneity of the electrode is represented by a constant phase element (CPE, Q dl ). The Warburg element (W d ) and the pseudocapacitive charging (Q ps ) arise due to the diffusion limitation and electron transport inside the nanoporous framework, respectively.</p><p>At higher frequencies, the diffusion of ions inside the nanopores was restricted due to a shorter time period. 31 The 'knee frequency', the frequency at which the diffusion of ions begins, was 121 Hz for PANI@TCOF-2 compared to 82 Hz for pristine PANI. The high knee frequency of PANI@TCOF-2 indicates a facile ion transport inside the porous networks. The Bode plots also suggest facile diffusion of electrolyte ions throughout the nanoporous surfaces of PANI@TCOFs. The transition point between the resistive and the capacitive behaviour was obtained by the characteristic frequency, f 0 , at the phase angle of -45°. 32 The dielectric relaxation time constant (τ 0 = 1/f 0 ) represents the time required for a supercapacitor to deliver half of its power. 33 The estimated τ 0 for PANI@TCOF-2 was 87 ms. The Ragone plots for PANI@TCOFs are presented in Fig. 3j. The maximum energy density and power density for PANI@TCOF-2 were 24.4 W h kg -1 and 4000 W kg -1 , respectively, promising for the device fabrication.</p><p>As discussed above, the encouraging results prompted us to fabricate a solid-state asymmetric capacitor using PANITCOF-2 and acetylene black as the electrode materials. A homogeneous slurry containing PANI@TCOF-2 (70%), carbon black (15%), and polyvinylidene fluoride (PVDF) binder (15%) in Nmethylpyrrolidone (NMP) was coated on a platinum foil (1 cm 2 ). The electrodes were assembled in a two-electrode set-up using a Whatman filter paper as a separator with a gel electrolyte (polyvinyl alcohol and H 2 SO 4 ). The four individual solid-state devices derived from PANI@TCOF-2 were connected in a series to enhance the working potential window. After charging the device for 10 s, a green light-emitting diode (2.2-3.3 V) can be powered for ∼1 min.</p><!><p>In summary, we successfully synthesized conducting polymer modified triazine-based covalent organic frameworks (PANI@TCOF-1 and PANI@TCOF-2) through in situ polymerization of aniline using chemical oxidation method. The characteristic stretching bands and the variation in the pore size distribution of PANI@TCOFs implied the inclusion of PANI within the porous frameworks of TCOFs. The SEM and TEM images showed a well-distributed intricate network structure attributing to the loading of polyaniline with the porous framework that resulted in significantly enhanced electrical conductivity (1.4-1.9 x 10 -2 S cm -1 ). More interestingly, the whisker-like morphology of PANI in PANI@TCOF-2 enhanced the ion diffusion within the framework. The synergistic effect of high electrical conductivity, faster ion diffusion, and reversible redox reactions of PANI led to the 10-fold enhancement in specific capacitance of composite materials compared to pristine TCOFs. Owing to significant energy (24.4 W h kg -1 ) and power (4000 W kg -1 ) density, PANI@TCOF-2 was used as a cathode material to devise a solid-state asymmetric supercapacitor that can light up a green LED. Thus, the present study delineates a cost-effective in situ polymerization strategy for doping the conducting polymer into the porous framework for large-scale production of all-organic electrode materials for the futuristic energy storage devices.</p><!><p>There are no conflicts to declare.</p>

ChemRxiv

Inorganic Synthesis Based on Reactions of Ionic Liquids and Deep Eutectic Solvents

AbstractIonic liquids and deep eutectic solvents are of growing interest as solvents for the resource‐efficient synthesis of inorganic materials. This Review covers chemical reactions of various deep eutectic solvents and types of ionic liquids, including metal‐containing ionic liquids, [BF4]−‐ or [PF6]−‐based ionic liquids, basic ionic liquids, and chalcogen‐containing ionic liquids. Cases in which cations, anions, or both are incorporated into the final products are also included. The purpose of this Review is to raise caution about the chemical reactivity of ionic liquids and deep eutectic solvents and to establish a guide for their proper use.

inorganic_synthesis_based_on_reactions_of_ionic_liquids_and_deep_eutectic_solvents

<!>Introduction<!><!>Reactions of Metal‐Containing Ionic Liquids<!><!>Reactions of Metal‐Containing Ionic Liquids<!><!>Reactions of Metal‐Containing Ionic Liquids<!>Reactions of [BF4]−‐ or [PF6]−‐Based Ionic Liquids<!><!>Reactions of [BF4]−‐ or [PF6]−‐Based Ionic Liquids<!>Reactions of Basic Ionic Liquids<!><!>Reactions of Basic Ionic Liquids<!>Reactions of Chalcogen‐Containing Ionic Liquids (Including Reactions of Ionic Liquids with Chalcogens)<!><!>Reactions of Chalcogen‐Containing Ionic Liquids (Including Reactions of Ionic Liquids with Chalcogens)<!><!>Reactions of Chalcogen‐Containing Ionic Liquids (Including Reactions of Ionic Liquids with Chalcogens)<!><!>Reactions of Chalcogen‐Containing Ionic Liquids (Including Reactions of Ionic Liquids with Chalcogens)<!>Reactions of Ionic Liquids Whose Cations, Anions, or Both Are Incorporated into the Final Products<!>Metal Halide Compounds<!><!>Zeolites<!><!>Zeolites<!>Metal‐Organic Frameworks (MOFs)<!>Polyanionic/Polycationic Compounds<!><!>Polyanionic/Polycationic Compounds<!><!>Polyanionic/Polycationic Compounds<!>Reactions of Deep Eutectic Solvents<!><!>Reactions of Deep Eutectic Solvents<!><!>Reactions of Deep Eutectic Solvents<!>Other Types of Reactions of Ionic Liquids<!>Summary and Outlook<!>Conflict of interest<!>Biographical Information

<p>T. Zhang, T. Doert, H. Wang, S. Zhang, M. Ruck, Angew. Chem. Int. Ed. 2021, 60, 22148.</p><!><p>Ionic liquids (ILs), first reported by Paul Walden in 1914, [1] are defined as molten salts with melting points below 100 °C. Nowadays, ILs are widely applied in a broad variety of fields, including catalysis, separations, synthesis, and many others.[ 2 , 3 , 4 , 5 ] Compared to the broad applications of ILs in organic chemistry, which have already been explored for about 40 years, inorganic syntheses in ILs, especially the so‐called ionothermal syntheses,[ 6 , 7 , 8 ] have been actively investigated only since the early 2000s. Since then, various inorganic compounds (e.g. metals and non‐metals, metal oxides and chalcogenides, metalates and framework compounds) have been prepared using, or in the presence of, ILs.[ 9 , 10 , 11 , 12 , 13 , 14 ]</p><p>ILs provide several unique properties for the preparation of inorganic materials. For example, ILs can facilitate the dissolution of versatile precursors, including both inorganic and organic compounds, which is fundamental for the synthesis of most materials.[ 15 , 16 ] ILs create a special microphasic separation of the hydrophilic and hydrophobic fragments, typically with imidazolium ILs with their long alkyl chains. [17] This heterogeneity of ILs provides the ability to control nucleation and growth rates, particle sizes, and morphologies in materials synthesis. [17] Furthermore, some other characteristics, such as good thermal stability in ionothermal synthesis, high polarizability for microwave synthesis, or wide electrochemical windows and high conductivity for electrodeposition, make ILs an attractive alternative to conventional organic solvents for the synthesis of inorganic materials as well as to high‐temperature reactions in melts or the solid state.</p><p>In 2003, the concept "deep eutectic solvent" was first coined by Abbott et al. [18] Deep eutectic solvents (DESs) are acknowledged as a new class of IL‐analogue solvents and share many characteristics of traditional ILs, such as low vapor pressure, high polarity, and tunable chemical properties. However, DESs are easier to access synthetically. In most cases, a DES is obtained by mixing a quaternary ammonium or phosphonium salt with a hydrogen‐bond donor (HBD), thereby generating a new liquid phase with a melting point below that of either individual component.[ 19 , 20 ] No purification is usually needed. Furthermore, most DESs are quite inexpensive because of the low cost of their constituents, such as urea and choline chloride. Therefore, IL analogues as well as the more accessible DESs are increasingly being used in the synthesis of inorganic materials.</p><p>The thermal and chemical stabilities of ILs are usually highlighted as advantageous for inorganic synthesis. Previous investigations have indicated that the actual degradation temperature of ILs is overestimated by the onset decomposition temperature (T onset) derived from the ramped temperature in thermogravimetric analysis.[ 21 , 22 , 23 ] Therefore, the concept of long‐term thermal stability is utilized to obtain more accurate information on the decomposition of ILs at high temperature. The thermal stability of ILs, including the characterization methods, mechanism of decomposition, and kinetics of thermal degradation, has been extensively explored by several research groups,[ 24 , 25 , 26 , 27 , 28 , 29 ] and is not within the scope of this Review.</p><p>ILs are readily accessible as inert reaction media for inorganic synthesis. Dai and co‐workers demonstrated that several imidazolium ILs containing [NTf2]− anions could be used as the flux medium for the direct recycling of spent cathode materials or as effective structure‐directing templates for the synthesis of advanced catalysts and anode materials because of their good thermal and chemical stability.[ 30 , 31 , 32 ] In all cases, the [NTf2]−‐containing imidazolium ILs can be readily reused and recycled after the reaction, thus providing new strategies for designing sustainable ILs for advanced inorganic synthesis.</p><p>However, many ILs contain reactive moieties in either the cation or anion. Thus, the ILs themselves can take part in reactions. For example, an IL can be tailored for a specific task, such as to release one component of the desired product upon its decomposition. Thus, the IL acts as solvent, template, and reactant, thereby simplifying the reaction system significantly. In other circumstances, the IL cation and anion can separate during the reactions, thereby leading to incorporation of the IL cation or anion in the final products. Such reactions are often used in the synthesis of some framework compounds (e.g. zeolites, MOFs, and polycationic/polyanionic compounds).[ 13 , 33 , 34 ] The IL cation or anion serves as a counterion to balance the charge of the framework as well as a template. IL decomposition or cation/anion separation during the ionothermal synthesis may cause a change in the properties of the IL (e.g. viscosity, conductivity, and dissolving capacity) and further influence the formation of the target product. [35] Thus, the reaction mechanism of ILs should be considered. Furthermore, the ionothermal approach, in particular, which exploits the chemical reactivity of ILs or DESs, provides new options for the synthesis of inorganic materials.</p><p>Several inspiring reviews on this young and fast‐growing subject of inorganic synthesis in ILs or DESs have been published, with an emphasis on selected themes.[ 7 , 8 , 9 , 10 , 11 , 36 , 37 , 38 , 39 , 40 ] However, most of them lack a comprehensive understanding of the chemical reactivity of the ILs or DESs in the reactions. To date, there are only a few case studies on the detailed reaction mechanisms.[ 16 , 41 , 42 , 43 , 44 , 45 ] In comparison, the chemical reactivity of ILs in organic synthesis has been discussed in several reviews.[ 35 , 46 , 47 , 48 ]</p><p>Herein, we attempt to systematically and comprehensively summarize this fascinating research area from the point of view of inorganic synthesis based on the chemical reactions of ILs or DESs. It includes reactions of metal‐containing ILs, fluorine‐containing ILs, basic ILs, chalcogen‐containing ILs, and DESs. Moreover, reactions of ILs whose cations, anions, or both are incorporated into the final products are also included. Table 1 shows a summary of all the abbreviations used in this Review. The decomposition and reaction mechanism of some IL/DES‐based reactions are discussed. This Review aims to illustrate a promising synthetic approach based on the reactivity of ILs/DESs and to provide a better understanding of the fundamental chemistry of ILs/DESs in the reactions.</p><!><p>The abbreviations of the IL cations and anions as well as other reagents used in this Review.</p><p>Abbreviation</p><p>Full name</p><p>[MIm]+</p><p>1‐methylimidazolium cation</p><p>[MMIm]+</p><p>1,3‐dimethylimidazolium cation</p><p>[EMIm]+</p><p>1‐ethyl‐3‐methylimidazolium cation</p><p>[BMIm]+</p><p>1‐butyl‐3‐methylimidazolium cation</p><p>[PMIm]+</p><p>1‐pentyl‐3‐methylimidazolium cation</p><p>[OMIm]+</p><p>1‐methyl‐3‐octylimidazolium cation</p><p>[C16MIm]+</p><p>1‐hexadecyl‐3‐methylimidazolium cation</p><p>[BMMIm]+</p><p>1‐butyl‐2,3‐dimethylimidazolium cation</p><p>[C12MMIm]+</p><p>1‐dodecyl‐2,3‐dimethylimidazolium cation</p><p>[BMPyr]+</p><p>1‐butyl‐1‐methylpyrrolidinium cation</p><p>[DMPyr]+</p><p>1‐decyl‐1‐methylpyrrolidinium cation</p><p>[P4444]+</p><p>tetrabutylphosphonium cation</p><p>[P66614]+</p><p>trihexyltetradecylphosphonium cation</p><p>[NTf2]−</p><p>bis(trifluoromethylsulfonyl)imide anion</p><p>[OTf]−</p><p>triflate anion</p><p>TBAH</p><p>tetrabutylammonium hydroxide</p><p>TEAH</p><p>tetraethylammonium hydroxide</p><p>BTMAH</p><p>benzyltrimethylammonium hydroxide</p><p>TBPH</p><p>tetrabutylphosphonium hydroxide</p><p>Pbis</p><p>1,5‐bis(3‐methylimidazole‐2‐selone)pentane</p><p>Me</p><p>methyl group</p><p>Et</p><p>ethyl group</p><p>t‐Bu</p><p>tert‐butyl group</p><p>DBU</p><p>1,8‐diazabicyclo(5.4.0)undec‐7‐ene</p><!><p>An overview of all the discussed studies reporting the synthesis of inorganic materials using, or in the presence of, ILs is presented in Table 2.</p><!><p>A summary of all the studies on inorganic materials prepared from ionic liquids discussed in this Review.</p><p>Material composition</p><p>Ionic liquid</p><p>Refs.</p><p>CuCl</p><p>[C5H5N‐C12H25][CuCl4]</p><p>[50]</p><p>ZnO</p><p>Zn(L)4(NTf2)2 (L=alkylamine)</p><p>[51]</p><p>CuO</p><p>[C16MIm]2[CuCl4]</p><p>[52]</p><p>CuS</p><p>[BMIm]2[Cu2Cl6]</p><p>[53]</p><p>ZnS</p><p>[C n MIm][ZnCl3] (n=4, 8, and 16)</p><p>[54]</p><p>FeS2</p><p>[C12MMIm][ FeCl4]</p><p>[55]</p><p>A2SiF6 (A=Li, Na, K, Rb, and Cs)</p><p>[BMIm][PF6]</p><p>[68]</p><p>MnF2</p><p>[BMIm][BF4]</p><p>[69]</p><p>iron fluoride</p><p>[BMIm][BF4]</p><p>[70, 71, 72, 73]</p><p>NaYF4</p><p>[BMIm][BF4], [BMIm][PF6]</p><p>[74]</p><p>Ce3+‐, Tb3+‐, Eu3+/Sm3+‐doped BaLuF5</p><p>[OMIm][PF6]</p><p>[75]</p><p>Eu2+‐doped BaFCl</p><p>[BMIm][BF4]</p><p>[76]</p><p>Yb3+‐, Er3+/Tm3+‐doped NaYF4</p><p>[BMIm][BF4]</p><p>[77]</p><p>MF x , M=Fe, Co, Pr, Eu, Gd, and Er</p><p>[BMIm][BF4]</p><p>[78, 79, 80]</p><p>turbostratic boron nitride (t‐BN)</p><p>[BMMIm][BF4]</p><p>[81]</p><p>ZnO</p><p>TBAH, TEAH, and BTMAH</p><p>[83, 84, 85, 86], [89, 90]</p><p>metal (hydr)oxides, metal=Fe, Co, Mn, Ni, Cu</p><p>TBAH</p><p>[87, 88]</p><p>SrTiO3</p><p>TBAH, TBPH</p><p>[91]</p><p>CdSe, Bi2Se3, ZnSe, and PbSe</p><p>Pbis</p><p>[109]</p><p>NiSe2, ZnSe, Bi2Te3, Ag2Te, and Te nanostructures</p><p>[P66614]Cl, [P66614][decanoate], and [P66614][N(CN)2]</p><p>[110, 111, 112]</p><p>CdS</p><p>[BMIm][SCN]</p><p>[113]</p><p>ZnSe, Cu2−x Se, and CdSe</p><p>[BMIm][SeO2(OCH3)]</p><p>[115, 116, 117]</p><p>ZnSe</p><p>selenoether‐based ILs[a]</p><p>[118]</p><p>BiOCl</p><p>[C16MIm]Cl</p><p>[122]</p><p>Li4B7O12X (X=Cl, Br)</p><p>[P66614]X (X=Cl, Br)</p><p>[123]</p><p>A2B5O9X (A=Sr, Ba, X=Cl, Br)</p><p>mixture of [P66614]X and LiNTf2 (X=Cl, Br)</p><p>[124]</p><p>Pb2B5O9X (X=Cl, Br)</p><p>[P66614]X without adding LiNTf2 (X=Cl, Br)</p><p>[125]</p><p>SIZ‐1, SIZ‐3, SIZ‐4, and SIZ‐5</p><p>[EMIm]Br</p><p>[6]</p><p>metal phosphates (metal=Be, Al, Zn, and Fe)</p><p>[MIm][H2PO4]</p><p>[134, 135]</p><p>[Si48O96]F4(C8N2H15)2(C2H7O)2</p><p>[BMIm]OH0.65Br0.35</p><p>[136]</p><p>porous TiNb2O7 and MnCeOx</p><p>[BMIm][NTf2]</p><p>[30, 31]</p><p>[EMIm][Cd(btc)][b]</p><p>[EMIm]Br</p><p>[138]</p><p>[Cd3F(ina)4(4‐pic)3][BF4], [Cd3F(ina)3(4,4′‐bpy)2(4‐pic)2][BF4]2⋅(4,4′‐bpy)⋅2 H2O,</p><p>and [Cd3F(ina)3(4,4′‐bpy)3][BF4]2⋅(4,4′‐bpy)⋅2 H2O[c]</p><p>[BMIm][BF4]</p><p>[149]</p><p>[PMIm][Zn2(btc)(OH)Br]</p><p>[PMIm]Br</p><p>[151]</p><p>[BMIm][Zn2(btc)(OH)I]</p><p>[BMIm]I</p><p>[152]</p><p>[BMPyr]2[Br20]</p><p>mixture of [DMPyr]Br and [BMPyr][OTf]</p><p>[158]</p><p>[P4444]2[Br24]</p><p>mixture of [P4444]Br and [P66614][NTf2]</p><p>[159]</p><p>[BMMIm]24[Sn36Ge24Se132] and [BMIm]24[Sn32.5Ge27.5Se132]</p><p>([BMMIm][BF4] and [BMIm][BF4]</p><p>[164]</p><p>[Sb10Se10][AlCl4]2</p><p>[BMIm]Cl⋅n AlCl3</p><p>[166]</p><p>[Sb2Se2][AlCl4] and [Sb13Se16][AlCl4]6Al2Cl7</p><p>[BMIm]Cl⋅4.7 AlCl3</p><p>[167, 168]</p><p>[Sb13Se16Br2][AlX4]5</p><p>[BMIm]Br⋅5.1 AlCl3</p><p>[169]</p><p>[Sb7Se8Br2][Sb13Se16Br2][AlBr4]8</p><p>[BMIm]Br⋅4.7 AlBr3</p><p>[169]</p><p>[Sb7Se8Br2][AlX4]3</p><p>[BMIm]Br⋅4.7 AlBr3 (a small amount of NbCl5)</p><p>[169]</p><p>(CuBi8)[AlCl4]2[Al2Cl7] and (CuBi8)[AlCl4]3</p><p>[BMIm]Cl⋅4 AlCl3</p><p>[170]</p><p>Ni2P and Ni12P5</p><p>[P4444]Cl</p><p>[188]</p><p>Co2P</p><p>[P66614]2[CoCl4]</p><p>[189, 190]</p><p>[a] ILs of N‐[(phenylseleno)methylene]pyridinium, N‐(methyl)‐ and N‐(butyl)‐N′‐[(phenylseleno)methylene]imidazolium with [NTf2]− anions. [b] btc=benzene‐1,3,5‐tricarboxylate. [c] ina=isonicotinate, 4,4′‐bpy=4,4′‐bipyridine, 4‐pic=4‐methylpyridine.</p><!><p>In this Review, metal‐containing ionic liquids (M‐ILs) represent a subclass of ILs that contain a metal atom as part of the cation and/or anion. In addition to the general fluidic properties of ILs, the incorporated metal ions endow M‐ILs with some new functions, such as luminescent, catalytic, or magnetic properties. Recently, M‐ILs have gained increasing research attention in a variety of fields (e.g. catalysis, optical devices, and magnetic components). [49]</p><p>M‐ILs that serve as metal sources in inorganic synthesis have been widely investigated. In 2004, Taubert reported that CuCl nanoplatelets were synthesized from a Cu‐containing IL (Figure 1 a) and 6‐O‐palmitoyl ascorbic acid (Figure 1 b). [50] It was found that the mixture of the two compounds forms thermotropic liquid crystals with lamellar self‐assembled structures. The layered structures then template the formation of CuCl nanoplatelets as the temperature is increased (Figure 1 c). The Cu‐containing IL can be regarded as an "all‐in‐one" IL because it acts as solvent, reactant, as well as template. After this study, a number of M‐ILs were designed and applied for the synthesis of inorganic nanomaterials, including metal oxides and metal sulfides.</p><!><p>a) A Cu‐containing IL. b) 6‐O‐palmitoyl ascorbic acid. c) SEM image of CuCl nanoplatelets precipitated from a 1:1 (w/w) mixture of (a) and (b) at 85 °C. Reproduced with permission. [50] Copyright 2004, Wiley‐VCH.</p><!><p>Dai and co‐workers reported that hierarchical ZnO structures with diverse morphologies were obtained under an ionothermal synthesis when employing the Zn‐containing IL Zn(L)4(NTf2)2 (L=alkylamine, NTf2=−N(SO2CF3)2) as both the solvent and Zn source. [51] The solvent properties can be tailored by varying the IL ligand structures, thereby resulting in various morphologies of ZnO. In addition, CuO nanorods can be prepared in the presence of the Cu‐IL [C16MIm]2[CuCl4] under solvothermal conditions. [52]</p><p>Zheng and co‐workers demonstrated that three‐dimensional (3D) hierarchical CuS microspheres assembled from nanosheets were produced from the Cu‐containing IL precursor [BMIm]2[Cu2Cl6] by a solvothermal method. [53] The Cu‐IL plays a significant role in directing the final CuS structures. On one hand, the crystal growth along the [001] direction is inhibited because the [BMIm]+ prefers to adsorb onto the (001) facets of CuS. On the other hand, the assembly of CuS microspheres is influenced by the alkyl chain of the Cu‐IL. A tight hierarchical CuS structure is the preferred form when a short‐chain IL is used. ZnS quantum dots were prepared using the Zn‐containing IL [C n MIm][ZnCl3] (n=4, 8, and 16) as a precursor, template, and solvent. [54] FeS2 microspheres wrapped by N‐doped reduced graphene oxide were synthesized from the Fe‐based IL [C12MMIm][ FeCl4]. [55] The Fe‐IL can be used as the metal and nitrogen source, an assembly medium, and surfactant.</p><p>Another major application of M‐ILs is for the low‐temperature electrodeposition of various metals and alloys.[ 56 , 57 , 58 , 59 ] One well‐studied example involves the electrodeposition of Al in the IL‐AlCl3 system, whose Lewis acidity depends on the molar ratio of the organic salts to metal halides. [56] In recent years, ILs with metal‐containing cations have been developed for the electrodeposition of metals at high current densities because of the easy access of cationic metal complexes to the electrode surface. [60] Investigations into the electrodeposition of metals (e.g. Ni, Co, Cu, Al, and rare earth metals) by various cationic metal‐containing ILs have been reported by a series of research groups.[ 60 , 61 , 62 , 63 , 64 , 65 ]</p><!><p>Inorganic metal fluorides are well‐studied for their applications in photonics, catalysis, biosensing, lubricants, electrochemical energy storage, and high‐temperature superconductor devices.[ 66 , 67 ] In traditional syntheses of metal fluorides, the toxic and harmful HF, NaF, or NH4F is usually utilized as a fluorine source. Recent studies have shown that fluorine‐containing ([BF4]− or [PF6]−) ILs can be used as fluorine sources, thereby opening a safe pathway to prepare metal fluorides with novel morphologies and functions. The hydrolysis of the [BF4]− anion occurs in the presence of a small amount of residual water in the IL or the water of crystallization in metal salts upon heating, thereby forming BF3⋅H2O and F−. The reaction of fluoride ions with metal ions under the given conditions contributes to the in situ crystallization of metal fluorides. [67] Similarly, [PF6]− may also decompose to release F− under specific conditions. [68]</p><p>Wen and co‐workers synthesized a nanostructured MnF2 by using Mn(CH3COO)2⋅4 H2O as a manganese source and [BMIm][BF4] as a fluorine source (Figure 2 a). [69] The resulting MnF2 nanoparticles could be promising anode materials for lithium batteries with a long cycle life. Li et al. reported that a variety of hydrated Fe‐based fluoride nanoparticles could be successfully synthesized using Fe(NO3)3⋅9 H2O and [BMIm][BF4] as the precursors (e.g. Figure 2 b).[ 70 , 71 , 72 , 73 ] In the process, [BMIm][BF4] serves as the solvent, template, and fluorine source. The as‐synthesized iron‐based fluoride and its composite materials can be used as cathodes for lithium or sodium batteries.</p><!><p>SEM or TEM images of metal fluorides obtained in (or in the presence of) [BF4]−‐ or [PF6]−‐based ILs. a) The as‐prepared MnF2 sample. Reproduced with permission. [69] Copyright 2015, Wiley‐VCH. b) An orthorhombic FeF3⋅0.33H2O electrode fabricated in [BMIm][BF4]. Reproduced with permission. [70] Copyright 2010, Wiley‐VCH. c) Na2SiF6 nanoparticles obtained from a mixture of [BMIm][PF6] and ethanol under microwave irradiation. Reproduced with permission. [68] Copyright 2017, Wiley‐VCH. d) A BaLuF5 sample synthesized in a mixture of ethylene glycol and [OMIm][PF6]. Reproduced with permission. [75] Copyright 2017, Elsevier Inc. e) BaFCl:Eu2+ nanoparticles. Reproduced with permission. [76] Copyright 2018, Wiley‐VCH. f) NaY0.78Yb0.20Er0.02F4 nanocrystals. Reproduced with permission. [77] Copyright 2009, Royal Society of Chemistry.</p><!><p>Wickleder and co‐workers synthesized a series of ternary fluoridosilicates A2SiF6 (A=Li, Na, K, Rb, and Cs) with particle sizes of a few tens of nanometers (Figure 2 c) by using [BMIm][PF6] as both the solvent and fluoride source in a microwave‐assisted ionothermal synthesis at low temperatures. [68] This approach is very simple, energy‐efficient, and time‐saving since it avoids the use of harmful and toxic HF or its derivatives. The nanoparticles of this series of ternary fluoridosilicates A2SiF6 are regarded as possible host materials for future LEDs.</p><p>Rare‐earth fluorides are an interesting family of compounds because of their optical properties, and their syntheses in ILs have been widely investigated. Yan and co‐workers reported that novel spherical NaYF4 nanoclusters were prepared in [BMIm][BF4] or [BMIm][PF6] with the assistance of microwave radiation. [74] ILs serve as reaction solvents, fluorine sources, and microwave absorbents. They found that some NaF particles were formed when [BMIm][PF6] was used to prepare NaYF4 nanocrystals. This result is probably due to the easier breaking of the P−F bond in [BMIm][PF6] compared to the B−F bond in [BMIm][BF4]. Thus, a high concentration of fluoride ions in the [BMIm][PF6] reaction system leads to the formation of NaF. The morphology of the NaYF4 nanoparticles obtained in [BMIm][PF6] is different from that obtained in [BMIm][BF4]. The main reason is that the higher viscosity of [BMIm][PF6] might prevent the assembly and aggregation of small nanoparticles, which affects the final morphology. Finally, NaYF4 nanoparticles doped with lanthanide ions (Ln3+) display superior upconversion properties.</p><p>Zou and co‐workers reported that BaLuF5:Ce,Tb,Eu(Sm) sub‐microspheres with tunable sizes and morphologies could be synthesized from IL/ethylene glycol dual‐solvent systems (Figure 2 d). [75] The results show that the [OMIm][PF6] IL used serves as both the fluoride source and capping agent. The aggregation manner of the nanoparticles and the size of the particles can be tuned by changing the ratio of the IL and ethylene glycol. Doping with Ce3+, Tb3+, or Eu3+/Sm3+ ions allows the photoluminescence colors to be tuned from green, through yellow, to orange. Wickleder and co‐workers reported a novel IL‐assisted synthesis of Eu2+‐doped BaFCl nanoparticles by using [BMIm][BF4] as both the solvent and fluorine source under sonochemical and microwave conditions (Figure 2 e). [76] Additionally, the [BMIm][BF4] IL coordinates the Eu2+ ions and stabilizes their oxidation state. Kong and co‐workers reported the ionothermal synthesis of pure‐phase NaYF4:Yb3+,Er3+/Tm3+ nanoparticles (Figure 2 f). [77] The key in the synthesis is the use of [BMIm][BF4], which serves as the solvent, template, and fluorine source. The obtained nanocrystals can be directly dispersed in water and present—because of the hydrophilic overlayer of [BMIm][BF4] on the crystal surface—a strong positive charge as well as strong upconversion luminescence.</p><p>Janiak and co‐workers reported that a variety of transition‐metal and rare‐earth metal fluoride nanoparticles (MF x ‐NPs, M=Fe, Co, Pr, Eu, Gd, and Er) could be obtained by the microwave‐induced decomposition of their metal amidinate complexes [M{MeC(N(iPr)2)} n ] (M(amd) n ; M=MnII, FeII, CoII, PrIII, GdIII, ErIII) and tris(2,2,6,6‐tetramethyl‐3,5‐heptanedionato)europium(III) (Eu(dpm)3) in [BMIm][BF4].[ 78 , 79 , 80 ] The M(amd) n and Eu(dpm)3 are suspended in dried [BMIm][BF4] under argon. An increased temperature drives the decomposition of M(amd) n /Eu(dpm)3 to release the metal species and the hydrolysis/decomposition of the [BF4]− anion to release F−. The reaction of metal species with F− results in the formation of metal fluorides.</p><p>Interestingly, it is found that the degraded components of [BF4]− anions can be utilized in the presence of moisture or heat as a boron precursor in the reaction. Turbostratic boron nitride nanoflakes (t‐BN) were obtained on using [BMMIm][BF4] as the boron source. [81] The hydrogen‐bond‐co‐π‐π stack mechanism is responsible for the self‐assembly of the [BMMIm][BF4] IL for the formation of the flake‐like t‐BN.</p><!><p>Conventional inorganic bases, such as NaOH, KOH, or Na2CO3, have many disadvantages such as being corrosive and producing waste. Basic ILs, which combine the advantages of inorganic bases and ILs, have great potential to replace them. They are noncorrosive, nonvolatile, flexible, and immiscible with many organic solvents. Therefore, basic ILs are often applied in some base‐catalyzed processes in organic chemistry [82] and nanomaterial preparation. In inorganic synthesis, basic IL anions provide the required basic environment of the reactions, while the organic cations may play an important role in the crystal nucleation and growth.</p><p>Li et al. designed a variety of tetraalkylammonium hydroxide ILs to synthesize metal oxides of various sizes and shapes. For example, several unusual ZnO nanostructures, including flower‐like particles, "lotus‐leaf‐like" ZnO plates, and porous ZnO plates, can be produced from tetrabutylammonium hydroxide (TBAH). [83] In these reactions, TBAH serves as an efficient IL precursor for the synthesis of ZnO nanoparticles with controlled sizes and morphologies.[ 84 , 85 ] Interestingly, not only regular nanocrystals, but also special hollow ZnO mesocrystals can be obtained in TBAH by varying the concentration of the zinc acetate precursor (Figure 3 a). [86] The resulting tubular microstructures are composed of smaller nanosized ZnO primary particles with a high degree of order, which classifies them as mesocrystals. It is assumed that the large tetrabutylammonium cation of TBAH reverses the polarity of the negatively charged surfaces of the small particles, thus preventing further growth of these small primary particles and aiding their aggregation into larger structures. A wide variety of uniform metal (hydr)oxide particles (Figure 3 b–e) was successfully prepared from water/TBAH liquid precursor mixtures simply by using metal acetates (M(OAc)2, M=Fe, Co, Mn, Ni, and Cu) as the metal sources.[ 87 , 88 ] Moreover, some other types of hydroxide‐based IL precursors, including tetraethylammonium hydroxide (TEAH) and benzyltrimethylammonium hydroxide (BTMAH), were used instead of TBAH for the efficient synthesis of ZnO particles with controlled sizes and morphologies.[ 89 , 90 ]</p><!><p>SEM and TEM images of metal oxide and hydroxide samples obtained in (or in the presence of) TBAH. a) Hollow ZnO mesocrystals. Reproduced with permission. [86] Copyright 2008, Wiley‐VCH. b–d) γ‐Fe2O3/Fe3O4 cubes and spheres, β‐Ni(OH)2 plates, and Co(OH)2 plates, respectively. Reproduced with permission. [87] Copyright 2008, Wiley‐VCH. e) CuO nanoplates. Reproduced with permission. [88] Copyright 2008, American Chemical Society. f) Hierarchically structured SrTiO3 microparticles. Reproduced with permission. [91] Copyright 2017, Royal Society of Chemistry.</p><!><p>Ruck and co‐workers successfully extended the use of TBAH for the fabrication of a perovskite‐type oxide SrTiO3 (Figure 3 f). [91] Hierarchical desert‐rose‐like SrTiO3 microstructures with a high surface area of up to 186 m2 g−1 are obtained by using TBAH as the alkali under solvothermal conditions mediated by ethylene glycol. It was found that TBAH or tetrabutylphosphonium hydroxide (TBPH) can replace the ethylene glycol and act as both the solvent and reactant to yield polyhedral SrTiO3 nanoparticles.</p><!><p>This section covers reactions of imidazolium or phosphonium salts with chalcogens (S, Se, or Te) to generate the corresponding imidazole‐2‐chalcogenones or trialkylphosphane chalcogenides, respectively. The reaction mechanisms are also discussed. The utilization of chalcogenones or trialkylphosphane chalcogenides as chalcogen sources for the synthesis of metal chalcogenides is also summarized in this section.</p><p>It has been recognized that the C2‐position of the 1,3‐dialkylimidazolium cation contains an acidic proton. Deprotonation of this position leads to the formation of a stable carbene, which is often used as an intermediate in organic synthesis. [92] It is found that reactions of inorganic reactants (e.g. chalcogens) with imidazolium salts at their C2‐position can also take place, especially in the presence of a base. Rogers and co‐workers reported that 1,3‐dialkylimidazolium acetates can react with elemental S or Se to afford the corresponding imidazole‐2‐chalcogenones directly, even in the absence of an additional base (Scheme 1). [93] Investigations show that the imidazolium acetate IL serves as both the carbene source and base to generate, in situ, the carbenes, which then react with S to form the thione.</p><!><p>Reactions of the imidazolium acetate ionic liquid with chalcogens. [93] .</p><!><p>However, when other anion‐based (e.g. Cl−, [HSO4]−, [SCN]−, [NTf2]−, and [BF4]−) imidazolium ILs were tested, no reactions with the chalcogens were observed. In these cases, ultrasound irradiation or strong bases are usually required. Ansell et al. demonstrated that [MMIm]I can readily react with elemental S to yield 1,3‐dimethylimidazole‐2‐thione in the presence of K2CO3 in methanol. [94] This "MeOH/K2CO3" method has since been utilized to prepare a variety of chalcogenones, including 1,3‐dialkylimidazole‐2‐thione/selone,[ 95 , 96 ] bridged bis(imidazoline‐2‐thione/selone),[ 97 , 98 , 99 ] and bridged mixed bidentate N‐heterocyclic carbene (NHC)/sulfur ligands, [100] starting from the corresponding imidazolium salts and chalcogens. Other "base/solvent" systems such as MeOH/pyridine/DBU, [101] pyridine/Et3N, [102] and THF/KO(t‐Bu) [103] have also been investigated for the synthesis of imidazole‐2‐chalcogenones by the reaction of quaternary imidazolium salts with chalcogens.</p><p>The use of refluxing methods has also been reported. For example, Wasserscheid and co‐workers found that 1,3‐dialkylimidazolium halide salts can react with elemental S in refluxing methanol in the presence of NaOMe to afford 1‐alkyl‐3‐methylimidazolium‐2‐thiones. [104] Tian et al. prepared a variety of selenones by refluxing the respective imidazolium salts with Se and Na2CO3 in water. [105]</p><p>In addition, Inesi and co‐workers developed an efficient combined electrochemical and ultrasound method for the synthesis of imidazole‐2‐thiones. [106] In this reaction, the imidazolium IL is first electrochemically reduced to the corresponding carbene, which then reacts with elemental S under ultrasound irradiation to give the target thiones in high yields. Lei and co‐workers reported that reactions of imidazolium salts with potassium thioacetate/thiocyanate as the S source yield imidazole‐2‐thiones rapidly and efficiently under microwave radiation. [107]</p><p>In contrast to thiones and selenones, the synthesis of tellurones derived from their corresponding imidazolium salts is much more difficult because of the relatively weak C−Te bond compared to C−S/Se bond. Singh and co‐workers developed a new approach for the high‐yielding synthesis of benzimidazolin‐2‐tellurones by the reaction of Te nucleophiles Na2Te/Na2Te2 with various benzimidazolium salts. [108] Compared to Te powder, the stronger Te2−/Te2 2− nucleophiles facilitate chemical reactions with benzimidazolium salts under mild conditions.</p><p>The use of imidazole‐2‐chalcogenones as chalcogen precursors for the synthesis of metal chalcogenides is rare. [109] Shi and co‐workers utilized 1,5‐bis(3‐methylimidazole‐2‐selone)pentane (Pbis) as a novel Se precursor to successfully synthesize a series of metal selenides, including ZnSe, CdSe, PbSe, and Bi2Se3. [109] Pbis is easily obtained and air‐stable. Moreover, the significantly positively charged 1,5‐bis(3‐methylimidazole)pentane and the negative Se valence in Pbis lead to the rapid and efficient reaction of Pbis with metal cations. Thus, it constitutes a facile and general method to prepare various metal selenide nanoparticles.</p><p>In comparison to the popularly investigated imidazolium ILs, phosphonium ILs have been less studied. Despite their higher thermal and chemical stability, phosphonium ILs are not completely inert, and decomposition can occur under certain conditions. [35] Ruck and co‐workers showed that quaternary phosphonium cations of ILs can undergo decomposition in the presence of Se/Te above 220 °C.[ 110 , 111 ] A series of dissolution tests, in which the solute Se/Te species were tracked by nuclear magnetic resonance (NMR) spectroscopy, was applied to systematically investigate the decomposition mechanisms. These studies indicate that one alkyl substituent of the quaternary phosphonium cations is eliminated through an SN2 decomposition pathway, leading to dissolution of Se/Te through the formation of the corresponding trialkylphosphane selenides/tellurides (Figure 4). However, the decomposition mechanism of the phosphonium IL in the presence of Te is much more complicated than that in the presence of Se. The 1 J PTe coupling, which indicates a P−Te bond is formed, is only observed in the NMR spectra when a sufficient amount of Te (e.g. Te/IL=1:1) is present (Figure 4 d). The use of smaller amounts of Te results in the 125Te satellites in the 31P NMR spectra disappearing and the doublets in the 125Te NMR spectra collapsing to one broad doublet (Figure 4 c) or a single line (Figure 4 b). In addition, the existence of a parallel, competitive IL decomposition route to the SN2 reaction is regarded as the side reaction for the dissolution of Te. This may at least partially explain the relatively lower solubility of Te compared to Se in phosphonium‐based ILs.</p><!><p>a) 77Se NMR spectrum of the reaction solution with a molar ratio of Se/[P66614][decanoate]=1:4 at 220 °C under Ar. Reproduced with permission. [110] Copyright 2017, Royal Society of Chemistry. 125Te NMR spectra of Te solutions with a molar ratio of Te/[P66614][decanoate]=1:7.6 (b), 1:2 (c), and 1:1 (d) at 220 °C under Ar. Reproduced with permission. [111] Copyright 2018, Wiley‐VCH.</p><!><p>These preformed trialkylphosphane selenides/tellurides can serve as Se/Te reservoirs for the preparation of nanostructured metal selenides/tellurides, such as octahedral NiSe2 particles, ZnSe nanocrystal aggregates, or 3D intergrown Bi2Te3 crystals (Figure 5 a,b,e,f). Additionally, Te single crystals and various Te microstructures, including 3D hierarchical fern‐leaf‐like Te structures, 3D Te fusiform assemblies, and 3D aloe‐like Te microarchitectures, are obtained when using a reactive Te solution in dried commercial [P66614]Cl as the Te precursor (Figure 5 c,d,g–i).[ 111 , 112 ] These IL‐based synthetic methods provide convenient and efficient strategies for the preparation of Se/Te‐based materials compared to the conventional complicated solution methods which typically need a reductant (e.g. NaBH4) in the presence of a surfactant (e.g. polyvinylpyrrolidone or cetyltrimethylammonium bromide).</p><!><p>SEM and TEM images of Se/Te‐based nano‐/microparticles obtained in phosphonium ILs. a,b) NiSe2 and ZnSe nanoparticle aggregates. Reproduced with permission. [110] Copyright 2017, Royal Society of Chemistry. c) Leaf‐like Te microstructure. d) Te single crystal. e) Bi2Te3 nanoplate. f) Flower‐like Bi2Te3 particle. Reproduced with permission. [111] Copyright 2018, Wiley‐VCH. g–i) 3D complex Te microstructures. Reproduced with permission. [100] Copyright 2020, Royal Society of Chemistry.</p><!><p>Some examples also document the direct synthesis of chalcogenides from chalcogen‐containing ILs. This case is similar to the use of metal‐containing ILs as metal sources, as mentioned above, since both the chalcogen and metal species can be incorporated in the IL anion or cation. Wu and co‐workers reported that the thiocyanate IL [BMIm][SCN] can serve as both the solvent and sulfur source for the preparation of CdS nanocomposites. [113] Zheng and co‐workers designed the Se‐containing IL [BMIm][SeO2(OCH3)] as a novel Se source. [114] The IL anion ([SeO2(OCH3)]− ion) shows similar reactivity as the commonly used Na2SeO3 system. However, precipitates form in some systems from the reaction of Na2SeO3 with metal ions. The use of the [SeO2(OCH3)]− anion avoids this precipitation problem, because of its weaker polarizing capability, and the metal ions exist as free ions in the solutions. Moreover, particle growth is influenced by the adsorption of the [BMIm]+ cation on the formed crystal surfaces, leading to nanoparticles with diverse shapes. As a consequence of these distinct characteristics of this Se‐containing IL precursor, various metal selenides with special morphologies, including CuSe nanoflakes, [115] Cu2−x Se nanocrystals, [115] ZnSe hollow nanospheres, [116] and CdSe nanospheres and nanodendrites, [117] have been successfully prepared. Selenium can also be incorporated into the cation of the ILs. Janiak and co‐workers synthesized several selenoether‐functionalized ILs with the [NTf2]− anion, and these were used as both the reaction media and Se reagents for the preparation of ZnSe nanoparticles under irradiation with microwaves at 220 or 250 °C. [118] It is assumed that the proximity of the in situ generated carbene precursor complex to the Zn2+ ions leads to an interaction and the formation of an intramolecular coordinative Zn−Se bond. The decomposition of these NHC(Se)‐Zn complexes under microwave heating yields the corresponding ZnSe nanoparticles. Furthermore, the same group reported the synthesis of a variety of metal selenide (e.g. CdSe, PbSe, and Pd17Se15) nanoparticles by decomposing the corresponding metal‐Se‐based molecular complexes in ILs.[ 119 , 120 , 121 ]</p><!><p>ILs allow the creation of several types of open‐framework materials such as zeolites or metal‐organic frameworks (MOFs) as well as polyanionic/polycationic compounds. In many reactions, the IL cation or anion, as the counterion, is incorporated into the final structures as a result of the charge of the framework. In this section, reactions of ILs whose cations, anions, or both are incorporated into the final products are summarized.</p><!><p>As new types of halide sources, halide‐based ILs exhibit distinctive features compared to the commonly used halide sources. Zheng and co‐workers reported that various BiOCl nanostructures, such as ultrathin BiOCl nanoflakes, curved nanoplates, and nanoplate arrays, could be successfully prepared using [C16MIm]Cl as the solvent, template, and chloride source. [122] The [C16MIm]+ cation with its long alkyl chain tends to adsorb on the (001) plane of BiOCl, and crystal growth along the c‐axis direction is inhibited, which leads to the formation of thin BiOCl nanoflakes. The obtained BiOCl nanoplates show potential applications for the removal of heavy metal ions in the field of wastewater treatment.</p><p>Ruck and co‐workers investigated the ionothermal synthesis of several borate halide compounds using quaternary phosphonium halide ILs as both the solvent and halide source. The Li‐ion‐conductive polycrystalline Li4B7O12Cl can be obtained in [P66614]Cl in the presence of lithium acetate dehydrate and boric acid at a temperature of 200 °C for 48 h. [123] The borate framework, constructed from [BO4] tetrahedra and [BO3] triangles, forms interpenetrating channels, within which Li+ and Cl− are trapped (Figure 6 a). Similarly, Li4B7O12Br can also be synthesized using [P66614]Br as the bromide source. Three nanostructured borate halides of the A2B5O9X type (A=Sr, Ba, X=Cl, Br)—Sr2B5O9Cl nanorods, Sr2B5O9Br nanoneedles, and Ba2B5O9Cl nanosheets—were synthesized by the reaction of acetates A(OAc)2 and boric acid B(OH)3 in a mixture of [P66614]X and LiNTf2. [124] As shown in Figure 6 b, the [BO4] tetrahedra and [BO3] triangles form the anionic [B5O9]3− framework with large channels. Halide anions, from the phosphonium halide ILs, and metal cations are trapped in these channels. Investigations further show that the reactivity of [P66614]X is promoted by adding the metal salt LiNTf2 to the reaction system, as it weakens the cation–anion interactions of the ILs. However, microcrystalline Pb2B5O9X (X=Cl, Br), with an average diameter of 1 μm, was obtained in [P66614]X without adding LiNTf2. [125] Both Pb2+ and X− are incorporated into the channels of Pb2B5O9X compounds. These borate halides show efficient second harmonic generation, even as microcrystalline powders.</p><!><p>a) The crystal structure of Li4B7O12Cl. Reproduced with permission. [123] Copyright 2019, American Chemical Society. b) The crystal structure of Sr2B5O9Cl. Reproduced with permission. [124] Copyright 2020, Wiley‐VCH. The [BO4] tetrahedra and [BO3] triangles in the borate framework are highlighted in red.</p><!><p>Zeolites are a family of porous materials that are widely applied in adsorption and catalysis. [126] As an example, the industrially significant aluminosilicate zeolite frameworks are comprised of corner‐sharing [SiO4/2] and [AlO4/2]− tetrahedra linked through bridging oxygen atoms. Thus, the overall framework bears a negative charge caused by the negatively charged [AlO4/2]− units. A wide range of cations, such as Na+, K+, Ca2+, Mg2+, and others, can be accommodated in the zeolite cavities as counterions to balance the anionic charge. If a zeolite is prepared in an IL, the organic cations are incorporated into the zeolite channels to balance the charge and also act as structural templates. [34]</p><p>The first IL‐based synthesis of zeolites in ILs was reported by Morris and co‐workers in 2004. [6] Several aluminophosphates (e.g. SIZ‐1, SIZ‐3, SIZ‐4, and SIZ‐5, Figure 7 a) are produced in [EMIm]Br. In SIZ‐1 (Figure 7 a), the hexagonal prismatic units are joined to form layers, and the neighboring layers are linked by four tetrahedral centers into a 3D framework. The framework is negatively charged due to the presence of terminal P−O bonds. [EMIm]+ cations are present in the pores, balancing the negative framework charges as well as templating the formation of the zeolite structure.</p><!><p>Aluminophosphate materials synthesized in ILs and DESs. a) SIZ‐1, SIZ‐3, SIZ‐4, and SIZ‐5 are obtained ionothermally in [EMIm]Br. b) SIZ‐2 and AlPO‐CJ2 synthesized in a ChCl/urea mixture. [6] Reproduced with permission from the Nature Publishing Group. Copyright 2004.</p><!><p>Since then, various framework types have been successfully obtained for aluminophosphates, including AEL,[ 127 , 128 , 129 ] LTA,[ 130 , 131 ] CHA, [132] and layered structures, [133] by the ionothermal synthetic route. In these reactions, the organic cations of the IL have been demonstrated to be effective templates that often reside in the cavities of the obtained zeolites to compensate for the negative charges of the frameworks. Very recently, Lin and co‐workers designed the multifunctional IL [MIm][H2PO4] (MIm=N‐methylimidazolium) for the ionothermal synthesis of crystalline metal phosphates (metal=Be, Al, Zn, and Fe).[ 134 , 135 ] Interestingly, [MIm][H2PO4] provides the phosphorus source to build the framework unit without phosphoric acid, as well as being a template and solvent. The obtained aluminum phosphate has a 2D structure with 8‐membered‐ring windows and the beryllium phosphate contains extra‐large 24‐membered‐ring channels. The [MIm]+ cations are located within the large channels in the case of beryllium phosphate or within the interlayer region in the case of aluminum phosphate.</p><p>Despite much success in the ionothermal synthesis of aluminophosphates, the application of ILs in the preparation of silica‐based zeolites still faces a major obstacle because silica is poorly soluble in ILs. Morris and co‐workers designed a task‐specific IL [BMIm]OH0.65Br0.35 for the first ionothermal synthesis of siliceous zeolites. [136] The hydroxide component in the IL anion leads to a better dissolution of silica, while the cation templates the formation of the MFI framework. Fluoride is added to promote the dissolution of silicate precursors and the crystallization of the zeolite. The fluorine atoms are present in the final product as part of SiO4F within the pentasil units. The [BMIm]+ cations are incorporated inside the pores to balance the negative charge of the fluoride.</p><p>The IL‐templating effects were also used by Dai and co‐workers to synthesize porous transition‐metal oxides.[ 30 , 31 ] Unlike the incorporation of the IL cation or anion into the zeolite framework, the IL template (e.g. [BMIm][NTf2]) used for the fabrication of porous transition‐metal oxides can be easily extracted and removed by organic solvents. Based on this [BMIm][NTf2] templating method, well‐defined nanoporous TiNb2O7 and mesoporous MnCeO x have been synthesized that exhibit superior performance for fast‐rechargeable lithium‐ion batteries and high activity for the selective oxidation of hydrocarbons at low temperature (100–120 °C), respectively.</p><!><p>MOFs, as a group of porous crystalline materials consisting of metal ions (clusters) coordinated to organic ligands, have received much research interest in many applications (e.g. gas storage, gas separation, catalysis, drug delivery, and sensing) because of their diverse structures, high porosity, and controllable chemical structures. [137] In an ionothermal synthesis of MOFs, the IL cation, anion, or both may be incorporated in the open cavities of MOFs.</p><p>When anionic MOF frameworks are created in ILs, it is common for the IL cation to be incorporated into the MOF structure to balance the negative charge of the framework and to also act as a template. [34] For example, the metal‐organic framework [EMIm][Cd(btc)] (btc=benzene‐1,3,5‐tricarboxylate) was obtained by the reaction of Cd(NO3)2⋅4 H2O with H3btc using [EMIm]Br as a reaction medium. The framework is composed of Cd2 units with six btc ligands coordinated to each Cd2 moiety, while each btc is linked by three Cd2 units. This leads to the formation of an anionic [Cd(btc)]− framework. The [EMIm]+ cations are located in the void space of the framework. [138] If H3btc is reacted with other metal precursors (e.g. Mn(OAc)2⋅4 H2O, Ni(OAc)2⋅4 H2O, or Co(OAc)2⋅4 H2O) in [C n MIm]X (n=2 or 3; X=Cl−, Br−, or I−) the corresponding anionic frameworks ([Mn(btc)]−, [Ni3(btc)2(OAc)2]2−, or [Co3(btc)2(OAc)2]2−) are obtained, with 1‐alkyl‐3‐methylimidazolium cations within the channels.[ 139 , 140 ] A series of MOFs containing other linking groups, such as bdc (benzene‐1,4‐dicarboxylate),[ 141 , 142 ] iso‐bdc (benzene‐1,3‐dicarboxylate),[ 143 , 144 ] 1,4‐ndc (naphthalene‐1,4‐dicarboxylate), [145] btetc (benzene‐1,2,4,5‐tetracarboxylate), [146] and d‐cam (d‐camphorate)[ 147 , 148 ] has been successfully prepared in various imidazolium ILs, with the cations residing in the open regions of the frameworks.</p><p>The anions of an IL can also be incorporated in the voids of the MOF as charge‐compensating species. Huang and co‐workers reported that three Cd3F‐based compounds with cationic frameworks, namely [Cd3F(ina)4(4‐pic)3][BF4], [Cd3F(ina)3(4,4′‐bpy)2(4‐pic)2][BF4]2⋅(4,4′‐bpy)⋅2 H2O, and [Cd3F(ina)3(4,4′‐bpy)3][BF4]2⋅(4,4′‐bpy)⋅2 H2O (ina=isonicotinate, 4,4′‐bpy=4,4′‐bipyridine, 4‐pic=4‐methylpyridine) were prepared in [BMIm][BF4]. [149] The [BF4]− anion serves as a charge‐balancing unit located in the voids of the frameworks. However, the F− ions formed by in situ hydrolysis of the [BF4]− anion are trapped within the MOF framework through the formation of a [Cd3F]5+ unit. Thus, the [BMIm][BF4] IL serves as solvent, fluoride source, and structure‐directing agent.</p><p>In another case, the anion of an IL could be utilized as the reactant through coordinating with metal ions to form a negatively charged framework, while the cation was trapped in the channels as a counterion and a template, that is, both the anion and cation of the IL can be incorporated in the voids of MOF frameworks during ionothermal syntheses. [150] Kwon and co‐workers reported the synthesis of [PMIm][Zn2(btc)(OH)Br] in [PMIm]Br and [BMIm][Zn2(btc)(OH)I] in [BMIm]I by using Zn(NO3)2⋅6 H2O and H3btc as starting materials.[ 151 , 152 ] In both cases, the IL anions (bromide or iodide) form Zn−X (X=Br or I) bonds to become part of the anionic frameworks. The imidazolium cations are incorporated in the channels and appear to show strong interactions with the frameworks.</p><!><p>Polyanions and polycations constitute an interesting class of compounds as a consequence of their diverse structures and chemical bonding. The ionothermal approach is promising for the formation of new polyanionic and polycationic compounds with unique structures that are not accessible using well‐established hydro‐/solvothermal techniques. Recently, the application of room‐temperature ILs for the preparation of polycationic or polyanionic cluster compounds was extensively explored by the groups of Ruck, Dehnen, Kanatzidis, Feldmann, and Riedel.[ 13 , 36 , 153 , 154 , 155 , 156 , 157 ]</p><p>To date, several types of polyanionic compounds, such as polyhalides,[ 158 , 159 , 160 ] metal carbonyl cluster anions,[ 161 , 162 , 163 ] and anionic chalcogenide frameworks,[ 36 , 164 , 165 ] have been synthesized in ILs. For example, polybromides were usually limited to a maximum of 10 atoms because of their increasing vapor pressure and reactivity before the new IL‐based approaches were developed. [13] The use of ILs has resulted in several new polybromides. Feldmann and co‐workers reported the first 3D bromine‐rich polybromide network, namely [BMPyr]2[Br20], through an ionothermal synthesis using a mixture of [DMPyr]Br and [BMPyr][OTf] (Figure 8 a). [158] [DMPyr]Br acts as a "bromide donor" to bromine molecules. [BMPyr][OTf] is used as a "liquifier" to form a eutectic mixture with [DMPyr]Br, thereby establishing a liquid state of the mixture at or even below room temperature for convenient isolation of the solid product. Later, Maschmeyer and co‐workers discovered that the higher‐order polybromide [P4444]2[Br24] can be obtained from an equimolar mixture of [P4444]Br and [P66614][NTf2] (Figure 8 b). [159] Investigations show that the IL cation is the dominant factor in the product‐selective synthesis. The large number of close H⋅⋅⋅Br interactions between the butyl chains of [P4444]+ and the [Br24]2− are assumed to stabilize and direct the formation of this high‐nuclearity species. In [P4444]2[Br24], the "central" bromine atom is five‐coordinate, whereas that of [BMPyr]2[Br20] is six‐coordinate (Figure 8). This arises primarily from the required space for an octahedral building block being occupied by a butyl chain of [P4444]+, which prevents coordination of a sixth dibromine molecule.</p><!><p>a) Structure of [Br20]2− in [C4Mpyr]2[Br20]. Cations are omitted for clarity. Reproduced with permission. [158] Copyright 2011 Wiley‐VCH. b) Structure of the highest known polybromide anion [Br24]2− in [P4444]2[Br24]. Cations are omitted for clarity. Reproduced with permission.[ 159 , 160 ] Copyright 2015 and 2020 Wiley‐VCH.</p><!><p>In another case reported by Dehnen and co‐workers, two polyanionic compounds [BMMIm]24[Sn36Ge24Se132] and [BMIm]24[Sn32.5Ge27.5Se132] were synthesized by the reaction of [K4(H2O)3][Ge4Se10] with SnCl4⋅5 H2O in tetrafluoridoborate ILs ([BMMIm][BF4] and [BMIm][BF4], respectively) in the presence of DMMP (DMMP=2,6‐dimethylmorpholine). [164] These two compounds contain the largest known discrete polyanion [Sn36−x Ge24+x Se132]24− (x=0 or 3.5) with an outer diameter of 2.83 nm and an inner diameter of 1.16 nm. The IL cations [BMMIm]+ and [BMIm]+ serve as counterions that surround the anions and partially penetrate them. This research further shows that the addition of a small amount of an amine promotes phase formation and phase selectivity of the products.</p><p>Polycationic compounds are commonly carried out in Lewis‐acidic ILs, which usually combine alkylimidazolium halides with more than equimolar amounts of aluminum or gallium trihalides (MX3). [13] This leads to the high solubility of metals (e.g. Se, Te, Sb, and In) and their metal halides in highly polar Lewis‐acidic systems. Moreover, the self‐drying ILs protect the formed polycations from hydrolysis. The structures of the polycationic compounds can be tuned by using different Lewis acids.</p><p>For example, Ruck and co‐workers reported that several Sb‐Se heteropolycations can be accessed in the Lewis‐acidic room‐temperature ILs [BMIm]X⋅n AlX3 (X=Br, Cl; n=1.2–5.2) (Figure 9).[ 166 , 167 , 168 , 169 ] If Lewis‐acidic ILs [BMIm]Cl⋅n AlCl3 are used in the synthesis, some binary Sb‐Se polycations are obtained. Reactions of Sb with Se in [BMIm]Cl⋅4.7 AlCl3 yield, depending on the reaction temperature, the two cluster compounds [Sb2Se2][AlCl4] and [Sb13Se16][AlCl4]6Al2Cl7.[ 167 , 168 ] When SeCl4 is added to Se and Sb in [BMIm]Cl⋅n AlCl3, [Sb10Se10][AlCl4]2 crystallizes at room temperature. [166] If [BMIm]Br⋅n AlX3, however, is used as the reaction medium, ternary Sb‐Se‐Br polycations with the general formula [Sb4+3n Se4+4n Br2](2+n)+ are produced. [169] These cationic clusters are spiro‐heterocubanes with two terminal bromide ions, and their structures can be tuned by varying the bromine‐to‐chlorine ratio in the ILs. The use of the chlorine‐rich IL [BMIm]Br⋅5.1 AlCl3 at 160 °C results in the precipitation of [Sb13Se16Br2][AlX4]5. However, [Sb7Se8Br2][Sb13Se16Br2][AlBr4]8 crystallizes if the purely bromine IL [BMIm]Br⋅4.7 AlBr3 is used for the reaction. Finally, [Sb7Se8Br2][AlX4]3 is produced in [BMIm]Br⋅4.7 AlBr3 in the presence of a small amount of NbCl5. NbCl5 is added to modify the Lewis acidity.</p><!><p>Structures of Sb‐Se heteropolycations obtained in Lewis‐acidic ILs. Copyright 2016, MDPI: Multidisciplinary Digital Publishing Institute. [13] a) [Sb10Se10]2+ in [Sb10Se10][AlCl4]2; [166] b) 1∞ [Sb2Se2]+ in [Sb2Se2][AlCl4]; [167] c) [Sb13Se16]7+ in [Sb13Se16][AlCl4]6Al2Cl7;[ 167 , 168 ] d) [Sb13Se16Br2]5+ in [Sb13Se16Br2][AlX 4]5; [169] e) [Sb7Se8Br2]3+ in [Sb7Se8Br2][AlX 4]3; [169] and f) [Sb7Se8Br2]3+ and [Sb13Se16Br2]5+ coexisting in [Sb7Se8Br2][Sb13Se16Br2][AlBr4]8. [169] .</p><!><p>In another synthesis, the two compounds (CuBi8)[AlCl4]2[Al2Cl7] and (CuBi8)[AlCl4]3 were accessed by reacting stoichiometric amounts of Bi, BiCl3, with CuCl in the IL [BMIm]Cl⋅4 AlCl3 at 180 °C; (CuBi8)3+ is the first intermetallic bismuth polycation with a 3d metal atom. [170] The Lewis‐acidic IL takes part in the reaction, whereby [AlCl4]− or/and the [Al2Cl7]− ion becomes part of the final product to balance the (CuBi8)3+ cluster cation. Moreover, one of the chloride ions of the [AlCl4]− group coordinates to the copper atom, thereby completing its 18‐electron count. Further investigations have shown that the (CuBi8)3+ cluster cannot be obtained either in molten AlCl3 or in ILs containing deficient/excess AlCl3, thus showing the indispensable role of the IL and a suitable Lewis acid content.</p><!><p>DESs exhibit some similar physical and chemical properties as ILs. Thus, they also lead to significant successes in inorganic synthesis, especially in large‐scale applications because of their inexpensive constituents and easy preparation. This DES‐based synthetic strategy opens up many new opportunities for the synthesis of zeolites and other inorganic nanomaterials. In many cases, however, one or more components can decompose in the DESs upon moderate heating. This section summarizes some examples of the preparation of inorganic materials by utilizing the unstable properties of DESs. An overview is given in Table 3.</p><!><p>A summary of all the studies on inorganic materials prepared in DESs discussed in this review.</p><p>Materials composition</p><p>Deep eutectic solvents</p><p>Refs.</p><p>Al2(PO4)3⋅3 NH4</p><p>choline chloride/urea</p><p>[6]</p><p>[Zn(O3PCH2CO2)]⋅NH4</p><p>choline chloride/urea</p><p>[172]</p><p>Al(PO4)2⋅(CH3NH3)2(NH4), Al(HPO4)2F⋅(CH3NH3)2,</p><p>tetraethylammonium bromide (choline chloride)/1,3‐dimethyl urea</p><p>[173]</p><p>Al(PO4)2⋅(NH3(CH3)2NH3)(NH4), Al(PO4)2⋅(NH3(CH3)2NH3)(NH4)</p><p>choline chloride/ethylene urea</p><p>[173]</p><p>Al3(PO4)4⋅n((NH3(CH2)3NH3), Al(HPO4)(PO4)(NH3(CH2)3NH3),</p><p>Al3(PO4)4⋅n((NH3(CH2)3NH3), Al2(OH)(PO4)2⋅2 H2O⋅NH4</p><p>choline chloride/N,N′‐trimethylene urea</p><p>[173]</p><p>[Al3CoClP4O16][C5H13NOH]2</p><p>choline chloride/succinic acid, choline chloride/glutaric acid, or choline chloride/citric acid</p><p>[177]</p><p>C5H14NO⋅ZnCl(HPO3)</p><p>choline chloride/urea</p><p>[180]</p><p>Ni(NH3)6Cl2, NiCl2, α‐Ni(OH)2, NiO</p><p>choline chloride/urea</p><p>[181]</p><p>α‐Co(OH)2, Co3O4</p><p>choline chloride/urea</p><p>[182]</p><p>Fe2O3</p><p>choline chloride/urea</p><p>[183]</p><p>MnCO3</p><p>choline chloride/urea</p><p>[171]</p><p>CoFe layered double hydroxide and CoFe oxide</p><p>choline chloride/urea</p><p>[184, 185]</p><p>Bi2S3, Sb2S3, CuS, ZnS, PbS, Ag2S, and CdS</p><p>choline chloride/thioacetamide</p><p>[186]</p><p>iron alkoxide of glycerol</p><p>choline chloride/glycerol</p><p>[187]</p><!><p>To date, the 1:2 mixture of choline chloride and urea (ChCl/urea, melting point 12 °C) is the most widely investigated DES in the literature. Investigations have shown that heating at 125–225 °C usually leads to decomposition of the urea in ChCl/urea, thereby resulting in the reactivity of ChCl/urea at high temperatures. [171] Thus, ChCl/urea is usually used as a reactive reagent for the preparation of various inorganic compounds with unusual structures. In 2004, Morris and co‐workers first reported that a novel zeolite‐type framework (SIZ‐2, Al2(PO4)3⋅3 NH4) was produced in a ChCl/urea eutectic mixture (Figure 7 b). [6] The ammonia stemming from the partial decomposition of the urea templates the structure and balances the charge of the framework that forms the interrupted structure of SIZ‐2. Similarly, a new zinc organophosphate was synthesized in a ChCl/urea mixture by Liao et al. and, again, the ammonia acts as a template. [172] Later, Morris and co‐workers studied several eutectic mixtures based on quaternary ammonium halides (e.g. choline chloride and tetraethylammonium bromide) and urea derivatives (e.g. 1,3‐dimethylurea, ethylene urea, and N,N′‐trimethyleneurea) as the reaction media for the ionothermal synthesis of new zeolites. [173] As expected, the breakdown of the various urea derivatives of the DESs at high temperatures gives rise to the corresponding organic species (e.g. methylammonium, ethylene diammonium, and propylene diammonium), which serve as templates and enable controlled delivery to the reaction mixture. Nine aluminophosphate materials including five unknown compounds have been prepared in this way.</p><p>Metal phosphate MPO4 (M=Ga, Zr, Co, Fe, and Mn) frameworks can also be successfully synthesized using various DESs.[ 174 , 175 , 176 ] The organic template is delivered to the reaction mixture by decomposition of one or more components of the DES mixture. Some new metal phosphate frameworks have been produced using the unique and flexible properties of DESs.</p><p>Clearly, in these ChCl/urea‐based reaction systems, it is the urea portion that provides the better template. In fact, choline itself can be a very attractive template. To avoid the competition between these two ammonium cations as templates, Morris and co‐workers synthesized a series of DESs based on choline chloride/carboxylic acid for the ionothermal synthesis of cobalt aluminophosphate (CoAlPO) materials, [177] including an unusual layered zeolite material ([Al3CoClP4O16][C5H13NOH]2, SIZ‐13). The choline cations fill the interlayer space without significant chemical modification. However, the chloride ions from choline chloride are incorporated into the structure of SIZ‐13 through the formation of covalent Co−Cl bonds. Such metal–chlorine bonds have not been found in the hydrothermal synthesis because of their sensitivity to hydrolysis, but the water in DESs tends to be less reactive because of strong interactions with anions of the DESs.[ 178 , 179 ] Similarly, Harrison reported that the compound C5H14NO⋅ZnCl(HPO3), which contains covalent Zn−Cl bonds, was obtained by reacting choline chloride with Zn2+ and hydrogen phosphite precursors in ChCl/urea. [180]</p><p>DESs are also applied as reactive reagents for the preparation of various functional materials such as metal hydroxides, metal oxides, metal chalcogenides, and organic–inorganic hybrids. [39] Gu and co‐workers synthesized various nanostructured transition‐metal complexes and layered transition‐metal hydroxides, as well as their derivatives in ChCl/urea through an ionothermal strategy at a relatively high temperature (120–210 °C). Octahedral [Ni(NH3)6]Cl2 crystals with an open structure can be obtained when the Ni2+:ChCl/urea solution is heated in a sealed vessel (Figure 10 a). [181] Nanosheet‐like NiCl2 is produced by annealing the [Ni(NH3)6]Cl2 precursor. When the Ni2+:ChCl/urea solution is thermally treated under an open system, however, the ammonia released from the urea is removed from the reaction solution, which leads to the formation of the flower‐like α‐Ni(OH)2 when a small amount of water is added to the solution under heating (Figure 10 b). NiO with the same flower‐like morphology is synthesized through annealing the as‐obtained α‐Ni(OH)2 (Figure 10 c). Similarly, α‐Co(OH)2 and Co3O4 can also be accessed by this water injection method. [182] In the case of Fe, however, it is the Fe‐oxide phase Fe2O3 that is directly obtained from a Fe3+:ChCl/urea solution. [183] If a Mn2+:ChCl/urea solution is heated in a closed system, a calcite‐type MnCO3 phase is produced. [171] Moreover, a "two‐stage water injection" strategy is applied to synthesize the cobalt iron layered double hydroxide (CoFe LDH) with different interlayer spacings. [184] Investigations have shown that the volume of injected water at each stage plays an important role in determining the structure of the CoFe LDH. When a small amount of water is injected in the first stage, the ChCl‐DES maintains its superstructure. In this case, CoFe LDH with an expanded interlayer spacing of 11.3 Å in its (003) plane is obtained. Further calcination of the CoFe LDHs results in porous CoFe oxide nanosheets with a large specific surface area of 79.5 m2 g−1. [185]</p><!><p>a) SEM image of the Ni[NH3]6Cl2 octahedron with exfoliated facets. b,c) TEM image and corresponding SEAD patterns of α‐Ni(OH)2 and NiO. Reproduced with permission.[ 39 , 181 ] Copyright 2013 and 2017, Royal Society of Chemistry. d–j) SEM images of ZnS, CuS, Bi2S3, Sb2S3, PbS, Ag2S, and CdS, respectively, which are obtained from ChCl/TAA‐based DESs. Reproduced with permission. [186] Copyright 2017, Wiley‐VCH. k) SEM image of the as‐synthesized iron glycerate hybrid in ChCl/glycerol based DES. Reproduced with permission. [187] Copyright 2019, American Chemical Society.</p><!><p>In addition to the well‐studied ChCl/urea‐based DES, other types of DESs may also be used as reactive reagents for the formation of inorganic materials. A family of binary metal sulfides, such as Bi2S3, Sb2S3, CuS, ZnS, PbS, Ag2S, and CdS, have been successfully synthesized by Ruck and co‐workers by using a DES based on ChCl/thioacetamide (TAA; Figure 10 d—j). [186] The proposed reaction mechanism consists of: 1) a metal salt is dissolved or dispersed in the ChCl/TAA‐based DES and the corresponding metal‐DES is formed; 2) the final sulfide is formed by thermal decomposition of the metal–DES complex. This ChCl/TAA‐based DES serves as both the solvent and sulfur source, providing an ideal "all‐in‐one" reaction medium for the efficient synthesis of sulfide nanoparticles.</p><p>Li and co‐workers found that a Fe‐based organic–inorganic hybrid—a hierarchical 3D iron alkoxide of glycerol—was synthesized using Fe(NO3)3⋅9 H2O as a starting material in a ChCl/glycerol (1:2) DES (Figure 10 k). [187] The ChCl/glycerol DES serves not only as a benign reaction medium but also as a reactant. Fe3+ is chelated by glycerol and is integrated into the DES matrix through coordinative bonding and hydrogen bonding. Thus, the reactants are effectively brought together by the DES through a prestructuring effect, which results in the formation of the 3D hierarchical seedlike iron alkoxide of glycerol nanospheres. The as‐synthesized Fe‐based organic‐inorganic hybrid exhibits an enhanced oxygen evolution reaction performance, with a low overpotential of 280 mV at a current density of 10 mA cm−2.</p><!><p>In some IL‐based reactions, the IL itself may undergo complete decomposition. As mentioned in Section 5, phosphonium ILs are partly decomposed into trialkylphosphanes, and these intermediates then react with Se/Te to form the corresponding trialkylphosphane selenides/tellurides. Interestingly, phosphonium ILs can be further used as a phosphorus source for the synthesis of metal phosphides through complete decomposition of the quaternary phosphonium cations at higher temperatures (>350 °C). Li and co‐workers reported that nanostructured Ni2P and Ni12P5 nanoparticles were fabricated using [P4444]Cl as both the phosphorus source and reaction medium upon microwave heating at 350 °C for 1–2 minutes. [188] The as‐synthesized Ni2P nanocrystals show an enhanced electrocatalytic hydrogen evolution performance in an acidic medium. Moreover, phosphonium ILs can be designed to be metal‐containing ILs and can thus be utilized as both the metal and phosphorus source for the preparation of metal phosphides. For example, [P66614]2[CoCl4] was used to synthesize Co2P by a one‐step phosphidation at 400 °C without adding other reagents.[ 189 , 190 ] The obtained Co2P/carbon nanotube (CNT) composite shows high activity for the hydrogen evolution reaction. This strategy based on phosphonium ILs provides a remarkable advantage for the efficient synthesis of metal phosphides. Ruck and co‐workers reported the synthesis of copper‐deficient Cu3−x P (0.1<x<0.7) from elemental precursors in halide ILs (e.g. [P66614]Cl).[ 42 , 191 ] Investigations have shown that the halide anions drastically promote the reactivity of red or white phosphorus and kinetically suppress the formation of Cu2P by‐products. Based on mechanistic studies, it was found that chloride ions act as strong nucleophiles that attack the phosphorus network, thereby resulting in degradation of the phosphorus. At a high concentration of chloride ions, the P−P bonds are sufficiently activated, leading to a drastic increase in the formation of the Cu3−x P phase.</p><p>Recently, there has been a lot of interest in using ILs (including DESs) as versatile carbon precursors, rather than conventional polymers, for the preparation of carbon materials because of the unique properties of ILs, such as negligible vapor pressure, carbon‐rich nature, and structural diversity. In the carbonization processes, ILs are completely decomposed, and the corresponding ILs are converted into carbon residues. The yield, properties, and structure of the obtained carbons depend on the structure of the IL precursors. The relevant studies have been comprehensively reviewed elsewhere,[ 5 , 39 , 192 , 193 ] and will not be the focus of this Review.</p><!><p>Great developments have been made in inorganic syntheses using, or in the presence of, ILs, which has led to the formation of diverse compounds with interesting properties. However, the chemical reactivity of the ILs and DESs in the reactions is usually neglected or not explicitly discussed. This Review gives an overview of the chemical reactions of ILs or DESs in inorganic synthesis.</p><p>Metal‐containing ILs represent a promising class of metal sources. The IL properties can be tailored by varying the ligand structures and the incorporated metal ions, which enables the formation of inorganic nanoparticles with diverse sizes and morphologies. Basic ILs are usually employed to replace the traditional bases, such as NaOH, KOH, or Na2CO3, to provide the required basic environment for the production of metal (hydr)oxide particles. Moreover, the organic cations of the basic ILs can act as a template to control the crystal nucleation and growth. The hydrolysis of [BF4]− or [PF6]− anions gives a fluorine source for the synthesis of metal fluorides, thereby avoiding the use of toxic and harmful HF, NaF, or NH4F. The C2‐position of the 1,3‐dialkylimidazolium cation shows reactivity towards chalcogens (e.g. S, Se, and Te) for the formation of the corresponding imidazole‐2‐chalcogenones, especially in the presence of a base. In addition, phosphonium ILs can react with chalcogens at high temperatures to generate the corresponding trialkylphosphane chalcogenides. These formed imidazole‐2‐chalcogenones or trialkylphosphane chalcogenides can be further applied as chalcogen sources for the preparation of metal chalcogenides. When some porous materials such as zeolites, MOFs, and polycationic/polyanionic compounds are synthesized by an ionothermal approach, either the cation or anion of the IL can be incorporated in the channels of the products to balance the charged frameworks and can also serve as structural templates. In the case of DESs, one or more of their components may decompose to form in situ templates for the formation of metal phosphate frameworks or act as reactive species for the synthesis of functional nanoparticles (e.g. metal hydroxides, metal oxides, metal chalcogenides, and organic–inorganic hybrids).</p><p>The chemical reactivity of ILs and DESs in inorganic synthesis should not be underestimated. Furthermore, the reactive properties of the ILs and DESs in the reactions can be used as a synthetic tool to prepare inorganic materials that are difficult or even impossible to obtain by traditional synthetic routes. In this regard, it is important to have an in‐depth understanding of the interaction of ILs or DESs with reactants and solutes, and thus fully understand the reaction mechanism for the directed use of ILs and DESs for the preparation of inorganic materials.</p><!><p>The authors declare no conflict of interest.</p><!><p>Tao Zhang obtained his PhD in inorganic chemistry from the Technische Universität Dresden in 2018 under the supervision of Prof. Michael Ruck. He then joined to the Institute of Process Engineering, Chinese Academy of Sciences in 2019 as an Associate Professor. His research interests are concerned with the synthesis of functional materials in ionic liquids.</p><p></p><p>Thomas Doert received his PhD from the University of Düsseldorf in 1994 with Prof. Peter Böttcher. He then moved to the Technische Universität Dresden, where he is now Adjunct Professor. He was visiting scientist at Stockholm University as well as Guest Professor at Strasbourg University and Shanghai University of Engineering Science. His research interests cover crystallographic aspects of solid‐state chemistry, solid‐state chemistry of chalcogenides, layered materials, and frustrated magnets.</p><p></p><p>Hui Wang is a professor at the Institute of Process Engineering of the Chinese Academy of Sciences. She received her PhD from the University of the Chinese Academy of Sciences in 2010 with Prof. Zengxi Li. In the same year, she joined Professor Robin D. Rogers' Group at the University of Alabama as a postdoctoral research fellow. In 2015, she moved to the Institute of Process Engineering, Beijing. Her research interests focus on the properties of ionic liquids and the applications of ionic liquids in green chemical processes.</p><p></p><p>Suojiang Zhang received his PhD from Zhejiang University in 1994 with Prof. Shijun Han. He has been a professor at the Institute of Process Engineering of the Chinese Academy of Sciences since 2001, and was elected as a member of the Chinese Academy of Sciences in 2015. He is currently Director General of the Institute of Process Engineering and Fellow of the Royal Society of Chemistry. His research interests focus on the designed synthesis of ionic liquids and their applications in green chemistry and green process engineering.</p><p></p><p>Michael Ruck received his PhD from the University of Stuttgart in 1991 with Prof. Arndt Simon. After his habilitation at the University of Karlsruhe in 1997, he was a Heisenberg fellow of the German Science Foundation. Since 2000 he has been Full Professor of Inorganic Chemistry at the Technische Universität Dresden, where he has served as Dean of the Faculty of Science and Vice Rector. He has repeatedly been appointed a Fellow of the Max Planck Society and awarded the Steinhofer Research Award and the Will Kleber Commemorative Coin. His research interests include solid‐state chemistry and sustainable material syntheses.</p><p></p>

PubMed Open Access

Dissolving used rubber tires

Used automobile tires present an enormous environmental burden. Efficient methods for degradation of the sulfur crosslinks in organic elastomers have proven elusive. We show that the reductive silylation of RS-SR bonds to silyl thio ethers RSSiR'' 3 in up to 90% yield using a variety of hydrosilicones occurs in the presence of <1 mol% B(C 6 F 5 ) 3 for model compounds. Sulfur-cured automotive rubber required 10 wt% catalyst for efficient sulfide cleavage. At temperatures ranging from room temperature to 100 °C recoveries of organic polymers as oils from tires using this one step process ranged from 56% for complex mixtures of rubber crumb from ground tires to 93% for butyl rubber (bicycle inner tubes; 87% yield at 100 °C over 30 minutes). After removal of inorganic materials by simple filtration, the recovered polymeric oils were radically or oxidatively crosslinked to generate new elastomers that can be optionally reinforced with the solids recovered in the initial reduction procedure. This mild process constitutes a facile route to reutilize the organic polymers found in automobile and other sulfur-crosslinked rubbers. † Electronic supplementary information (ESI) available: Tables of reactivity of diand tetrasulfides, rubber components, efficiency of repeated reduction and GPC data of produced oils. Figures showing NMR data of starting rubbers, product oils, TGA data or starting rubbers, and products oils, photographs showing the process from rubber to oils to new rubbers. See

dissolving_used_rubber_tires

Introduction<!>Model reductions of dibenzyl disulfide and tetrasulfide<!>Reduction of used automotive rubbers<!>Methods<!>Preparation of dibenzyl tetrasulfide<!>Titration of disulfides to establish relative reactivity of functional groups<!>Reduction of rubbers: general procedure<!>Reduction with inexpensive tetramethyldisiloxane M H M H<!>Desilylation of product polymeric oils to give 11<!>Crosslinking of recovered oils<!>Conclusions<!>Conflicts of interest

<p>The radically induced 1 vulcanization of alkene-containing hydrocarbon polymers, reported by Goodyear in 1844, 2 is a technological advancement that remains an integral part of modern life; sales of automobile tires prepared using this process are expected to reach 3 billion units in 2019. 3 The elastomer products, crosslinked with sulfur oligomers, are incredibly robust products, which pose a major challenge; they are far too stable to be readily recycled.</p><p>Automobile tires exemplify polymers derived from fossil fuels that are destined for single use. Used tires constitute a significant environmental burden, particularly because of the scale of production. 4 In large part, used tires are simply placed in stockpiles, 5 from which leaching into the environment of their many (toxic) constituents occurs. 6 Dangerous, highly polluting, difficult-to-arrest tire fires at such storage facilities are not uncommon. 7 Some automotive rubber is exploited as fuel in the cement industry; capturing SO 2 during combustion may be problematic. Some tires are turned into crumb and used as fillers, 8 for example, in asphalt, cement or turf replacements from which, however, leaching of contaminants may still occur. 9 There is a longstanding need to recover the organic materials from tires for sustainable reuse. Although the S-S bond strength is only ∼280 kJ mol −1 , 10 practicable processes for S-S cleavage in vulcanized tires have not been reported. Aggressive chemical approaches, for example, reactive reduction with LiAlH 4 11,12 or amines 13 have not proven commercially viable.</p><p>Reuse strategies therefore typically involve energetically intensive, relatively inefficient pyrolytic conversion into fuel gas, low grade carbon black and other low value materials. 14 The world is in dire need of new efficient methods for recycling waste tire rubber, especially in ways that allow the recovery and reuse of the basic building blocks. The weak Si-H bond in hydrosilanes makes them excellent reducing agents, particularly when strong Si-heteroatom bonds are formed in the process. 15 Thus, reductive hydrolysis/ alcoholysis 16 and CvO hydrosilylation, 17 among others, are efficient processes. Key to the work described here is the Lewis acid-catalyzed (typically B(C 6 F 5 ) 3 = BCF) reduction of carbonyls (Fig. 1a), ethers, silanols, alkoxysilanes (the Piers-Rubinsztajn reaction [18][19][20] Fig. 1b), benzylic sulfides, and thioacetals (Fig. 1c) using HSi functional groups. 21 These reactions are normally easy to control, often work at room temperature, and the main experimental issues are associated with managing the co-products when they are flammable gases, including hydrogen or alkanes.</p><p>We report that, catalyzed by B(C 6 F 5 ) 3 , hydrosilanes effectively reduce S-S bonds of model organic disulfides, tetrasulfides and, more importantly, complex sulfur-crosslinked solid automotive rubbers in the forms of bicycle inner tubes, solid tires or tire crumb in good to excellent yield. The products are polymeric, silyl-protected thiolated organic oils that are readily separated from the accompanying, unreactive solids, such as fillers, fiber and metal reinforcements, pigments, etc. simply by filtration or centrifugation. The products, sulfur-containing polymeric oils, may be converted back into (reinforced) rubbers using simple oxidative or radical processes.</p><!><p>Model reductions of dibenzyl disulfide 1 (n = 1) were undertaken with bis(trimethylsiloxy)methylsilane 2 (HSiMe (OSiMe 3 ) 2 , MD H M) (the common nomenclature used for silicones is Q: SiO 4/2 ; T: MeSiO 3/2 ; D: MeSiO 2/2 and M: Me 3 SiO∼) as reducing agent in the presence of BCF (Fig. 1d, e, Fig. 2, Table S1, and Fig. S1, ESI †). With less than one equivalent of hydrosiloxane, residual starting material and only product 3 were recovered, demonstrating that the reaction of SiH with S-H, e.g., in compound 4, is faster than that with S-S bonds. Complete reduction of 1 → 3 required 2 equiv. of the hydrosiloxane and occurred in 90% yield using only 0.8 mol% BCF; the other sulfur-based product 5 was removed under reduced pressure. Reduction of the analogous tetrasulfide 6 to 3 using HSiMe 2 OSiMe 3 7 required five equivalents of MD H M and demonstrated that both C-S a -S a and S a -S b -S b linkages undergo efficient reductive silylation (Fig. 1f, 2 and Fig. S2, ESI †).</p><!><p>Automotive tires contain a complex variety of constituents, including (spent) catalysts for their formation, antioxidants, colorants, particulate reinforcing agents like carbon black and/ or organosulfur-modified silica, and fibrous reinforcing agents including nylon cord and woven steel. 14 Holding together this complex assortment of excipients is the sulfur-crosslinked elastomer. We reasoned that the (oligo)sulfide linkages in rubber tires could be reductively cleaved using hydrosilanes in analogy to the reactions with the oligosulfide model compounds (Fig. 1d-f).</p><p>Scrap rubber from automobile tires is available in large quantity in the form of 'rubber crumb'. It is formed by shredding tires from multiple sources to remove metal wires and polyester cord and grinding the resulting product to various crumb sizes. The typical organic constituents in crumb mixtures include isobutene isoprene (IIR), butadiene rubber (BR), styrene-butadiene rubber (SBR), isoprene rubber (IR) and natural rubber (NR). Thermal degradation profiles allow one to determine the constitution of sulfur-crosslinked elastomers; depending on their structure, the polymers degrade between 300-485 °C; 22,23 (Fig. S5, and S6, Table S3 †) while inorganic carbon (carbon black) thermally decomposes from 560-800 °C in oxygen. 24 In the rubbers tested, the organic rubber content was approximately 60 wt% (Table 1, Fig. S11 †).</p><p>Two separate sources of commercial crumb were compared for their reactivity under the reducing silylation conditions. Much more BCF catalyst (10 wt% compared to the rubber start- S2. † ing material) was required to achieve reasonable yields of reduction with rubbers than with the model compounds (<1 mol%), which is not surprising given the complexity of the mixed rubber starting materials and the fact that they have been exposed to degradation and various environments during use.</p><p>With 7 as reducing agent, organic oils were recovered in 36% yield from one crumb rubber source and a moderate 56% yield from another (2 lots, Fig. 3g). Use of second and third reduction steps with fresh catalyst and hydrosilane significantly improved overall conversion of elastomer to oils (steps (1) 56% → (2) +29.6% → (3) +2.5% (total 88% organic recovery) the inorganic constituents were removed by centrifugal separation (Tables 2 and 3, Fig. S14 †)). In retrospect, in a practical sense, only the improvement in overall efficiency of the second step could be justified. The reduction process was readily visible by eye, as black dispersions were converted to yellow oils (Fig. 3b → f,g, Fig. S7 and S8, ESI †).</p><p>Improved recoveries of organic polymers were observed with single composition rubbers. For example, about 60% of an EPDM elastomer (ethylene propylene diene terpolymer, 'pond liner') was converted to a soluble oil using reductive silylation with 7 and B(C 6 F 5 ) 3 in toluene at 60 °C. Automotive rubbers were efficiently reduced to oils in one step in yields ranging from 52-93%. These included: IIR from a (used) bicycle inner tube; IR/NR from the outermost section of truck tire tread; a mixture of IIR, IR/NR and BR from a snow tire side wall; and IR/NR, BR from a snow tire tread (Tables 2 and 3). The process is easily seen from the reduction of a bulk section of snow tire tread (Fig. 3a). Shortly after the reduction reaction started, the reinforcing steel wires separated from the bulk rubber and were collected on the magnetic stir bar (Fig. 3c); accompanied by particulate formation to give a black dispersion (Fig. 3b)the bulk rubber underwent shrinkage, but not complete disintegration (Fig. 3d, Fig. S19 †). Filtration allowed separation of a black solid mass comprised primarily of inorganic excipients and a yellow solution of silylated organic oils in toluene (Fig. 3e, f, Fig. S7 and S18 †).</p><p>Oligosulfides were converted into silyl thiol ethers during reduction (Fig. 3g and Fig. S7 †). Therefore, the residual solids and recovered non-volatile liquids often exhibited a weight gain when compared to the starting rubber mass (Table S4 †). The product oils 8 (Fig. 3, for 1 H NMR data, see Fig. S15 †) typically exhibited a bimodal distribution of molecular weight, with a low fraction centered near 10 000 g mol −1 and a broad peak centered near 1 million g mol −1 (Table S5, Fig. S16 †).</p><p>Several factors were manipulated to improve the efficiency of the reduction process. A variety of hydrosilicones are commercially available that vary in the density of SiH groups. Model studies on the reduction of the organic sulfides or auto- motive rubbers were undertaken with 2 or 7, respectively, because the use of small molecules facilitated characterization of the reaction products. Either compound is too expensive for practical use. Attempts to facilitate reduction of rubbers with the inexpensive, high SiH density polymer Me 3 Si (OSiMeH) n SiMe 3 9 were unsuccessful (Fig. S17 †) because the silicone product of the reduction is a network polymer, which led to the formation of intractable tars. By contrast, the use of inexpensive, high SiH density HMe 2 SiOSiMe 2 H 10 led to efficacious, rapid reduction of rubbers (Tables 2 and 3, Fig. S17 †). Unlike the model compounds above, relatively large quantities of the BCF catalyst, 10 wt% against the rubber, were c Organic yield = (total organic-recovered organic)/total organic × 100. Organic composition established using TGA (Fig. S6, S9, S11, Table S3, † and Table 1). d Cumulative yield for 3 process steps. e Process utilized hydrosilicone 10 (1.5 mL) at 100 °C for 30 min. f External road contacting component only. g Cross-section of entire tread from core to external surface. required for the reduction of rubbers to occur efficiently. Preswelling the rubber in commonly used organic solvents like acetone, 25 or an initial Soxhlet extraction using acetone to remove potential catalyst inhibitors, e.g., amines, free sulfur, acetone soluble colorants, antioxidants, processing rubber additives, etc., did not appreciably increase either the rate or yield of the reduction (Table 3, Fig. S14 †). We continue to work on process optimization to reduce the quantities of catalyst required. Several other factors were found to the affect the efficiency of the reduction of rubbers, including surface area, process temperature and reducing agent. Unsurprisingly, the reduction of bulk elastomers, including cylindrical sections of bicycle inner tube (diameter ∼16.9 mm, thickness ∼0.82 mm, IIR, Fig. S7 †), and a cross-section of an automobile snow tire (IR/NR + BR, Fig. 3), were slow to occur and low yielding at 60 °C using 7 (Fig. S6 †). In both cases, the objects underwent significant shrinkage (Fig. S19 †), and increased crosslink density as shown by an increase in Young's modulus (Table S4 †), but maintained their shape. Cryogenic grinding of the starting rubbers to increase rubber surface area (to particle size ∼330 μm, Fig. 4) led to significant enhancements in yield;</p><p>an increase from 52 to 93% was observed in the case of the inner tube (IIR, Table 3).</p><p>The tire reductive silylation studies were initially undertaken using relatively mild temperatures because literature suggests that the B(C 6 F 5 ) 3 catalyst undergoes degradation at temperatures above 80 °C in the presence of moisture. 26 Current studies with rubber reduction, however, showed this not to be problematic. A 93% yield of recovered organic polymer (IIR from inner tube) was achieved at 100 °C after 18 hours using 10, but an 87% yield had already been achieved in the first 30 minutes (Table 3). This result suggests that reduction processes at 100 °C or higher could be adapted to a continuous process.</p><p>Initial studies for reusing/re-crosslinking the recovered oil focused initially on the regeneration of thiols from silyl thio ethers. There are few reports of the reactivity of Si-S compounds. Me 3 Si-S-SiMe 3 is very labile, undergoing rapid degradation simply in the presence of water (vapor) to form H 2 S and Me 3 SiOH. 27 By contrast, the hydrolysis of silicone-based thio ethers was much less facile. Alcoholysis of 3 (Fig. 1g) yielded 12% product only once acetic acid was added to isopropanol solutions (the less sterically hindered thio ether PhCH 2 SSiMe 2 OSiMe 3 underwent rapid, quantitative cleavage under the same conditions). The silylated polymeric oils derived from elastomers 8 were yet less reactive. It was necessary to use more aggressive nucleophiles for silicon, such as Bu 4 NF 28 to regenerate the silyl free thiols 11 (Fig. 3h and Fig. S20 †).</p><p>Once cleaved, the freed thiols on the organic polymers could be crosslinked into a new elastomer 12 by oxidative coupling using iodine in isopropyl alcohol 29 (Fig. 3i and Fig. S21 †). However, it was also discovered that, if silylated polymers 8 were derived from IR/NR or BR and possessed residual alkenes, re-crosslinking did not require removal of the silyl groups; simply adding a radical initiator such as benzoyl peroxide (BPO) and heating led to new elastomers 13 (Fig. 3j).</p><p>The ability to create new elastomers from the recovered polymeric oils was demonstrated by creating a new automotive tire (for a child's toy) using 8. A mold of the tire was made in silicone rubber (Fig. 4a and b). Silylated oil 8 derived from IR/NR (tire tread) was placed in the mold in the presence of BPO and heated to give a new, soft elastomer (Fig. 4b + c → e,f durometer Shore OO 68, Table S6 †). Adding to 8 the inorganic excipients (recovered from the production of 8, Fig. 4h), and then curing oxidatively, led to harder, more brittle elastomers (Shore A 91; original rubber Shore A 60).</p><p>Reductive silylation processes have shown synthetic merit in many arenas. The relatively weak SiH bond, 30 particularly in the in the presence of B(C 6 F 5 ) 3 and related Lewis acids, readily reduces a variety of bonds 15 driven, in large part, by the thermodynamic benefit of forming Si-heteroatom bonds (heteroatomvO, N, S, etc.). 31 We have demonstrated that this type of process works effectively with S-S bonds to form thio silyl ethers. The key finding of this work is that, in addition to clean model compounds, the process works with complex and dirty samplesused automotive tiresto convert sulfur-cured elastomers into polymeric oils, in up to ∼90% yield. The process can be rendered practicable at temperatures as low as 100 °C and the product oils can find new utility in elastomeric objects using at least two different cure modes. We are currently establishing the quality of elastomers that can be produced from different used tire feedstocks. The recovered inorganic mass can also be repurposed as a filler in those new elastomers. Thus, reductive silylation provides a new opportunity to find commercial value in materials that are environmentally problematic. It is not possible to effectively 'reuse' automotive rubber, but reductive silylation is worthy of consideration as a strategy for recycling and reuse. n M) 9, were purchased from Gelest and used after drying over molecular sieves overnight. Dibenzyl disulfide, benzyl bromide, tetrabutylammonium fluoride trihydrate (TBAF), and iodine (I 2 ) were obtained from Sigma Aldrich and used as received. Benzoyl peroxide (BPO) was purchased from BDH. Sodium tetrasulfide was purchased from Dojindo. B(C 6 F 5 ) 3 (BCF) was prepared by Grignard reaction following a literature procedure; 32 we acknowledge with gratitude Prof. David Emslie, McMaster University, for providing this sample. Rubber samples: bicycle inner tube (Chaoyang 700 × 38/45C bicycle inner tube, China), EPDM ( pond liner, purchased at a local garden centre, producer unknown), Crumb-1 (Als-RC, Amazon, Canada), Crumb-2 (Canadian Eco Rubber Ltd, Emterra, Canada), were used as received. Truck tread 1: a piece of truck tread, not part of a complete tire, was found at a local garbage dump (origin unknown). Truck tread 2: (Sailun 225/ 70R19.5). In both cases, samples were cut only from the external, road contacting tread part (Fig. S3 †); both rubbers were based on polyisoprene. The tread and side wall samplescross-sectionswere cut from different parts of a used car tire (snow tire, Cooper 185/65R4, Fig. 5). Naphthalene (internal standard) was purchased from Fisher. Toluene (solvent) received from Caledon (HPLC grade) was dried over activated alumina before use. Deuterated NMR solvents were obtained from Cambridge Isotope Laboratories. Glass apparatus were dried overnight at 120 °C and cooled under a dry nitrogen atmosphere for 30 min prior to use.</p><!><p>NMR. 1 H, 13 C and 29 Si NMR spectra were recorded on a Bruker Avance 600 MHz nuclear magnetic resonance spectrometer using deuterated solvents chloroform-d and actone-d 6 .</p><p>For 29 Si NMR, chromium(III) acetylacetonate was used as paramagnetic relaxation agent.</p><p>GC-MS. Analyses were performed using an Agilent 6890N gas chromatograph (Santa Clara, CA, USA), equipped with a DB-17ht column (30 m × 0.25 mm i.d. × 0.15 μm film, J&W Scientific) and a retention gap (deactivated fused silica, 5 m × 0.53 mm i.d.), and coupled to an Agilent 5973 MSD single quadruple mass spectrometer. One microliter of sample was injected using an Agilent 7683 autosampler in splitless mode. The injector temperature was 250 °C and carrier gas (helium) flow was 1.1 mL min −1 . The transfer line was 280 °C and the MS source temperature was 230 °C. The column temperature started at 50 °C and was increased to 300 °C at 8 °C min −1 , then held at 300 °C for 15 min to give a total run time of 46.25 min. Full scan mass spectra between m/z 50 and 800 mass units were acquired after a solvent delay of 8 min.</p><p>LC-MS. Analyses were undertaken using an Agilent Technologies 1200 LC coupled to an Agilent 6550 QTOF mass spectrometer. An injection volume of 2 μL was separated on a Phenomenex Luna C18(2) (150 mm × 2.0 mm, 3 μm) column with 100 Å pore size (Phenomenex, CA, USA). The mobile phases were LC-MS-grade 45/55 water/methanol with 0.5% acetic acid (A) and methanol with 0.5% acetic acid (B) at a flow rate of 300 μL min −1 . The column temperature was maintained at 40 °C, and the autosampler storage tray was set at 10 °C. The mobile phase gradient eluted isocratically with 10% B for 1.0 min followed by a gradient to 100% B over 17 min. The gradient was maintained at 100% B for 2 min and decreased to 10% B over 0.1 min. The gradient was then followed by a 5 min re-equilibration prior to the next injection. The total time for an HPLC run was 25 min. The MS parameters (for LC-MS) chosen were as follows: ESI, gas temperature at 225 °C, drying gas at 13 L min −1 , nebulizer pressure at 20 psi, sheath gas temperature at 400 °C, sheath gas flow at 12 L min −1 , VCap at 3500 V, nozzle voltage at 1000 V, fragmentor at 375 V, and Oct 1 RF Vpp at 750 V. The data were acquired in electrospray positive mode from m/z 50 to 1000 at a scan rate of 1.5 Hz. The mass was auto recalibrated using reference lock mass from Agilent ESI-T Tuning Mix (for Ion Trap).</p><p>GPC. Molecular weight of recovered organic oil and polydispersity index (PDI) were estimated from gel permeation chromatography (GPC) using a Waters 2695 Separations Module equipped with a Waters 2996 photodiode array detector, a Waters 2414 refractive-index detector, and two Jordi Labs Jordi Gel DVB columns. Polystyrene standards were used for calibration, and THF was used as the eluent at a flow rate of 1.0 mL min −1 .</p><p>The polymer constituents of rubber samples were estimated from carbon high-resolution magic angle spinning ( 13 C HR-MAS) NMR spectroscopy (Fig. S5 †). TGA: Thermogravimetric analysis (TGA) according to ASTM D 6370-99 (American Society for Testing and Materials) was used to measure the organic carbon (polymer), carbon black content and inorganic residue of the component. 24 A small amount of test sample (2 to 5 mg) was placed into the alumina pan of the calibrated Thermogravimetric Analyzer (Mettler Toledo TGA/DSC 3+). A 100 cm 3 min −1 argon purge was applied and the furnace was heated from 50 °C to 560 °C at 10 °C min −1 . Then, the furnace was cooled to 300 °C and the purge gas was changed to air at 100 cm 3 min −1 . The temperature was allowed to equilibrate for 2 min before the furnace was heated to 800 °C at 10 °C min −1 .</p><p>The constituents of sulfur-cured rubbers have well-defined thermal decomposition profiles (Table S3 †). Within the thermal decomposition range of the organic polymers, there are further distinctions. TGA and DTA data (Fig. S6 †) show the constituents of the rubber samples tested (Table 1).</p><p>Young's modulus. The Young's moduli of rubber samples were determined using a MACH-1 micromechanical system (Biomomentum Instruments) with a 0.500 mm hemispherical indenter radius, and Poisson ratio of 0.3. All measurements were conducted at 22 °C and in triplicate, with error bars representing the standard deviation of the replicate measurements.</p><p>Preparation of powdered rubber. Raw rubber samples of different shapes and sizes were obtained. The rubber crumb samples were of broad dispersity, with average particles sizes of 1.30 ± 0.09 mm for Crumb-1 and 2.14 ± 0.06 mm for Crumb-2. As noted above, more efficient reduction occurred with higher surface area samples. A cryogenic grinding progress was used to obtain rubber powder samples with homogeneous particle sizes for comparable experiments. Liquid nitrogen was employed to cool the rubber samples below their glass transition temperature before they were pulverized with a coffee grinder (KitchenAid) to give powders with an average particle size of ∼330 mm (Fig. 5g, i and Fig. S4 †).</p><!><p>To a pre-dried 200 mL round-bottomed flask purged with dry N 2 were added sodium tetrasulfide (0.098 g, 0.562 mmol), benzyl bromide (0.209 g, 1.22 mmol) and dry THF (50 mL, 44.45 g, 0.616 mol) as solvent. The mixture was stirred for 23 d and collected by vacuum filtration. The mixture was purified using column chromatography; the low polarity impurity (S 8 ) was removed using hexanes, after which the elution solvent was switched to dichloromethane, to give a yield of 77% (137 mg, based on the different sulfides found in the product. Note: it was not possible to detect tetrasulfide or higher polysulfide linkages in the GC-MS, which may be due to thermal degradation of polysulfide bond (when n > 3). 33</p><!><p>Conversion in the reaction was shown by peak area of the hydrogens on the carbon adjacent to the disulfide bond (-CH̲ 2 SS) in 1 H NMR, which was plotted against different ratios of hydrosilane (SiH̲ ) to disulphide. Analogous techniques were used to follow the reduction of the tetrasulfide (Fig. 2).</p><!><p>The reaction conditions for the reduction of rubber powder are given in Tables 2 and 3. The general experimental procedure is as follows. The cryogenically ground rubber powder was allowed to swell in dry toluene (12 mL) for 30 min. Pentamethyldisiloxane 7 was added to the reaction mixture. The stock catalyst solution was added to initiate the reaction. The suspension was heated in a 60 °C oil bath for 48 h. The residual undissolved rubber powder was washed with toluene and separated by centrifugation (Eppendorf, Centrifuge 5424, at 12 000 rpm for 20 min). The extraction process was repeated twice to completely remove soluble compounds. The supernatants were mixed, and the solvents were removed by rotary evaporation. Any residual (organic) volatiles were removed under a stream of N 2 over 24 h. The recovered organic liquid was characterized by NMR. The recovered rubber powders were dried at 100 °C overnight and then examined by TGA. The bulk sample were reduced by the similar protocol and more details were provided in ESI. †</p><!><p>Cryogenically ground inner tube powder (300 mg) was placed in dry toluene (12 mL), followed by tetramethyldisiloxane M H M H (1.5 mL, 8.5 mmol). The stock catalyst solution (600 µL, stock catalyst concentration: 50 mg mL −1 in toluene, catalyst concentration in reaction: 10 wt%/inner tube) was added immediately afterwards to initiate the reaction. The suspension was heated in a preheated 100 °C oil bath for 30 min. The reaction flask was put into a room temperature water bath to quench the reaction and followed by a separation process using the same protocol stated in the former paragraph. The organic yield was 87%.</p><!><p>The silylated organic oil 8 (based tire tread, derived from IR/ NR, 0.50 g) was desilylated by treatment with TBAF solution (0.5 g TBAF, 1.92 mmol TBAF, dissolved in 10 mL THF containing 0.1 mL methanol) for 24 h at 80 °C. The solvent and siloxane fragments were removed by using a rotary evaporator, followed by kugelrohr distillation; loss of silicone was clearly seen in the 1 H NMR (200 °C, 3 h, Fig. S20 †).</p><!><p>Oxidative coupling of thiols using iodine. The desilyated organic oil 11 (derived from truck tread IR/NR, 0.2 g) was allowed to react with an iodine solution (50 mM dissolved in 1/1 v/v toluene/isopropanol) for 12 h at room temperature. Solvents associated with the resulting elastomer 12 were removed under a stream of N 2 over 12 h (Fig. S21a †). Crosslinking was confirmed using a swelling test. Compound 12 (0.1 g) was swelled in 10 mL of hexane; the degree of swelling was 209 wt%. The precursor oil 8 dissolved readily in hexane easily while 12 remained an elastomeric solid even after 1 h sonication (Fig. S21b †).</p><p>Preparation of toy tire using a peroxide initiator (Fig. 4)</p><p>A silicone mode was prepared with a two-part liquid component kit (Sylgard 184). Two components were mixed at the recommended ratio of 10 parts (10.0 g) base to 1 part curing agent (1.0 g). The mixing process was performed using a planetary centrifugal mixer (FlackTek Inc.) with a duration of 5 min at a speed of 3000 rpm. In order to fabricate bubble free elastomer, the mixed uncured PDMS was thoroughly degassed in a vacuum desiccator at low pressure for 30 min.</p><p>The right front tire of a toy car was removed from the toy and placed in the degassed, uncured mixture. The mixture was cured in an 80 °C oven overnight. The tire was removed from the cured mold.</p><p>Used rubber powder from the truck tread (Sailun) (2.0 g) was reduced by pentamethyldisiloxane 7 in a 100 °C oil bath for 18 h to give a polymeric oil 8 (81.5% yield). Solvents in the supernatants, after centrifugation, were removed by rotary evaporation; any residual volatiles and silicone by-products were removed using a stream of N 2 over 24 h. The oil was accompanied by residual undissolved rubber powder that was washed with toluene and separated by centrifugation (Eppendorf, Centrifuge 5424, at 12 000 rpm for 20 min), dried an 80 °C oven for 18 h, and ground into powder using a mortar and pestle (Fig. 4d).</p><p>The silylated organic oil 8 from the former step (comprised of IR/NR derivatives, 0.707 g) was dissolved in hexanes (10 mL), benzoyl peroxide (BPO, 0.01 g, 1 wt%, 0.0413 mmol) and, optionally, ground residual inorganic solids (from the preparation of 8, 0.3010 g), were added sequentially and mixed to give a homogeneous dispersion. After the solvent was removed by rotary evaporation, the mixture was placed in the silicone mold and degassed under vacuum in a desiccator for 30 min. The curing process was performed at 100 °C for 18 h. The formulations for rubber with different residual solid are listed in Table S6. †</p><!><p>Utilization of the reactions described here to create polymeric oils by the reductive silylation of used automotive rubbers, and subsequent oxidation to new elastomers, permits the completion of a full cycle of use for these organic materials. Mild, efficient reduction of S-S bonds of organic disulfides, including those found in used, sulfur-cured rubbers permits the formation of silylated polymers 8already in 87% yield at 100 °C within 30 minutesthat can be separated from the inorganic constituents. Radical cure of the oils if alkenes are present, or following desilylation, oxidative cure of thiolated polymers leads to new elastomers 12, 13 that, optionally, can be reinforced by the inorganic constituents recovered during the reduction process. The practice of this recycling process has the potential to reduce the environmental impact of used, sulfur-crosslinked elastomers.</p><!><p>There are no conflicts to declare.</p>

Royal Society of Chemistry (RSC)

Chemical biology tools for probing transcytosis at the blood–brain barrier

Absorptive-and receptor-mediated transcytosis (AMT/RMT) are widely studied strategies to deliver therapeutics across the blood-brain barrier (BBB). However, an improved understanding of the mechanism surrounding trafficking is required that could promote delivery. Accordingly, we designed a flexible platform that merged AMT and RMT motifs on a single scaffold to probe various parameters (ligand, affinity, valency, position) in a screening campaign. During this process we adapted an in vitro BBB model to reliably rank transcytosis of the vehicle library. Our results demonstrate heightened uptake and trafficking for the shuttles, with a structure-activity relationship for transcytosis emerging. Notably, due to their small size, the majority of shuttles demonstrated increased permeation compared to transferrin, with the highest performing shuttle affording a 4.9-fold increase. Consequently, we have identified novel peptide conjugates that have the capacity to act as promising brain shuttles.

chemical_biology_tools_for_probing_transcytosis_at_the_blood–brain_barrier

Introduction<!>Results and discussion<!>Secondary structure determination of brain shuttles<!>In vitro binding capacity of shuttles towards TfR<!>Evaluation of the BBB-shuttle properties of the conjugates in brain endothelial cells<!>Evaluation of the internalisation capacity in brain endothelial cells<!>Intracellular location of BBB shuttles<!>Permeability experiments<!>Optimisation and adaptation of BBB in vitro model<!>Permeability screen of BBB shuttles<!>Conclusions<!>Conflicts of interest

<p>A major hurdle hindering the diagnosis and treatment of neurological disorders is the difficulty for biotherapeutics to enter the central nervous system (CNS). This is due to the blood-brain barrier (BBB), which is comprised of tightly connected polarized endothelial cells that limit the passage of hydrophilic components and prevents the accumulation of material for transport at the BBB. 1 Despite these limitations, specialized endogenous transport mechanisms exist to allow the transcytosis of nutrients and ions, thus enabling CNS homeostasis. Of these, absorptive mediated-(AMT) and receptor mediated-transcytosis (RMT) pathways are key vesicular based transport systems which have become long-standing approaches for drug delivery to the CNS. 2 While these routes have become widely exploited by conjugating molecules restricted by the barrier to those which have this capacity, transcytosis at the BBB is more complex than initially thought, and delivery of therapeutics and biologicals remains modest. 3,4 Whilst there is limited experimental data surrounding the molecular basis of uptake and trafficking for CNS delivery, it has been demonstrated that dissociation from receptors on the brain side is essential for trafficking mediated by the transferrin receptor (TfR), the quintessential receptor for RMT. 5 Therefore transcytosis is more likely when the overall affinity towards the receptor is moderate to low, 5 or when bivalent engagement of the receptor is discouraged to limit avidity. 6 Here we present the design, synthesis and validation of a diverse shuttle library to identify key physicochemical properties for transcytosis. We based our approach on two essential modular components to develop the chemical tool kit: (i) sweetarrow-peptide (SAP), an isolated sequence derived from the Nterminal proline rich domain of g-zein, and an innate CPP with a dened PPII secondary structure to act as a scaffold, 2,7 and; (ii) a variety of validated RMT ligands to enable targeted delivery, 8,9 Fig. 1 and Table 1. It was anticipated that uptake at the BBB could be probed through exploring ligand type (i.e. targeted receptor, affinity) and arrangement (valency, position) of the ligands fused to the scaffold, since previous studies have indicated that brain exposure is directly affected by these parameters. 3,5,6,10,11 We provide compelling evidence that uptake and brain trafficking can be improved by combining AMT and RMT motifs on a single shuttle and that better understanding of receptor mediated trafficking within the brain endothelium is required at an individual and ligand-by-ligand basis, with ligand type, number and position effecting permeability in endothelial cells.</p><!><p>Design and synthesis of brain delivery shuttles SAP, the core of our delivery vehicles, is formed from a short repeating sequence of (VRLPPP) 3 that is readily accessible in high yield through general automated solid-phase peptide synthesis (SPPS). It is noteworthy to mention that SAP retains a PPII conformation if 50% of the sequence remains as proline, forming a le-handed helix of 3.0 residues per turn, which affords three distinct 'faces' in aqueous solution, Fig. 1B. 7,12,13 Consequently, spatial parameters of the BBB shuttle could be readily studied through simple mutation of valine, arginine, or leucine amino acids to orthogonal residues at specic sites. For this, we included lysine (K) or propargylglycine (X) on the PPII scaffold to conjugate relevant RMT motifs through amide bond forming and copper alkyne-azide chemistry (CuAAC) conjugations respectively. The full scaffold library we generated is shown in Table 2. Mutations to the SAP primary sequence follow standard convention and, when conjugated to the specied ligand, the attachment point on the scaffold is given in brackets and in order of conjugation. All peptide scaffolds were made with the chemically inert C-terminal amide in place of a carboxylic acid and FAM was conjugated to the N-terminus as cargo and to allow in vitro study.</p><p>To enable targeted brain delivery, we chose to functionalise our scaffold with established ligands that engage the transferrin receptor (TfR) and low-density lipoprotein-receptor related protein (LRP1). The relative abundance of these receptors on brain endothelial, alongside their high transport capacity, made them ideal targets for RMT mediated delivery and both have been actively explored in research. 8,[14][15][16] We focused on three main peptide ligands for these receptors, as shown in Table 1. Both TfRL1 and TfRL2 have been shown to interact with TfR at alternative sites compared to transferrin (Tf), and have either nanomolar (TfRL1; 15.0 nM) or micromolar affinity (TfRL2; 440 mM) towards the receptor. 9 Furthermore, branched BBB-shuttles incorporating dimers of TfRL1 have recently shown a non-linear increase in uptake within cellular models of the BBB. 11 Alternatively, Angiopep2 (APep2) displays a high transcytosis potential mediated by LRP1. 8 While less is known about traf-cking of LRP1, APep2-conjugates have shown demonstrable success in clinical trials of neurological disease models. 8 Peptide ligands were synthesised by general SPPS on rink-amide resin (see Table S1 † for characterisation data and yields). It is important to note that the stability of the peptide vehicles was not considered in this proof-of-concept screen. Recent investigations with retro-enantio sequences of both TfRL1 and APep2 have shown that metabolic limitations can be overcome without the loss of function, offering a plausible solution to degradation. 14,17 This is also apparent for the D-amino acid analog of SAP. 18 Chemoselective synthesis of brain delivery shuttles Four conjugation approaches were used to decorate the scaffold in various formats, Scheme 1A-D. Solution phase CuAAC chemistry with microwave heating was most effective for affording PPII-ligand conjugates in either monovalent or bivalent formats that included identical ligands (Scheme 1A). Alternatively, for the attachment of orthogonal ligands to the scaffold, TfRL2 was rst incorporated via continuation of Fmoc/ tBu SPPS on the lysine side chain of PPII scaffolds, as generalised in Scheme 1B. Following FAM conjugation, unreactive Nterminal PPII was capped to ensure no unwanted reactions, before the lysine protecting group (ivDde) was selectively removed on resin. Resin was re-submitted to automated SPPS to afford the product following deprotection and cleavage. If propargylglycine (X) was also incorporated on the scaffold, bivalent conjugates with alternative ligands were afforded through subsequent reaction by CuAAC (Scheme 1C). While this approach afforded conjugates in high yield, it was somewhat limited, since conjugates of both TfRL1 and APep2 were unattainable through continuation of SPPS on resin. To overcome this limitation, CuAAC could be used to attach one ligand to the PPII scaffold, which was followed by subsequent coupling of small molecule pentynoic acid to lysine under amide bonding forming reactions. A secondary CuAAC reaction afforded bivalent scaffolds with alternative ligands in moderate yield (Scheme 1D). As demonstrated here and elsewhere, CuAAC mediated convergence provided rapid access to versatile bioconjugates that was reproducible. 19 Detailed characterisation data and yields for all conjugates are reported in Table S2. †</p><!><p>We determined structural integrity of the PPII helix by circular dichroism (CD) spectroscopy, where spectra of unmodied SAP and the N-terminal FITC-conjugated analogue were collected as controls, Fig. 2A. Importantly, the results suggest preservation of the secondary PPII structure following modication to the backbone since all peptides demonstrated strong absorption at 203 nm, Fig. 2B-D. Specically, PPII helicity was insensitive to the conjugate linkage chemistry, ligand type and number of additions to the PPII scaffold.</p><!><p>To evaluate functionality of the conjugated peptides, in vitro analyses were performed. TfR binding was assessed through an ELISA format, whereby FAM-conjugated shuttles were serially diluted and added to plates that were coated with TfR protein.</p><p>FAM-labelled vehicles were detected by an anti-FAM HRPconjugated antibody. As predicted, TfRL1-vehicles contained a higher affinity towards the TfR than the equivalent vehicle incorporating TfRL2 (PPII(L3X;L15X)TfRL1: 0.26 AE 0.17 nM; PPII(L3X;V13X)TfRL1: 0.31 AE 0.26; cf. PPII(L3X;L15X)TfRL2: 11.8 AE 1.11 nM; PPII(L3X;V13X)TfRL2: 12.7 AE 1.37), Fig. 2E. 9 Notably, TfRL2 bivalent shuttles contained a similar affinity towards TfR compared to Tf-biotin when assayed in a similar format (EC 50 : 20.9 AE 2.1 nM). It is assumed that an avidity effect is demonstrated by the bivalent vehicles since monovalent TfRtargeting shuttles contained too low-affinity towards TfR to be detected. Further, positioning of the ligand did not affect TfR binding affinity since scaffolds that incorporated ligands on the same face afforded affinity values similar to those with ligands fused on different sides.</p><!><p>Having demonstrated preservation of receptor binding, we next used bEnd.3 cells to screen the uptake capacity of the shuttle library by ow cytometry. We determined peptide uptake and internalisation in a conuent monolayer of bEnd.3 cells which is an immortalised mouse brain endothelial cell line that has similar characteristics to the BBB. TfR is an established receptor for clathrin dependent uptake of Tf, favoured in an iron bound form when at physiological pH (i.e. holo-Tf). 20 Flow cytometry (FC) and microscopy data obtained with human derived Alexa-Fluor 647-conjugated Tf (Tf-A647) conrmed functional, membrane resident TfR (see Fig. S1 and S2 †).</p><!><p>For library screening, cells were exposed to equal concentrations (500 nM) of FAM labelled molecules in media for 3 h to allow binding, internalisation and sorting, and external FAM uorescence was quenched by addition of trypan blue. 21 Low temperature was shown to inhibit cellular uptake of the vehicles (Fig. S3 †), demonstrating an energy-driven internalisation process. Furthermore, using DAPI as a viability indicator, no cytotoxicity of the vehicles was demonstrated during these experiments and over a concentration range of up to 100 mM, where viability remained over 98% (Fig. S4 †).</p><p>It can be seen from Fig. 2F that, in general, brain shuttles contained heightened, non-additive, uptake compared to their unconjugated counterparts, implying a synergistic effect for uptake. TfRL1 demonstrated the highest capacity for endocytosis when incubated alone, however appreciable difference was shown for the monovalent vehicle in this format (TfRL1 cf. PPII(R8X)TfR1 P ¼ 0.798; TfRL2 cf. PPII(R8X)TfR2 P ¼ 0.018; APep2 cf. PPII(R8X)APep2 P ¼ 0.002). Notably, when arranged in a bivalent format, TfRL1 modied vehicles showed a dramatic increase in cellular internalisation that exceeded additive contributions. Interestingly, endocytosis of TfR-targeting bivalent vehicles was not signicantly inuenced by its positional arrangement on the scaffold (PPII(L3X;L15X)TfRL1 cf. PPII(L3X;V13X)TfRL1 P ¼ 0.408; PPII(L3X;L15X)TfRL2 cf. PPII(L3X;V13X)TfRL2 P ¼ 0.050), compared to those targeting LRP-2 which favoured ligands attached to opposite faces of the scaffold (PPII(L3X;L15X)APep2 cf. PPII(L3X;V13X)APep2 P ¼ 0.0059). This phenomenon could be attributed to increased steric strain of the larger APep2 peptide when incorporated on the same face, or possibly linked to the ligands ability to inuence receptor clustering on the extracellular membrane. Again, higher uptake was demonstrated for scaffolds that combined alternative transcytosis ligands on the same molecule compared to individual ligands. However, uptake was not improved compared to bivalent scaffolds containing identical ligands.</p><!><p>To obtain information on the intracellular fate of the shuttles, bEnd.3 cells were pulsed with bivalent vehicles, Tf-A647 and unconjugated TfRL1 and APep2 ligands for 3 h, unbound compounds were removed and cells xed and stained for immunouorescence microscopy. Images were processed as described in Fig. S5. † Fig. 3A-C shows colocalisation of PPII(L3X;V13X)TfRL1 with DAPI, Tf-A647 or LAMP1 respectively and represents a typical image generated from the screen following deconvolution and segmentation. It can be seen from Fig. 3D that all vehicles gave highest colocalisation with Tf-A647 as opposed to LAMP1, indicating uptake into endosomal vesicles with minimal degradation through lysosomal sorting. In addition, Tf-A647 colocalisation values were higher for vehicles compared to the individual RMT ligand components. Notably, the degree of Tf-A647 and LAMP1 colocalisation was comparable for TfRL1 vehicles incorporating this ligand on the same or opposite face. In addition, Tf-A647 colocalisation for bivalent TfRL1 vehicles and PPII(L3X;L15X)TfRL2 was also comparable. However, increased levels of LAMP1 colocalisation was observed for TfRL2 bivalent vehicle, indicating that the higher affinity TfR ligand is more efficient for non-degradative cellular uptake in this system. Colocalisation of APep2 bivalent vehicles with Tf-A647 were affected by the position of the ligand, demonstrating a similar trend to that shown with endocytotic capacity, Fig. 2F. Trivalent vehicles decorated with TfR ligands demonstrated similar colocalisation with Tf-A647 to those in a bivalent format. Whilst vehicle PPII(L3X;L15K)TfRL2(K) TfRL1(X) demonstrated highest Tf-A647 colocalisation, it also had heightened association in the lysosome. Notably,</p><!><p>To study transcytosis potential of the vehicles in vitro we optimised a BBB model using a 3D-transwell format. We found that a bEnd.3/mesenchymal stem cell (MSC) co-culture reproducibly afforded the highest resistance to paracellular diffusion through assessment with transepithelial electrical resistance measurements (TEER) and small molecule permeability studies (Fig. S6 †). This is similar to results shown by others. 16 Immunostaining for TJ protein ZO-1 in the bEnd.3 cell line conrmed its presence and trafficking towards the cell junctions (Fig. S7 †). While this indicates that an adequate and reproducible barrier was formed from the bEnd.3/MSC co-culture model, we found that it was of paramount importance to distinguish transcytosis from the expected background of paracellular ow since others have reported issues distinguishing these values. 22</p><!><p>Oen in vitro assays of the BBB overlook contributions of paracellular ux that can lead to overestimations of brain exposure, and offer no valid comparison between the individual molecules being screened. We believed that without a quantiable probe for paracellular diffusion, it would be difficult to accurately screen the transcytosis capacity of our vehicles.</p><p>It seemed plausible that a similar sized marker (such as TexasRed labelled dextran (dex-TexR; 3000 g mol À1 )) that is incapable of AMT or RMT uptake could serve as an internal standard to quantify paracellular contribution of the vehicles. As exemplied in Fig. 4A, experiments are performed in parallel with probes (both dex-TexR and FAM-PPII vehicle or Tf-A647) fed to insets containing either the cell monolayer (PS t ) or lter alone (PS f ). The corresponding P app for dex-TexR in both scenarios is rst calculated and the extent in which movement is reduced by presence of the monolayer (dened here as the transport ratio (TR)) is used to determine the expected paracellular diffusion rate of the individual vehicles. We employed Tf-A647 and a similarly sized TexasRed labelled dextran (dex-TexR; 70 000 g mol À1 ) as a positive control for transcytosis. Typical clearance proles when compounds are incubated with and without cells, are shown in Fig. 4B-C. As expected, due to their similar molecular weight, both dex-TexR and Tf-A647 afforded comparable clearance when incubated without cells (PS f ). When incubated in the presence of cells, dex-TexR was detected in the basolateral compartment indicating background paracellular ux. However, Tf-A647 was cleared at a faster rate in comparison, conrming transcytosis of the molecule.</p><p>Corrected values were subsequently calculated to internally rank the transcytosis capacity of BBB-shuttles. In addition, to verify validity of the model, a selection of high permeability vehicles were selected for further studies at low temperature, where uptake and transcytosis should be inhibited. In agreement, shuttles showed permeability similar to dex-TexR (3000 g mol À1 ), demonstrating negligible true transcytosis in this condition (Fig. S8 †).</p><!><p>Our screen shows that the majority of functionalised vehicles had higher rates of permeability than Tf-A647 with PPII(R8X) TfRL1, PPII(L3X;L15X)APep2 and both bivalent TfRL2 vehicles being the exception, Fig. 4D. TfRL1 modied vehicles preferred a bivalent format (PPII(L3X;V13X)TfRL1: 3.5-fold increase relative to Tf-A647; PPII(L3X;L15X)TfRL1: 3.0-fold), whereas TfRL2 and APep2 monovalent vehicles experienced higher levels of basal transport (PPII(R8X)TfRL2: 1.4-fold; PPII(R8X)APep2: 4.9- fold), with the latter vehicle showcasing the best permeability of those screened. In agreement with the endocytosis and microscopy studies, TfR targeting bivalent vehicles showed a moderate or no clear positional preference for ligands attached to alternative faces, whereas Apep2 highly favoured ligands attached to alternative faces of the scaffold with PPII(L3X;L15X)Apep2 showing limited transcytosis. In agreement with the microscopy data, trivalent vehicles where both ligands are directed towards the TfR demonstrated improved transcytosis when ligands were incorporated on opposite sides of the scaffold. In addition, the data implies that affinity towards the TfR does not govern transport ability for our peptidic BBB shuttles since trivalent vehicles combining high and low affinity ligands showed lower permeability than the corresponding bivalent high affinity TfRL1 vehicles.</p><p>Whilst vehicle PPII(L3X;V13K)TfRL2(K);APep2(X) gave the highest level of transcytosis for a highly functionalised shuttle (3.8-fold increase relative to Tf-A647), the increased permeability may be attributed to the ligands interacting independently with their receptors, since both ligands enjoyed higher transport in a monovalent form. In line with this hypothesis, transcytosis for PPII(L3X;L15K)TfRL2(K);APep2(X) was lowest for trifunctional vehicles with ligands merged to the same face (1.8-fold), implying that the ligands are not working in synergy. This result is in contrast to when ligand TfRL1 and APep2 are combined, which showed improved permeability when ligands are arranged on the same face.</p><!><p>Here we report the rst use of CPP SAP as a scaffold for developing targeted BBB penetrable shuttles, which constitutes one of the most restrictive barriers in the body. In this regard, we designed a versatile vehicle library by a convergent approach, strategically introducing mutations within the sequence to gra RMT ligands in a selective manor using either amide bond forming or CuAAC mediated reactions. As noted elsewhere, CuAAC provided a superior and exible reaction for these modications. Notably, SAP retained a helical PPII structure aer modication, and vehicles screened in biologically relevant assays demonstrated uptake and trafficking of cargo at the BBB. It was shown that AMT and RMT motifs worked in synergy to encourage cellular uptake, with certain molecular characteristics such as affinity, position and valency inuencing both uptake and transcytosis for individual ligands. Notably, the majority of vehicles screened demonstrated heightened transcytosis rates compared to Tf in a BBB model. Here we believe the small size of the peptide conjugates, compared to Tf and other macromolecule shuttles, afford them an advantage for targeted transcytosis due to higher diffusion rates. Within our permeability screening campaign, dex-TexR was successfully included as an internal standard for quantifying paracellular and non-specic movement. Consequently, the results presented demonstrate that PPII derived shuttles represent novel, exciting and promising classes of bioconjugates for enhancing uptake at the BBB. The exibility of the screening approach could be readily adopted to investigate other ligands for AMT and RMT uptake at the BBB to validate and identify optimal ligands and shuttles for delivery.</p><!><p>There are no conicts of interest to declare.</p>

Royal Society of Chemistry (RSC)

Insulin Hexamer-caged Gadolinium Ion as MRI Contrast-o-phore

High-relaxivity protein-complexes of Gd(III) are being pursued as MRI contrast agents in hope that they can be used at much lower doses that would eliminate toxic-side effects of Gd(III) release from traditional contrast agents. We construct here a new type of protein-based MRI contrast agent, a proteinaceous cage based on a stable insulin hexamer in which Gd(III) is captured inside a water filled cavity. The macromolecular structure and the large number of \xe2\x80\x98free\xe2\x80\x99 Gd(III) coordination sites available for water binding lead to exceptionally high relaxivities per one Gd(III) ion. The Gd(III) slowly diffuses out of this cage, but this diffusion can be prevented by addition of ligands that bind to the hexamer. The ligands that trigger structural changes in the hexamer, Cl\xe2\x88\x92 and phenols, modulate relaxivities through an outside-in signaling that is allosterically transduced through the protein cage. Contrast-o-phores based on protein-caged metal ions have potential to become clinical contrast agents with environmentally-sensitive properties.

insulin_hexamer-caged_gadolinium_ion_as_mri_contrast-o-phore

<p>Differences in the magnetic properties of water hydrogens in tissues can be exploited to obtain three-dimensional images in magnetic resonance imaging (MRI). The sensitivity of the method can be improved by injection of contrast agents (CAs), which increase these differences. At the moment of this writing, 30 million doses of Gd(III)-containing CAs are administered annually worldwide - approximately 1-in-3 of MRI scans.[1]</p><p>Contrast agents are most often based on Gd(III) complexes. While these complexes are very stable in vivo, they need to be used at very high concentrations, which raises concerns due to potential toxicity of released free Gd(III). Thus, there is a substantial effort to optimize the properties that lead to strong contrast in order to minimize the amounts of contrast agent used. Contrast agents impact relaxation time constants, longitudinal T1 and transverse T2, which reflect the rates at which proton spins in external magnetic fields return to equilibrium from excited states achieved by irradiation with radio-frequency. Dependence of these two time constants on the concentration of contrast agents is characterized by two relaxivity constants, r1 and r2, which are systematically being targeted for optimization[2-7].</p><p>Current clinical gadolinium-based CAs are small molecules with rapid tumbling times and usually only one coordinated water. These properties lead to relaxivity values that are generally below 10 mM−1 s−1 at current clinical magnetic field strengths (1.5T and 3T) and, consequently, 0.1-0.3 millimole/Kg quantities of administered doses. Hence, there is a drive to develop MRI contrast agents with high relaxivity - upwards of 100 mM−1 s−1 per Gd(III) ion, which would reduce doses. One possible way to achieve this is to use macromolecules, because large size and slow tumbling times result in dramatic increases in relaxivity.</p><p>Proteins can provide a scaffold for incorporating the Gd(III) ion into so-called ProCAs/[8] The incorporation of the Gd(III) ion in proteinaceous structures has been achieved in several ways: 1) replacement of a protein's native metal ions by the Gd(III) ion or complexes (cf. in apo-ferritin cage.[9]);[10] 2) bioconjugating a small molecule gadolinium complex to a protein;[2] 3) engineering a gadolinium site into the protein or self-assembled peptides, either by modifying a native protein[5] or by de novo.[11] The modified native protein approach led not only to stable Gd(III) ion capture with exceptionally high relaxivity in MRI applications, but also to successful preclinical demonstrations of applications.[9,12-15]</p><p>We now report an adaptation of a process of natural self-assembly of a peptide insulin to synthesize a stable hexameric cage that firmly captures one Gd(III) ion at its low-affinity, low-coordination number metal-ion binding site. This caged Gd(III) has exceptional contrast properties due to both substantial size and a large number of impacted water molecules, and represents a novel lead for creation of environment-responsive[16] protein-based contrast agents.</p><p>Insulin spontaneously oligomerizes in solution, and in the presence of metal ions, such as Zn(II) and Co(II), this equilibrium is shifted towards hexamer at higher monomer concentrations (e.g., > 1 μM),.[17] This hexamer is not stable upon dilution, and although it can be observed by size exclusion chromatography (FPLC), it cannot be isolated without re-equilibrating back to monomer. During our research in oligonucleotide-based devices triggering insulin release,[18] we were familiarized with a protocol for an in situ oxidation of Co(II) to Co(III) to form a stable Ins6Co(III)2 hexamer as an HPLC standard for quantification of less stable hexamers, e.g., Ins6Zn(II)2).[16] The hexamer, when locked together with two Co(III) ions, has no detectable release of monomer units in buffer, with an estimated half-life for ligand exchange of two years; it releases insulin more quickly in vivo, with t1/2 in hours, presumably via a metabolic reduction of Co(III) to Co(II).[19]</p><p>In crystal structures[20] of various insulin hexamers, metal ions hold two interdigitated tris-alpha-helical bundles via coordination with histidine side chains (2×3xHisB10), creating a water-filled cavity inside the hexamer. The cavity is lined up by hydrophilic residues, and in its central region there is a metal ion binding site, defined by three pairs of γ-carboxylic groups of Histamates (3×2xGluB13). Glutamate pairs are too far away from one another to all bind to the same metal ion at the same time; therefore, this site has relatively low affinity to metal ions, with X-ray crystallography showing binding to Cd(II), and Pb(II), and with KD for Ca(II) and Cd(II) ions of ~80 and <10 μM, respectively.[21] The observed number of metal ions inside the cavity at this site ranges from one (Cd(II)[22]) to three ions (Pb(II)[22], Zn(II)[23] and Na(I)[24]). The site was also previously characterized via its impact on the fluorescence of lanthanides (i.e., Eu(III)) with an experimental observation that the lanthanide at that site has properties consistent with four water molecules coordinating this ion for Ins6Zn(II)2Eu(III), and with fluorescent lifetimes indicating that up to three Eu(III) ions can be captured at the site.[25] `</p><p>Based on all these considerations, we decided to form a stable Co(III) complex in the presence of a variable number of equivalents of Gd(III), expecting that this cation, when caged within the cavity, will exhibit unique contrast properties. The hexamer formed in the pressence of various amounts of GdCl3 could be purified with chelating resin to remove excess Gd(III) ions. When adding greater than one equivalent of Gd(III) per hexamer, there is very little change in the relaxation rate before and after passing through the chelation column, thus supporting the formation of a 1:1 complex. The product was then isolated by size exclusion chromatography (Figure S1), and, in agreement with previous reports with no added Gd(III), was stable (as a hexamer) indefinitely in HEPES buffer (time-course monitored by FPLC analysis and relaxivity; cf. Figure S2 for Mw determination).</p><p>The purified hexamer was crystalized and its X-ray structure determined at 1.8 Å resolution (Figures 1, 2, 4 and Supporting Information). Based on the B-chain configuration, it has a classical T6 hexamer conformation.[26-28] In it, the two Co(III) ions each bridge three insulin monomers via three HisB10 side chains. The resulting two sets of tris-alpha-helical bundles are inter-digitated, defining a central cavity lined with hydrophilic residues, capped at each end by the Co(III) ions (Figure 2a).</p><p>We recognize two halfes of the cavity, defined by two B-chain trimers, D, I, N and B, L, G (Figure 2a). Asymmetrically positioned between the two Co(III) ions (Figure 2a), we observe major electron density for Gd(III) that is equally distributed (i.e., each modeled with 1/3 occupancy) between three disordered carboxylates from GluB13 on individual B-chains D, I, N (i.e., DIN plane) that have more than one conformation (Figure 2a). There is a precedent for this interpretation in a Cd(II) containing hexamer (Ins6Cd(II)2Cd(II)).[22]</p><p>At the nearly identical position in the other half of the cavity, defined by the B, L, G B-chains (i.e., in BLG plane), but rotated by 44° on C3 axis) we observe a density that can be attributed either to three water molecules, or to a lower level occupancy of Gd(III). We cannot resolve this solely through crystallographic means, but the latter situation is consistent with Gd(IIII) distributed between six GluB13equally in solution, with the occupancy difference being a reflection of the asymmetry in the T3 structures</p><p>In insulin, with approximately 2:1 preference for occupancy of BLG over the DIN plane, or about 0.5 kcal.mol−1. This means that our X-ray structure is a superposition between six different conformations in which Gd(III) can exist, three with higher (in DIN plane) and three with lower (in BLG plane) occupancy. When Gd(III) is in one of these positions, the other five are likely occupied with water molecules, however, coordinated waters were not identified due to the partial occupancy of the Gd(III) ions.[22] This dynamic distribution of a single Gd(III) ion over six glutamates is consistent with saturation of water proton relaxivity at one equivalent of Gd(III) ion during formation of the hexamer.</p><p>We also observe weak electron density on the 3-fold crystallographic symmetry axis within bonding distance to the Gd(III) ion close to DIN plane. This was previously assigned to a partial (e.g., ½) occupancy by a chloride ion for Ins6Cd(II)2Cd(II),[22] but we cannot exclude the possibility that it is another water molecule.</p><p>The Ins6Co(III)2Gd hexamer, at pH 7.0, 37 °C, and in a magnetic field of 1.4 T, had relaxivity values of r1 = 138 ± 5 mM−1s−1, and r2 = 260 mM−1s−1 ± 10 mM−1s−1, regardless if one, two, three, or four equivalents of Gd(III) were added at the assembly stage. Therefore, the ratio of one Gd(III) ion per hexamer exists in solution as well (Figure 3a, b). Changes in relaxation time (Ti) were even detected at sub-μM concentrations (Figure 3 upper inset, LoD[29] for T1 and T2 of 500 and 200 nM respectively), indicating the potential for minimization of quantities of Gd(III) administered, and potential, as well as limitations, as a targeted-MRI contrast agent.[13,14,30,31]</p><p>The r1 and r2 relaxivities are amongst the highest reported per Gd(III) ion (cf., Table). In the case of the hexamer, the tumbling time is calculated at ~20 ns (50 MHz), a close match to the Larmor precession time for the water proton at 1.4T (60 MHz), which is a requirement to maximize relaxivity through an increase in molecular size (i.e. tumbling time). This effect is limited to lower fields for r1. Indeed, when we characterized the hexamer at higher fields (1.5T vs 3, 7, and 9.4T, all at room temperature) r1 values decreased as magnetic field strength increased, while r2 values remained exceptionally high (Table). Relaxivity plots are shown in Figure S3.</p><p>The relaxivity values increased with an increase above room temperature, a property that is favorable for in vivo use. Typically, this suggests that the system is limited by slow rates of water exchange.[32] While the exact cause of this phenomenon awaits more precise biophysical characterization, the tempterature could impact the rate of diffusion of water, conformational equilibrium of hexamer resulting in shifts towards cavities with a larger number of water molecules, and/or a change in KD value for Gd(III). It is difficult to give a precise estimation of the KD of the complex that is based on sterically hindered capture, as opposed to classical coordination, but it's koff can be approximated by experiments in which phosphate ion is added to remove generated free Gd(III) from solution, leading to the loss of contrast (see below). This koff was (2.4±0.2)x10−6 s−1, which is slower than one that characterizes streptavidin-biotin interactions.45</p><p>Another known contributor to high relaxivity is the number of water molecules bound to the metal ion.[33-35] Typically, Gd(III) has a coordination number of 8 or 9, but in the hexamer reported here, it is only bound, in any of the major conformations, to at most just one of the glutamates and a chloride ion as non-water ligands, with six or seven other coordination sites available for water molecules (Figure 2b). In a fluorescence study of Eu(III) in a Zn(II)-insulin hexamer, the number of water molecules bound was estimated as four.[25] We could identify twelve water molecules in the cavity of our crystal structure forming two planes with six waters in each. These are primarily hydrogen-bonded to the side chains of SerB9, HisB10 and GluB13 on the B-chain alpha helices. Another five water molecules could be present based on the assumption that when Gd(III) is fixed at one position, the other five positions are occupied by water molecules. Overall, the Gd(III) ion is caged within a cavity with as much as seventeen water molecules, and with a low number of its coordination sites filled by non-water ligands; which is a unique situation for a gadolinium-MRI contrast agent.</p><p>In order for the hexamer to be useful as a contrast agent, the high relaxivity must be maintained in vivo. Stability also prevents toxicity of free Gd(III) and of insulin monomer components, with the latter readily addressed by inactive insulin analogs. Stability of the hexamer itself is measured by FPLC, but this method would not detect that Gd(III) left the cavity in the absence of concomitant degradation. In contrast, because 1/T is directly proportional to the concentration of Gd(III) in the central cavity, the relaxivity monitoring captures all changes in the cations molecular environment.</p><p>Purified Ins6Co(III)2Gd(III) hexamer is stable indefinitely by FPLC and relaxation measurements at pH 7.0 at room temperature and at a concentration less than 200μM, which is consistent with exchange experiments with radio-labeled insulin that measured half-lives of over two years.[19] At higher concentrations a precipitate is observed, in both concentration- and temperature-dependent manner (Supporting Information). We observed a decrease in relaxation rate (1/T2) of <3% per day for Ins6Co(III)2Gd(III) at 37°C, without any decomposition of hexamer by FPLC even after several weeks of monitoring. In the presence of 150mM Cl− ions, the behavior was similar. In contrast, the phosphate anion, known to form an insoluble complex with Gd(III), removes it from the cavity at a higher rate (Figure 4a), while also not degrading the hexamer (Figure S4). Ca(II) ions at 10mM can also interfere with the Gd(III) - likely via transmetalation (Figure 4a).</p><p>These results can be rationalized by the presence of six major tunnels in T6-hexamers that lead from the protein surface to the cavity hosting Gd(III). Based on the previous studies measuring the escape of Cd(II),[21] the channels represent pathways for a potential escape of a metal ion from the cavity (Figure 4c, and Figure S5), and as an entrance of other agents to reach it. In a control experiments, we monitored relaxivity of a preformed 'Ins6Co(III)2' in the presence of free Gd(III), observing a slow rate of increase with an estimated t1/2 of ~40 hours (Figure S6). This rate is significantly faster than the reported rate of exchange of monomeric units,[19] and is consistent with the potential for equilibration between cavity and environment through these channels.</p><p>In vitro stability of the Gd(III) hexamer was also tested in the presence of ascorbic acid (Figure 4a and Figure S4), confirming that Co(III) hexamer is degraded to monomers by reducing agents (cf., release in vivo of insulin with t1/2 in hours[19]). Phenolic ligands, used for stabilization of hexamers in formulations, such as resorcinol (and tylenol), also degrade the hexamer (Figure S4), as expected, based on their redox activity.</p><p>Insulin hexamers are known to undergo gross structural rearrangements of the cavity in the presence of various ligands,[36] and we hoped to take advantage of these to achieve sensing. For example, when a T6-hexamer is converted to a R6-hexamer form, the channels leading from the bulk solvent to the central cavity collapse (cf., Figure 4c with 4d). Because Co(III) is not expected to fully switch in both positions to a tetrahedral ligand field arrangement (as observed in R6-hexamers), we decided to study Ins6Zn(II)2Gd(III) formed in situ, which is expected to have full flexibility. This formulation is stable at 10 μM concentrations of monomeric insulin, and has similar and stable relaxivity, comparable to that of Ins6Co(III)2Gd(III), which makes it suitable to measure relaxivities of otherwise unstable hexamers. Indeed, in the presence of SCN−, and also Cl− (Figure 4b, and Figure S7, respectively), we observed a 1/T2 increase of over 20%, consistent with the change in the structure that was previously proposed to occur at the high concentrations of this anion.[37] At the same time, we observe complete protection from the degradation by ascorbic acid, indicating that the complex does not equilibrate any more to release dimers and Zn(II). Interestingly, addition of resorcinol, caused a downward shift by approximately 15% (Figure 4b), while still protecting fully from ascorbic acid (i.e., indicating conformational change, rather than re-equilibration). These are very large changes in absolute numbers, indicating outstanding potential to use allosteric effects in proteins to achieve 'smart' behaviors of ProCAs (cf. Figure 4c and d). Further, we observed that resorcinol and thiocyanate provided substantial protection from complexation agents such as ascorbic acid.</p><p>In conclusion, we report synthesis, structure, and properties of a Gd(III)-containing peptide-cage based on insulin. This cage has several unique properties: It contains Gd(III) captured in a cavity with a large number of water molecules, while coordinated to only one-to-two non-water ligands. Second, both high molecular mass and high-water occupancy contribute to exceptionally high relaxivity values. Third, the stability of the hexamer is extraordinary, with the exception of reducing conditions. And, fourth, the hexamer provided us with the first example of an allosteric change in a protein impacting contrast properties.[43, 44] The hexamer system is in this respect similar to GFP, a barrel protein with chromophore centrally positioned, but which can be sensitized to the outside environment. Thus, while this specific hexamer, as described in our work, is not likely to be directly used in clinics due to bioactivity of insulin monomers, it represents the first-in-class contrast agent on which new targeted and environmentally sensitive systems can be built.</p>

PubMed Author Manuscript

Design and Development of Stable, Water-soluble, Human Toll-like Receptor 2-Specific, Monoacyl Lipopeptides as Candidate Vaccine Adjuvants

Antigens in modern subunit vaccines are largely soluble and poorly immunogenic proteins inducing relatively short-lived immune responses. Appropriate adjuvants initiate early innate immune responses, amplifying subsequent adaptive immune responses. Agonists of TLR2 are devoid of significant pro-inflammatory activity in ex vivo human blood models, and yet potently adjuvantic, suggesting that this chemotype may be a safe and effective adjuvant. Our earlier work on the monoacyl lipopeptide class of TLR2 agonists led to the design of a highly potent lead, but with negligible aqueous solubility, necessitating the reintroduction of aqueous solubility. We explored several strategies of introducing ionizable groups on the lipopeptide, as well as the systematic evaluation of chemically stable bioisosteres of the ester-linked palmitoyl group. These studies have led to a fully optimized, chemically stable, and highly water-soluble, human TLR2-specific agonist, which was found to have an excellent safety profile and displayed prominent adjuvantic activities in rabbit models.

design_and_development_of_stable,_water-soluble,_human_toll-like_receptor_2-specific,_monoacyl_lipop

Introduction<!>Results and Discussion<!>Chemistry<!>Synthesis of Compound 6: (2S,2\xe2\x80\xb2S)-Dimethyl 2,2\xe2\x80\xb2-(((2R,2\xe2\x80\xb2R)-3,3\xe2\x80\xb2-disulfanediylbis(2-acetamidopropanoyl))bis(azanediyl))bis(6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) hexanoate)<!>Synthesis of Compound 9: (9S,12R)-12-Acetamido-1-(9H-fluoren-9-yl)-9-(methoxycarbonyl)-3,11-dioxo-2-oxa-14-thia-4,10-diazahexadecan-16-yl palmitate<!>Synthesis of Compound 10: 2-(((R)-2-Acetamido-3-(((S)-6-amino-1-methoxy-1-oxohexan-2-yl)amino)-3-oxopropyl)thio)ethyl palmitate<!>Synthesis of 12: 2-(((R)-2-Acetamido-3-(((S)-3-(((S)-2,6-diaminohexanoyl)oxy)-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)ethyl palmitate<!>Synthesis of 13: 2-(((R)-2-Acetamido-3-(((S)-1-methoxy-1-oxo-3-(sulfooxy)propan-2-yl)amino)-3-oxopropyl)thio)ethyl palmitate<!>Synthesis of 14: (7S,10R)-10-Acetamido-7-(methoxycarbonyl)-4,9,16-trioxo-5,15-dioxa-12-thia-8-azahentriacontan-1-oic acid<!>Synthesis of 15: (S)-2-((R)-2-Acetamido-3-((2-(palmitoyloxy)ethyl)thio)propanamido)-3-methoxy-3-oxopropyl nicotinate<!>Synthesis of 19: (S)-Methyl 2-((R)-3-((2-azidoethyl)thio)-2-((tert-butoxycarbonyl)amino) propanamido)-3-(tert-butoxy)propanoate<!>Synthesis of 20: (S)-Methyl 2-((R)-3-((2-aminoethyl)thio)-2-((tert-butoxycarbonyl)amino) propanamido)-3-(tert-butoxy)propanoate<!>Synthesis of 21: (S)-Methyl 3-(tert-butoxy)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-((2-palmitamidoethyl)thio)propanamido)propanoate<!>Synthesis of 22: (S)-Methyl 2-((R)-2-acetamido-3-((2-palmitamidoethyl)thio)propanamido)-3-hydroxypropanoate<!>Synthesis of Compound 23: (S)-methyl 3-(tert-butoxy)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-((2-(4-tridecyl-1H-1,2,3-triazol-1-yl)ethyl)thio)propanamido)propanoate<!>Synthesis of Compound 24: (S)-Methyl 2-((R)-2-acetamido-3-((2-(4-tridecyl-1H-1,2,3-triazol-1-yl)ethyl)thio)propanamido)-3-hydroxypropanoate<!>Synthesis of Compound 27: (S)-Methyl 2-((R)-2-acetamido-3-((2-(1-hexadecyl-1H-1,2,3-triazol-4-yl)ethyl)thio)propanamido)-3-hydroxypropanoate<!>Synthesis of Compound 30: (2S,5R)-methyl 5-amino-2-(hydroxymethyl)-4,11-dioxo-10-oxa-7-thia-3,12-diazaoctacosan-1-oate<!>Synthesis of Compound 31: (2S,5R)-Methyl 5-acetamido-2-(hydroxymethyl)-4,11-dioxo-10-oxa-7-thia-3,12-diazaoctacosan-1-oate<!>Synthesis of Compound 32: (2S,5R)-Methyl 5-((tert-butoxycarbonyl)amino)-2-(tert-butoxymethyl)-4,11-dioxo-12-oxa-7-thia-3,10-diazaoctacosan-1-oate<!>Synthesis of Compound 33: (2S,5R)-Methyl 5-amino-2-(hydroxymethyl)-4,11-dioxo-12-oxa-7-thia-3,10-diazaoctacosan-1-oate<!>Synthesis of Compound 34: (2S,5R)-Methyl 5-acetamido-2-(hydroxymethyl)-4,11-dioxo-12-oxa-7-thia-3,10-diazaoctacosan-1-oate<!>Synthesis of Compound 35: (2S,5R)-5-((tert-Butoxycarbonyl)amino)-2-(tert-butoxymethyl)-4,11-dioxo-12-oxa-7-thia-3,10-diazaoctacosan-1-oic acid<!>Synthesis of Compound 36: tert-Butyl hexadecyl ((8S,11R)-8-(tert-butoxymethyl)-2-methyl-7,10-dioxo-13-thia-2,6,9-triazapentadecane-11,15-diyl)dicarbamate<!>Synthesis of Compound 38: Hexadecyl ((8S,11R)-11-acetamido-8-(hydroxymethyl)-2-methyl-7,10-dioxo-13-thia-2,6,9-triazapentadecan-15-yl)carbamate<!>TLR2-specific NF-\xce\xbaB induction<!>Immunoassays for cytokines and chemokines<!>Flow-cytometric immunostimulation experiments<!>Immunization and safety evaluation in rabbits

<p>The overall goal of vaccination is the generation of specific, robust, and durable immune responses against the antigen to provide long-term protection against pathogens. Early vaccines which frequently utilized killed whole organisms are reactogenic, and are associated with local and systemic adverse reactions, whole cell pertussis vaccines being an example.1–4 Modern vaccines such as acellular pertussis vaccines1 use highly purified antigens. Such 'subunit vaccines' have a much more defined composition, facilitating not only the ease of production and quality control, but importantly, are also considerably less reactogenic. However, subunit antigens are largely soluble proteins which are intrinsically poorly immunogenic, and do not induce long-lived immune responses. The recent reemergence of pertussis in the United States5–7 has served to highlight the rapid waning of protective immunity following vaccination with acellular subunit pertussis vaccines,8,9 and emphasizes the need for safe and effective adjuvants.</p><p>Adjuvants initiate early innate immune responses which subsequently lead to the induction of robust and long-lasting adaptive immunity.10 Aluminum salts (primarily phosphate and hydroxide), discovered by Glenny and coworkers,11 have been the only adjuvants in clinical use until the recent introduction of 3-O-desacyl-4′-monophosphoryl lipid A (MPL).12 Aluminum salts (used as adjuvants in acellular pertussis vaccines) are weak adjuvants for antibody induction, promoting a T helper 2 (Th2)-skewed, rather than a Th1 response,13,14 and are virtually ineffective at inducing cytotoxic T lymphocyte or mucosal IgA antibody responses. They also appear to promote the induction of IgE isotype switching which has been associated with allergic reactions in some subjects.13,14</p><p>Our knowledge of the molecular mechanisms of innate immunity has expanded rapidly since the discovery of Toll-like receptors (TLRs)15–17 and of their role in induction and amplification of adaptive immune responses.18,19 Innate immune afferent signals activated by vaccine adjuvants include those originating from Toll-like receptors (TLRs), as well as RIG-I-like receptors20 and NOD-like receptors (NLRs).21,22 There are 10 functional TLRs encoded in the human genome, which are trans-membrane proteins with an extracellular domain having leucine-rich repeats (LRR) and a cytosolic domain called the Toll/IL-1 receptor (TIR) domain.23 The ligands for these receptors are highly conserved molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double stranded RNA (TLR3), CpG motif-containing DNA (recognized by TLR9), and profilin present on uropathogenic bacteria (TLR11).24 TLR1, -2, -4, -5, and -6 recognize extracellular stimuli, while TLR3, -7, -8 and -9 function within the endolysosomal compartment.23 The engagement of TLRs by their cognate ligands lead to the production of inflammatory cytokines, and up-regulation of major histocompatibility complex (MHC) molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer (NK) cells (innate immune response). These responses result in the priming and amplification of T-, and B-cell effector functions (adaptive immune responses).25–28</p><p>We have been systematically exploring detailed structure-activity relationships (SAR) of several immunostimulatory TLR agonists,29–37 with a particular focus on TLR2 agonists. Unlike other TLR-active compounds, agonists of TLR2, first identified in a mycoplasmal lipopeptide, S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-R-cysteinyl-GNNDESNISFKEK, termed Macrophage-Activating Lipopeptide-2 1 (MALP-2)38–40 (Fig. 1) and exemplified by the S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine 2 (PAM2CS) chemotype30,36 are of particular interest to us, for although the lipopeptide is devoid of any detectable pro-inflammatory activity in ex vivo human blood models (as defined by the production of detectable levels of TNF-α, IL-1β, IL-6, or IL-8),41 or of local reactogenicity and pyrogenicity in rabbit models,31 it is potently adjuvantic in murine models of immunization,41 suggesting that this chemotype may be a safe and effective adjuvant.</p><p>The exposure of bone marrow-derived dendritic cells of C57/BL6 mice to TLR2-agonistic lipopeptides results in an upregulation of MHC Class II and CD80/CD86 costimulatory molecules, with enhanced expression of CD11b and CD11c, associated with the production of TNF-α and IL-12.42 These results have been confirmed in BALB/c mice using 1, and extended to show that the lipopeptide also upregulates immunoproteasome (LMP2, LMP7 and MECL1) activity in a dose-dependent manner, suggesting that TLR2 agonists may indirectly enhance MHC Class I-restricted responses by accelerated antigen processing and peptide presentation.43 In a recent report comparing the adjuvanticity of several ligands of TLRs in Chlamydia major outer membrane protein vaccine constructs, TLR2 agonists were found to be superior in inducing protective responses against a challenge of Chlamydia trachomatis.44</p><p>Importantly, the effects of lipopeptides on APC maturation and antigen presentation have also been demonstrated in human dendritic cells (DCs).45 Plasmacytoid and myeloid dendritic cells (pDCs and mDCs, respectively) constitute the most potent professional antigen presenting cells, and play a pivotal role in the induction and polarization of antigen-specific immune responses.46 Human myeloid dendritic cells comprise of two major subsets: CD1c+ mDCs and CD141+ mDCs, both of which express TLR2.47,48 Furthermore, TLR2 agonists induce isotypic switching and differentiation of naïve human B lymphocytes to IgG-secreting plasma cells,49 indicating a functional association between BCR stimulation and TLR activation.49</p><p>It is pertinent to highlight anecdotal human data on TLR2 agonists in the context of tolerability and safety of these compounds. In one study, ten patients with incompletely resectable pancreas carcinomas were injected intra-tumorally during surgery with 20–30 μg of 1 followed by postoperative chemotherapy. As expected, the investigators observed influx of lymphocytes and monocytes in wound secretions, but no systemic side effects were noted. The study concluded that "up to 20 μg of 1 was well tolerated".50 In another study, 0.125–1.0 μg of 1 was directly applied on punch biopsy lesions of the skin in twelve patients; other than transient and self-limited erythema, no systemic side effects were noted.51 The lack of proinflammatory activity, excellent safety profile in animal models and human subjects, the expression of the TLR2 in multiple DC subsets, and its strong adjuvantic activity have been compelling reasons for our focus on agonists of TLR2.</p><p>Our SAR studies on the 2 series of compounds36 led to a simplified second-generation monoacyl lipopeptide 3 (Fig. 1), in which the spacing between the ester-linked acyl group and the thioether was found to play a crucial role in determining activity;30 further SAR studies led to the identification of a cysteine N-acetyl analogue 4, which retained exquisite human TLR2 (hTLR2)-specificity, with a substantial gain in potency, rivaling that of 2.52 Although highly potent and hTLR2-specific, the acetylation of the cysteine amine in 4 led to loss of the lone ionizable group and, consequently, to the complete loss of aqueous solubility. An important component of our work on vaccine adjuvant design and development has been to engineer complete water solubility into our candidate adjuvants so as to obviate the need for any excipients, and it therefore became necessary to reintroduce aqueous solubility in 4. Drawing from our previous SAR studies that the sidechain of the terminal amino acid (Ser) of the dipeptide unit was not a key determinant of TLR2 activity,30,36,52 an obvious and straightforward approach was to replace it with lysine, the ε-amine of which was anticipated to restore solubility to the lipopeptide. However, we observed hydrolytic lability in the analogue, with consequent deterioration of activity. This observation prompted us to explore various strategies of introducing ionizable groups on the lipopeptide, as well as the systematic evaluation of stable bioisosteres of the ester-linked palmitoyl group. Analogues with the serine hydroxyl functionality of the lipopeptide esterified with L-lysine, succinic acid or nicotinic acid retained hTLR2-specific agonistic activity, but progressively hydrolyzed to 4 upon prolonged storage, indicating that ester groups were contributing to lability. These initial observations prompted us to first explore replacing the ester-linked palmitoyl group with amide, triazole, and carbamate linked long-chain alkyl groups. A carbamate derivative was found to be more potent than the parent compound. Aqueous solubility in this analogue was restored by appending a N1,N1-dimethylpropane-1,3-diamine moiety to the carboxyl group of serine via an amide linkage, culminating in a fully optimized, stable, and highly water-soluble, human TLR2-specific agonist with very high potency. The optimized lead compound was found to have an excellent safety profile in rabbit models, and displayed prominent adjuvantic activities.</p><!><p>To restore water solubility to 4, we synthesized an analogue with the terminal serine methyl ester replaced with a lysine methyl ester (Compound 10, Scheme 1). While the lysine analogue 10 was indeed as active as 4 (EC50 = 1.50 nM, Table 1, 10Fig. 2) and highly water soluble, we noticed significant loss of activity of aqueous stocks in less than a week. Mass spectrometry revealed hydrolysis of the methyl ester, presumably via an intramolecular attack of the ε-amine of lysine, which presaged inadequate shelf-life of vaccine constructs incorporating as a candidate adjuvant. We next explored analogues with the serine hydroxyl functionality of 4 esterified with L-lysine (12), sulfated (13), converted to the hemisuccinate (14) or esterified with nicotinic acid (15) (Scheme 2). All the compounds retained hTLR2-specific agonistic activity (Table 1, 12Fig. 2), and the lysine conjugate (EC50 = 1.50 nM), the sulfate 13 (EC50 = 5.84 nM), as well as hemisuccinate 14 (EC50 = 0.65 nM) were found to be highly water soluble. As observed for compound 10, aqueous stocks of compound 12 were also found to be unstable, and a white precipitate of its parent compound 4 was observed (confirmed by TLC and LC-MS) upon prolonged storage, indicating that compound 12 was behaving as a water-soluble, but relatively unstable prodrug of compound 4.</p><p>Given that the mono-acyl lipopeptides are human TLR2-specific, murine models of immunization that we had used previously34 to benchmark adjuvantic activity were inappropriate, and it was of importance to verify whether the rabbit model34,53 that we had subsequently adopted as a screen would be suitable to evaluate the adjuvanticity of this chemotype. We elected first to evaluate the adjuvanticity of the highly water-soluble 12 using bovine α-lactalbumin as a model subunit antigen34,53 and under excipient-free conditions, reasoning that degradation in vivo of 12 would yield 4, which would retain TLR2-agonistic activity. We were gratified to find that the water soluble monoacyl lipopeptide 12 showed excellent induction of anti-bovine α-lactalbumin IgG responses in rabbits using a prime+dual-boost model (Fig. 3), validating the relevance of the animal model, and demonstrating excellent adjuvanticity.</p><p>Having observed lability of two ester groups, both of which undermine and compromise the activity and shelf-life, we set out to design out all of the labile groups in our lead compound 4, beginning with the replacement of the ester-linked palmitoyl group with amide- (compound 22, Scheme 3), triazole- (compounds 24 and 27, Scheme 4) and carbamate- (compounds 31 and 34, Scheme 5) linked long-chain alkyl groups.</p><p>Starting from L-cystine, the advanced intermediate 18 was synthesized as reported by us earlier.30,52 The hydroxyl functionality in 18 was mesylated and displaced by sodium azide to furnish the azido compound 19 (73% over two steps, Scheme 3). A Staudinger azide reduction protocol was used for the synthesis of amino compound 20, which was N-palmitoylated to obtain 21. Global deprotection and controlled acetylation (yielding predominantly the cystine N-acetylated product) furnished the desired amide-linked derivative 22. The triazole linked derivatives 24 and 27 were designed based on the fact that substituted 1,2,3-triazoles are popular functionalities used in drug discovery for the bioisosteric replacement of key functional groups. They are also known to readily associate with biological targets through hydrogen-bonding and dipole interactions.54,55 The azido intermediate 19 described above was a convenient synthon for the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction for the synthesis of the 4-alkyl-1H-1,2,3-triazol-1-yl compound 24. Pentadec-1-yne was selected as an alkyne component in this reaction to maintain the overall length of the molecule. The regioisomeric 1-alkyl-1H-1,2,3-triazol-4-yl analogue 27 was also synthesized by swapping the alkyne and azide functionalities in the respective synthons (Scheme 4); the alkyne component 25 was synthesized by alkylation of mercapto-dipeptide 17 with 4-bromobut-1-yne, while 1-azidohexadecane was synthesized from 1-bromohexadecane using sodium azide in DMF. The synthesis of the carbamate-linked analogue 29 was achieved by treatment of the primary alcohol functionality in compound 18 with 1,1′-carbonyldiimidazole (CDI) in dichloromethane (DCM), and reacting in situ the imidazolecarboxylate intermediate 28 with hexadecylamine (Scheme 5). The desired derivative 31 was synthesized as reported for the synthesis of compound 22. The regioisomeric carbamate derivative 34 was synthesized simply by the reaction of 20 with cetylchloroformate and further elaboration as reported for compounds 22 or 31.</p><p>The triazole derivatives 24 (EC50 = 0.29 μM, Table 1, 27Fig. 4) and (EC50 = 1.02 μM, Table 1, 22Fig. 4) were found to be the least active in TLR2 agonism assay. The loss in activity was also observed in amide derivative (EC50 = 12.78 nM, Table 1, 31Fig. 4). We were delighted to observe in our cell-based reporter gene assays that the carbamate derivative was as potent (EC50 = 0.32 nM, Table 1, 4Fig. 4) as , and its regioisomer 34 was highly active (34: EC50 = 65 pM; 4: EC50 = 0.66 nM; Table 1, Fig. 4).</p><p>Aqueous solubility in the highly potent and stable carbamate derivative 34 was restored by appending a N1,N1-dimethylpropane-1,3-diamine moiety to the carboxyl group of serine via a stable amide linkage (compound 38, Scheme 6), an approach that is similar to attaching a polar polyethylene glycol unit to the terminal amino acid residue.56 Our initial attempts at hydrolyzing the methyl ester in compound 32 for coupling the N1,N1-dimethylpropane-1,3-diamine using conventional lithium hydroxide protocols resulted in epimerization of the stereocenters, yielding the target compound in its racemic form (EC50 = 1.29 nM). We successfully utilized the mild and selective trimethytin hydroxide ester hydrolysis method developed by Nicolaou57 and obtained compound 35 in enantiopure form. Subsequent amidation of 35 using N1,N1-dimethylpropane-1,3-diamine and further elaboration resulted in the highly water soluble (>10 mg/mL) and highly potent (EC50 = 0.25 nM, Table 1) enantiopure final compound 38, which retained excellent chemical stability under accelerated stability testing conditions.</p><p>The combination of highly desired attributes of high potency (in primary screens), excellent aqueous solubility, and chemical stability in 38 warranted a careful evaluation of this compound in secondary screens. Consistent with our earlier findings that 2 induced virtually undetectable levels of proinflammatory mediators such as TNF-α, IL-1β and IL-18,31 we could not detect any significant proinflammatory cytokine signatures in human PBMCs or human whole blood stimulated with 38 using our standard 5-plex cytometric bead assays35,41 (data not shown). In an effort to understand in greater detail the basis of the potent adjuvanticity of TLR2 agonists that appears to be entirely dissociated from the induction of proinflammatory cytokines, we examined 38 in a 41-plex immunoassay that we have recently implemented. We observed prominent biphasic induction of the chemokines monocyte chemotactic protein-1 (MCP-1), and MCP-3, macrophage-derived chemokine (MDC), as well as interleukin-8 (IL-8, also a chemokine; Fig. 6). The rather unexpected finding of strong macrophage-derived or -targeted chemokine induction prompted us to examine markers of monocytic activation in ex vivo flow cytometric assays using whole human blood. As previously reported,31 TLR2 and TLR4 agonists induce CD11b upregulation in human granulocytes, and we found that 38 indeed upregulated CD11b very potently, relative to 2 (Fig. 6, left panel) in neutrophils; however, we also observed strong monocytic CD11b upregulation (Fig. 6, right panel), which has not been previously reported for agonists of TLR2. Although we cannot as yet establish a causal relationship between monocytic activation and chemokine production on the one hand, and adjuvanticity on the other, these biomarkers will likely prove useful in evaluating several other chemotypes which we are currently examining.</p><p>Cautious of the very high potency of 38, we elected to first evaluate the safety of this lipopeptide in rabbit models. A dose-escalation study in rabbits up to 100 μg/animal (administered intramuscularly, or as an intravenous bolus) did not result in any observable adverse effects. Whereas a dose of 1 μg of LPS evoked prominent leucopenia, lymphocytopenia (Fig. 7a–b) and febrile responses (Fig. 7c) in the animals, 100 μg of 38 did not induce any such effects (Fig. 7).</p><p>Encouraged by the excellent safety profile, we proceeded to evaluate the adjuvanticity of 38. Non-alum-adsorbed, toxoided pertussis antigens are not commercially available, and we therefore chose CRM197, a nontoxic mutant of diphtheria toxin as a test-antigen.58 The excellent solubility of the lipopeptide allowed excipient-free formulation of the test-antigen and adjuvant in sterile saline. Pre-immune test-bleeds were first obtained via venipuncture of the marginal vein of the ear. Rabbits were then immunized intramuscularly on Days 1, 15, and 28 with antigen-sparing doses (10 μg/dose) of unadjuvanted CRM197 in saline, or adjuvanted with 100 μg/dose of 38 in a total volume of 0.2 mL. A test bleed was performed on Day 25 (ten days after the first boost) and a final bleed was performed via the marginal vein of the ear on Day 38. Sera were stored at −80 °C until used. CRM197-specific IgG titers in sera were quantified by conventional antibody-capture ELISA techniques performed in 384-well format using automated liquid handling methods as described by us.34,53 We were gratified to find rapid and robust anti-CRM197 IgG titers in animals receiving the adjuvant (Fig. 8), as compared to unadjuvanted controls.</p><p>We have been fortunate in being able to successfully apply principles of classical medicinal chemistry and rational drug optimization to an unexpected problem of chemical stability, achieving in the process augmented potency, excellent aqueous solubility and, importantly, preserving safety and efficacy. The identification of key chemokine biomarkers for TLR2 agonists will likely prove useful as we continue to develop this and other chemotypes as candidate vaccine adjuvants.</p><!><p>All of the solvents and reagents used were obtained commercially and used as such unless noted otherwise. Moisture- or air-sensitive reactions were conducted under nitrogen atmosphere in oven-dried (120 °C) glass apparatus. The solvents were removed under reduced pressure using standard rotary evaporators. Flash column chromatography was carried out using RediSep Rf 'Gold' high performance silica columns on CombiFlash Rf instrument unless otherwise mentioned, while thin-layer chromatography was carried out on silica gel CCM pre-coated aluminum sheets. Purity for all final compounds was confirmed to be greater than 97% by LC-MS using a Zorbax Eclipse Plus 4.6 mm × 150 mm, 5 μm analytical reverse phase C18 column with H2Oisopropanol or H2O-CH3CN gradients and an Agilent ESI-TOF mass spectrometer (mass accuracy of 3 ppm) operating in the positive ion (or negative ion, as appropriate) acquisition mode.</p><!><p>To a solution of L-cystine (500 mg, 2.08 mmol) in water (10 mL) were added triethylamine (870 μL, 6.24 mmol) and di-tert-butyldicarbonate (1.35 g, 6.24 mmol). The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10% HCl. The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (15% MeOH/CH2Cl2) to obtain compound Nα,Nα′-di-Boc-L-cystine (870 mg, 95%). To a solution of Nα,Nα′-di-Boc-L-cystine (500 mg, 1.13 mmol) in anhydrous DMF (15 mL) were added H-Lys( Fmoc)-OMe·HCl (1.05 g, 2.50 mmol), HOBt (338 mg, 2.50 mmol), and pyridine (411 μL, 4.50 mmol). The reaction mixture was stirred at 0 °C for 30 min, followed by addition of EDCI (958 mg, 5.00 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in ethyl acetate and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (50% EtOAc/hexanes) to obtain compound 5 (1.10 g, 83%). MS (ESI-TOF) for C60H76N6O14S2 [M+H]+ Found 1169.5011, Calculated 1169.4934; [M+Na]+ Found 1191.4834, Calculated 1191.4753. Compound 5 (600 mg, 0.51 mmol) was dissolved in hydrogen chloride solution (10 mL, 4M in dioxane) and the reaction mixture was stirred at room temperature for an hour and the volatilities were removed to afford (2S,2′S)-dimethyl 2,2′-(((2R,2′R)-3,3′-disulfanediylbis(2-aminopropanoyl))bis(azanediyl))bis(6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanoate) dihydrochloride salt (535 mg, 0.51 mmol). The crude product was dissolved in CH2Cl2 (5 mL) and pyridine (5 mL). Acetic anhydride (291 μL, 3.08 mmol) was added and the reaction mixture was stirred at room temperature for 2h, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in ethyl acetate and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (50% EtOAc/hexanes) to obtain compound 6 (425 mg, 79% over two steps). 1H NMR (500 MHz, CDCl3) δ 8.46 (d, J = 7.1 Hz, 2H), 7.74 (d, J = 7.5 Hz, 4H), 7.57 (d, J = 7.4 Hz, 4H), 7.38 (t, J = 7.4 Hz, 4H), 7.29 (t, J = 7.5 Hz, 4H), 6.72 (d, J = 9.1 Hz, 2H), 5.45 (td, J = 9.9, 3.2 Hz, 2H), 4.94 (t, J = 5.4 Hz, 2H), 4.48 – 4.32 (m, 6H), 4.18 (t, J = 6.8 Hz, 2H), 3.71 (s, 6H), 3.20 – 3.03 (m, 6H), 2.94 – 2.83 (m, 2H), 2.02 (s, 6H), 1.91 – 1.68 (m, 4H), 1.58 – 1.31 (m, 8H). 13C NMR (126 MHz, CDCl3) δ 172.4, 170.7, 156.5, 144.1, 141.4, 127.8, 127.2, 125.1, 120.1, 66.6, 53.2, 52.7, 52.5, 47.4, 46.2, 40.8, 31.2, 29.5, 23.5, 22.8. MS (ESI-TOF) for C54H64N6O12S2 [M+H]+ Found 1053.4234, Calculated 1053.4096; [M+Na]+ Found 1075.4048, Calculated 1075.3916.</p><!><p>To a solution of 6 (375 mg, 0.356 mmol) in CH2Cl2 (5 mL) were added water (100 μL) and tributylphosphine (356 μL, 1.43 mmol). The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction, the solvent was removed under reduced pressure to obtain the crude product, which was purified using column chromatography (50% EtOAc/hexanes) to obtain compound 7 (250 mg, 66%). MS (ESI-TOF) for C27H33N3O6S [M+H]+ Found 528.2232, Calculated 528.2163; [M+Na]+ Found 550.2061, Calculated 550.1988. To a solution of compound 7 (245 mg, 0.465 mmol) in DMF (5 mL) were added 2-iodoethanol (182 μL, 2.33 mmol) and potassium carbonate (320 mg, 2.33 mmol). The reaction mixture was stirred at room temperature for an hour. After completion of the reaction, the solid potassium carbonate was filtered out and the solvent was removed under reduced pressure to obtain the crude product, which was purified using column chromatography (5% MeOH/CH2Cl2) to obtain compound 8 as a white solid (111 mg, 42%). MS (ESI-TOF) for C29H37N3O7S [M+H]+ Found 572.2427, Calculated 572.2425; [M+Na]+ Found 594.2255, Calculated 594.2244. Compound 8 (100 mg, 0.175 mmol) was then dissolved in CH2Cl2 (1 mL) and pyridine (1 mL). Palmitoyl chloride (80 μL, 0.262 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction, the solvents were removed under reduced pressure to obtain the crude product, which was purified using column chromatography (50% EtOAc/hexanes) to obtain compound 9 (133 mg, 94%). 1H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.07 (d, J = 7.7 Hz, 1H), 6.54 (d, J = 7.2 Hz, 1H), 5.12 (t, J = 5.7 Hz, 1H), 4.63 – 4.50 (m, 2H), 4.46 – 4.35 (m, 2H), 4.31 – 4.16 (m, 3H), 3.73 (s, 3H), 3.17 (td, J = 13.6, 6.9 Hz, 2H), 2.92 (qd, J = 14.0, 6.4 Hz, 2H), 2.82 (td, J = 6.6, 1.9 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 2.01 (s, 3H), 1.89 (ddd, J = 14.9, 10.8, 5.7 Hz, 1H), 1.79 – 1.66 (m, 1H), 1.65 – 1.56 (m, 2H), 1.56 – 1.47 (m, 2H), 1.45 – 1.33 (m, 2H), 1.33 – 1.21 (m, 24H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.05, 172.33, 170.53, 170.38, 156.76, 144.11, 144.08, 141.44, 127.81, 127.17, 125.17, 120.11, 66.67, 62.76, 52.68, 52.64, 52.39, 47.39, 40.53, 34.34, 34.26, 32.06, 31.64, 31.15, 29.83, 29.79, 29.76, 29.62, 29.50, 29.42, 29.36, 29.28, 25.02, 23.20, 22.83, 22.40, 14.27. MS (ESI-TOF) for C45H67N3O8S [M+H]+ Found 810.4754, Calculated 810.4722; [M+Na]+ Found 832.4591, Calculated 832.4541.</p><!><p>To a solution of compound 9 (81 mg, 0.1 mmol) in DMF (1 mL) was added polymer-bound piperazine (1–2 mmol/g loading) (333 mg, ~ 0.5 mmol) and the reaction mixture was stirred at room temperature for 4 h. After completion of the reaction, the resin was filtered out and the solvents were removed under reduced pressure to obtain the crude product, which was purified using column chromatography (20% MeOH/CH2Cl2) to obtain compound 10 as a white solid (32 mg, 55%). 1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 7.9 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 4.84 (td, J = 8.0, 5.5 Hz, 1H), 4.54 (dd, J = 13.8, 7.4 Hz, 1H), 4.31 – 4.15 (m, 2H), 3.73 (s, 3H), 3.06 (ddd, J = 19.2, 13.6, 6.2 Hz, 3H), 2.92 – 2.72 (m, 3H), 2.34 – 2.26 (m, 2H), 2.06 (s, 3H), 1.97 – 1.88 (m, 1H), 1.85 (dd, J = 15.0, 7.7 Hz, 2H), 1.77 – 1.67 (m, 1H), 1.59 (dd, J = 14.3, 7.2 Hz, 4H), 1.40 – 1.17 (m, 24H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.01, 172.17, 171.42, 170.90, 63.08, 52.63, 52.40, 39.05, 34.97, 34.36, 32.07, 31.19, 30.21, 29.85, 29.82, 29.81, 29.79, 29.66, 29.51, 29.46, 29.33, 26.58, 25.06, 23.41, 22.84, 22.07, 14.28. MS (ESI-TOF) for C30H57N3O6S [M+H]+ Found 588.4086, Calculated 588.4041; [M+Na]+ Found 610.3914, Calculated 610.3860.</p><!><p>To a solution of N,N′-di-boc-L-lysine (63 mg, 0.183 mmol) and compound 4 (50 mg, 0.091 mmol) in anhydrous CH2Cl2 (5 mL), were added N-methylmorpholine (20 μL, 0.183 mmol) and catalytic amount of DMAP. The reaction mixture was stirred at 0 °C and EDCI (28 mg, 0.183 mmol) was added after 15 min. The reaction mixture was then stirred at room temperature for 4 h. After the completion of reaction, water (10 mL) was added and the product was extracted in CH2Cl2. The organic layer was washed with water (10 mL × 2), brine (10 mL) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was purified using column chromatography (5 % MeOH/CH2Cl2) to obtain the diboc protected intermediate 11 as white solid (40 mg, 50%). 1H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 7.6 Hz, 1H), 5.23 (d, J = 6.6 Hz, 1H), 4.83 (dt, J = 7.7, 3.9 Hz, 1H), 4.77 (s, 1H), 4.66 (d, J = 6.3 Hz, 1H), 4.58 (d, J = 10.0 Hz, 1H), 4.45 (dd, J = 11.2, 3.9 Hz, 1H), 4.32 – 4.14 (m, 3H), 3.78 (s, 3H), 3.18 – 3.07 (m, 2H), 2.97 (qd, J = 13.9, 6.4 Hz, 2H), 2.85 – 2.74 (m, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.07 (s, 3H), 1.86 – 1.55 (m, 6H), 1.55 – 1.41 (m, 18H), 1.41 – 1.20 (m, 24H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 173.95, 172.59, 170.84, 170.57, 169.19, 156.49, 155.86, 80.25, 79.42, 63.83, 62.93, 53.54, 53.15, 52.66, 52.15, 39.84, 34.35, 33.95, 32.07, 31.67, 31.11, 29.84, 29.80, 29.77, 29.63, 29.51, 29.44, 29.30, 28.59, 28.50, 25.04, 23.25, 22.84, 22.48, 14.28. MS (ESI-TOF) for C43H78N4O12S [M+H]+ Found 875.5417, Calculated 875.5410; [M+Na]+ Found 897.5232, Calculated 897.5229. Compound 11 was then dissolved in HCl/dioxane (4 M solution, 2 mL) and the reaction mixture was stirred at room temperature for 15 min, followed by removal of the solvent under vacuum to obtain compound 12 as a white solid in quantitative yield. 1H NMR (500 MHz, DMSO) δ 8.80 (d, J = 8.1 Hz, 1H), 8.63 (s, 3H), 8.31 (d, J = 8.3 Hz, 1H), 7.95 (s, 3H), 4.72 (ddd, J = 7.9, 6.3, 4.9 Hz, 1H), 4.46 (ddd, J = 15.7, 10.0, 4.7 Hz, 2H), 4.38 (dd, J = 11.2, 6.4 Hz, 1H), 4.18 – 4.11 (m, 2H), 3.97 (bs, 1H), 3.73 – 3.69 (m, 1H), 3.69 – 3.64 (m, 4H), 3.52 – 3.48 (m, 1H), 3.46 (ddd, J = 6.1, 3.9, 1.2 Hz, 1H), 2.93 (dd, J = 13.7, 4.8 Hz, 1H), 2.83 – 2.72 (m, 4H), 2.67 (dd, J = 13.7, 9.4 Hz, 1H), 2.28 (t, J = 7.4 Hz, 2H), 1.88 (s, 3H), 1.84 – 1.77 (m, 2H), 1.62 – 1.32 (m, 8H), 1.30 – 1.18 (m, 24H), 0.85 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 172.81, 170.76, 169.68, 169.23, 168.91, 72.17, 70.53, 63.95, 62.88, 60.18, 52.44, 51.67, 50.78, 43.63, 38.23, 33.53, 33.41, 31.31, 30.01, 29.11, 29.06, 29.03, 29.02, 29.00, 28.90, 28.72, 28.46, 26.19, 24.44, 22.55, 22.12, 21.13, 13.99. MS (ESI-TOF) for C33H62N4O8S [M+H]+ Found 675.4181, Calculated 675.4361.</p><!><p>To a solution of compound 4 (60 mg, 0.110 mmol) in anhydrous pyridine was added sulfur trioxide pyridine complex (175 mg, 1.10 mmol). The reaction mixture was heated at 80 °C for 16 h. The solvent was removed under reduced pressure to obtain the residue which was purified using column chromatography (10% MeOH/CH2Cl2), to furnish compound 13 as a white solid (23 mg, 33%). 1H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 5.3 Hz, 1H), 7.30 (d, J = 1.6 Hz, 1H), 4.92 – 4.79 (m, 2H), 4.32 (dd, J = 33.8, 6.7 Hz, 2H), 4.26 – 4.14 (m, 2H), 3.76 (s, 3H), 3.00 (d, J = 9.1 Hz, 1H), 2.89 (dd, J = 12.8, 6.8 Hz, 1H), 2.78 (t, J = 6.4 Hz, 2H), 2.36 – 2.26 (m, 3H), 2.02 (s, 3H), 1.59 (dd, J = 14.3, 7.2 Hz, 2H), 1.35 – 1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.00, 172.28, 171.43, 170.29, 67.45, 62.92, 53.15, 52.60, 52.19, 34.36, 32.08, 30.97, 29.87, 29.86, 29.82, 29.71, 29.52, 29.51, 29.38, 25.08, 23.01, 22.85, 14.28. MS (ESI-TOF, Negative Mode) for C27H50N2O10S2, [M−H]− Found 625.2633, Calculated 625.2834.</p><!><p>To a solution of compound 4 (50 mg, 0.091 mmol) in anhydrous THF were added triethylamine (25 μL, 0.183 mmol) and succinic anhydride (18 mg, 0.183 mmol). The reaction mixture was stirred at 50 °C for 2 h. The solvent was removed under vacuum to obtain the residue which was purified using column chromatography (10% MeOH/CH2Cl2), to furnish compound 14 (37 mg, 62%). 1H NMR (500 MHz, CDCl3) δ 7.25 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 4.87 – 4.78 (m, 2H), 4.65 (dd, J = 11.4, 3.5 Hz, 1H), 4.39 (dd, J = 11.4, 3.4 Hz, 1H), 4.34 – 4.19 (m, 2H), 3.79 (s, 3H), 2.99 (dd, J = 14.0, 5.9 Hz, 1H), 2.87 (dd, J = 14.0, 7.2 Hz, 1H), 2.81 (td, J = 6.8, 1.0 Hz, 2H), 2.75 – 2.53 (m, 4H), 2.37 – 2.29 (m, 2H), 2.08 (s, 3H), 1.66 – 1.56 (m, 2H), 1.35 – 1.21 (m, 24H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.28, 174.54, 171.84, 171.65, 170.25, 169.41, 63.29, 63.04, 53.10, 52.40, 52.03, 34.58, 34.39, 32.06, 30.90, 29.84, 29.80, 29.76, 29.67, 29.61, 29.50, 29.41, 29.39, 29.27, 25.00, 23.36, 22.83, 14.27. MS (ESI-TOF) for C31H54N2O10S [M+H]+ Found 647.3649, Calculated 647.3572; [M+Na]+ Found 669.3476, Calculated 669.3391.</p><!><p>To a solution of compound 4 (50 mg, 0.091 mmol) and nicotinic acid (23 mg, 0.183 mmol) in anhydrous CH2Cl2 (5 mL), were added N-methylmorpholine (20 μL, 0.183 mmol) and catalytic amount of DMAP. The reaction mixture was stirred at 0 °C and EDCI (28 mg, 0.183 mmol) was added after 15 min. The reaction mixture was then stirred at room temperature overnight. After completion of the reaction, water (10 mL) was added and the product was extracted in CH2Cl2. The organic layer was washed with water (10 mL × 2), brine (10 mL) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was purified using column chromatography (5 % MeOH/CH2Cl2) to obtain product 15 as a white solid (41 mg, 68%). 1H NMR (500 MHz, CDCl3) δ 9.19 (dd, J = 2.2, 0.8 Hz, 1H), 8.80 (dd, J = 4.9, 1.7 Hz, 1H), 8.30 – 8.23 (m, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.41 (ddd, J = 8.0, 4.9, 0.8 Hz, 1H), 6.44 (d, J = 7.1 Hz, 1H), 4.95 (dt, J = 7.7, 3.8 Hz, 1H), 4.77 – 4.65 (m, 2H), 4.61 (td, J = 7.2, 5.7 Hz, 1H), 4.32 – 4.18 (m, 2H), 3.82 (s, 3H), 2.99 (dd, J = 14.0, 5.6 Hz, 1H), 2.93 – 2.75 (m, 3H), 2.34 – 2.24 (m, 2H), 2.01 (s, 3H), 1.62 (s, 3H), 1.62 – 1.53 (m, 2H), 1.36 – 1.18 (m, 24H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.05, 170.51, 170.44, 169.36, 164.88, 154.04, 151.18, 137.36, 125.39, 123.56, 64.41, 62.61, 53.28, 52.55, 52.23, 34.35, 33.99, 32.07, 31.20, 29.84, 29.80, 29.77, 29.62, 29.51, 29.42, 29.29, 25.02, 23.21, 22.84, 14.28. MS (ESI-TOF) for C33H53N3O8S [M+H]+ Found 652.3672, Calculated 652.3626; [M+Na]+ Found 674.3472, Calculated 674.3446.</p><!><p>To a solution of compound 18 (500 mg, 1.185 mmol) in CH2Cl2 (5 mL) were added triethylamine (0.5 mL, 3.56 mmol) and methanesulfonyl chloride (276 μL, 3.55 mmol) and the reaction mixture was stirred at room temperature for 3 h. After the completion of the reaction, water (10 mL) was added and the product was extracted in CH2Cl2. The organic layer was washed with water (10 mL × 2) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude mesylate. This crude product was dissolved in DMF (5 mL) and sodium azide (385 mg, 5.925 mmol) was added and the reaction mixture was stirred at 60 C for 4 h. After completion of the reaction, water (20 mL) was added to the reaction and the product obtained was extracted in EtOAc. The organic layer was washed with water (10 mL × 3) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (20% EtOAc/hexanes) to obtain compound 19 (390 mg, 73% over two steps). 1H NMR (500 MHz, CDCl3) δ 7.14 (d, J = 7.8 Hz, 1H), 5.49 (s, 1H), 4.66 (dt, J = 8.2, 3.0 Hz, 1H), 4.32 (d, J = 4.9 Hz, 1H), 3.83 (dd, J = 9.1, 2.9 Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.51 (ddd, J = 12.5, 9.0, 6.2 Hz, 2H), 3.02 (dd, J = 14.0, 5.1 Hz, 1H), 2.90 (dd, J = 14.0, 7.0 Hz, 1H), 2.79 (ddd, J = 12.1, 9.5, 3.7 Hz, 2H), 1.46 (s, 9H), 1.14 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 170.59, 170.41, 155.40, 80.47, 73.70, 61.76, 53.93, 53.31, 52.60, 51.14, 34.89, 31.86, 28.43, 27.41. MS (ESI-TOF) for C18H33N5O6S [M+Na]+ Found 470.1919, Calculated 470.2044.</p><!><p>To a solution of compound 19 (237 mg, 0.53 mmol) in dry THF (5 mL) was added triphenylphosphine (208 mg, 0.795 mmol) and the reaction mixture was heated to reflux for 3 h. Water (1 mL) was added and the heating was continued for 2 more hours. After the completion of reaction, the solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (10% MeOH/CH2Cl2) to obtain compound 19 (200 mg, 90%). 1H NMR (500 MHz, CDCl3) δ 7.57 (bs, 1H), 5.73 (d, J = 7.5 Hz, 1H), 4.74 – 4.61 (m, 1H), 4.35 (bs, 1H), 3.82 (dd, J = 9.1, 3.0 Hz, 1H), 3.73 (s, 3H), 3.57 (dd, J = 9.1, 3.3 Hz, 1H), 3.03 – 2.80 (m, 4H), 2.79 – 2.63 (m, 2H), 1.45 (s, 9H), 1.13 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 170.7, 170.6, 155.5, 80.3, 73.6, 61.9, 54.0, 53.3, 52.5, 41.4, 36.4, 34.9, 28.5 (3C), 27.4 (3C). MS (ESI-TOF) for C18H35N3O6S [M+H]+ Found 470.1919, Calculated 470.2044.</p><!><p>To a solution of compound 20 (100 mg, 0.238 mmol) in dry CH2Cl2 (2 mL) were added triethylamine (50 mL, 0.356 mmol) and palmitoyl chloride (109 mL, 0.356 mmol) and the reaction mixture was stirred at room temperature for 30 min. After the completion of reaction, water (10 mL) was added to the reaction and the product obtained was extracted in CH2Cl2. The organic layer was washed with water (10 mL × 3) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (40% EtOAc/hexanes) to obtain compound 21 (105 mg, 69%). 1H NMR (500 MHz, CDCl3) δ 7.14 (d, J = 8.1 Hz, 1H), 6.25 (s, 1H), 5.48 (d, J = 4.1 Hz, 1H), 4.66 (dt, J = 8.2, 3.1 Hz, 1H), 4.32 (d, J = 5.6 Hz, 1H), 3.83 (dd, J = 9.1, 3.0 Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.48 (td, J = 9.3, 6.0 Hz, 2H), 2.97 (dd, J = 13.9, 5.5 Hz, 1H), 2.88 (dd, J = 13.9, 6.9 Hz, 1H), 2.82 – 2.66 (m, 2H), 2.24 – 2.13 (m, 2H), 1.67 – 1.56 (m, 2H), 1.45 (s, 9H), 1.37 – 1.19 (m, 24H), 1.14 (s, 9H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 173.53, 170.71, 170.50, 155.50, 80.47, 73.75, 61.76, 53.88, 53.30, 52.62, 38.58, 36.86, 34.55, 32.51, 32.06, 29.84, 29.80, 29.78, 29.71, 29.67, 29.54, 29.50, 28.45, 27.43, 25.88, 22.83, 14.27. MS (ESI-TOF) for C34H65N3O7S [M+H]+ Found 660.4560, Calculated 660.4616; [M+Na]+ Found 682.4373, Calculated 682.4435.</p><!><p>To compound 21 (95 mg, 0.183 mmol) was added TFA (2 mL). The reaction mixture was stirred at room temperature for 30 min and then dried by blowing nitrogen through the solution. The crude product was used directly for the next step. To a solution of the crude intermediate in CH2Cl2 (2 mL) was added pyridine (16 μL, 0.2 mmol) and acetic anhydride (19 μL, 0.2 mmol). The reaction mixture was stirred at room temperature for 30 min and then concentrated. The residue was purified by a flash chromatography (5% MeOH/CH2Cl2) to give product 22 (70 mg, 84%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 7.8 Hz, 1H), 6.75 (d, J = 7.3 Hz, 1H), 6.07 (s, 1H), 4.71 (d, J = 7.1 Hz, 1H), 4.62 (dd, J = 7.7, 3.3 Hz, 1H), 4.01 (s, 2H), 3.78 (s, 2H), 3.63 – 3.48 (m, 2H), 2.95 (dd, J = 22.3, 6.2 Hz, 2H), 2.85 (dt, J = 14.2, 5.7 Hz, 1H), 2.72 (dt, J = 14.5, 7.4 Hz, 1H), 2.23 – 2.19 (m, 1H), 2.06 (s, 2H), 1.63 – 1.56 (m, 2H), 1.34 – 1.22 (m, 18H), 0.88 (t, J = 6.9 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 175.10, 170.66, 170.61, 170.55, 62.88, 55.43, 52.78, 52.28, 39.22, 37.01, 35.36, 32.75, 32.07, 29.85, 29.84, 29.81, 29.77, 29.63, 29.51, 29.45, 29.40, 25.80, 23.35, 22.84, 14.28. MS (ESI-TOF) for C27H51N3O6S [M+H]+ Found 546.3474, Calculated 546.3571; [M+Na]+ Found 568.3293, Calculated 568.3391.</p><!><p>To a stirred solution of compound 19 (60 mg, 0.134 mmol) and pentadec-1-yne (39 μL, 0.147 mmol) in THF (2 mL), were added CuSO4.5H2O (3 mg in 0.25 mL water, 0.013 mmol) and sodium ascorbate (5 mg in 0.25 mL water, 0.003 mmol) and the reaction mixture was stirred at room temperature for overnight. After the completion of reaction, water (10 mL) was added and the product obtained was extracted in EtOAc. The organic layer was washed with water (10 mL × 2) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product. The residue was further purified by a flash chromatography (5% MeOH/CH2Cl2) to give product 23 (48 mg, 55%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 7.43 (s, 1H), 7.12 (d, J = 7.6 Hz, 1H), 5.51 (d, J = 4.1 Hz, 1H), 4.64 (dt, J = 8.2, 3.1 Hz, 1H), 4.54 (t, J = 7.0 Hz, 2H), 4.32 (d, J = 4.4 Hz, 1H), 3.82 (dd, J = 9.1, 3.0 Hz, 1H), 3.73 (s, 3H), 3.56 (dd, J = 9.1, 3.2 Hz, 1H), 3.12 (dd, J = 13.3, 6.5 Hz, 1H), 3.02 (ddd, J = 14.1, 13.1, 5.9 Hz, 2H), 2.84 (dd, J = 14.1, 7.2 Hz, 1H), 2.74 – 2.64 (m, 2H), 1.71 – 1.59 (m, 3H), 1.46 (s, 9H), 1.39 – 1.21 (m, 20H), 1.13 (s, 9H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.56, 170.36, 155.36, 148.63, 121.30, 80.54, 73.75, 61.67, 53.85, 53.35, 52.62, 49.64, 34.44, 32.24, 32.07, 29.84, 29.82, 29.80, 29.72, 29.62, 29.55, 29.50, 29.47, 28.44, 27.42, 25.86, 22.84, 14.28. MS (ESI-TOF) for C33H61N5O6S [M+H]+ Found 656.4403, Calculated 656.4415; [M+Na]+ Found 678.4223, Calculated 678.4235.</p><!><p>The global deprotection of compound 23 using TFA and further N-acetylation was carried out similarly as described earlier (synthesis of 22) to furnish compound 24 as a white solid. 1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 7.8 Hz, 1H), 7.38 (s, 1H), 6.78 (d, J = 6.8 Hz, 1H), 5.21 (s, 1H), 4.92 (ddd, J = 14.2, 8.9, 4.1 Hz, 1H), 4.63 (ddd, J = 7.7, 4.6, 3.0 Hz, 1H), 4.61 – 4.52 (m, 2H), 4.03 (dd, J = 12.2, 3.7 Hz, 2H), 3.75 (s, 3H), 3.24 (ddd, J = 14.8, 5.9, 4.2 Hz, 1H), 3.07 (dd, J = 9.0, 4.7 Hz, 1H), 2.90 (dd, J = 14.5, 4.1 Hz, 1H), 2.72 (dd, J = 14.5, 9.1 Hz, 1H), 2.67 (dd, J = 8.5, 6.8 Hz, 2H), 2.03 (s, 3H), 1.73 – 1.54 (m, 2H), 1.39 – 1.17 (m, 20H), 0.87 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.40, 170.23, 169.96, 149.51, 121.78, 62.52, 55.79, 53.11, 52.67, 50.05, 36.03, 34.69, 32.06, 29.83, 29.81, 29.79, 29.69, 29.50, 29.48, 29.39, 29.37, 25.57, 23.31, 22.84, 14.28. MS (ESI-TOF) for C26H47N5O5S [M+H]+ Found 542.3378, Calculated 542.3371; [M+Na]+ Found 564.3201, Calculated 564.3190.</p><!><p>To a solution of compound 17 (200 mg, 0.529 mmol) in dry DMF (5 mL) were added 4-bromobut-1-yne (248 μL, 2.65 mmol) and triethylamine (147 μL, 1.06 mmol) and the reaction mixture was heated to 90 °C for 1 h. After the completion of reaction, water (20 mL) was added to the reaction and the product obtained was extracted in EtOAc. The organic layer was washed with water (10 mL × 3) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product 25 as a white solid. The crude product was further washed with hexanes to remove excess 4-bromobut-1-yne, dried and used as it is for the next step. To a stirred solution of compound 25 (150 mg, 0.35 mmol) and 1-azidohexadecane (139 mg, 0.52 mmol) in THF (3 mL), were added CuSO4.5H2O (9 mg in 0.5 mL water, 0.035 mmol) and sodium ascorbate (14 mg in 0.5 mL water, 0.07 mmol) and the reaction mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added and the product obtained was extracted in EtOAc. The organic layer was washed with water (10 mL × 2) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product 26. MS (ESI-TOF) for C36H67N5O6S [M+H]+ Found 698.5075, Calculated 698.4885. The product 26 was used as it is for the next step. The global deprotection using TFA and further N-acetylation was carried out similarly as described earlier for synthesis of 22 to furnish compound 27 as white solid. 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 7.5 Hz, 1H), 7.38 (s, 1H), 6.93 (d, J = 7.2 Hz, 1H), 4.66 (td, J = 7.6, 4.5 Hz, 1H), 4.62 (dt, J = 7.3, 3.5 Hz, 1H), 4.29 (t, J = 7.3 Hz, 2H), 4.27 (bs, 1H), 4.02 (bs, 2H), 3.77 (s, 3H), 3.16 (ddd, J = 13.9, 7.7, 5.4 Hz, 1H), 3.09 (dd, J = 14.3, 4.4 Hz, 1H), 3.06 – 2.90 (m, 3H), 2.79 (dd, J = 14.3, 7.9 Hz, 1H), 2.06 (s, 3H), 1.92 – 1.82 (m, 2H), 1.30 (bs, 2H), 1.25 (bs, 20H), 0.87 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.61, 170.56, 170.36, 146.09, 121.88, 62.41, 55.56, 52.76, 52.68, 50.67, 35.02, 32.56, 32.07, 30.37, 29.85, 29.83, 29.80, 29.75, 29.68, 29.53, 29.51, 29.14, 26.64, 25.54, 23.37, 22.85, 14.29. MS (ESI-TOF) for C29H53N5O5S [M+H]+ Found 584.3889, Calculated 584.3840; [M+Na]+ Found 606.3692, Calculated 606.3660.</p><!><p>To a solution of compound 18 (100 mg, 0.237 mmol) in CH2Cl2 (1 mL) was added carbonyldiimidazole (58 mg, 0.355 mmol) and the reaction mixture was stirred at room temperature. Hexadecylamine (146 mg, 0.593 mmol) and DMF (1 mL) were added after 4 h and the reaction was kept stirring for 1 h. After the completion of reaction, water (10 mL) was added and the product obtained was extracted in CH2Cl2. The organic layer was washed with water (10 mL × 3) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product. The residue was further purified by a flash chromatography (5% MeOH/CH2Cl2 to yield product 29 (110 mg, 67%) as a white solid. MS (ESI-TOF) for C35H67N3O8S [M+H]+ Found 690.4659, Calculated 690.4722; [M+Na]+ Found 712.4478, Calculated 712.4541. The global deprotection of compound 29 using TFA resulted in compound 30 as a TFA salt in quantitative yield. 1H NMR (500 MHz, CDCl3) δ 8.53 (d, J = 7.6 Hz, 1H), 5.41 (t, J = 5.7 Hz, 1H), 4.72 – 4.63 (m, 1H), 4.36 (t, J = 6.5 Hz, 1H), 4.22 (ddd, J = 23.1, 11.5, 5.7 Hz, 2H), 3.89 (dt, J = 11.7, 7.2 Hz, 2H), 3.75 (s, 3H), 3.24 – 2.96 (m, 4H), 2.80 (t, J = 5.5 Hz, 2H), 1.46 (m, 2H), 1.25 (s, 26H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.45, 168.42, 156.76, 63.91, 61.89, 55.41, 52.94, 52.89, 41.32, 33.41, 32.08, 31.40, 29.94, 29.87, 29.86, 29.82, 29.77, 29.52, 26.97, 22.84, 14.28. MS (ESITOF) for C26H51N3O6S [M+H]+ Found 534.3524, Calculated 534.3571.</p><!><p>To a solution of compound 30 (56 mg, 0.087 mmol) in CH2Cl2 (1 mL) were added pyridine (8 μL, 0.1 mmol) and acetic anhydride (9 μL, 0.1 mmol). The reaction mixture was stirred at room temperature for 30 min and then concentrated. The residue was purified by a flash chromatography (5% MeOH/CH2Cl2) to yield product 31 (32 mg, 64%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 7.48 (d, J = 7.6 Hz, 1H), 6.66 (d, J = 7.1 Hz, 1H), 5.25 (t, J = 5.5 Hz, 1H), 4.67 (dd, J = 14.1, 7.1 Hz, 1H), 4.63 (dd, J = 7.5, 3.6 Hz, 1H), 4.32 (dt, J = 12.2, 6.2 Hz, 1H), 4.25 (dt, J = 11.7, 6.0 Hz, 1H), 3.97 (q, J = 11.4 Hz, 2H), 3.79 (s, 3H), 3.31 (bs, 1H), 3.15 (dd, J = 13.4, 6.8 Hz, 2H), 2.96 (qd, J = 14.1, 6.6 Hz, 2H), 2.89 – 2.78 (m, 2H), 2.05 (s, 3H), 1.52 – 1.44 (m, 2H), 1.34 – 1.20 (m, 26H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.83, 170.68, 170.63, 156.79, 64.06, 62.80, 55.23, 52.91, 52.78, 41.31, 34.86, 32.07, 31.87, 29.98, 29.85, 29.81, 29.76, 29.72, 29.51, 29.46, 26.94, 23.29, 22.84, 14.28. MS (ESI-TOF) for C28H53N3O7S [M+H]+ Found 576.3594, Calculated 576.3677; [M+Na]+ Found 598.3414, Calculated 598.3496.</p><!><p>To a solution of compound 20 (100 mg, 0.238 mmol) in dry CH2Cl2 (5 mL) were added triethylamine (50 μL, 0.356 mmol) and cetyl chloroformate (117 μL, 0.356 mmol) and the reaction mixture was stirred at room temperature for 30 min. After the completion of reaction, water (10 mL) was added to the reaction and the product obtained was extracted in CH2Cl2. The organic layer was washed with water (10 mL × 2) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (50% EtOAc/hexanes) to obtain compound 32 (120 mg, 74%). 1H NMR (500 MHz, CDCl3) δ 7.15 (d, J = 8.0 Hz, 1H), 5.47 (s, 1H), 5.26 (s, 1H), 4.66 (dt, J = 8.2, 3.1 Hz, 1H), 4.32 (s, 1H), 4.03 (t, J = 6.6 Hz, 2H), 3.82 (dd, J = 9.1, 3.0 Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.39 (d, J = 5.4 Hz, 2H), 2.97 (dd, J = 13.9, 5.4 Hz, 1H), 2.87 (dd, J = 13.8, 6.8 Hz, 1H), 2.80 – 2.65 (m, 2H), 1.63 – 1.54 (m, 2H), 1.45 (s, 9H), 1.33 – 1.23 (m, 26H), 1.14 (s, 9H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.68, 170.52, 156.92, 155.44, 80.44, 73.73, 65.28, 61.78, 53.79, 53.30, 52.60, 40.15, 34.50, 32.73, 32.06, 29.84, 29.82, 29.80, 29.75, 29.71, 29.50, 29.47, 29.16, 28.44, 27.42, 26.01, 22.83, 14.27. MS (ESI-TOF) for C35H67N3O8S [M+Na]+ Found 712.4461, Calculated 712.4547.</p><!><p>To compound 32 (110 mg, 0.159 mmol) was added TFA (2 mL). The reaction mixture was stirred at room temperature for 30 min and then dried by blowing nitrogen through the solution. The residue was purified by a flash chromatography (10% MeOH/CH2Cl2) to yield product 33 in quantitative yield. 1H NMR (500 MHz, DMSO) δ 8.96 (d, J = 7.8 Hz, 1H), 8.09 (s, 2H), 7.98 (t, J = 5.7 Hz, 1H), 5.26 (t, J = 5.4 Hz, 1H), 4.47 – 4.41 (m, 1H), 4.02 (dd, J = 8.9, 4.6 Hz, 1H), 3.79 (dt, J = 10.4, 5.1 Hz, 1H), 3.65 (s, 3H), 3.64 – 3.60 (m, 1H), 3.24 (tt, J = 13.7, 7.0 Hz, 2H), 3.03 (dd, J = 14.4, 4.5 Hz, 1H), 2.74 (dd, J = 14.4, 8.9 Hz, 1H), 2.65 (td, J = 6.6, 2.6 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.51 – 1.44 (m, 2H), 1.32 – 1.18 (m, 24H), 0.85 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 172.57, 170.35, 167.92, 61.06, 54.76, 52.13, 51.68, 38.12, 35.41, 32.82, 31.36, 31.32, 29.08, 29.04, 28.98, 28.84, 28.73, 28.70, 25.28, 22.12, 13.99. MS (ESI-TOF) for C26H51N3O6S [M+H]+ Found 534.3530, Calculated 534.3571; [M+Na]+ Found 556.3351, Calculated 556.3391.</p><!><p>To a solution of compound 33 (60 mg, 0.093 mmol) in dichloromethane (2 mL) was added pyridine (8 μL, 0.102 mmol) and acetic anhydride (10 μL, 0.102 mmol). The reaction mixture was stirred at room temperature for 30 min and then concentrated. The residue was purified by a flash chromatography (5% MeOH/CH2Cl2) to yield product 34. 1H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 7.4 Hz, 1H), 6.75 (d, J = 6.7 Hz, 1H), 5.20 (d, J = 5.2 Hz, 1H), 4.74 – 4.58 (m, 2H), 4.10 – 3.89 (m, 4H), 3.79 (s, 3H), 3.42 (dd, J = 12.5, 6.2 Hz, 3H), 2.94 (d, J = 6.1 Hz, 2H), 2.83 – 2.67 (m, 2H), 2.05 (s, 3H), 1.66 – 1.53 (m, 2H), 1.36 – 1.20 (m, 26H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.87, 170.66, 157.61, 65.69, 62.77, 55.23, 52.90, 52.58, 40.34, 34.48, 32.92, 32.07, 29.85, 29.84, 29.81, 29.75, 29.71, 29.51, 29.45, 29.09, 26.00, 23.27, 22.84, 14.28. MS (ESI-TOF) for C28H53N3O7S [M+H]+ Found 576.3602, Calculated 576.3677; [M+Na]+ Found 598.3416, Calculated 598.3496.</p><!><p>To a solution of compound 32 (100 mg, 0.145 mmol) in dichloroethane (2.5 mL) was added trimethyltin hydroxide (79 mg, 0.435 mmol) and the reaction mixture was heated to reflux for 6 h. After the completion of reaction, the solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (50% CH2Cl2/EtOAc) to obtain compound 35 (72 mg, 73%). 1H NMR (500 MHz, CDCl3) δ 7.23 (d, J = 6.8 Hz, 1H), 5.55 (d, J = 7.3 Hz, 1H), 5.28 (s, 1H), 4.70 – 4.60 (m, 1H), 4.34 (s, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.90 (dd, J = 9.0, 3.5 Hz, 1H), 3.57 (dd, J = 8.5, 4.9 Hz, 1H), 3.36 (dd, J = 16.2, 10.1 Hz, 2H), 2.93 (qd, J = 13.9, 6.2 Hz, 2H), 2.78 – 2.61 (m, 2H), 1.64 – 1.54 (m, 2H), 1.45 (s, 9H), 1.35 – 1.22 (m, 26H), 1.19 (s, 3H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 172.38, 171.01, 157.13, 155.65, 80.69, 74.63, 65.46, 61.26, 53.86, 52.90, 40.17, 34.31, 32.65, 32.07, 29.85, 29.81, 29.76, 29.72, 29.51, 29.48, 29.15, 28.44, 27.45, 26.01, 22.84, 14.28. MS (ESI-TOF) for C34H65N3O8S [M+Na]+ Found 698.4272, Calculated 698.4385.</p><!><p>To a solution of acid 35 (563 mg, 0.833 mmol) and N1,N1-dimethylpropane-1,3-diamine (115 μL, 0.916 mmol) in DMF (5 mL) were added triethylamine (232 μL, 1.67 mmol) and N-hydroxybenzotriazole (HOBt, 56 mg, 0.417 mmol). The reaction mixture was cooled to 0 °C and EDCI (258 mg, 1.67 mmol) was added after 30 min. The resulting mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added to the reaction and the product obtained was extracted in EtOAc. The organic layer was washed with water (10 mL × 3) and brine (10 mL), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (10% MeOH/CH2Cl2) to obtain compound 36 (530 mg, 84%). MS (ESITOF) for C39H77N5O7S [M+H]+ Found 760.5486, Calculated 760.5616.</p><!><p>To compound 36 (521 mg, 0.685 mmol) was added TFA (5 mL). The reaction mixture was stirred at room temperature for 30 min and then dried by blowing nitrogen through the solution. The crude product was used directly for the next step. To a solution of the crude intermediate 37 in CH2Cl2 (5 mL) were added pyridine (61 μL, 0.754 mmol) and acetic anhydride (71 μL, 0.754 mmol). The reaction mixture was stirred at room temperature for 30 min and then concentrated. The residue was purified by a flash chromatography (20% MeOH/CH2Cl2) to give product 38 as a white solid. 1H NMR (500 MHz, MeOD) δ 4.49 (dd, J = 8.0, 5.9 Hz, 1H), 4.28 (t, J = 5.0 Hz, 1H), 4.02 (t, J = 6.6 Hz, 2H), 3.88 (dd, J = 11.0, 5.1 Hz, 1H), 3.79 (dd, J = 11.0, 5.0 Hz, 1H), 3.40 – 3.31 (m, 4H), 3.17 – 3.10 (m, 2H), 3.01 (dd, J = 13.7, 5.7 Hz, 1H), 2.89 (s, 6H), 2.83 (dd, J = 13.7, 8.1 Hz, 1H), 2.68 (td, J = 13.5, 6.6 Hz, 2H), 2.03 (s, 3H), 1.93 (dt, J = 13.2, 6.4 Hz, 2H), 1.68 – 1.55 (m, 2H), 1.42 – 1.25 (m, 26H), 0.90 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 173.97, 173.33, 173.25, 159.35, 66.05, 62.44, 57.50, 56.40, 54.92, 43.59, 41.16, 36.65, 33.94, 33.09, 33.02, 30.80, 30.78, 30.72, 30.49, 30.44, 30.23, 27.00, 25.97, 23.75, 22.48, 14.45. MS (ESI-TOF) for C32H63N5O6S [M+H]+ Found 646.4426, Calculated 646.4572.</p><!><p>The induction of NF-κB in a human TLR2-specific reporter gene assay was quantified using HEK-Blue™ cells as previously described by us.30,36,52 Occupancy of TLR2 by cognate ligands leads to nuclear translocation of NF-κB in a MyD88-dependent manner,59–61 and consequent transactivation of the secreted alkaline phosphatase (seAP) reporter gene. Extracellular seAP in the supernatant is proportional to NF-κB induction, and was quantified spectrophotometrically.</p><!><p>Fresh human peripheral blood mononuclear cells (hPBMC) were isolated from human blood obtained by venipuncture with informed consent and as per institutional guidelines on Ficoll-Hypaque gradients as described elsewhere.62 Aliquots of PBMCs (105 cells in 100 μL/well) were stimulated for 12 h with graded concentrations of test compounds. Supernatants were isolated by centrifugation, and were assayed in duplicates for 41 chemokines and cytokines (EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1ra, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, PDGF-AA, PDGFAB/BB, RANTES, TGFα, TNF-α, TNF-β, VEGF, sCD40L) using a magnetic bead-based multiplexed assay kit (Milliplex MAP Human Cytokine/Chemokine kit). Data were acquired and processed on a MAGPIX instrument (EMD Millipore, Billerica, MA) with an intra-assay coefficients of variation ranging from 4–8% for the 41 analytes.</p><!><p>CD11b upregulation was determined by flow cytometry using protocols published by us previously,41 modified for rapid-throughput using an automated liquid-handling system. Briefly, heparin-anticoagulated whole blood samples were obtained by venipuncture from healthy human volunteers with informed consent and as per guidelines approved by the University of Kansas Human Subjects Experimentation Committee. Serial dilutions of 38 (as well as 2 and LPS, used as reference compounds) were performed using a Bio-Tek Precision 2000 XS liquid handler in sterile 96-well polypropylene plates, to which were added 100 μL aliquots of anticoagulated whole human blood. Negative (fRPMI) controls were included in each experiment. The plates were incubated at 37 °C for 1 h. Following incubation, 10 μL of each fluorochrome-conjugated antibody (CD11b-PE, CD14-APC, Becton-Dickinson Biosciences, San Jose, CA) were added to each well with a liquid handler, and incubated at 4 °C in the dark for 30 min. Following staining, erythrocytes were lysed and leukocytes fixed by mixing 200 μL of the samples in 2 mL prewarmed Whole Blood Lyse/Fix Buffer (Becton-Dickinson Biosciences, San Jose, CA) in 96 deep-well plates. After washing the cells twice at 200 g for 8 minutes in saline, the cells were transferred to a 96-well plate. Flow cytometry was performed using a BD FACSArray instrument with acquisition on 100,000 gated events. Granulocytes were gated by forward- and side-scatter, while monocytes were gated on the basis of CD14−APC staining.</p><!><p>All experiments were performed at Harlan Laboratories (Indianapolis, IN) in accordance with institutional guidelines (University of Kansas IACUC permit # 119-06). In experiments designed to evaluate the adjuvanticity of 12, cohorts of adult female New Zealand White rabbits were immunized intramuscularly in the flank region with either 100 μg of bovine α-lactalbumin in 0.2 mL saline (unadjuvanted control), or 100 μg of bovine α-lactalbumin plus 100 μg of 12 in 0.2 mL saline. The adjuvantic property of 38 (100 μg/dose) was evaluated using CRM197 as antigen (10 μg/dose). Pre-immune test-bleeds were first obtained via venipuncture of the marginal vein of the ear. Animals were immunized on Days 1, 15 and 28. A test bleed was performed on Day 25 and a final test-bleed was performed via the marginal vein of the ear on Day 38. Sera were stored at −80 °C until used. Antigen-specific ELISAs were performed in 384- well format using automated liquid handling methods as described by us.34,53 Safety evaluation in rabbit models included examination of clinical hematology parameters (total and differential leukocyte counts, enumeration of band-forms (immature neutrophils), hemoglobin, and mean corpuscular hemoglobin), as well as core-temperature monitoring. Cohorts of animals (n=3 per group) received either 38 (100 μg/dose), LPS (1 μg/dose, positive control) or vehicle (saline) formulated in sterile saline without any excipients in a volume of 0.5 mL, administered as an intravenous bolus in the marginal vein of the ear. Blood was obtained at 0h (pre-challenge), and at 2h and 6h via venipuncture on the contralateral ear. Core temperature was monitored by rectal thermistor probes connected to a temperature logging device as described by us.31</p>

PubMed Author Manuscript

Iron and Mechanisms of Emotional Behavior

Iron is required for appropriate behavioral organization. Iron deficiency results in poor brain myelination and impaired monoamine metabolism. Glutamate and GABA homeostasis is modified by changes in brain iron status. Such changes not only produce deficits in memory/learning capacity and motor skills, but also emotional and psychological problems. An accumulating body of evidence indicates that both energy metabolism and neurotransmitter homeostasis influence emotional behavior, and both functions are influenced by brain iron status. Like other neurobehavioral aspects, the influence of iron metabolism on mechanisms of emotional behavior are multifactorial: brain region-specific control of behavior, regulation of neurotransmitters and associated proteins, temporal and regional differences in iron requirements, oxidative stress responses to excess iron, sex differences in metabolism, and interactions between iron and other metals. To better understand the role that brain iron plays in emotional behavior and mental health, this review discusses the pathologies associated with anxiety and other emotional disorders with respect to body iron status.

iron_and_mechanisms_of_emotional_behavior

1. Introduction<!>2. Brain iron<!>3. Iron deficiency<!>4. Iron overload<!>5. Potential mechanisms underlying the influence of iron status on emotional behavior<!>5.1. Dopamine<!>5.2. Serotonin and norepinephrine<!>5.3. Glutamate<!>5.4. Gaba<!>5.5. Oxidative stress<!>6. Sex differences in iron metabolism and emotional behavior<!>7. Other metals<!>7.1. Zinc and selenium<!>7.2. Manganese and copper<!>7.3. Lead and mercury<!>8. Conclusions

<p>Iron is required for numerous vital functions, including oxygen transport, cellular respiration, immune function, nitric oxide metabolism and DNA synthesis [1]. Iron deficiency is the most prevalent single nutrient deficiency worldwide [1,2] and results in anemia, decreased immune function, retarded growth, and impaired thermoregulation [1,3]. The metal also plays a critical role in proper brain morphology, neurochemistry, and bioenergetics [4]. Poor brain myelination resulting from iron deficiency in early development has long-lasting effects on behavioral functions [5-9]. Iron is vital in neurochemical circuits including monoaminergic systems [10-20] and glutamate and γ-aminobutyric acid (GABA) homeostasis [21]. Energy metabolism is also altered by brain iron status [21]. For example, cytochrome c oxidase is reduced in prenatal iron deficiency, leading to impaired hippocampal metabolic function [22]. Iron is a cofactor for tyrosine hydroxylase and tryptophan hydroxylase, enzymes that are responsible for dopamine and serotonin synthesis, respectively. Monoamine oxidase activity is also lower in humans and rats with iron deficiency anemia [23-26]. Since monoamines and GABA are involved in the regulation of mood, neuronal activity and anxiety [26-28], it is reasonable to assume that emotional behaviors are strongly affected by brain iron levels, and especially by iron deficiency conditions. Since many studies have evaluated the role of iron in motor coordination and learning/memory function, the current review focuses on emotional behavior in the context of iron status and the potential underlying molecular mechanisms.</p><!><p>Brain iron concentrations are highest in the substantia nigra, globus pallidus, nucleus caudate, red nucleus and putamen [29]. The rapid accumulation of iron in these areas is required for the development of the brain and may significantly contribute to behavioral organization [29]. Within the brain, iron is particularly concentrated in the basal ganglia, an area highly influenced by dopamine and GABA metabolism [26-28]. Therefore, the functions of this brain region are very susceptible to changes in iron status. Not only is the distribution of iron region-specific, the sensitivity of various brain regions to iron deficiency differs during different stages of neurodevelopment [30,31]; during the mid and late neonatal periods in rodents (equivalent to human ages 6-12 months), iron content significantly decreases by 25% in the cortex, striatum and hindbrain after a short period of feeding a low-iron diet, whereas the thalamus shows only a 5% reduction [1]. During postweaning iron deficiency, the thalamus becomes more sensitive to dietary iron, suggesting that there is a prioritization of brain iron distribution during development [1].</p><!><p>Given the susceptibility of the developing brain along with the huge prevalence of iron deficiency, it is important to understand the role of iron in behavioral and mental health. There is strong evidence that iron deficiency causes developmental delays in young children [32]. Iron deficiency is also associated with cognitive alterations in adolescents [33]. Although the condition of iron deficiency can be later corrected by supplementation, behavioral alterations persist [10]. Iron-deficient children have increased anxiety and/or depression with social and attentional problems [34]. An accumulating body of evidence has demonstrated that iron deficiency is closely associated with altered brain homeostasis in both myelination and neurotransmission, especially monoamine metabolism [1]. Studies using rodent models of iron deficiency have also revealed a strong relationship between behavioral abnormalities and altered dopamine metabolism in the striatum [10,35]. Finally, early iron deficiency can also influence the glutamatergic system and energy metabolism [22,36,37].</p><p>Human studies link anxiety-driven behavior to poor iron status. For example, increased fearfulness is found in anemic infants despite iron therapy [38]. Infants with marginal iron deficiency also have increased fearfulness [39]. However, mixed results have been reported for behavior in iron-deficient rats; anxiety is elevated [40] or unchanged in adult rats that were formerly iron-deficient [41]. Beard et al [40] examined anxiety-related behaviors in a light/dark box study of iron-deficient rats at 6-wk of age. This group found that iron-deficient rats moved four times more rapidly into the dark compartment, but the time spent in the dark compared with the light compartment was not different from that of control rats. The iron-deficient rats also spent less time in the center of in open field box, indicating increased fearfulness and anxiety [40]. The authors argue that the young rat is a model organism for behavioral studies to explore the influence of iron deficiency on brain function, particularly given the similarity of behaviors observed in iron-deficient children [38,42]. When examined by elevated plus maze, young male rats with dietary iron deficiency demonstrate reduced time in and entry to the open arms [20], supporting the idea that iron deficiency increases anxiety.</p><p>Ultrasonic vocalization also has been used to test anxiety in rat pups born to iron-deficient or control dams [41]. Maternal iron deficiency was associated with more distress calls, consistent with the idea that postnatal iron deficiency induces anxiety-like behaviors [40]. When supplemented with dietary iron on PND 10, the formerly iron-deficient rats did not show anxious responses on PND 99, suggesting that iron repletion can normalize anxiety-like behavior [41]. This observation contrasts with findings that early iron deficiency during the pre-weaning period followed by iron repletion results in persistent hypoactivity in an open field on PND 21 or PND 49 [17,40,43]. Eseh and Zimmerman offer potential explanations [41]. First, these differences may be explained by inadequate time for compensatory neural processes. Second, it is possible that different neural systems (e.g., dopamine and GABA) have different requirements or timing for plasticity. Finally, it must be recognized that results obtained using different methods, different levels of dietary iron, and at different developmental time periods will be inherently difficult to reconcile. For example, a decrease in stereotypic movements induced by postnatal iron deficiency was reversed by 4 weeks of iron repletion in one study [17], but not completely in another [44].</p><p>It is generally thought that the critical time window for altered emotional behaviors due to iron deficiency may be later than gestation or within the first 10 days of life in rats, equivalent in brain development to a full human gestation [41]. Neural damage during gestation may recover, whereas continued iron deficiency past a critical point could produce a lack of effect on the recuperative or compensatory process needed to reverse anxiogenic effects [41]. It is notable that iron-deficient rats given iron therapy at PND 10 still weigh significantly less than the controls on both PND 55 and PND 75 [41]. Whether the small size of the low iron group is due to decreased food intake or altered metabolism (or both) has yet to be fully examined. Ultimately, it is critical to keep in mind that the period of development during which nutritional deficiency occurs can later produce both behavioral and physiological consequences [45] – whether iron repletion can reverse the behavioral influence of iron deficiency and the mechanism(s) underlying restoration to normalcy remain important questions to be studied [46].</p><!><p>Excess iron in the brain is implicated in the development and pathogenesis of neurodegenerative disorders [47-51]. Iron levels in the brain increase with age [52-54]; this has been shown to occur mainly in brain regions that are affected in the disease states, including Alzheimer's, Parkinson's, and Huntington's diseases [54]. Increased iron levels promote the generation of reactive oxygen species, leading to cellular and tissue damage [55,56]. With respect to emotional behaviors, iron overload appears to alter anxiety-like behavior and mood [57-59]. Anxious responses, determined by the elevated plus maze, are observed in adult rats receiving daily intraperitoneal injections of iron [59]. Other behavioral impairments have been found in rats fed carbonyl iron diet containing 20,000 ppm iron [57]. These findings support the idea that imbalanced iron metabolism plays a pivotal role in modulating anxiety and emotional behaviors.</p><p>Neuronal damage due to iron overload may be incurred during the postnatal period and aging because the rate of iron influx into the brain is increased in both early and late stages of life [60-62]. Iron overload has been clearly shown to disrupt neurotransmitter homeostasis. Iron infusions into the substantia nigra perturb monoaminergic systems, especially the dopaminergic pathway, to promote impaired motor function resembling Parkinson's disease [63-65]. The effects of iron overload on learning and memory deficits are also well documented during postnatal development in mice [66-70]. However, there are only a few studies that have characterized the influence of iron loading on emotional behavior. In humans, iron supplementation in anemic women has been reported to alter emotional processes such as anxiety or depression [58]. Intraperitoneal injection of iron affects the emotional behavior of Wistar rats [59]; in the elevated plus maze, these iron-loaded rats spent more time in the closed arms and entered the open arms less frequently than controls, indicating that iron-treated rats display elevated anxiety. In addition, the total entries into the closed arms and activity in the maze were significantly reduced in iron overload rats compared with controls, reflecting a reduced activity and exploratory drive. Moreover, iron-loaded rats have a lower locomotion and reared less frequently in the open field [59]. These results demonstrate increased anxiety/emotional reactivity upon iron overload, which is similar to the behavioral phenotype of iron deficiency [10,19,40,71]. Since activity or exploration drive are also affected by iron overload during postnatal development [68], behavioral methods that are more anxiety-specific and less exploration/activity-dependent (e.g., conditioned place avoidance, social interaction or taste aversion tests) have been suggested to more reliably examine the effect of iron overload on anxiety [59].</p><p>The influence of iron loading on emotional behavior is dose-dependent. In contrast to intranigral iron dose of 3.0 mg/kg body weight, rats intranigrally injected with 1.5 mg iron/kg did not show a significant difference in behavioral functions compared with control rats [59]. It can be speculated that the organism, particularly the adults, can compensate for a small dose of iron supplementation [59]. Other studies reported similar results [57,68]. Sobotka et al [57] demonstrated a significant accumulation of iron in the brain only at the highest dose of iron overload (i.e., 20,000 ppm in diet for 12 weeks) but not at lower doses (350 and 3,500 ppm). A mechanism that protects the brain against iron overload until a certain point, and that cannot compensate when the load exceeds this limit, has been proposed [59].</p><!><p>Despite a large body of evidence about iron's effect on behavioral functions from experiments using iron-deficient rodent models and observations from iron-deficient infants and children, the molecular information about the role of iron in emotional behavior is scarce. A correlation study has found interesting relationships among iron, dopaminergic system and anxiety-like behavior. Nosepokes and rate of habituation are associated with prefrontal cortical iron concentrations, whereas spontaneous activities, including locomotion and repeated movements, are better correlated with iron levels and dopamine receptor density in the ventral midbrain [40]. The regression analysis also revealed that iron levels in the ventral midbrain and prefrontal cortex are important for anxiety-like behaviors. It should be noted that other potential mechanisms could be involved in iron deficiency-related emotional behaviors and that other neurotransmitters could play significant roles in these behaviors. For example, iron deficiency alters serotoninergic [72] and GABAergic functions [73]. It is also possible that these effects are not due to direct effect by iron but may be a consequence of other factors such as metal interactions. These mechanisms are discussed below and represented in Figure 1.</p><!><p>A large body of evidence has indicated that impaired emotional behaviors are associated with iron deficiency through altered dopamine metabolism [14,20,74,75]. In general, there is a universal negative effect of iron deficiency on dopamine functions [11,76,77], which are specific to brain region and the stage of neural development.</p><p>Dopamine receptor 1 (D1R) expression is down-regulated in the caudate putamen and prefrontal cortex of iron-deficient rats [78]. In contrast, Beard et al [40] did not observe any effect of iron deficiency on D1R in both brain areas, possibly due to different specificity of ligands for D1R. Iron deficiency decreases the density of D2R in rat striatum [78] and prefrontal cortex as measured by radioactive tracer binding [40]. Western blot analysis, however, revealed no difference in D2R in the prefrontal cortex between iron-deficient and control rats [20]. While this discrepancy may reflect different approaches used to detect D2R, the region-specific response of iron deficiency to dopamine receptor density could also be due to different sensitivity to dietary iron deficiency [30,31]. Another possibility is the differential expression of receptor subtypes in different brain regions along with less specific ligands. For example, the ratio of D4R to D2/3R is greater in the prefrontal cortex than in the striatum [79]. Hence the greater binding of ligand with poor subtype specificity could result in different interpretations.</p><p>Since D2R autoreceptor regulates dopamine clearance in the synaptic cleft via dopamine transporter (DAT) [80,81], the negative effect of iron deficiency on D2R could result in decreased DAT activity and increased striatal dopamine [80]. A positive correlation between D2R density and DAT density appears to support this possibility [78]. It has been proposed that iron deficiency-related "desensitization" could occur such that more dopamine is needed to stimulate DAT [78]. Iron deficiency decreases DAT density in the striatum and nucleus accumbens [16]. Effects of cocaine on DAT are reduced upon iron deficiency, implying changes in both DAT density and functioning [16]. In contrast, DAT levels are unchanged in the prefrontal cortex upon iron deficiency [20]. These observations may reflect brain region-specific regulation of the transporter by iron and/or different methods of measurement [20].</p><p>Iron is a cofactor for tyrosine hydroxylase, a critical enzyme in dopamine production; whether its activity is specifically affected in the prefrontal cortex and striatum during iron deficiency should be better explored [82]. Extracellular dopamine is elevated in the caudate putamen and nucleus accumbens upon iron deficiency [14-16,75], most likely due to decreased DAT density [15], and returns to normal levels when brain iron levels are corrected [15,75]. In contrast, extracellular concentrations of dopamine in the prefrontal cortex, determined from microdialysis samples, are lower in iron-deficient rats compared with control rats, whereas the amphetamine-evoked response is similar between iron-deficient and control rats, suggesting that the reduced basal dopamine in the prefrontal cortex could be reflected by elevated anxiety upon iron deficiency [20]. Alternatively, the amount of neurotransmitter available for evoked release may be limiting in the prefrontal cortex of iron-deficient rats [20].</p><!><p>Alterations in serotonin signaling might also be responsible for emotional behaviors since it has an important role in mediating affective behaviors. However, conflicting results exist on the effects of iron deficiency on serotonin levels in rats. Iron deficiency may decrease levels of serotonin due to a down-regulation of synthetic enzymes in juveniles [13]. On the other hand, serotonin levels are elevated upon iron deficiency in adults, possibly reflecting a down-regulation of serotonin metabolism [83]. Serotonin transporter densities are reportedly reduced in the striatum of iron-deficient mice [72]. Likewise, moderate and severe gestational iron deficiency reduces serotonin uptake by brain synaptic vesicles in offspring, and this effect can be normalized with 4 weeks of iron repletion [84]. In other studies, however, iron deficiency had no effect on serotonin levels or metabolism in newborns or adults [12] and serotonin levels in the prefrontal cortex of the iron-deficient rats did not differ from controls [20].</p><p>Extracellular norepinephrine (NE) concentrations are elevated under iron deficiency states whereas tissue levels are unchanged compared with controls [43,76,77]. Bianco et al found increased caudate NE concentration and further demonstrated that the activity of dopamine-β-hydroxylase, the enzyme to produce NE from dopamine, is elevated by 75% in caudate homogenates from iron-deficient rats [85]. This evidence suggests a shift to increase NE production upon brain iron deficiency, possibly compensating for altered dopamine response [85]. In addition, the brain NE transporter is down-regulated in iron deficiency [18]. The observations in serotonin and NE homeostasis are not necessarily conflicting but may hint at underlying mechanisms of the iron-monoamine relationship since the distribution of neurotransmitters between intracellular and extracellular pools appears to be the primary site of influence of brain iron deficiency [1].</p><p>Ceruloplasmin (Cp) is a ferroxidase that converts Fe2+ to Fe3+ and contributes to cellular iron efflux. Texel et al have demonstrated that Cp-deficient mice exhibit increased iron deposition in the liver and spleen, whereas brain iron is reduced, especially in the hippocampus, which is accompanied by increased anxiety-like behavior with no changes in motor function or learning and memory [86]. This increased anxiety is associated with elevated levels of plasma corticosterone and decreased levels of serotonin and NE, as well as brain-derived neurotrophic factor (BDNF) and its receptor [86]. Altered hippocampal BDNF signaling is linked to changes in serotonin and NE levels. BDNF promotes the survival and differentiation of serotonin neurons [87], while NE increases BDNF and receptor activation [88]. Reduced BDNF and increased corticosterone are associated with increased anxiety [89,90]. Also, mice with hippocampus-specific deletion of BDNF display anxious behavior [91]. Tran et al have shown that BDNF is reduced in iron-deficient rats [92,93]. Since iron is a cofactor for the rate-limiting enzymes involved in serotonin and NE synthesis, decreased iron in the hippocampus due to Cp deficiency could result in impaired production of these monoamines and down-regulate BDNF signaling, which together promote an anxiety phenotype. These results indicate that redox properties of iron could contribute to emotional behavior by altering monoamine metabolism and BDNF homeostasis [86].</p><!><p>There are few studies about the role of iron and glutamate/GABA concerning emotional behavior. Iron deficiency in both prenatal and postnatal periods is associated with decreased activities of glutamate decarboxylase, glutamate dehydrogenase and GABA transaminase [73,94]. Brain glutamate levels are elevated in iron-deficient rats [37], suggesting either increased synthesis or decreased release from the neurons. Since excitatory glutamatergic neurotransmission accounts for >80% of the total energy expenditure in the brain [95], Rao et al postulated that glutamate-mediated neurotransmission is decreased due to inefficient energy metabolism in perinatal iron deficiency, leading to high intracellular glutamate levels [37]. Furthermore, glutamate binding to synaptic membranes is reduced in iron-deficient brains [96]. This evidence supports the idea that emotional responses are attenuation of glutamatergic signaling under low iron conditions.</p><!><p>Altered GABA metabolism is associated with decreased iron concentrations [27,28,37,73,77,94,96]. Iron deficiency results in elevated concentrations of GABA in several brain regions, including hippocampus, striatum and globus pallidus [37,97]. The elevated GABA levels suggest an increased inhibitory drive for reducing the overall neurotransmission rates and brain activity due to insufficient energy [37]. In addition, iron deficiency enhances GABA binding to synaptic membranes [96]. Activities of GABA shunt enzymes (e.g., glutamate dehydrogenase and GABA transaminase) are reduced in rats made iron-deficient during pregnancy and lactation, and this effect is not restored by iron repletion of dams [94]. Notably, these enzymes are also decreased in postweaning iron deficiency, but this loss can be corrected upon iron supplementation [98,99].</p><!><p>Iron overload enhances reactive oxygen species [55], and iron-treated rats show increased iron in brain regions involved in emotional processes, including the frontal cortex, basal ganglia, hippocampus, and cerebellum [59]. To account for the effects of iron exposure on behavior, Maaroufi et al [59] proposed the hypothesis that increased oxidative stress due to iron overload impairs monoamine functions. Administration of a small amount of iron for only a few days (3.0 mg/kg for 5 consecutive days) promotes iron accumulation in the substantia nigra of an adult brain, where iron may generate cytotoxic free radicals. Such oxidative stress can impair dopaminergic signaling and monoamine function, consequently leading to the behavioral impairment [100,101]. However, while several studies have shown an association between brain iron loading and oxidative stress in neurodegenerative disorders [47-51], the exact role of iron overload in emotional behavior remains to be determined.</p><!><p>There is a significant influence of sex on anxiety behavior in adulthood; females are less anxious than males based on elevated plus maze results [41]. Other studies reached similar conclusions [102,103]. Estrogen effects on dopaminergic function in the context of iron deficiency have been reported [78], possibly by transcriptional regulation of DAT and receptors [19]. Estrogen also modifies D2R expression [104,105]. Sex differences also exist in spatial memory performance [106].</p><p>Latency to startle response is attenuated in female, but not in male, iron-deficient rats [19]. The expression of monoamine transporters and D1R is also different between male and female [18,72]. In males, iron-deficient rats have lower DAT levels in several brain regions, including caudate putamen and nucleus accumbens. However, female rats do not show a difference in DAT levels between iron deficiency and control diets. D1R is another example: while iron-deficient rats show a significant reduction in D1R in nucleus accumbens and substantia nigra, female rats increase D1R compared with control of the respective gender [19]. Overall, it appears that male mice are more sensitive to the effect of iron deficiency than are female mice [72]. Studies using rats also showed a similar pattern [19]. Finally, it has been noted that brain iron levels are also dependent upon gender and iron diet [43].</p><!><p>Although iron has been most studied in emotional behavior and neurotransmitter metabolism, abnormal levels of other metals, whether essential or not, also significantly contribute to mental disorders and here we briefly discuss several metals.</p><!><p>As an essential metal, zinc plays an important role in brain function and energy metabolism. The metal is involved in controlling emotional behavior, and mood disorders and depression are associated with reduced zinc concentrations in serum [107] while zinc supplementation improves anger and depression [108]. Likewise, zinc-deficient rats display anxiety-like behavior [109] and zinc treatment provides anxiolytic activity in rodents [110]. The role of zinc in cognitive and emotional behavior mediated by glutamate and glucocorticoid signaling under stressful circumstances has been reviewed [111]. Selenium, a metalloid mineral, provides anti-oxidant activity to protect the body from oxidative stress. Human studies show that selenium supplementation improves mood [112-114].</p><!><p>Manganese is required for several critical enzymes, including superoxide dismutase, glutamine synthetase and arginase [115,116]. When over-deposited in the brain, however, manganese promotes neurotoxicity characterized by memory loss, impaired motor coordination and psychotic behavior resembling Parkinson's disease [117-119]. Impaired dopamine and cholinergic systems participate in manganese-mediated psychological disorders [120-122]. Similarly, abnormal tissue accumulation of copper, observed in Wilson's disease, is also associated with impaired emotional behavior and neurological problems [123,124].</p><!><p>Heavy metals have long been recognized as neurotoxicants. Emotional problems associated with lead exposures in neonates [125] or adults [126] affect mental health, although not always [127]. Social and emotional dysfunction in children correlate with pre- and postnatal lead exposures [128]. Studies in mice show lead-associated behavioral effects could be mediated, at least in part, by increased corticosterone levels [129]. Other possibilities include disruption of monoamine metabolism in the basal ganglia [130] and formation of hydroxyl radicals [131].</p><p>Behavioral deficits are reported in rats upon perinatal methylmercury exposures [132]. In humans, chronic subtoxic levels of inorganic mercury produced heightened distress, anxiety and psychoticism without alterations in general intellectual functioning and motor skills [133]. In studies using zebrafish, Maximino et al have proposed that oxidative stress induced by methylmercury produces mitochondrial dysfunction and inhibits tryptophan hydroxylase, thereby altering serotoninergic systems [134].</p><!><p>A strong body of evidence demonstrates altered metabolism of iron and other metals modifies emotional behaviors. Conversely, people with psychological disorders appear to have reduced iron status; for example, serum iron levels are lower in schizophrenics than in controls [135]. Subjects with major depression have lower hematocrit and serum transferrin [136]. Interestingly, these patients also display lower zinc levels [137]. Moreover, the amount of selenium in the diet is inversely associated with reports of anxiety, depression, and tiredness, which are improved by selenium supplementation [113]. These findings suggest a possibility that individuals with mood symptoms may have insufficient vitamins and minerals [113] and therefore may need more micronutrient supplementation than healthy subjects [138].</p><p>Effects of iron in emotional behavior are determined by many physiological/biological properties and spatial/temporal factors; these include intracellular and extracellular concentrations of neurotransmitters, brain iron levels, different brain regions (e.g., density and affinity of neurotransmitter receptors/transporters/enzymes), regulation of these molecules, iron exposure period and timing, route of exposure, animal species, sex, nutritional status and disease state. Different methods of behavioral measurements and the influence of other metals produce different behavioral outcomes. More systematic and controlled studies are warranted to better understand the underlying mechanism of iron-associated emotional behavior and mental health. It is necessary to improve our understanding of the pathologies associated with anxiety and other psychiatric disorders to develop therapies to alleviate emotional dysfunction.</p>

PubMed Author Manuscript

Supertetrahedral polyanionic network in the first lithium phosphidoindate Li3InP2 – structural similarity to Li2SiP2 and Li2GeP2 and dissimilarity to Li3AlP2 and Li3GaP2†

Phosphide-based materials have been investigated as promising candidates for solid electrolytes, among which the recently reported Li9AlP4 displays an ionic conductivity of 3 mS cm−1. While the phases Li–Al–P and Li–Ga–P have already been investigated, no ternary indium-based phosphide has been reported up to now. Here, we describe the synthesis and characterization of the first lithium phosphidoindate Li3InP2, which is easily accessible via ball milling of the elements and subsequent annealing. Li3InP2 crystallizes in the tetragonal space group I41/acd with lattice parameters of a = 12.0007(2) and c = 23.917(5) Å, featuring a supertetrahedral polyanionic framework of interconnected InP4 tetrahedra. All lithium atoms occupy tetrahedral voids with no partial occupation. Remarkably, Li3InP2 is not isotypic to the previously reported homologues Li3AlP2 and Li3GaP2, which both crystallize in the space group Cmce and feature 2D layers of connected tetrahedra but no supertetrahedral framework. DFT computations support the observed stability of Li3InP2. A detailed geometrical analysis leads to a more general insight into the structural factors governing lithium ion mobility in phosphide-based materials: in the non-ionic conducting Li3InP2 the Li ions exclusively occupy tetrahedral voids in the distorted close packing of P atoms, whereas partially filled octahedral voids are present in the moderate ionic conductors Li2SiP2 and Li2GeP2.

supertetrahedral_polyanionic_network_in_the_first_lithium_phosphidoindate_li3inp2_–_structural_simil

Introduction<!>Synthesis and structure of Li3InP2<!>MAS-NMR spectroscopy<!>Comparison of Li3InP2 with the lighter homologues Li3AlP2 and Li3GaP2<!>Comparison of Li3InP2 with the phosphidotetrelates Li2SiP2 and Li2GeP2<!>Impedance spectroscopy<!>Conclusions<!>Author contributions<!>Conflicts of interest

<p>All-solid-state batteries (ASSB) have recently become the focus of research as an attractive alternative to state-of-the-art liquid-based batteries due to their enhanced safety combined with high energy/power density and mechanical stability.1–7 One of the main obstacles for the commercialization of ASSBs is the difficulty to develop superionic solid conductors, which are crucial for fast ionic diffusion in ASSBs. Recently, our group investigated new classes of lithium ion conductors based on phosphides. Starting with Li8SiP4 in 2016, we introduced phosphidosilicates with an ionic conductivity of 4.5 × 10−5 S cm−1.8 Lately, in the Li-richer compound Li14SiP6 the conductivity was even higher with up to 1 × 10−3 S cm−1.9 Furthermore, we extended the system to the heavier tetrel (group-14) homologues, phosphidogermanates, with two Li-ion conducting modifications of Li8GeP4 that show ionic conductivities of up to 8.6 × 10−5 S cm−1 and with Li14GeP6, which achieves an ionic conductivity of 1.7 × 10−3 S cm−1.10,11 The structural building units in these phosphides are [TtP4]8− tetrahedra surrounded by lithium atoms (Tt = Si, Ge). They exhibit a huge structural variety, and by decreasing the amount of lithium, condensed and covalently connected tetrahedra are formed, thereby offering different polyanionic networks: Li10Si2P6 features pairs of edge-sharing SiP4 tetrahedra,12 in Li2SiP2/Li2GeP2 and LiSi2P3, respectively, SiP4 and GeP4 tetrahedra are condensed to networks of supertetrahedra.8,13,14 Layered structures have been reported as well: in Li3Si3P7, vertex-sharing SiP4 tetrahedra form double layers,12 and LiGe3P3 is built up by a two dimensionally extended polyanion comprising GeP4 and Ge(P3Ge) tetrahedra.13</p><p>Phosphide-based materials as lithium ionic conductors originated from the aliovalent substitution of [TtS4]4− tetrahedra, which are the main building block in sulfide-based conductors. This leads to analogous structures with more negatively charged [TtP4]8− tetrahedra, which can therefore accommodate more lithium than the well-known sulfur-based analogues. In recent investigations we expanded this class of compound further to phosphidoaluminates, which contain tetrahedral AlP4 building units, and we discovered the fast lithium ion conductor Li9AlP4, which shows ionic conductivities of 3 × 10−3 S cm−1.15 In addition, we also obtained Li3AlP2, which is built up by layers of corner- and edge-sharing AlP4 tetrahedra,16 and we then also introduced the isotypic gallium compound Li3GaP2 as the first phosphidogallate.16 Both trielate (Tr = Al, Ga) compounds do not show moderate lithium ion conductivity but unexpectedly turned out to be direct band gap semiconductors with optical band gaps of 3.1 and 2.8 eV, respectively.</p><p>Prior to the present work, no ternary Li–In–P phase has been described in the literature, and only one ternary Na–In–P phase was mentioned: Na3InP2 is built up by a distorted hcp of P atoms with all octahedral voids filled by Na, whereas the tetrahedral voids are occupied by Na and In, giving rise to a polyanionic network of corner-sharing InP4 tetrahedra.17</p><p>In the present work, we report the first lithium phosphidoindate, Li3InP2, synthesized via ball milling of the elements and subsequent annealing. The compound retains the principal structural building unit of TrP4 tetrahedra, but their arrangement is notably different from that of Li3AlP2 and Li3GaP2. In Li3InP2, the InP4 tetrahedra are condensed to supertetrahedra in a three-dimensional framework. The structure is determined by single crystal X-ray diffraction and analyzed by complementary solid-state NMR experiments and first-principles computations. The knowledge of the lithium ion mobilities of the now-completed series of phosphidotrielates allows us to suggest a structural design rule linked to ionic conductivity, namely, the presence (or absence) of partially occupied Li-containing octahedral sites between which the ions can move rapidly.</p><!><p>Li3InP2 was synthesized from the elements via a two-step procedure. At first, stoichiometric amounts of lithium, indium and phosphorus were ball milled resulting in a reactive mixture. Besides small amounts of the desired phase, Li3InP2, the polycrystalline powder contains considerable amounts of InP and Li0.3In1.7 (see Fig. S4†). Subsequently, pellets of the mixture were annealed in sealed niobium ampules at 1023 K for 22 h. Afterwards, the ampoules were rapidly cooled to room temperature by quenching in an ice-water mixture yielding almost phase-pure Li3InP2 with 3.3(1) % Li0.3In1.7 as an impurity according to Rietveld analysis (Fig. S3†). Annealing at lower temperatures such as 673 K or slow cooling rates led to impurities such as InP. Powdered Li3InP2 is brick-red. Complete data of the Rietveld refinement are given in the ESI; Tables S5 and S6.†</p><p>Red single crystals of Li3InP2 were obtained after reacting the elements with the formal stoichiometry "Li3In2P3" at 1073 K in tantalum ampoules. Besides Li3InP2, the resulting product contains InP and at least one more, so far unknown phase according to unassigned reflections in the powder X-ray diffractogram (see Fig. S5†). Details of the structure refinement of the single crystal X-ray diffraction data of Li3InP2 are listed in the ESI in Tables S1–S4.†</p><p>According to the single crystal structure determination, Li3InP2 crystallizes in the tetragonal space group I41/acd (no. 142) with seven independent crystallographic positions (one for In, three each for Li and P; Table S2†). Considering that the crystal structure is based on a tetragonally distorted cubic close packing of phosphorus atoms, the multiplicity of the phosphorus Wyckoff positions (32g + 16e + 16e) leads to a total of 128 tetrahedral voids and 64 octahedral voids. One quarter of these tetrahedral voids is filled by the indium atoms (Wyckoff position 32g). The remaining 96 tetrahedral voids are occupied by lithium (3 × 32g). Hence, the tetrahedral voids are fully occupied, whereas all octahedral voids are empty. The unit cell determined by single crystal X-ray diffraction is displayed in Fig. 1a.</p><p>Indium and phosphorus form InP4 tetrahedra, and four corner-sharing InP4 tetrahedra build a T2-supertetrahedron. These T2-supertetrahedra are interconnected via corners, yielding two independent adamantane-like networks, which are shown in red and blue colors in Fig. 1a and c.</p><p>The In and P atoms are covalently connected to four and two atoms, respectively, resulting in a formal negative charge for both In and P of (−1). Since the P atoms at the corner of the supertetrahedron are shared with the next supertetrahedron, one such unit can be written as [In4P6P4/2]12− (Fig. 1b), which leads to an electronically balanced formula Li3InP2 (≡(Li+)12[In4P6P4/2]12− or Li12In4P8).</p><p>The InP4 units slightly deviate from an ideal tetrahedron with P–In–P angles ranging from 107.20(1) to 111.55(1)°. The bond lengths within the InP4 tetrahedra are in the narrow range between 2.5676(5) and 2.5899(5) Å and are very similar to those in compounds with strong In–P interactions like InP (2.5412(1) Å)20 and Na3InP2 (2.592(3)–2.682(3) Å)17 and in excellent agreement with DFT computations after full structural optimization (2.57–2.58 Å). The Li–P bonds in Li3InP2 range from 2.526(2) to 2.673(2) Å and are in good agreement with those in other binary or ternary phases containing Li and P.8–10,12,13,15 DFT optimization yields 2.51–2.67 Å, again practically superimposable with the experimental results.</p><p>Considering each center of gravity of the supertetrahedra, the arrangement of the independent networks of the T2-supertetrahedra corresponds in a hierarchical relationship to the arrangement of the Cu and Fe cations in the chalcopyrite structure, which is highlighted in Fig. 1d. The concept of supertetrahedra is already known in the literature, including supertetrahedral sulfides,21,22 which show structures with huge cavities, and also phosphidosilicates.8,23</p><!><p>6Li and 31P MAS-NMR measurements (Fig. 2) support the results of the crystal structure determination. The 6Li NMR spectrum shows only one signal with a chemical shift of 3.85 ppm. As expected, the NMR experiment cannot distinguish between the three crystallographically different lithium atoms, all of which are tetrahedrally coordinated by phosphorus in a very similar chemical environment. The chemical shift of the Li atoms is in the same range as those for related compounds like Li9AlP4 (4.2 ppm), Li3AlP2 (4.0 and 3.0 ppm), Li3GaP2 (4.1 and 3.4 ppm), Li2SiP2 (2.1 ppm from 7Li MAS-NMR spectroscopy), and Li2GeP2 (3.6 and 2.4 ppm).8,13,15,16 Compared to the above-mentioned compounds with two signals in the 6Li NMR spectrum, the difference in local coordination, which is expressed by the P–Li–P angles, is the lowest for Li3InP2 (Li3InP2: 104.99(8)–113.25(8)°, Li3AlP2: 100.0(3)–116.647(1)°, Li3GaP2: 102.258(1)–115.2(3)°, Li2GeP2: 84.68(1)–158.89(2)°). The 31P NMR spectrum displays a very broad, asymmetric signal in the range of −260 to −360 ppm. This range is typical for chemical shifts of two-fold connected P1− atoms such as in Li3AlP2 (−300 and −308.7 ppm) or Li3GaP2 (−234.8 and −280.5 ppm).16 However, the signals of two-fold connected P1− atoms in the related phosphidotetrelates are much more downfield shifted (Li2SiP2: −129.1 and −241.5 ppm and Li2GeP2: −59.9, −164.8 and −178.4 ppm) due to the deshielding of the more electronegative tetrel elements compared to indium.8,13</p><p>Interestingly, only one 31P NMR signal is observed for Li3InP2, whereas two signals are obtained for all other related compounds. This correlates with the fact that the smallest distortion of the E–P–E bond angles is observed for Li3InP2 [106.411(9)–111.41(1)°] if compared to the others with E = Al, Ga, In, Si, Ge such as Li3AlP2 [78.298(1)–111.709(1)°], Li3GaP2 [79.943(1)–110.253(1)°], Li2SiP2 [102.669(9)–114.937(9)°], and Li2GeP2 [101.726(7)–112.609(7)°].</p><!><p>Recently, we described the two isotypic phases Li3AlP2 and Li3GaP2,16 which crystallize in a distorted orthorhombic packing of phosphorus atoms in the space group Cmce (no. 64) with lattice parameters a = 11.5138(2), b = 11.7634(2), c = 5.8202(1) Å and a = 11.5839(2), b = 11.7809(2), c = 5.8129(2) Å, respectively, both determined by Rietveld refinement at room temperature. The crystal structures are built up by corner- and edge-sharing TrP4 (Tr = Al, Ga) tetrahedra in two-dimensional layers. Based on a close packing of P atoms, the lithium atoms are located in all tetrahedral voids (Fig. 3). By contrast, Li3InP2 crystallizes in a tetragonal distorted phosphorus lattice in the space group I41/acd (no. 142) with lattice parameters of a = 12.03049(8) and c = 23.9641(3) Å, determined by Rietveld refinement at room temperature, and as mentioned above, the single crystal structure determination reveals a three-dimensional structure with exclusively corner-sharing InP4 tetrahedra for t-Li3InP2 (Fig. 1).</p><p>In order to gain additional insight into the experimentally observed structure types, we performed DFT-based structural optimizations for the Al, Ga and In compounds using the PBEsol functional24 as implemented in CASTEP25 (computational details are given in the ESI†). In addition to the experimentally determined unit cells we performed a substitutional "cross-check": both modifications, orthorhombic o-Li3TrP2 and tetragonal t-Li3TrP2, were used for Tr = Al, Ga and In, starting either from the experimentally determined structure or from a hypothetical one obtained by substituting the Tr species. The DFT-optimized cell parameters are in excellent agreement with the experiment for the title compound (we obtained aDFT = 11.96 Å and cDFT = 23.74 Å); full results are listed in Table S7.†Fig. 4a shows the resulting energies, relative to the respective binary phosphides similar in spirit to our recent work on Li9AlP4.15 We compute the DFT electronic energy, E, for the relaxed ternary structure as well as for Li3P and the respective zinc blende-type phase of AlP, GaP or InP; the difference (in the sense of a "reaction energy") then allows us to estimate the stability of the ternary phase:ΔE = E(Li3TrP2) − [E(Li3P) + E(TrP)]</p><p>Negative values of ΔE therefore indicate that the ternary phase is stable with respect to the binaries (Fig. 4a).</p><p>The compounds Li3TrP2 are energetically favored over their respective binary components Li3P and AlP, GaP and InP. The latter all adapt the cubic zinc blende type. The energy gain is significant considering the known stability of the zinc blende type that is most frequent among III–V semiconductors. More importantly, the difference in pairs of ΔE values allows us to compare the tendency for assuming either the Cmce or the I41/acd structure for all of the Li3TrP2 phases. For the Al and Ga compounds, the Cmce structure is favored by about 0.06 and 0.03 eV per formula unit (f.u.), respectively; by contrast, the I41/acd structure is preferred for Li3InP2 (by about 0.06 eV f.u.−1), all in agreement with experiments. The stabilization of the title compound compared to the constituent binary phosphides is computed to be 0.31 eV f.u.−1 (indicated by a negative sign in the convention of Fig. 4a), which represents a significant gain in stability and explains the synthetic accessibility of the ternary compound. Whilst there will always remain a certain error due to the DFT approximation and the neglect of thermal effects, we do trust that the computed trends shown in Fig. 4a are robust, and we note that they are fully consistent with the experimental observations.</p><p>As expected, the unit cell volume for the heavier homologues increases, however the In compound shows a much stronger increase: 788.30 Å3 for Al and 793.28 Å3 for Ga if compared to 867.10 Å3 (=3468.39 Å3:4) for In. This correlates with a larger increase of the size of the InP4 tetrahedron (8.8857 Å3) compared to AlP4 (7.0897 Å3) and GaP4 (7.1334 Å3).</p><p>The trends of the interatomic Tr–Tr (Tr = Al, Ga, In) distances in Li3AlP2, Li3GaP2 and Li3InP2 are listed in Table 1. Regarding the different orthorhombic (Li3AlP2, Li3GaP2) and tetragonal structures (Li3InP2), the interatomic distances of the metal atoms are shorter in the orthorhombic structures, where edge-sharing tetrahedra occur compared to the tetragonal structure, where only corner-sharing tetrahedra are present. One may ask for the origin of the preference of one structure type over the other when comparing all three phosphidotrielates side-by-side. Interestingly, the results of the calculations are in agreement with Pauling's third rule. At least qualitatively and within the limits of such empirical concepts,26 edge-sharing tetrahedra are disfavored on account of the repulsion of positively charged central atoms (Fig. 4b and c). This effect might be expected to be strongest in the In compound, where not only the ionic radius is the largest of the three, but the computed Mulliken charges for the series of Cmce structures (Al: +0.42e, Ga: +0.57e, hypothetical In structure: +0.65e) appear to be consistent with an increasing repulsion of Tr atoms in the case of edge-sharing tetrahedra. Note that the Mulliken charges, derived from quantum-mechanical computation, are not to the same as the formal negative charge of the Tr atom using the Lewis valence model (Fig. 1b). Accordingly, a structure containing edge-sharing tetrahedra is observed for Li3AlP2 and Li3GaP2, but not for Li3InP2 (Fig. 4c). This trend of the differences of the different metal to metal distances by DFT calculation is confirmed by the experimental interatomic Tr–Tr (Tr = Al, Ga, In) distances (Table 1). The experimental In–In distance is significantly longer than the Al–Al or Ga–Ga distances (4.116(3) Å (In) vs. 3.028(5) Å (Al) and 3.089(2) Å (Ga)).</p><!><p>The crystal structure of Li3InP2 is related to the structure of Li2SiP2 and Li2GeP2.8,13 The two latter isotypic phases also crystallize in the space group I41/acd (no. 142), with lattice parameters of a = 12.1111(1) and c = 18.6299(4) Å for Li2SiP2 and a = 12.3070(1) and c = 19.0307(4) Å for Li2GeP2 and a slightly longer a, but much shorter c parameter as compared to Li3InP2. A full comparison of the lattice parameters and the tetrahedral volumes in Li3InP2, Li2SiP2 and Li2GeP2 is given in Table 2.</p><p>Assuming an average volume of 18 Å3 per heavy atom, the increase in cell volume corresponds approximately to the volume of 32 additional lithium atoms in the unit cell of Li2SiP2. Besides the change in the number of Li atoms, also the larger volume of the InP4 tetrahedra compared to SiP4/GeP4 (see Table 2) contributes to an overall increase of the volume. However, this increase is highly anisotropic, since in Li3InP2 the lattice parameter c increases strongly, whereas the lattice parameter a is even slightly shorter compared to the one in Li2SiP2 and Li2GeP2.</p><p>Fig. 5 shows a comparison of the structures of Li3InP2 and Li2SiP2 viewed along the a and c direction. In Li3InP2 the InP4 tetrahedra respectively the T2-supertetrahedra are aligned in an almost parallel fashion, whereas in Li2SiP2 the T2-supertetrahedra are rotated along the tetragonal axes. Interestingly, the parallel alignment in Li3InP2 leads to a slight decrease of the a and b axes despite the higher lithium content, but to a significant increase of the c axes.</p><p>In Table 3 the Wyckoff positions in Li3InP2 and Li2SiP2 are compared (Li2GeP2 is omitted since it is isotypic to Li2SiP2). The higher Li content of the In compound arises from the occupation of two 32g Wyckoff sites instead of two 16f sites in the tetrelates. As a consequence, the coordination environments of the lithium atoms in the structures are different. The coordination of the lithium atoms in Li3InP2 and Li2SiP2 by phosphorus is illustrated in Fig. S2 and S8,† respectively. The positions Li1 and Li3 are similarly coordinated by four phosphorus atoms forming a distorted tetrahedron. By contrast, Li2 fills a strongly distorted octahedral void of phosphorus atoms with significant longer Li–P distances compared to Li1 and Li3. Here, the lithium atom Li2 is not located in the center of gravity of the octahedron but shows two much longer distances to neighboring P atoms of the distorted octahedron, resulting in a butterfly-type coordination of four P atoms. Interestingly, despite the smaller amount of Li atoms in Li2SiP2, not all the tetrahedral voids are occupied. In both compounds 25% of the tetrahedral voids are occupied by In or Si. Whereas all of the remaining 75% tetrahedral voids in Li3InP2 are filled with Li, only 37.5% are occupied by Li in Li2SiP2. In the latter, however, Li atoms occupy 25% of the distorted octahedral voids.</p><p>The different occupation of voids in Li3InP2 and Li2SiP2 also results in a different coordination of the supertetrahedra by lithium, which is shown in Fig. 6.</p><p>The different Li coordination arises from the different charges of the supertetrahedra Si4P88−/Ge4P88− and In4P812− (Fig. 1b). In Li3InP2 the lithium atoms form an almost regular octahedron around the indium atom with In–Li distances in the narrow range of 3.041 to 3.131 Å with an average of 3.075 Å, whereas in Li2SiP2 the octahedron formed by lithium atoms around silicon is strongly distorted with longer average distances of 3.222 Å and values between 2.958 and 3.556 Å. As a consequence, also octahedral voids of P atoms are filled with Li ions in Li2SiP2.</p><!><p>For Li3InP2 two impedance measurements were performed to determine the ionic conductivity. The results are shown in Fig. S10.† The semi-circle can be described as parallel circuit element of a resistor and a constant phase element (R/Q). For the constant phase element the fit of the data acquired at 298 K resulted in α values of ≈0.99 and Q parameters of ≈2 × 10−8 F s(α − 1); the conductivity was determined to σ(Li3InP2) = 2.8(2) × 10−9 S cm−1 at 298 K (obtained from two independently measured cells). DC polarization measurements in the range from 50 to 150 mV reveal an electronic conductivity of 2.7(3) × 10−9 S cm−1 at 298 K (based on the standard deviation of two cells). The conductivity value obtained by DC polarization measurements is in the same range as the value obtained by PEIS measurements. Hence, the Nyquist plot shows only the semi-circle of the electronic conductivity, and no semi-circle for the ionic conductivity appears.</p><!><p>Li3InP2 is the first lithium phosphidoindate and can be described as a tetragonally distorted fcc lattice of P atoms (space group I41/acd), in which the In atoms occupy tetrahedral voids, thus forming a polyanionic framework of InP4 supertetrahedra. The lithium atoms occupy the remaining tetrahedral voids. The structure of the compound is not isotypic to the previously reported ones of the lighter homologues, the orthorhombic compounds Li3AlP2 and Li3GaP2 (space group Cmce), which feature 2D layers of connected tetrahedra. First-principles DFT computations confirm the trend for the Al and Ga (In) compounds to crystallize in the orthorhombic (tetragonal) structure, respectively, which might originate in the different repulsive cation⋯cation interactions in both structures. Impedance spectroscopy reveals a very low electronic, but no ionic conductivity, whereas Li2SiP2 and Li2GeP2 show a moderate ionic mobility (2.2(3) × 10−7 S cm−1 at 293 K and 1.5(3) × 10−7 S cm−1 at 300 K, respectively).8,13 The geometrical analysis of the Li positions shows that in Li3InP2 all tetrahedral voids are fully occupied by lithium, whereas in Li2SiP2 and Li2GeP2 tetrahedral voids remain empty, and especially strongly distorted octahedral voids are filled. In accordance with the observations in fcc phosphide-based lithium ion conductors such as Li9AlP4,15 lithium diffusion preferably appears on pathways via partially occupied octahedral sites.</p><p>Overall, these results demonstrate that even though crystal structures of phosphide compounds can contain complex polyanionic networks, a relatively simple description in terms of distorted close-packed arrangements of phosphorus atoms gives better insight for the description of lithium ion mobility. The title compound Li3InP2 provides a missing link in two respects: (i) it shows the structure changes in the series Li3TrP2 for Tr = Al, Ga, In, and (ii) it shows changes in lithium ion mobility in the series Li3InP2, Li2SiP2 and Li2GeP2.</p><!><p>TMFR carried out the crystal structure determination by single crystal and powder X-ray diffraction, performed the impedance spectroscopy measurements and wrote the manuscript draft. VLD carried out the DFT computations and provided discussion. JM contributed to the synthesis and data evaluation. GRS performed NMR experiments. TF designed research, provided guidance, and critically reviewed the manuscript.</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Generating Non-Linear Concentration Gradients in Microfluidic Devices for Cell Studies

We describe a microfluidic device for generating non-linear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for co-flowing multiple aqueous solutions, a main co-flow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the co-flow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H2O2) concentrations.

generating_non-linear_concentration_gradients_in_microfluidic_devices_for_cell_studies

INTRODUCTION<!>Gradient formation<!>Numerical calculation<!><!>Cell culture and cytotoxicity test<!>Gradient formation<!>Cell culture and cytotoxicity test<!>CONCLUSION<!>

<p>Microscale technologies have proved to be a powerful tool for minimizing reagent volumes and reaction times in many biological and chemical applications.1 Microfluidic methods are particularly well compatible with biological materials such as proteins and cells and allow researchers to precisely control the cellular environment in culture and to miniaturize assays for high-throughput applications. This is especially true for device materials2, 3 such as poly(dimethylsiloxane) (PDMS) and room temperature vulcanizing (RTV) silicones, which are commonly used to fabricate active and passive fluidic channels and storage-reaction chambers. Standard applications include protein crystallization4–6, nanoliter-volume PCR7, microfabricated fluorescence activated cell sorting (uFACS)8, 9, single-cell enzyme screening10 and cell-based screening applications 11–14.</p><p>In the context of biological analysis, generation of (non-linear) chemical gradients and efficient mixing of the components within the integrated system is crucial for testing and analyzing biological responses to different analyte concentration levels15–18. The native microenvironment across cell membranes19, 20 and for cellular responses during growth, differentiation and proliferation21, for example, rarely includes linear concentration gradients. This has been shown in various cell studies including chemotaxis of breast cancer cells22 that could not be induced by linear concentration gradient of the epidermal growth factor (EGF). Nonlinear gradients of EGF, however, showed marked chemotaxis of breast cancer cells. The role of non-linear23, mainly exponential concentration gradients has also been studied in the context of cell proliferation24, and ligand binding25, and other cellular responses26. Hence, a platform that can generate nonlinear concentration gradients, and specifically exponential gradients, is required.</p><p>The reliability of these concentration gradients directly determines the experimental outcome.27, 28 Concentration gradients for biological and chemical applications can be generated by diffusion, convection, and by adjusting the resistance of fluidic channels.8, 28–30 A fundamental challenge in generating gradient profiles in microfluidic devices, in particular non-linear gradients, is establishing good spatial and temporal control over multi-component laminar flows with different solute concentrations. At the same time, it is desirable to maintain a small device footprint, to avoid external elements such as pressure controllers and eliminate fluidic channels solely dedicated to gradient generation, for example on-chip mixers and multiplexers. The need for handling such individual or multiple gradients has enabled the development of large-scale integrated microfluidic devices10, 31, resulting in computer-controlled, programmable chips capable of parallel analysis and high-throughput screening of optimal experimental conditions32–34. In particular, acquiring the ability to rapidly and reliably generate concentration gradients in simple device structures may provide a useful tool in screening the complexity of biological microenvironment for stem cell and tissue engineering research1, 35–37. In this paper we describe a robust microfluidic design for creating nonlinear (exponential and sigmoidal) concentration gradients in high-throughput, well-containing microfluidic networks for biological analysis. Furthermore, we demonstrate the capabilities of the device for cell based experimentation.</p><p>Popular microfluidic gradient designs include the tree-like structure, originally developed by Jeon et al28, the discrete gradient achieved by mixing of solutions via a multiplexer and mixer10, and nonlinear gradient structures like the logarithmic structure described by Kim et al8. Both the tree-like and multiplexer designs include a series of fluidic channels dedicated to gradient formation, which minimizes the space available for analyte storage. Our device structure, in contrast, requires little space, as it does not rely on dedicated mixing channels, and only a single dual-port syringe pump as external support, such that the majority of the chip space can be used for performing experiments. The simplicity of this device contributes to its robustness, as there are virtually no microfluidic elements that can fail. Furthermore, unlike the logarithmic design, our device can generate multiple non-linear gradients. In our structure, two fluids containing different concentrations of various chemicals are injected into the main fluidic channel separately, via a single syringe pump. The fluid branches off into several side channels that lead to an asymmetrical channel grid, which serves as a storage region. The grid feeds 512 storage wells placed in regular intervals along the branches (Figure 1a), so that the wells are filled with different mixtures of the two aqueous streams. As the solutions reach the side channels branching from the inlet channel, more and more of the main flow is removed, such that the interface between the two solutions inside the main channel repositions toward the grid (Figure 1b,c). For example, one branch may contain 90% clear solution and only 10% dye, and another branch may contain 50% clear solution and 50% dye etc. These mixtures then feed directly into the storage wells. In other words, when the two co-flowing streams reach the first branch, the total flow in the main channel is reduced, leading to a redistribution of the aqueous streams inside the channel. Therefore, if the ratio of the input flow rates is 1:1 inside the co-flow channel, and the first branch carries for example 25% of the total flow, then the ratio of the two solutions after this branch will change to 1:2. However, the total flow rate along all flow paths from the input to the exit (i.e. along any branch) is equal, as the path length and therefore resistance is constant (Figure 1e). More specifically, in the inlet or gradient generation region, the asymmetric channel geometry and the specific position of each branch combined with diffusion in the main channel lead to the formation of a one-dimensional concentration gradient in the side branches. An example is shown in the fluorescence image in Figure 1b. The corresponding data (dye concentration profiles measured along the red lines in Figure 1b) is plotted in Figure 1c.</p><p>A similar design for generating concentration gradients was developed by Holden et al.38 In their work, the two aqueous solutions were co-flowed at low flow rates (up to 30 μl/hr) to induce mixing via diffusion as they were distributed into an array of narrow channels (without subsequent redistribution of flows inside the main channel), such that each channel carried a solution of a different final concentration. Our gradient generator is also based on this principle, with some differences in the geometrical features of the design. Furthermore, the gradient generator is merged with a microwell array that enables the use of the generated concentration gradients for performing high throughput cell studies. In addition, Holden et al38 sampled a different experimental range, in that their flow rates were one to two orders of magnitude smaller, which enhanced mixing via diffusion and thus resulted in non-linear concentration gradients with different fit parameters.</p><p>As stated above, the main driving factor in our device is diffusion of the two aqueous solutions. In this paper we characterize the gradient generator by modifying this diffusion effect in two ways: a) by controlling the flow rates and b) using species of different diffusion constants. Finally, we demonstrate the usefulness of the device in analyzing cell viability upon exposure to various concentrations of a toxin.</p><!><p>The microfluidic device consists of two PDMS (Sylgard 184, Dow Corning) layers and is fabricated according to a standard soft lithography protocol (ESI). A schematic of the device structure is shown in Figure 1a. Two input channels are provided for the two solutions to be mixed; the solutions then combine and co-flow in a main fluidic channel (200 μm wide and 15 μm tall) before they enter the channel grid and subsequently fill the storage wells. The storage region houses 512 circular wells (200 μm height, 150 μm radius), placed at 800 μm intervals along a branch and at 1600 μm and 2200 μm intervals in the direction of the main flow, and it occupies an area of 1.25 cm × 1.1 cm. Grid channels are 100 μm wide and 15 μm deep with a rounded profile. There are 40 channels that branch off the inlet channel. In our experiments and simulations, however, we gather our measurements from wells placed along sixteen of these branches that are 2d and 2.75d apart, as the storage wells are ultimately the feature of the device we are most interested in. From here on, we number these channels sequentially from 1 to 16 (where 1 is closest to the input and 16 is closest to the output). We note that in this version of the device the storage wells are connected not only via the long side branches, but also via short, perpendicular channels (1d in length). These channels are currently passive, i.e. they are not actively used and do not contribute to the gradient generation. They are designed, however, to serve as part of a future perfusion system that will allow for long-term cell culture.</p><p>To establish an exponential concentration gradient we rely on a volume-driven system (dual Harvard PhD 2000 syringe pump). We demonstrated the repeatability of the device and tested the effect of different input flow rates by using clear PBS solution and fluorescein sodium dye (Aldrich) solution (1 mg/ml in PBS, phosphate buffered saline, Invitrogen) as the two co-flowing aqueous solutions. The flow rates of both aqueous streams were equal in all experiments. Fluorescence images were collected on a microscope (Nikon TE2000-U, 2x achromat objective) and analyzed by using ImageJ software (rsbweb.nih.gov). The device was primed by filling it completely with PBS solution prior to the introduction of the dye. In all experiments, the clear solution entered the device through the input on the left (close to the channel grid) and the fluorescent dye was pumped through the opposite input (far from the channel grid). To study the fluid flow rates and stability, the width of each aqueous stream in the input channel was visualized for least one hour. When studying the effect of the flow rate on the resulting concentration gradient, we applied equal flow rates of 10, 50, 100 and 200 μl/hr to both aqueous streams. In assessing the reproducibility of the experiments, we conducted each experiment three times. The time to generate and stabilize the gradient was longest at 10 μl/hr (10 minutes) and shorter at higher flow rates. We measured the intensity of the fluorescence signal in each well and normalized it by comparing it to the fluorescence at the input channel (100% fluorescent dye solution), then averaged our results from the three sets of experiments. Last, we fit an exponential or sigmoidal curve to each averaged data set and determined the function parameters using the Microcal Origin plotting and data analysis software.</p><p>We also studied the effect of diffusion constant on the resulting concentration gradient. We compared the fluorescein sodium dye experiment at 100 μl/hr/stream with concentration gradients of two fluorescently labelled Dextran compounds, FITC-Dextran 10kDa (Sigma) and FITC-Dextran 70kDa (Sigma).</p><!><p>To support our experimental data we performed a full scale simulation of our experimental setup. This is a 2D simulation in the x–y plane. Since the gradient generation is independent of time (aside from a short stabilization period), the governing equations can be written as follows: (1)(u→·∇)u→=−∇p+1Re∇2u→ (2)u→·∇φ=1Pe∇2φ (3)∇·u→=0, where u→ is the velocity vector, p is pressure, φ is analyte concentration density, Re and Pe represent Reynolds and Peclet numbers, respectively. Main parameters are d, the characteristic length (width of the main co-flow channel, 100 μm), v, kinematic viscosity of the suspension medium (10−6 m2/s for water at room temperature) and D, mass diffusivity of the analyte (4.9 · 10−10 m2/s for fluorescein sodium dye39). Another factor is U, the average velocity in the main flow channel (used in the simulation as reference velocity). For 10 μl/hr (and fluorescein sodium dye), U = 0.000926 m/s; for 50 μl/hr, U = 0.00463 m/s; for 100 μl/hr, U = 0.00926 m/s; and for 200 μl/hr, U =0.0185 m/s. The other diffusion constants are on the order of 2.9 · 10−11 m2/s for FITC-Dextran 10kDa40 and 2.3 · 10−11 m2/s for FITC-Dextran 70kDa41.</p><p>The flow field of our device was determined by solving steady-state Navier-Stokes equations with the continuity equations expressed in Eq. (1) and (3). Analyte concentration was obtained by solving the convection-diffusion equation as shown in Eq. (2). In the flow field calculation, fixed velocity (see above) and pressure conditions were imposed at the inlet and outlet, respectively. To find the analyte concentration, the normalized concentration φ was set to 1 at the dye inlet and 0 at the clear solution inlet. At the outlet we used the Neumann boundary condition, that is the normal derivative of the concentration was set to 0. To evaluate the concentration distribution, a commercial solver (CFD-ACE+) was employed. The device geometry and dimensions used in the numerical calculation replicate precisely the experimental conditions.</p><p>The applied boundary conditions are as follows:</p><!><p>Inlet boundary condition un1=u0un2=u0φ1=0φ2=1</p><p>where un1 and un2 are magnitude of the normal velocities at inlet 1 and 2, respectively. u0 is equal to Q/A, where Q is flow rate and A is channel cross section area. φ1 and φ2 are normalized analyte concentration at inlet 1 and 2, respectively. These inlet boundary conditions show that the analyte is introduced only through inlet 2 and analyte-free, clear solution is introduced through inlet 1.</p><p>Wall boundary condition u=0D∂φ∂n|wall=0</p><p>where ∂∂nn is the normal derivative and u is the velocity vector. We imposed a no-slip boundary condition for the flow velocity field and the zero normal derivative condition for the analyte concentration (Neumann boundary condition), that is, the normal derivative of the analyte concentration at the wall is set to zero. The analyte flux N can be written as N=φu−D∇φ</p><p>and the normal flux of the analyte at the wall can be expressed as n·N=φu·n−n·D∇φ=0(∵u=0,n·D∇ϕ=D∂ϕ∂n=0)</p><p>where N is the analyte flux vector and n is the unit normal vector at the wall. Therefore, normal flux of analyte at the wall is zero, that is, a zero flux boundary condition is imposed.</p><p>Outlet boundary condition Pstatic=0D∂φ∂n|outlet=0</p><p>We imposed a constant pressure condition for the flow field and zero normal derivative of analyte concentration at the outlet (Neumann boundary condition). At the outlet, however, the normal flux of the analyte can be expressed as n·N=φu·n−n·D∇φ=φu·n(∵n·D∇ϕ=D∂ϕ∂n=0)</p><p>that is, a convective normal flux condition was imposed.</p><!><p>Cell experiments were conducted on NIH-3T3 mouse fibroblasts cultured in high glucose-Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin/streptomycin (Gibco, USA). Cells were grown in a humidified incubator at 37° C and supplemented with 5% CO2 for 3 days, then trypsinized with 0.25% trypsin-EDTA (Gibco, USA) and harvested. Finally, cells were resuspended in 1 ml medium after centrifuging.</p><p>To sterilize the device, we pumped a 70% ethanol solution through the channels for 1 hour, then rinsed it with phosphate buffered saline (PBS, Invitrogen, USA) for three hours and finally filled the device completely with culture medium. The temperature of 37° C was maintained for the duration of the experiment. Cells were loaded into the device at a concentration of 2 × 106 cells/ml using a hand-held 1 ml plastic syringe (Becton-Dickinson). Once the cells were stored in wells at a low density (up to 50 cells per well), resulting in a monolayer of cells at the bottom of each well, a concentration gradient (at 50 μl/hr per stream) between 10 mM hydrogen peroxide (H2O2) solution with DMEM and pure PBS was established as described above and maintained for 60 minutes at constant flow. Hydrogen peroxide is known to affect the viability of various cell types, and at high concentrations can lead to necrosis in 3T3 cells.42–44 After 60 minutes we gently disconnected the tubing containing hydrogel peroxide and PBS from the device to avoid disturbing the cells and plugged one of these two holes with a melted piece of tubing. There was no washing step. To determine the viability of 3T3 cells exposed to the gradient, we immediately stained the cells for 15 minutes inside the incubator with the Live/Dead Viability/Cytotoxicity Kit (Invitrogen, USA), calcein AM for live cells and ethidium homodimer (EthD-1) for dead cells. The excitation/emission wavelengths for calcein and ethidium homodimer are 494/517 nm and 528/617 nm, respectively. We introduced the kit using a hand-held Becton-Dickinson plastic syringe and then waited 15 minutes for the cells to be stained. We obtained fluorescence images of the cells (4x objective), in which red fluorescence indicates dead cells, while green fluorescence indicates live cells. We counted the number of red and green fluorescent cells in all occupied wells on each side channel, normalized it against the total number of cells and calculated the average fraction of dead and live cells. In a control experiment cells were immersed in PBS and loaded into the device. Their viability was recorded immediately after loading. The details of the experiment were the same as described above, except that no hydrogen peroxide was used.</p><!><p>Following the procedure described above we formed a concentration gradient of fluorescent dye on-chip. We observed a one-dimensional non-linear gradient of dye concentration across the device parallel to the main flow channel (Figure 2). We also observed a shallow concentration gradient along each branch, that is, normal to the main flow channel, but the difference between the lowest and highest concentration value in a branch was on average 0.05 and decreased with increasing flow rate. This variation may be due to the short orthogonally placed channels within the grid that connect two wells and that were originally designed for cell perfusion purposes, as described above (Figure 1a). In addition, we conducted all experiments three times (n=3) and plot here the averages of those measurements. The three concentration measurements differ from each other by at most 5%, which we contribute to experimental error (intrinsic variability in the flow rate applied by the syringe pump). Since we are interested in non-linear concentration gradients, a 5% difference in concentration measurement is acceptable, thus we consider our results robust and reproducible within our range of interest.</p><p>In both the experimental and simulation results (Figure 2), the higher the input flow rates, the steeper the initial slope of the exponential fit curve. This is because at high flow rates the two aqueous streams did not have sufficient time to mix completely via diffusion before the flow branches off into the side channels. Similarly, the maximum concentration of dye in channels close to the output (branch number 16) was proportional to the flow rate. At low flow rates, for example, the two streams were mixed well before they reached the last side channel, such that this last branch did not contain pure dye, but rather a mixture of clear solution and dye (e.g. 70% dye concentration and 30% clear solution). In contrast, at high flow rates, the two solutions did not mix significantly by the time they reached the last branch (well number 16), such that almost pure dye (95%) flowed through that branch.</p><p>This led to a special case at high flow rates (200 μl/hr per stream). Here, the concentration of dye did not increase exponentially, but approximated a sigmoidal curve in which branches 1–4 contained almost the same amount of dye. Branches 15 and 16 (and in some cases, branches 12 to 16) also had equal, albeit much higher dye concentrations. In branches 5–14 (or 5–11) the concentration of dye appeared to increase linearly (Figure 2b). A similar behavior was also evident in the simulation results (Figure 2c). Because of the large width of the linear region, this device has the potential to be used not only for generation of exponential and sigmoidal, but may also be used to generate linear concentration gradients. This observation can be explained by considering the extreme case, an extremely fast flow rate, in which the effect of diffusion is negligible. In this case the two solutions would flow much faster than they could mix via diffusion, such that the concentration gradient would be a sigmoidal function with a large slope, approximating a step function.</p><p>The effect of diffusion constant on the gradient generation is shown in Figure 3. Our experiments showed that as expected, the larger the radius and thus the molecular weight of the diffusing species, the slower the mixing via diffusion, and thus the steeper the initial slope of the exponential concentration gradient. We fit two types of non-linear curves to the experimental data: An exponential curve of the form</p><p> y=y0+Ae(−x/B) was fit to low flow rate data (10, 50, and 100 μl/hr/stream), and a sigmoidal Boltzman function,</p><p> y=[A1−A2/(1+e(x−x0)/dx)]+A2 was fit to high flow rate data (200 μl/hr/stream) collected from fluorescein sodium salt solution. (In case of FITC-Dextran solutions, most fits were sigmoidal.) In both equations the variable x indicates the branch number. The goodness of fit was determined visually – the low flow rate data (below 200 μl/hr/stream) is approximated better with an exponential decay curve, and high flow rate data is approximated better with a sigmoidal Boltzmann curve. Supplementary Table S1a contains the exponential fit parameters for experiments conducted with clear PBS solution and fluorescein sodium salt at flow rates ranging from 10 μl/hr to 200 μl/hr per stream and for the corresponding numerical simulations. The numerical results match our experimental results qualitatively, and the characteristic exponential and sigmoidal fit parameters are also comparable. Supplementary Table S1b contains the exponential fit parameters for experiments comparing the effect of different diffusion constants. Interestingly, there was a similarity between increasing the flow rate and lowering the diffusion constant. In both cases the defining parameter changed accordingly. In exponential fits to our experimental data, we view the initial slope A as the defining parameter that describes the concentration gradient: The higher the flow rate and the lower the diffusion constant, the larger A. With sigmoidal fits, we view the width of the saddle region dx as the defining factor: the higher the diffusion constant and the higher the flow rate, the smaller dx (see ESI, Table S1).</p><!><p>In the cell experiment, we first seeded 3T3 fibroblast cells in the microwells. To achieve uniform cell seeding across all 512 storage wells in the device, the cells were introduced separately into each branch containing wells, by punching an additional input and an exit hole into each of those branches. The exit hole of any branch was only open during the cell loading at that particular branch. After cells were loaded into a particular branch, the respective access holes were plugged with melted tubing and not reopened, so that they did not affect the concentration gradient formation.</p><p>The seeded cells were on average 15 μm in diameter, roughly the same diameter as the channel height. The percentage of filled wells was measured to be ~ 93% with between 3 and 50 cells per well (the average number of cells per well was 12).</p><p>In most wells the cells were organized in a monolayer and not in clusters, allowing us to count them accurately using phase-contrast images. When in addition cell clusters were present we only counted the cells that were in focus at the bottom of the well, even if they were part of a cluster. After loading the cells, we established a H2O2 concentration gradient by simultaneously supplying PBS and hydrogen peroxide to the channel grid at 50 μl/hr for the duration of the experiment. We inspected the occupied wells visually before, during and after the introduction of the gradient and did not observe any movement of the cells which had settled on the bottom of the 200 μm deep wells; these cells appeared to be undisturbed by the applied flow rate. Other cells, however, that had attached to the sides of the wells and were closer to the main flow channel were displaced by this flow. Forthcoming work from our laboratory analyzes the effect of shear and well depth on the stability of cell storage in this device.</p><p>The applied toxin gradient resulted in the highest fraction of dead cells in the top branch (1), and the highest fraction of live cells on the opposite end of the device (branch 16) (Figure 4a,b). Representative wells from each branch are shown in Figure 4b. We counted the number of dead (red) and live (green) cells in each well by using fluorescence images. Because of the large variation in the number of cells per well, we added the numbers of cells in sets of four wells each and treated those sets as individual populations, such that the number of cells in most sets varied between 20 and 70, with a statistically more reliable average number of 46. In other words, instead of 32 independent wells with an average of 12 cells we counted 8 sets of 4 wells each with an average of 46 cells. We normalized the number of live and dead cells in each set by comparing them to the total number of cells in that set to find the fraction of live and dead cells. The numbers were then averaged across the 8 sets on each branch and plotted (Figure 4c). The fraction of live cells increased non-linearly with branch number, confirming qualitatively our previous experimental and simulation results. Similarly, the fraction of dead cells decreased non-linearly with branch number.</p><p>We note that some cells fluoresced at both red and green wavelengths. Those cells were originally healthy, thus fluorescing green, but were damaged with prolonged exposure to the toxin, thus starting to fluoresce red. Whether we labelled these bi-signalling cells as live or dead, the resulting curves were always non-linear (exponential), but their fit parameters differed. As this group of cells was already damaged and was thus expected to die, we chose, however, to count them as dead cells.</p><p>The diffusion constant of hydrogen peroxide can be estimated at 10−9 m2/s45, 46 for a small molecule. We can infer from the 50 μl/hr data of FITC-300Da, which differs only by a factor of ½, that the H2O2 concentration at branch 16 should be 2.5mM. The percentage of dead cells at this branch is 40%. Literature values43 for a 20 minute long exposure to 2mM H2O2 indicate a 20% necrotic rate. As the cell death rate depends both on toxin concentration and exposure time, our observation is in accordance with the literature results.</p><p>In the control experiment, in which we counted the number of live and dead cells in all branches after loading and without the presence of toxin (Figure 4d), we treated each well separately, rather than in clusters. The average number of cells per well was 16. The result indicates that the viability is close to 100% in all branches. Also, the calculated maximum shear stress acting on a cell is 0.27 dyne cm−2 at 200 μl/hr47 is not large enough to contribute to cell death, which is in agreement with literature results48–51.</p><!><p>We developed a microfluidic device for generating exponential and sigmoidal concentration gradients by exploiting diffusion of two aqueous species in an asymmetrical design. The flow patterns of two miscible aqueous solutions generate concentration gradients parallel to the input line and were investigated with respect to the applied flow rates and diffusion constants of the two co-flowing solutions and species.</p><p>At low flow rates of water and fluorescent sodium dye (10, 50, and 100 μl/hr per stream) we observed exponential gradients with an increasingly steeper initial slope, which reached higher final concentrations with increasing flow rate. At a high flow rate (200 μl/hr per stream), however, we observed sigmoidal concentration gradients. The saddle region of such gradients could be approximated with a linear curve, making this device useful for applications requiring linear concentration gradients. Further, an increasing flow rate was imitated by using species with smaller diffusion constants. These experimental parameters were equivalent insofar as they weakened the mixing of the two species, leading to a steeper initial slope of the concentration gradient. For fluorescently labeled Dextran, for example, the sigmoidal behavior sets on at a lower flow rate (50 μl/hr per stream). Lastly, we demonstrated the applicability of the device in biological studies, namely a live-dead assay on fibroblast cells. The resulting concentration gradients of live and dead cells were non-linear, as expected from our device characterization study.</p><p>The main advantage of the microfluidic device presented here is its small footprint, as it does not include a dedicated mixing region, and it is capable of producing specific concentration gradients of different biochemical components solely by controlling the applied flow rates. With regard to external equipment, operation of this device only requires a dual syringe pump, a standard equipment item in most biology laboratories. As such, this device essentially has no fallible elements, increasing its robustness. Our microfluidic device therefore has the potential to become a useful tool for studying the effects of microenvironments on biological activities, e.g. cell behavior in response to various natural or synthetic stimuli, from basic biological applications to drug discovery studies. Our future work will focus on improving our cell seeding capabilities in this PDMS device by utilizing the short cross-channels between individual wells and introducing microfluidic valves to control the fluid delivery. We plan to conduct further cell studies, for example subject stem cells to different growth factors or drug gradients. For example, we envision first conducting a screening experiment using a sigmoidal concentration gradient, then using these results focusing on a particular concentration range by applying an exponential gradient. We will also work on modifying the device design to allow for long-term cell culture, for example by introducing a media perfusion system. Apart from cell studies, future applications could include protein crystallization, where protein droplets instead of cells will be captured in the storage wells and subjected to different pH gradients.</p><!><p>a) Device structure with key dimensions (d = 800 μm). The input line for the two co-flowing solutions is shown on the right; several channels branch off and lead to the channel grid left of it. Magnified images show storage wells (placed at some intersections of the grid channels) and the gradient generation section (main input channel with side branches). Gradient generation region: b) Fluorescence photograph showing eight side branches. Note that the device has originally been designed with an additional exit channel for flushing out potential clogs near the inputs. This channel points to the right of the first branch and is permanently closed in our experiments. The white dashed line outlines the edge of the channel, with clear solution on the left and fluorescent dye on the right. The two solutions mix via diffusion as they travel through the main channel, such that different amounts of either solution enter the side branches, giving rise to a non-linear concentration gradient along the input flow direction. The red lines indicate positions along the input line where the concentration profile was measured. A graph of these concentration profiles is shown in c). d) Sketch showing a single side branch, illustrating the redistribution of flow in the main channel and e) sketch of the full device, showing that different flow paths (e.g. the yellow and the green path, labeled Q2 and Q16) have almost equal dimensions and therefore equal resistance. (Online in color.)</p><p>a) 3D concentration profile of fluorescein sodium salt as a function of branch number and length, when introduced into the device together with a clear solution at 100 μl/hr per stream (data stems from a single experiment). b) Experimental data (symbols, averaged from three experiments) and corresponding exponential and sigmoidal fit curves (lines) for the co-flow of clear solution and a fluorescent dye solution. The listed flow rates are applied to each stream. For n=3 the standard deviation is below 9%, but we omit error bars to keep the graph readable. c) Corresponding simulation results. d) Stitched image of the microfluidic device at 50 μl/hr per stream and e) the corresponding simulation result. (Online in color.)</p><p>Comparison of a) experimental (n=3) and b) simulation results for concentration gradients of three molecular species with different diffusion constants at 100 μl/hr/stream. The standard deviation in a) is below 9%, but we omit here the error bars to keep the graph readable. c) 3D representation of a single 100 μl/hr/stream experiment with FITC_Dextran 70kDa. Experiment and d) simulation. (Online in color.)</p><p>Live-dead assay of 3T3 cells due to an exponential concentration gradient of H2O2. a) Stitched phase contrast photograph of the storage region with seeded cells (scale bar: 800 μm). Inset: typical wells (scale bar: 200 μm). b) Superposed red and green fluorescence images of cells stored in wells on branches 1 to 16, with decreasing H2O2 concentration (scale bar: 100 μm). c) Fraction of live and dead cells as a function of branch number, in response to the toxin gradient. d) Fraction of live and dead cells in the control experiment, immediately after loading and without the presence of hydrogen peroxide. The error bars in c) and d) are standard deviation values. For easier viewing, we only include one set of error bars for each data set.</p>

PubMed Author Manuscript

Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter

The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.

identification_of_a_novel_allosteric_modulator_of_the_human_dopamine_transporter

INTRODUCTION<!>In Silico Screening Successfully Identifies a Novel DAT Allosteric Modulator.<!>KM822 Is a Selective and Noncompetitive DAT Modulator.<!>Binding Site Characterization of KM822 Confirms the Location of the Allosteric Site Predicted by the in Silico Studies.<!>Interaction of KM822 with Inward (Y335A) versus Outward (Y156F) Equilibrium Shifting DAT Mutations.<!>KM822 Influences the Conformation of DAT Domains.<!>KM822 Affects Psychostimulant Activity in the in Vitro and ex Vivo Studies.<!>KM822 Suppresses Psychostimulant-Associated Behaviors in a Planarian Model of Addiction.<!>CONCLUSION<!>Animals and Materials.<!>Hybrid Structure-Based Method.<!>Chemical Synthesis of KM822.<!>Site-Directed Mutagenesis, Cell Culture, and Transfections.<!>Transport Kinetic Assays Using COS-7 Cells.<!>Transport Inhibition Assays Using MDCK or COS-7 Cells.<!>Biotinylation.<!>Synaptosomal Uptake Assay.<!>Ex Vivo Slice Voltammetry.<!>Drug Induced Locomotion in Planarians.<!>Data Analysis.

<p>The dopamine transporter (DAT) is a member of the solute carrier 6 (SLC6) family of transporters and is embedded in the plasma membrane of presynaptic terminals of dopaminergic neurons in the central nervous system.1,2 DAT plays a key role in controlling the signal amplitude and duration of dopaminergic neurotransmission by removing extracellular dopamine (DA) resulting in decreased levels of DA in the extracellular space. Pharmacological modulation of the DAT will as a result affect neuronal dopaminergic activity. Indeed, DAT is the primary site of action for a number of psychostimulants and recreational drugs, including cocaine, amphetamine, and methamphetamine, which all block or reverse the transport of DA, thereby increasing synaptic dopaminergic neurotransmission.3</p><p>DAT is a 12-transmembrane domain (TMD) protein mediating DA uptake through the coupling to the sodium and chloride gradients. The substrate-translocation is believed to follow an "alternating access" mechanism where the transporter sequentially transitions through several different conformations including outward-open, occluded and inward-open conformations to transport substrate from extracellular side to the intracellular milieu.4 The discoveries of homologous bacterial (Aquifex aeolicus) leucine transporter (LeuT),5 Drosophila melanogaster DAT (dDAT)6,7 and human serotonin transporter (SERT)8 crystal structures have been pivotal in enhancing our knowledge on the structural biology of this family of neurotransmitter transporters. The structure of these transporters includes 12 α-helical TMDs connected with flexible intracellular and extracellular loops. The N- and C- termini lie in the intracellular region. The high-affinity primary orthosteric substrate binding site, S1, lies at the core of the translocation pathway located between TMD1 and TMD6. Interestingly, besides the S1 site where substrates and psychostimulants bind, both structural and functional work has suggested that there might exist additional allosteric binding sites on these transporters.2</p><p>We are interested in developing nonclassical ways of targeting and modulating DAT activity by exploring the idea of employing allosteric modulators of DAT as potential antiaddiction therapeutics. Different from the well-established understanding and availability of selective competitive inhibitors of DAT, the current level of understanding of allosteric modulation of DAT function is very limited. However, convincing evidence of the presence of secondary or allosteric sites in these transporters is provided by the human SERT crystal structure bound with two escitalopram molecules, one in the S1 site and the other in the extra vestibular region.8</p><p>Prior to the discovery of the second citalopram molecule in the SERT crystal structure, we pursued the idea of the existence of allosteric sites on DAT and SERT. Through structure/function studies, molecular dynamics and comparative genomics techniques, we identified a unique allosteric domain in SERT located outside the central translocation pathway that dictated pharmacological differences between human transporters and transporters from the parasite Schistosoma mansoni.9,10 We used the hybrid structure-based (HSB) method11 to successfully identify an allosteric modulator of human SERT (ATM7) that interacts with this allosteric site.12 Intriguingly, the site we identified through computational and biochemical studies is similar to the site where the second citalopram molecule later was found in the SERT cocrystal structure. Other functional studies have also pointed to this region as a domain that could have allosteric activity.9 For example, in studies on the bacterial DAT homologue LeuT, a site (S2) was proposed to exist in the extracellular vestibular region that lies in the solvent-accessible pathway connecting the extracellular milieu of the transporter with the orthosteric S1 site.13 This site overlaps partially with the site we have identified in this study. Engaging S2 by substrates is thought to allosterically trigger a conformational change in the LeuT transporter from the substrate-bound occluded state to an inward open state, which facilitates the release of the substrate.</p><p>In the current study, we target a similar region in DAT that we previously targeted in SERT. Since this site is proximal to the orthosteric site and is networked through a series of hydrogen bonds, we hypothesize that conformational changes induced by small molecules at this site will produce allosteric effects. Indeed, we demonstrate that a small molecule, KM822, identified using the HSB screening, interacts at this site and causes significant changes in the structure and function of the DAT protein. We also show that this molecule affects cocaine potency in inhibiting DAT-mediated DA reuptake as well as cocaine-associated behaviors in a planarian model of psychostimulant activity.</p><!><p>The crystal structure of Drosophila DAT (dDAT) was used to model the outward-facing conformation of human DAT (hDAT) using the homology modeling program Modeler. Since cocaine binds to the outward-facing conformation of DAT, we rationalized that an allosteric modulator that can inhibit the binding of cocaine should bind to the outward-facing conformation of DAT and hence chose to model the outward-facing conformation using dDAT as a template. Among the 10 hDAT conformations generated by Modeler, the best ranking conformation with the lowest energy was chosen for molecular dynamics (MD) simulations. Analysis of the trajectory from MD simulations from the production run revealed that the allosteric pocket was formed in coordination with the membrane lipids and retained the pocket configuration throughout the simulation (Figure 1A). The allosteric site is in a similar site as previously identified in hSERT.12 The binding pocket is lined by several aromatic and hydropathic residues such as W84, Y88, F155, Y156, F472, and H477 and hydrophilic residues R85, K384, D385, T473, and D476. The fluctuations of residues W84, D385, D476, R544, and Y548 used to define the pharmacophore's distance restraints were measured during the last 10 ns of the production run of the MD simulation. Screening the in silico libraries for the five-point pharmacophore (Figure 1B) resulted in 598 hits that followed a minimum of four out of five pharmacophoric features. Screening the hits against the in silico model for blood-brain barrier (BBB) penetration resulted in 256 hits.14 Docking of the 256 hits to the binding pocket and rank ordering the complexes using customized scoring functions led us to designate compound KM822 as our lead molecule with a high docking score of 87.39. KM822, a substituted triazinoindole molecule (Figure 1C), occupied the entire volume of the allosteric site locking the transporter in an outward-facing conformation. Key interactions of KM822 at the binding pocket (Figure 1C) includes hydrogen bonding interactions of residues D385 and D476 with nitrogen atoms on the central indole ring of KM822. Other interactions that contribute to the high docking score of KM822 include hydrogen bond interactions between residue T473 and the sulfonamide group, residue D555 and the phenylacetamide group of KM822. The phenylacetamide group has additional interactions with residues R544 and Y548. The central triazinoindole ring also has aromatic cation interactions with residue D476, staggered stacking interaction with residue W84 and aromatic interactions with residue F155 that contribute to its high docking score.</p><!><p>To characterize the pharmacology and mechanism of action of KM822, dose–response and uptake saturation assays were performed. In dose–response assays against hDAT, hSERT, and the human norepinephrine transporter (hNET), KM822 was found to preferentially inhibit neurotransmitter reuptake in hDAT as compared to hNET and hSERT (Figure 2A). The potency for hDAT transport inhibition is 30 and 50 folds higher than for hNET and SERT, respectively (DAT IC50 = 3.7 ± 0.65 μM, NET IC50 = 119 ± 22.82 μM, and hSERT IC50 = 191.6 ± 34.45 μM). The selective but relatively low potency of KM822-mediated inhibition of hDAT could be a desirable feature as many known high potency hDAT inhibitors are known to display addictive properties. KM822 was further characterized for its effect on dopamine uptake kinetics (Figure 2B). KM822 was found to inhibit dopamine uptake in a noncompetitive manner with a decrease in Vmax in the presence of 1 μM (65.8 ± 8.71 μmol/min/well) and 5 μM KM822 (57.9 ± 4.15 μmol/min/well) compared to vehicle (71.5 ± 6.12 μmol/min/well), with no significant change observed for the apparent affinity for DA (KM) in the absence or presence of KM822 (6.2 ± 0.69, 7.0 ± 0.58, and 10.54 ± 0.78 μM in the presence of vehicle, 1 μM KM822, and 5 μM KM822, respectively). This result suggests a novel allosteric mechanism of action of KM822 acting through a site different from the orthosteric site.</p><!><p>To confirm the in silico predicted binding site and further characterize the location and structural determinants of the binding site of KM822 within DAT we employed DA uptake studies and a biotinylation-based biochemical assay. From the molecular model of DAT bound to KM822 and the previous studies of SERT it is clear that the allosteric binding site of KM822 is distinct from the substrate binding (S1) site. As seen in the molecular model (Figure 1) and described above, DAT residues that line this site include W84, R85, Y88, F155, I159, K384, D385, T465, T473, D476, H477, R544, Y548, A550, D555, and I564, where R85 and D476 form a salt bridge that has been proposed to form a gate to the extracellular space. The DA and cocaine orthosteric binding sites are located beneath this proposed salt bridge in closest proximity to residues F76, V152, and F326.6,15 In order to experimentally validate the KM822-bound DAT molecular model and physically localize the binding site of KM822, we employed structure/function dose–response uptake studies and the substituted cysteine-accessibility method (SCAM).16–18</p><p>Several DAT mutants were created in which the amino acids lining the proposed allosteric pocket were systematically replaced by cysteine via site-directed mutagenesis. The only cysteine in the native DAT that is accessible from the extracellular environment (C306) was replaced with an alanine to create a biotinylation insensitive mutant and this mutant (DAT-XC) was used to generate all the mutants.19 Only DAT mutants with significant expression levels and functional DA transport activity comparable to the WT DAT were progressed further for functional and SCAM studies. For example, mutants F155C, I159C, L474C, H477C, G549C, A550C, and L560C which showed less than 20% maximal DA transport activity (data not shown) as compared to the WT were not included in the functional or the SCAM analyses. As a result, mutants with >50% surface expression levels and >20% DA transport activity in comparison with the WT DAT included in this study were W84C, K384C, D385C, D476E, D476N, T473C, and R544C (Figure 3). In the functional uptake studies using dose− response inhibition assays, KM822's IC50 values were significantly altered in transiently expressed DAT mutants, W84C, D385C, and D476N, in comparison to WT, suggesting these residues are involved in the binding of KM822 to DAT (Figure 3A). For the W84C mutant we observed an increase in potency and for the D385C and D476N mutants, apparent potency was lowered by the mutation (Figure 3A). It is interesting to note that a previous study20 on a different mutant of W84 to a leucine found a decrease in apparent potency for DAT inhibitors benztropine and GBR12909 and increased apparent potency of cocaine suggesting interactions between this residue and the orthosteric site. To further validate the observations from structure/function studies we performed SCAM studies.</p><p>For the SCAM studies, the MTSEA-biotin reagent was used to assess accessibility and reactivity of the thiol (-SH) group of the introduced cysteine in the absence or presence of 20 μM KM822. If KM822 interacts with the respective cysteine residue, we hypothesize that it will physically protect the cysteine from being biotinylated. Following biotinylation, biotinylated DAT was affinity-purified using streptavidin beads, separated, and detected using immunoblotting employing an antibody against an HA-tag that is incorporated N-terminally in all the DAT constructs. The total DAT expression in the cells was measured in parallel using unpurified total cell lysates. As expected, the DAT-XC mutant was not biotinylated. We observed that there was no effect of KM822 coincubation on biotinylation of the cysteine mutants D385C, T473C, and R544C (Figure 4). Residue D385 is localized to the extracellular loop (EL) 4, T473 to EL5, and R544 is on the hinge region connecting EL6 which are flexible loop regions, and this could explain why they did not show a significant change in the level of KM822 treated biotinylated mutant DAT as compared to that of the vehicle. This is likely due to fluctuations in the conformation of the flexible loop and consequently the reversibly bound KM822 is unable to completely protect the residues from the covalent attachment of MTSEA-biotin. In case of W84C, its biotinylation is significantly decreased by KM822 coincubation, suggesting KM822 protects it from being biotinylated likely due to a proximal interaction of W84 with KM822 (Figures 3B and 4A, B). Furthermore, we also found that the labeling of this mutant with MTSEA-biotin is not affected by the coincubation of 100 μM cocaine suggesting W84 is not part of the orthosteric S1 site (Figure 4C and D). To further explain these results, we calculated the molecular surface accessibility of each of the investigated residues in the hDAT-KM822 complex. The molecular surface accessibility is calculated using the Connolly algorithm21 using a spherical water molecule of radius 1.4 Å and provides a score for the entire residue. According to this calculation, W84 has the least accessibility score of 1.47 when compared to the other residues in the SCAM experiment (Table 1). Because W84 has the least molecular surface accessibility in our molecular model of KM822 bound within hDAT, this suggests that W84 is the deepest buried residue among the ones examined and the most protected from biotinylation (Figures 1C and 3B). In addition, since cocaine does not protect W84 from covalent attachment of MTSEA-biotin, this suggests that the two compounds bind differently within the transporter: KM822 at the allosteric site and cocaine at the orthosteric S1 site.</p><p>To further assess the importance of W84 residue in the interaction of KM822 on hDAT, a dose–response experiment was performed. DAT-XC/W84C expressing HEK-293 cells were incubated with varying concentrations of KM822 prior to the addition of MTSEA-biotin. Coincubation with varying KM822 concentration resulted in a dose-dependent decrease in biotinylation signifying an increase in the degree of protection from labeling by increased concentrations of KM822 resulting in an IC50 value of 0.45 ± 0.08 μM (Figure 5). Importantly, the inhibition potency in the biotinylation experiment was similar to the potency of KM822 in inhibiting DA uptake in DAT-XC/W84C transfected COS-7 cells (Figure 3A), further supporting a direct role of W84 in the interaction between KM822 and DAT. In the W84C mutant, cysteine retains the electrostatic interactions with the triazineindole ring of KM822 but with a much less bulky side chain than tryptophan and hence can better accommodate KM822 in the binding pocket which may explain the increase in potency of the W84C mutant for KM822 (Figures 1C and 3B). Also, the comparable potency of KM822 against DAT-XC/W84C in the DA transport inhibition assay, strongly supports the robustness of our biotinylation assay.</p><p>These results also indicate that the MTSEA-biotin protection assay against W84C mutant can be used as an alternative to radioactive competition binding assays to assess the binding affinity of test compounds directly against this novel allosteric binding site.</p><p>Taken together, the structure/function uptake and SCAM studies supports the notion that KM822 interacts with the proposed allosteric site. It is important to note, that both types of studies are indirect evidence of this. Indirect effects of KM822 binding somewhere else producing long distance effects can as a result not be excluded. Only 3D crystal or cryo- EM studies can convincingly demonstrate the exact position of KM822 binding to DAT, and such studies are currently under way with our collaborators. Interestingly, in a recent study on the multihydrophobic substrate transporter MhsT from Bacillus halodurans, which is related to LeuT, it was found that biotinylation of the residue corresponding to W84 was protected by the substrate tryptophan in a similar manner as KM822 protects this site from biotinylation.22 As mentioned above the allosteric site described here shares some structural features with the proposed allosteric S2 site in LeuT and MhsT. Particular it shares the W84 residue in DAT with the corresponding residues in LeuT and MhsT. It could therefore be speculated that the structures mediating the allosteric effects of the substrates in bacterial LeuT and MhsT transporters are conserved in DAT and SERT and can be engaged by other types of compounds in these carriers to produce allosteric effects.</p><!><p>To further understand the mechanism of action of KM822, we examined whether KM822 preferentially binds to the outward- or the inward-facing conformation of DAT. We took advantage of two known mutations, i.e., Y156F and Y335A which are commonly used to determine the preferred DAT conformation for various DAT-interacting compounds.23–28 The mutation Y156F removes the interaction of the Y156 hydroxyl group with D79 in the S1 binding site of hDAT which results in an open-outward conformation of DAT. The binding affinity of cocaine and its analogs is unaffected by this mutation, but the potency of atypical DAT inhibitors such as JHW-007 and S-modafinil is significantly reduced.27 On the other hand, the Y335A mutation shifts the DAT conformation equilibrium toward an inward-facing orientation which markedly impairs the potency of cocaine and its analogs but causes only a slight loss in the potency of most benztropine analogs.27 KM822 potency was compared in DA uptake inhibition assay in COS-7 cells transiently expressing WT, Y156F or Y335A DAT mutants. We found that the potency of KM822 remained unchanged in the Y156F-DAT mutant as compared to the WT-DAT but was reduced by 3-fold in the Y335A mutant (Figure 6), suggesting KM822 preferably binds to an outward-facing conformation. To further support this observation, we modeled the inward-facing conformation of WT-DAT using the crystal structure of the inward-facing conformation of LeuT.29 Structural comparison of the outward-facing and inward-facing conformational models of DAT revealed significant conformational changes in the TMDs including hinge motions of several helices leading to an overall root mean squared deviation of ~9 Å. In the inward-facing conformation, TMD 1, 3, 10, and 11 have significant movement that clearly occlude the formation of the allosteric site (Figure 7A). A study by Cheng and Bahar found similar significant movements of TMDs during the transition from outward- to inward-facing conformations of DAT in their MD simulations.30 We attempted to dock KM822 to the inward-facing conformation, and as expected the triazineindole ring had steric hindrances from TMD 1, 10 and 11, while EL4, 5, and 6 had no interactions with the rest of the molecule (Figure 7B), suggesting that KM822 does not bind to the inward-facing conformation of DAT. A caveat to our model is that it is based on the inward-facing conformation of LeuT and no crystal structure of the inward-facing conformation of human DAT is available to validate the model. In addition, there are several differences in the secondary structure between DAT and LeuT, e.g., in the length and structure of EL4 which participates in binding of KM822 in DAT. Previous studies by Shan et al. have demonstrated that the S2 site in LeuT is similar to the S2 site in DAT despite the differences in the length of EL4 between the two proteins.31 Further, they conclude that binding of substrate at the S2 site likely initiates the conformational changes for transition of an outward or occluded DAT state toward the inward open conformations. However, due to lack of a crystal structure of the inward open conformation of DAT, these observations have not been fully validated. These caveats could limit the validity of our inward-facing DAT model, but we are encouraged by the fact that the experimental results are in agreement with the prediction of the model.</p><p>Taken together, these results indicate that KM822 prefers the outward-facing conformation of DAT similar to cocaine and its analogues19,27 but through a mechanism distinct from cocaine as the two compounds occupy different binding sites within DAT. Schmitt et al.32 also reports that another DAT mutant, W84L, shifts the DAT conformation equilibrium toward outward-facing conformation. It is possible that our W84C mutant also adopts a similar outward-open conformation which is preferred by KM822 which could also explain the increase in potency with the W84C mutant. In addition, although the outward-facing conformation is a preferred mode for cocaine and other analogs that have stimulatory effects, there are some DAT inhibitors that are known to prefer outward-facing conformation but produce atypical behavioral effects.33,34 Thus, despite KM822 displaying cocaine-like preference for DAT conformation, its low inhibitory potency with a noncompetitive mechanism of action could make this compound a prototype for another class of atypical DAT inhibitors with an allosteric mechanism of action and with therapeutic potential.</p><!><p>Previously, the SCAM method has also been used to characterize conformational states in DAT.19 In those studies, the residues T316 and A372 that are present in TMD6 and TMD7, respectively, and face the extracellular side of DAT were used to monitor DAT conformations as their accessibility was shown to be influenced by the binding of cholesterol and cocaine molecules which are known to promote the outward-facing conformation of DAT. We used this method to further study the effects of the KM822 interaction with DAT and compared it to that of cocaine. Previously, Hong and Amara19 showed that the DAT-XC/T316C mutant displayed increased labeling with a maleimide-based biotin molecule in the presence of cocaine, while DAT-XC/A372C showed no change in labeling when treated with cocaine. We observed the same results with MTSEA-biotin labeling with DAT-XC/T316C displaying increased labeling (Figure 8A) and DAT-XC/A372C showing no change in labeling in the presence of 100 μM cocaine (Figure 8B). When comparing these results with KM822-treated cysteine mutants, a similar effect was observed as the presence of 20 μM KM822 increased the MTSEA-biotin labeling of DAT-XC/T316C DAT while there was no significant change in the labeling of DAT-XC/A372C (Figure 8). These results indicate that KM822 and cocaine both orient the conformation of DAT in comparable ways and, thus, influence the position and accessibility of DAT mutants DAT-XC/T316C and DAT-XC/A372C toward MTSEA-biotin labeling to a similar extent.</p><!><p>To further study the allosteric effects of KM822, we examined if KM822 would affect cocaine inhibition of DAT by performing a dose–response curve of KM822 on cocaine–DAT interactions in DAT transfected COS-7 cells. As shown in Figure 8, KM822 dose-dependently decreased the potency of cocaine in DA transport inhibition assay. The potency of cocaine in the absence of KM822 was 1.2 ± 0.47 μM (IC50), whereas in the presence of 1 and 5 μM KM822, cocaine's potency decreased significantly to 4.2 ± 0.42 and 8.1 ± 2.62 μM, respectively (Figure 9A). In addition, KM822 effects were evaluated in ex vivo striatal synaptosomal preparations that contain DAT in its native environment. A similar trend was observed to that of the in vitro cell based assays with KM822 reducing the affinity of cocaine (IC50 is 0.18 ± 0.05 μM in the absence of KM822 versus 0.75 ± 0.31 μM in the presence of 5 μM KM822) toward DAT in native tissue (Figure 9B).</p><p>To further examine the effect of KM822 on DA release and uptake in an intact ex vivo model, fast scan cyclic voltammetry was performed on striatal slices containing the Nucleus accumbens. As shown in Figure 10A, KM822 did not affect cocaine-induced changes in DA release. However, KM822 significantly reduced the ability of cocaine at inhibiting the DAT as demonstrated in attenuated changes in apparent Km (app Km) when incubated with cocaine (Figure 10B). In conclusion, we show that KM822 dose-dependently decreased the potency of cocaine in inhibiting the transport of DA by DAT when evaluated in three different assays, i.e., the in vitro dose–response assay of DA transport in DAT-transfected COS-7 cells, the ex vivo assays of DA transport in striatal synaptosomes, as well as in the slice preparations containing DAT in its native environment. It is interesting to note that the effect of KM822 on the interaction of compounds with the orthosteric site differs as we do observe a decrease in apparent affinity for the substrate dopamine in the presence of KM822 but different from what we observe with cocaine this was not significant. This could be explained by the fact that dopamine is a substrate and cocaine is a nontransported inhibitor, but it could also suggest there are subtle differences in the way cocaine and dopamine interacts with the orthosteric site. These results prove the robustness of KM822 in serving as a potential starting point for developing molecules that could interfere with the addictive properties of psychostimulants like cocaine and therefore could have therapeutic potential.</p><!><p>Based on the results on cocaine–DAT interaction, we finally tested KM822 on a cocaine-associated behavior. Demonstrating an activity of KM822 in a behavioral assay further strengthens the idea of KM822 as a promising lead for the development of substance abuse disorder therapeutics.</p><p>As proof-of-concept, a planarian model of psychostimulant activity35 was employed to examine the effect of KM822 on a psychostimulant-associated behavior. Planaria have been shown to respond to psychostimulants with elevated locomotion and withdrawal symptoms. In addition, these behaviors have been demonstrated to be mediated by neurochemical signaling that is similar to what mediates comparable behaviors in mammalians.35 We found that KM822 specifically blocks the stimulated locomotion elicited by stimulants that interact with DAT (amphetamine and cocaine) resulting in locomotion comparable to vehicle treated animals (Figure 11). On the other hand, KM822 did not block nicotine-elicited locomotion that is not mediated through DAT but the nicotinic acetylcholine receptors. Furthermore, we also demonstrated that KM822 by itself does not affect locomotion in planaria. This nonstimulatory effect of KM822 is desirable as in the past, many DAT-interacting compounds that blocked cocaine's effects were also shown to be stimulatory by themselves. Our group will further evaluate the nonstimulatory and nonaddicting behaviors of KM822 in other animal models of addiction in future studies.</p><!><p>Observations from this and previous studies suggest additional binding sites do exist in DAT and open the door for research into exploring allosteric modulation of conformational changes and function of DAT. We used dynamic modeling and HSB virtual screening of an in silico chemical library to identify a novel allosteric modulator of hDAT. The compound, KM822, modulates the interaction of DAT with the highly addictive psychostimulant cocaine and it prevents the behavioral effects of cocaine in an in vivo model of addiction. The mechanistic studies herein suggest that KM822, similar to cocaine, interacts with an outward-facing conformation of DAT. A systematic evaluation of novel allosteric sites on DAT in the future should significantly advance our understanding of the relationship between the structure and function of this critical therapeutic target and provide valuable insight regarding the range and magnitude of possible modulatory activities. We speculate that molecules interacting with these sites could have therapeutic and clinical potential and that KM822 could serve as a lead for the development of molecules with antiaddictive properties.</p><!><p>All experiments using animal subjects were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments involving animal subjects were conducted as preapproved by Drexel University Institutional Animal Care and Use Committees. Radio-labeled substrates, [3H]-dopamine (32.6 Ci/mmol) and [3H]-serotonin (23.9 Ci/mmol), were purchased from PerkinElmer (Boston, MA). Cell culture media and supplements, including penicillin/streptomycin, Dulbecco's phosphate-buffered saline (D-PBS), Dulbecco's modified Eagle's medium (DMEM) with glucose, and scintillation fluid, were obtained from Thermo Fisher Scientific (Waltham, MA). Transfection reagents TransIT-LT1 and LipoJet reagent were from Mirus Bio LLC (Madison, WI) and SignaGen Laboratories (Rockville, MD), respectively. Reagents for uptake assays and nonradiolabeled substrates were purchased from Sigma-Aldrich (St. Louis, MO). MTSEA-biotin was purchased from Biotium, Inc. (Fremont, CA). Reagents for uptake assays and nonradiolabeled substrates were purchased from Sigma-Aldrich (St. Louis, MO).</p><!><p>The crystal structure of Drosophila melanogaster dopamine transporter (dDAT) (PDB code: 4M48) bound to nortriptyline,36 a tricyclic antidepressant and locked in the outer facing conformation was used for modeling the human DAT (hDAT) using MODELER software package (ver 9.1).37,38 The Modeler program's output was set to generate 10 low energy conformations, and the best ranking structure was optimized using energy minimization and constrained molecular dynamics simulations with a production run of 10 ns. The final structure from the production run was chosen for further membrane embedded simulations. The modeled hDAT structure was embedded in a POPC membrane patch using the Desmond program (D. E. Shaw Research, New York, NY) with a production run of 600 ns. Optimal positioning of the membrane was computed using the dDAT structures in orientations of proteins in the membrane (OPM) database.39 The results of the simulations were analyzed using inhouse trajectory analysis scripts and visualized using the VMD program.40 Structures from last 10 ns of the simulation was used to screen for cavities using the program VOIDOO41 and the results were rank ordered by the volume of the cavity. The best ranking allosteric cavity chosen was lined by several aromatic residues such as W84, Y88, F155, F472, Y548, W556, and Y551 and by hydrogen bonding residues such as R85, K384, D386, T473, D476, and R544. This pocket was similar to the ATM7 binding pocket that was identified for human SERT previously, thus implicating it as an allosteric site for hDAT.12 The composition of the allosteric binding pocket suggested that the pocket could be specific to the outward-facing conformation of DAT as it was significantly influenced by its interactions with the lipids from the membrane patch surrounding the pocket and thus maintaining the integrity of the pocket. A five-point receptor pharmacophore was designed using the residues W84, D385, D476, R544, and Y548 which included pharmacophoric features of an aromatic ring, hydrogen bond donor/acceptor pair, and hydrophobic interactions. The five-point pharmacophore was screened against a 3 × 106 compound library in a flexible mode. The hits were then filtered for blood-brain barrier permeability using an in silico model as described previously14 and the resulting hits were docked to the allosteric binding pocket of the hDAT using the genetic algorithm based docking program GOLD (ver 5.2).11,42,43 Docked complexes were ranked using goldscore and reranked using customized scoring scheme.11,43,44 Goldscore is a dimensionless score derived from the energy function that accounts for electrostatic, van der Waals, ligand torsion energy, and flexibility of binding pocket residues.42 Accordingly, the higher the goldscore, the better is the docking fit to the binding pocket; however, since the score approximates the energy terms, it cannot be used to calculate binding affinities or IC50 values. Customized scoring scheme is a knowledge-based scoring function that can be used to increase the weightage for favorable interactions and penalize unfavorable interactions. The method has been successfully used to screen activators of xenobiotic receptors and allosteric activators of the glutamate transporter.43,44 The method involves identifying all the residues within 6 Å from the pharmacophore and scoring the favorable and unfavorable interactions of the ligands with these residues. Stacking interactions with W84, F155, Y88, H477, Y548, W556, H547, and Y551 and hydrogen bonded interactions with D385, D476, K384, D555, R544, T465, and R85 contributed to higher weightage, while unfavorable interactions with opposing chemical features were negatively weighted. Based on the goldscore and customized scoring KM822 ranked the highest among all the ligands and hence was nominated as the lead hit molecule. The docked complex of hDAT and the lead compound KM822 was further subjected to minimization and 1 ns long MD simulation to ensure that the molecule docked stably in the binding pocket. A visual inspection of the simulation showed no significant change in the docked pose or position in the binding pocket which ensured stable docking.</p><p>To test whether KM822 prefers outward or inward-facing conformation of hDAT, we also modeled hDAT in the inward open conformation using the inward open confrmation crystal structure of LeuT as a template (PDB code: 3TT329) using the Modeler program. The structure was optimized with energy minimization and 3 ns of MD simulations as described above for modeling the outward-facing conformation of hDAT. The resulting hDAT structure in the inward open conformation was used to attempt docking KM822 using GOLD docking program as described above.</p><!><p>KM822 was obtained from a three-step synthetic scheme by utilizing 5-ethylindoline-2,3-dione and 4-acetamidobenzene-1-sulfonyl chloride as the starting materials. Details of KM822 synthesis and structural characterization are in the Supporting Information.</p><!><p>All single and double mutants were generated using the QuikChange (Stratagene, La jolla, CA) site-directed mutagenesis kit using human WT DAT and C306A-DAT as the background, respectively. The mutations were verified by sequencing (Genewiz, LLC). COS-7 and HEK-293 cells were maintained in DMEM (3.5 g/L glucose) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. Stably transfected MDCK cells expressing hDAT, hNET, or hSERT were maintained in DMEM (3.5 g/L glucose) supplemented with 10% FBS and 1% penicillin/streptomycin and blasticidin (5 μg/mL) at 37 °C with 5% CO2. For transient transfections, COS-7 cells were tranfected using the TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI), and HEK-293 cells were transfected using the Lipojet transfection reagent (SignaGen Laboratories, Rockville, MD).</p><!><p>Transport assays were performed as described previously.45 Briefly, COS-7 cells were transfected and plated in 96-well plates. Uptake experiments were performed 2 days after transfection. The media was removed, and the cells were washed with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) containing 0.1 mM CaCl2, 1 mM MgCl2, 5 mM RO 41–0960, and 100 mM ascorbic acid (PBS-CM). Following washing, various concentrations of [3H]-dopamine were added and the uptake was allowed to continue for 10 min at room temperature. The uptake was terminated by washing twice with PBS-CM. The cells were solubilized in scintillation cocktail and counted on a microplate scintillation and luminescence counter (PerkinElmer, Waltham, MA). Background was obtained from nontransfected cells and subtracted. Data were fitted to a Michaelis−Menten equation using nonlinear regression to obtain Km and Vmax</p><!><p>Stably transfected MDCK cells (expressing hDAT, hNET, or hSERT) or transiently transfected COS-7 cells were plated in 96-well plates. Uptake experiments were performed 2 days later. The media was removed, and the cells were washed with PBS-CM. Following washing, the cells were incubated for 10 min with various concentrations of KM822 and the uptake was initiated by adding [3H]-dopamine to a final concentration of 25 nM. The uptake was allowed to continue for 10 min at room temperature and was terminated by washing twice with PBS-CM. The cells were solubilized in scintillation cocktail and counted on microplate scintillation and luminescence counter (PerkinElmer, Waltham, MA). Data were fitted to a Hill equation by nonlinear regression analysis to obtain IC50 values.</p><!><p>HEK-293 cells were transiently transfected and cultured to confluency in a 6-well plate. The cells were washed with ice-cold PBS-CM (pH 7.4) twice, incubated at 4 °C with vehicle or KM822 (20 μM) for 10 min, followed by the addition of MTSEA- biotin (125 μM, final concentration). After 10 min incubation at 4 °C, the biotinylation was quenched by aspirating the liquid and washing the cells with 1 mM DTT in PBS-CM, followed by a final wash with PBS-CM only. The cells were lysed in 600 μL of TNE lysis buffer (pH 7.4) containing protease inhibitors (100×) for 40 min at 4 °C. The cells were collected and centrifuged at 12 000g for 10 min at 4 °C. The supernatant was collected of which 30 μL was mixed with DTT (0.5M, 5 μL) and LDS Nu-page (15 μL) for total lysate sample, and saved at −20 °C. 450 μL of the supernatant was incubated overnight at 4 °C with 50 μL of 50% slurry of high capacity neutravidin agarose resin. The beads containing "biotinylated" proteins were washed with ice-cold TNE lysis three times, followed by a final wash with PBS-CM, and then mixed with LDL Nu-page (15 μL), DTT (0.5M, 5 μL) and water (30 μL). Protein samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with HA antibodies against an N-terminal HA-tag in all the DAT cysteine constructs. Density of DAT bands were analyzed with an Odyssey imaging system and associated ImageStudio software (LI-COR, Lincoln, NE).</p><!><p>Dopamine uptake was measured by using synaptosomes prepared from rat striata. Adult male Sprague–Dawley rats were anesthetized for 3 min with 2.5% isoflurane before decapitation. Following decapitation, striata was immediately dissected, and homogenized in ice-cold Krebs–Ringer's buffer containing 0.32 M sucrose and protease inhibitors. The sample was centrifuged for 10 min at 1000g, the pellet was discarded, and the remaining supernatant was centrifuged for an additional 15 min at 16 000g. The resulting pellet (P2) was dissolved in uptake buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1 μM pargyline, 2 mg/mL glucose, 0.2 mg/mL ascorbic acid, pH 7.5). The uptake of radiolabeled dopamine (50 nM) was performed for 3 min at 25 °C in uptake buffer and terminated by filtering and extensive washes with uptake buffer using a PerkinElmer Filter Mate Universal Harvester. Background was estimated in the presence of cocaine (100 μM) or GBR12909 (1 μM). Radioactivity was measured with Wallac/PerkinElmer 1450–021 Microbeta Trilux Liquid Scintillation and Luminescence.</p><!><p>Voltammetry experiments were carried out as previously described.46 Adult male Sprague–Dawley rats were anesthetized for 3 min with 2.5% isoflurane before decapitation. Following decapitation, brains were quickly removed and transferred to ice cold oxygenated artificial cerebrospinal fluid (ACSF) containing, (in mM; NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), L-ascorbic acid (0.4), pH adjusted to 7.4. Coronal slices containing the NAc were prepared using a vibrating tissue slicer and were then transferred into a continuously oxygenated ACSF bath at room temperature. Following a 30 min recovery period, slices were transferred into a testing chamber flushed with ACSF (32 °C). A bipolar stimulating electrode (Plastics One, Roanoke VA) was placed on the surface of the slice and a carbon fiber microelectrode was placed within the NAc. DA release was elicited with single electrical stimulation (400 μA, 4 ms, monophasic) every 3 min. Baseline DA release and uptake were recorded until stability was achieved across three consecutive stimulations (<10% variation). Following baseline collections, slices were flushed with oxygenated ACSF containing 50 μM KM822 or just ACSF for control slices while collections continued. After 30 min, cocaine was applied cumulatively as previously described46 until each cocaine concentration elicited stable DA responding (approx. every 30 min).</p><p>Stimulated DA release and DA uptake measures (Vmax and Km) were assessed as described for the anesthetized FSCV experiments. Differences in baseline DA release and uptake were assessed using independent sample t tests, and differences in the effects of cocaine were assessed using a two-way ANOVA with drug as the between subject's variable and cocaine concentration as the repeated measures variable.</p><!><p>Planarians (Dugesia dorotocephala) were purchased from Carolina Biological Supply (Burlington, NC). Upon arrival in the laboratory, planarians were maintained in the aqueous solution provided by Carolina Biological Supply, acclimated to room temperature (21 °C), and tested within 3 days of receipt. Each planarian was used only once. Planarians were placed singly into a clear plastic Petri dish (14 cm diameter) containing room-temperature water (pH = 7.0) with or without KM822 (20 μM) and placed over grid paper (gridlines spaced 0.5 cm apart). Baseline locomotor velocity of each planarian was measured by counting the number of gridlines each planarian crossed over a 5 min observation period. The same planarian was transferred into another Petri dish containing cocaine (10 μM), amphetamine (5 μM), or nicotine (20 μM) with or without KM822 (20 μM), and drug-induced locomotion locomotor velocity of each planarian was again measured by counting the number of gridlines each planarian crossed over a 5 min observation period.</p><!><p>Data from experiments was analyzed and graphed using Prism 7.0 (GraphPad Software, Inc., La Jolla, CA). Specific details of statistical tests are given in each figure legend.</p>

PubMed Author Manuscript

Synaptotagmin-7 enhances calcium-sensing of chromaffin cell granules and slows discharge of granule cargos

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations \xe2\x80\x93 mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

synaptotagmin-7_enhances_calcium-sensing_of_chromaffin_cell_granules_and_slows_discharge_of_granule_

Introduction<!>Animals<!>Mouse chromaffin cells preparation and transfection<!><!>Western blotting<!>Reverse transcription and quantitative PCR<!>TIRF microscopy for observation of exocytosis<!>Cell stimulation<!>Image analysis<!>Immunocytochemistry<!>Electrophysiological recordings<!>Materials and Methods for Single granule/supported membrane fusion assay<!>PC12 Cell Culture<!>Dense core granule purification<!>Protein purification<!>Formation of planar supported bilayers with reconstituted plasma membrane SNAREs<!>TIRF microscopy for single granule/supported membrane fusion assay<!>Calcium-triggered single granule \xe2\x80\x93 planar supported membrane fusion assay<!>Statistical analysis<!>The localization of Syt-7 in mouse chromaffin cells<!>Endogenous Syt-7 slows dense core granule cargo release<!>Granule fusion probability and mobility in WT and Syt-7 KO cells<!>Stimulation of secretion in WT and Syt-7 KO cells with a physiological agonist<!>Sustained chromaffin cell secretory output relies on the presence of Syt-7<!>Syt-7 endows dense core granules with distinct Ca2+-sensing and fusion properties<!>Discussion

<p>Adrenomedullary chromaffin cells serve as a key effector arm of the sympathetic nervous system. In response to stimulation by preganglionic sympathetic nerves, they secrete a number of important hormones, including epinephrine, norepinephrine, neuropeptide Y (NPY), and tissue plasminogen activator (tPA) directly into the circulation (Guerineau 2019; Carmichael & Winkler 1985). The trigger for stimulus-evoked exocytosis in adrenal chromaffin cells is a rise in intracellular Ca2+. The level to which intracellular Ca2+ is elevated varies with the stimulus intensity and secretagogue (Augustine & Neher 1992; Fulop et al. 2005; Fulop & Smith 2007). Ca2+ regulates release by acting on the Ca2+-binding Syt-protein family (Chapman 2008; Geppert et al. 1994; Sudhof 2013) to drive its penetration into membranes that harbor anionic lipids (Bai et al. 2000; Bai et al. 2004). These actions result in subsequent zippering of the SNARE complex and opening of a fusion pore to permit diffusion or discharge of granule lumenal contents into the extracellular space (Murthy & De Camilli 2003).</p><p>Two Syts in particular – Syt-1 and Syt-7 –account for the vast majority of Ca2+-triggered release from chromaffin cells (Schonn et al. 2008). Although these proteins share a similar topology, they exhibit a number of biochemical differences. For example, in vitro, Syt-7 has a much higher affinity for Ca2+ in the presence of phospholipid membranes than Syt-1 (Bhalla et al. 2005; Sugita et al. 2002). In fact, Syt-7 stimulation of SNARE-mediated liposome fusion occurs with a 400-fold higher sensitivity to Ca2+ than Syt-1 (Bhalla et al. 2005). Once bound to Ca2+, Syt-7 also releases membranes at rates that are orders of magnitude faster than Syt-1(Bhalla et al. 2005; Sugita et al. 2002; Hui et al. 2005; Bendahmane et al. 2018). The biochemical distinctions between Syt-1 and Syt-7 have a strong bearing on their actions during chromaffin cell exocytosis. The much lower affinity of Syt-1 for Ca2+ compared to Syt-7 explains why small elevations in cytosolic Ca2+ levels from baseline are generally ineffective in driving fusion of granules bearing Syt-1, but much more effective at driving fusion of granules bearing Syt-7 (Rao et al. 2017a). Differences in the Ca2+ sensitivities of Syt isoforms, as well as their membrane association and dissociation rates, may also underlie the fast and slow kinetic components of the secretory response (Schonn et al. 2008). These components, revealed as a result of Ca2+ uncaging in combination with capacitance measurements, are differentially reliant on Syt-1 and Syt-7. The rapid phase of release is eliminated in the absence of Syt-1 (Schonn et al. 2008). The remaining delayed component exhibits a Ca2+-threshold that closely mirrors the low, micromolar-range sensitivity of Syt-7 for binding to phospholipids in the presence of Ca2+. Furthermore, deletion of Syt-7 in a Syt-1 knockout (KO) background, almost completely eliminates the slow phase of release (Schonn et al. 2008).</p><p>Thus, stimulation paradigms that elevate intracellular Ca2+ to varying degrees allow the impact of biochemical differences in the function of Syt-1 and Syt-7 on exocytosis to be readily appreciated. The purpose of this study is to delineate how such differences impinge on the release kinetics of cargos stored within dense core granules harboring Syt-7 and affect the secretory response to native, cholinergic stimulation. In order to define the localization and function of Syt isoforms – especially Syt-7 – in chromaffin cell exocytosis, multiple experimental preparations were employed. This includes primary chromaffin cells and an assay in which fusion of dense core granules with a planar membrane has been reconstituted (Kreutzberger et al. 2017b; Kreutzberger et al. 2017a; Kreutzberger et al. 2019). Immunolabeling of endogenous Syt proteins in mouse chromaffin cells as well as subcellular fractionation demonstrate that both Syt-1 and Syt-7 are principally sorted to intracellular organelles (including dense core granules). The role of Syt-7 in regulating the characteristics of exocytosis was ascertained by monitoring the discharge of fluorescently labeled lumenal cargo proteins in cells lacking endogenous Syt-7 (Syt-7 KO cells) using TIRF microscopy. These experiments revealed that cargos are discharged from Syt-7 KO cells at significantly faster rates than they are from WT cells, consistent with the idea that endogenous Syt-7 constrains fusion pore expansion (Jaiswal et al. 2004; Brahmachari et al. 1998; Rao et al. 2014; Rao et al. 2017a; Zhang et al. 2011).</p><p>The absence of Syt-7 has a pronounced detrimental effect on the secretory response to prolonged cholinergic stimulation. In fact, the likelihood of observing exocytosis at all declines sharply in KO cells after the first few seconds of perfusion with acetylcholine (ACh). In contrast, exocytotic events are evident throughout the stimulation period in WT cells, albeit with a lessening frequency as stimulation proceeds. Exocytosis is sustained in WT cells because of the high affinity of Syt-7 for Ca2+. This idea is underscored by reconstitution studies in which dense core granules consisting of only Syt-1 or Syt-7 were triggered to fuse with synthetic bilayers. Even in this setting, Syt-7-bearing granules fuse at significantly lower Ca2+ concentrations than Syt-1-bearing granules, with fusion pores that dilate at slower rates. Taken together, the data highlight clear functional distinctions in the roles of synaptotagmins. These properties have a direct bearing on the regulation of Ca2+-triggered exocytosis in cells and are likely to play a role in modulating adrenomedullary output, in situ.</p><!><p>Litters of adult male and female Syt-7 −/− (Catalogue # 004950, Jackson labs) (gift of Dr. Joel Swanson; (Chakrabarti et al. 2003) and Syt-7 +/+ (from a C57BL/6J background and obtained from Jackson Labs, Bar Harbor, ME, Catalogue # 000664) were used in these studies. Animals were group housed (2 to 5 per ventilated cage) with 24 hour (12/12 dark/light cycle) access to food and water. All animal procedures and experiments were conducted in accordance with the University of Michigan Institutional Animal Care and Use Committee protocol (PRO00007247). No randomization was performed to allocate subjects in the study.</p><!><p>Below, we describe a novel method for the isolation and culture of adult mouse chromaffin cells from the adrenal medulla. Although the protocol was adapted from previous studies (Kolski-Andreaco et al. 2007), it is different enough to warrant a more detailed description. Catalogue numbers and vendors of materials used can be found in Table 1. 6 to 16 week-old animals (male and female; 18 to 25 g) were gas anesthetized using an isoflurane drop jar technique and sacrificed by guillotine decapitation. Chromaffin cells are responsible for releasing catecholamines in response to stress, including hypoxia. Isoflurane is used to induce a faster loss of consciousness compared to CO2 euthanasia (30 s to 1 minute versus several minutes) and reduce animal stress. 3–4 mice are euthanized per plate to ensure proper cell density and health. Adrenal glands per condition were rapidly isolated and moved to dishes containing ice cold dissection buffer (148 mM NaCl, 2.57 mM KCl, 2.2 mM K2HPO4•3H2O, 6.5 mM KH2PO4, 10 mM glucose, 5 mM HEPES free acid, 14.2 mM mannitol). Under a dissection microscope, the cortex was rapidly and carefully removed using thin forceps (Dumont Swissmade, Switzerland Cat. # 72891-Bx) and thin micro scissors (World Precision Instruments, 14124-G). The isolated medullas were washed three times in 150 μL drops of enzyme solution containing (450 units/ml Papain (Worthington Biochemical. #LS003126), 250 μg/ml BSA, and 75 μg/ml dithiothreitol). The medullas were then digested for 15 minutes in 0.5 ml of the enzyme solution at 37°C. After 15 minutes, the digesting solution was carefully removed and replaced by 0.5 ml of fresh enzyme solution and left for a maximum of 15 extra minutes at 37°C. The digestion was stopped by transferring the glands into an antibiotic-free culture medium (Dulbecco's Modified Eagle's Medium (DMEM) (ThermoFisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS) (ThermoFisher Scientific)). The digested glands were then triturated by a push-pull movement through a 1 ml pipette tip (10 to 12 times). The suspension of digested and broken glands was spun at 1300 x g for 2.5 minutes. The supernatant was discarded, and the pellet was re-suspended in antibiotic-free medium and triturated again in a 200 μl pipette tip for a better cell dissociation (10 to 12 times). The suspension was spun again at 1300 x g for 2.5 minutes. After discarding the supernatant, the pellet was re-suspended in resuspension buffer (Invitrogen, Thermofisher Scientific) for transfection. The cells were rapidly counted and the desired plasmid was added (15 ng/106 cells). The suspended cells were then transiently transfected by electroporation with a single pulse (1050 mV, 40 ms) using the Neon transfection system (Invitrogen, Thermofisher Scientific). In parallel, 35 mm diameter dishes with 14 mm diameter glass-bottom dishes (MatTek Corporation, Ashland, MA. #P35G-1.5–14-C) were pre-coated with Matrigel (Corning, NY, Cat. #356230) diluted in DMEM (1:7) for two hours after which the dishes were washed with DMEM and let to dry. After electroporation, an antibiotic-free medium was added to cells to obtain a final concentration of 1 million cells per ml. Three hundred microliters of the final solution containing the electroporated cells were then deposited in each dish. The cells were stored in an incubator (37°C, 5% CO2) for three to five hours. Culture medium with antibiotics was then added to a final volume of 2 ml (DMEM supplemented with 10% FBS, 9.52 unit/ml Penicillin, 9.52 μg/ml Streptomycin and 238 μg/ml Gentamicin (ThermoFisher Scientific). The media was changed daily, and cells were used within 48 hours after plating. The method we describe here provides consistently healthy cells (Figure S1) which exhibit a high probability of secretion upon stimulation. As a matter of course, cell preps and experiments were usually performed during normal working hours (9 am – 5 pm).</p><p>Chromaffin cells were transfected with rat myc-Syt-7 or cargo proteins, including human Neuropeptide Y (NPY) and human tissue plasminogen activator (tPA). The mycSyt-7 plasmid was a gift from Dr. Thomas Sudhof. The fluorescent tag was located following the C-terminal region of the cargo proteins. NPY and tPA constructs (originally in pEGFP-N1 vectors) were provided by Dr. Ronald W. Holz.</p><!><p>Immunocytochemistry (Syt-7 + Syt-1): 4 KO and 4 WT</p><p>Immunocytochemistry (Syt-7 + PAI-1): 8 KO and 8 WT</p><p>Secretion (KCl): 46 KO and 46 WT</p><p>Secretion (ACh): 32 KO and 32 WT</p><p>Ca2+ imaging: 12 KO and 12 WT</p><p>Electrophysiology: 12 KO and 12 WT</p><p>Total: 114 KO and 114 WT (males and females, mixed)</p><!><p>Catalogue numbers, vendors of materials used and appropriate dilutions can be found in Table 1. Lysis buffer containing 8 M urea, 5% SDS, and 5 mM N-ethylmaleimide in water was heated to 65°C. 6 to 16 week-old animals (male and female; 18 to 25 g) were gas anesthetized using an isoflurane drop jar technique and sacrificed by guillotine decapitation. Adrenal glands were dissected from five months-old mice and immediately frozen in liquid nitrogen, then handed over to a colleague blinded to the genotype of the mice to complete the western blot. Four total adrenal glands from two mice of each genotype were dissolved into 200 μL of warm lysis buffer, and homogenized using a handheld, battery-operated homogenizer. The homogenate was incubated at 65°C for 20 minutes and mixed 1:1 with 5x PAGE buffer (5% (wt/vol) SDS, 25% (wt/vol) sucrose, 50 mM Tris pH 8, 5 mM EDTA, & bromophenol blue). The lysates were stored at −20°C until use. Samples (10 μL/well) were separated on a 4–12% NuPAGE Bis-Tris Gel in 1x NuPAGE MOPS SDS Running Buffer, for 1 hour at 175 mV. Transfer to nitrocellulose membrane occurred at 120 mV for 1.5 hours, on ice, in 1x NuPAGE transfer buffer. The membrane was blocked with blocking buffer containing 5% Bovine Serum Albumin (BSA) and 0.1% tween in TBS (TBS-T) for 1 hour, before incubation with primary antibodies (Rabbit anti Synaptotagmin-7 1:1000, Synaptic Systems, and Mouse anti alpha-tubulin, 1:10,000, Cedarlane; RRID:AB_10060319) at 4°C, overnight. The membrane was washed 3× 15 minutes with TBS-T and incubated for 1 hour with LiCor fluorescent secondaries (1:10,000) in blocking buffer (multiplexed). After washing 3× 15 minutes in TBS-T, the membrane was imaged using LiCor Odyssey Clx imager.</p><p>Chromaffin cells are isolated from bovine adrenal glands as previously described (Holz et al. 1982). Briefly, adrenal glands were obtained from a slaughterhouse and delivered to the lab. Surrounding fat and connective tissues were removed carefully and adrenal glands were perfused with physiological saline solution (PSS) through adreno-lumbar vein to remove residual blood. Glands were then digested at 37°C by perfusing with enzymatic solution A (Liberase TH) for 30 – 40 minutes. Digested medullas were collected and digested again at 37°C in enzymatic solution B (Liberase TH and TL mixture) for 30 minutes under constant agitation. This is then passed through 400 μm filter to obtain chromaffin cells. Subcellular fractionation of chromaffin cells was performed following a published protocol (Kreutzberger et al. 2019) with slight modifications. The fractions (400 μl) were collected for western blotting. Blots containing the transferred proteins were blocked in 5% milk and incubated overnight at 4°C with the following primary antibodies: Rabbit anti Synaptotagmin-7 (1:1000, Synaptic Systems, Cat.no. 105173; RRID: AB_887838), Mouse anti Synaptotagmin-1 (1:1000, Synaptic Systems,Cat # 105 011, 1:1200; RRID: AB_887832), Rabbit anti PAI-1 (1:500, Abcam, Cat. no. ab66705; RRID: AB_1310540), Rabbit anti LAMP-1 (1:1000, Abcam, Cat. no. ab24170; RRID: AB_775978). Following primary antibody incubation, blots were washed with TBS-T and incubated with HRP conjugated secondary antibodies (anti-mouse HRP 1:5000, Cat.no. NXA931V; RRID: AB_2721110 or anti-rabbit HRP 1:5000, Cat.no NA934V; RRID: AB_772206) diluted in 5% milk. Chemiluminescent substrate (Thermofisher) was used to visualize blots on iBright Imager (Invitrogen). Protein band intensity was quantified using Fiji software.</p><!><p>Reverse transcription was performed on mouse adrenal medullas dissected from adrenal glands and homogenized. Adrenal medullas from two animals in each WT and Syt-7 KO group are considered as one experiment. Specifically, four adrenal medullas from two animals were homogenized in one 1.5-ml Eppendorf tube on ice for ~45 seconds with motorized pestle mixer (Argos Technologies, Inc, Vernon Hills, IL). More than three experiments were performed for each target. RNeasy Mini (Qiagen, Valencia, CA) was used to isolate the RNAs. The first strand cDNA synthesis was performed with 400 ng of RNAs using the qScript cDNA SuperMix kit (Quanta Biosciences, Beverly, MA). The reverse transcription product was kept at −20°C until qPCR was performed. qPCR primers for the target genes were designed with online tools (GenScript PCR Primer Design and NCBI primer designing tool). The forward (fw) and reverse (rv) primer sequences are as follows: GAPDH fwCTGACGTGCCGCCTGGAGAAGAPDH rvCCCGGCATCGAAGGTGGAAGASyt-1 fwGGCGCGATCTCCAGAGTGCTSyt-1 rvGCCGGCAGTAGGGACGTAGCSyt-7 fwCCAGACGCCACACGATGAGTCSyt-7 rvCCTTCCAGAAGGTCTGCATCTGGNPY fwGTGTGTTTGGGCATTCTGGCNPY rvTGTCTCAGGGCTGGATCTCTtPA fwCTCGGCCTGGGCAGACACAAtPA rvAGGCCACAGGTGGAGCATGGTH fwGCGCCGGAAGCTGATTGCAGTH rvCCGGCAGGCATGGGTAGCAT</p><p>Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) was used as an endogenous control run in parallel with target genes. Each assay was performed in triplicate. For the qPCR, we used the PerfeCTa SYBR Green SuperMix (Quanta Biosciences, Beverly, MA). Ten microliters of reverse transcription product was added to the master mix with 10 μM of each primer. The directions for the PCR protocol were followed per the manufacturer's instructions. The qPCR was performed using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Melting curves were analyzed to verify that no primer dimers were produced.</p><!><p>TIRF imaging was performed using an Olympus cellTIRF-4Line microscope (Olympus, USA) equipped with a TIRF oil-immersion objective (NA 1.49) and an additional 2x lens in the emission path between the microscope and the cooled electron-multiplying charge-coupled device Camera (iXon 897; Andor Technology). The final pixel size of the images was 80 nm. Series of images were acquired at ~ 20 Hz using CellSense software with an exposure time of 30 ms and an EM gain of 100. pHl and GFP were excited using a 488 nm laser.</p><!><p>All TIRF experiments were performed at room temperature ~ 24° C. The culture medium was replaced by pre-warmed (37°C) physiological salt solution (PSS) (145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 15 mM HEPES, pH 7.4). Chromaffin cells were individually stimulated using a needle (100-μm inner diameter) connected to a perfusion system under positive pressure ALA-VM4 (ALA Scientific Instruments, Westbury, NY). To trigger exocytosis, cells were first perfused with PSS for 5 – 10 s and then stimulated with high potassium containing solution (100 mM KCl) for 70–75 s. For the acetylcholine experiment, 100 μM acetylcholine (Sigma-Aldrich) diluted in PSS was perfused for 120 s.</p><!><p>Fusion sites of granules containing GFP or pHl-tagged proteins undergoing exocytosis were identified. The abruptness with which NPY-GFP puncta disappear is taken as indication that fusion and release has occurred. In the event of slower cargo release, an increase in fluorescence often precedes the disappearance of GFP puncta as the granule moves closer to the brightest part of the evanescent field and the lumenal pH of the granule becomes more neutral (Taraska et al. 2003). Regions of interest (ROIs) measuring 0.8 μm diameter were manually selected at fusion sites and image sequences were analyzed using the Time Series Analyzer v3.0 plugin on Fiji software. For each ROI, the fluorescence intensity was measured for each frame. A nearby ROI of the same size where no fusion events were observed was selected for background subtraction. Using a custom program written in Interactive Data Language (IDL; ITT, Broomfield, CO) by Dr. Daniel Axelrod (University of Michigan), background subtracted intensity versus time curves were plotted and the duration of cargo release was calculated (Bohannon et al. 2017; Abbineni et al. 2019). Briefly, after a user selects fusion events from blinded files, the program generates fluorescence-versus-time curves for each one. A user selects a start time, tstart, just before fluorescence begins and an end time, tend, when fluorescence returns to baseline. The program determines the time of the maximum fluorescence tmax within this time window. The intervals (tstart, tmax) and (tmax, tend) are defined as the rise phase and fall phase, respectively. Each phase is fit with a fifth-degree polynomial function, and a weighted average slope is calculated (upward for the rising phase, and downward for the falling phase). The time period between the baseline intercept of the rising phase straight line and the falling phase straight line is considered to be the duration of the event (Bohannon et al. 2017). Each fusion event was further analyzed by the user to confirm that only events in which cargos were completely released were used in the analysis. Illustrations of the output of the curve-fitting procedure and examples of release durations calculated by the program, are reported in Bohannon et al. (2017) and in Bendahmane et al. (2018).</p><p>Cells transfected with GCaMP5G (Akerboom et al. 2012) were analyzed to determine the relative amount of calcium influx into the cell. Three ROIs measuring 1.68 μm diameter were manually selected at different points within each cell and fluorescence was measured for each ROI for each frame. The equation ΔF/F was applied to each ROI for each frame, then the values were averaged between the three ROIs to determine the overall ΔF/F for the whole cell. The values determined were plotted versus time. The exclusion criteria used to select which cells to analyze were based on proper fixation to the dish, no response to the control stimulation, and single response tracings that indicated calcium influx in response to the stimulus trigger.</p><p>As a matter of routine, imaging and analysis was performed by an experimenter who was blinded to the genotype of the cell. The individual who euthanized the mice assigned numbers to each groups and then handed over said groups to the experimenter who continued with the preparation. Whether the cells were harvested from WT or KO mice was revealed after analysis was completed. Chromaffin cells that appeared healthy in brightfield (Figure S1) with at least 10 docked granules, and responded to KCl or ACh stimulation with at least 1 fusion event, were included in data sets. No sample calculation was used.</p><!><p>Immunofluorescence imaging was performed to assess the distribution of endogenous Syt-1, Syt-7, LAMP-1 (Abcam cat# 25630; RRID: AB_470708), and PAI-1 in chromaffin cells. Mouse chromaffin cells were plated on the same Matrigel-precoated 14 mm glass-bottom dishes used for TIRF imaging. All incubations and washing steps were performed on ice unless otherwise stated. Twenty-four hours after plating, the cells were fixed with 4% paraformaldehyde in phosphate buffered solution (PBS) for 30 min. The fixed cells were quickly rinsed with PBS and quenched with 50 mM NH4Cl solution in PBS for 30 min. After a brief wash from the NH4Cl solution with PBS, the cells were permeabilized with methanol for 7 min at −20°C. Following the permeabilization, the cells were washed in Tris-buffered saline (TBS) and blocked in 0.01% gelatin solution for 30 minutes followed by another 30 min incubation in 4% donkey serum and 0.2% bovine serum albumin (BSA) prepared in TBS. Primary and secondary antibodies were diluted in TBS at 0.2%. Cells were incubated for two hours with a combination of polyclonal rabbit anti-Syt-7 antibody (Synaptic Systems, Göttingen, Germany. Cat # 105 173, 1:1200; RRID: AB_887838), and monoclonal mouse anti-Syt-1 (Synaptic Systems, Göttingen, Germany. Cat # 105 011, 1:1200; RRID: AB_887832). The cells were then washed and incubated for 70 minutes at room temperature with Alexa 488/561-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, Invitrogen). The double labeled cells were then washed and kept a 4°C until confocal imaging.</p><p>For co-labeling of endogenous PAI-1 and Syt-7, both the polyclonal anti-Syt-7 and polyclonal anti-PAI-1 (Abcam cat# 66705; RRID: AB_1310540) antibodies were made in rabbit. In this experiment, to avoid cross-labeling, the cells were first labeled with anti-Syt-7 following the protocol described above. Cells labeled for Syt-7 were then incubated for 60 minutes with polyclonal cross-adsorbed unconjugated F(ab')2-Goat anti-Rabbit IgG (Invitrogen, ThermoFisher, Cat. # A24539, 1:300; RRID: AB_2536007) diluted in TBS-0.2% BSA solution to bind the possibly remaining free primary antibody sites (anti-Syt-7) not bound by the Alexa 488conjugated donkey anti-rabbit.</p><p>Labeling for PAI-1 was performed separately. Briefly, the primary and secondary antibodies (anti-PAI-1, 1:600 – Alexa 561-conjugated anti rabbit, 1:600) were incubated for 70 minutes in reaction tube pre-adsorbed in dry milk 1% diluted in TBS (Kroeber et al. 1998). After incubation, normal rabbit serum (Invitrogen, ThermoFisher Scientific, Cat. # 016101; RRID: AB_2532937) was added to the tube (10% volume/volume dilution) to bind the free secondary antibody for 60 minutes. The mix was then added in the Syt-7 labeled dishes for two hours. In control dishes, either the Syt-7 or PAI-1 antibodies were omitted (Figure S2) to verify the absence of cross-labeling. Cells were subsequently imaged using a Zeiss 880 confocal microscope (Zeiss, Oberkochen, Germany) with a 63x oil immersion objective in airyscan mode. Images were analyzed using Imaris Software (Bitplane, Zurich, Switzerland) using the "spot" function module for puncta detection and colocalization analysis (Rao et al., 2017). Catalogue numbers, vendors of materials used and appropriate dilutions can be found in Table 1.</p><!><p>Primary cultures of mouse chromaffin cells were maintained on glass bottom dishes and mounted onto the stage of a Nikon Eclipse TE2000, as previously described (Rao et al. 2017a). A micro-manifold and polyamide-coated capillary was positioned in the field of view and bath solutions were exchanged through using a pressure-driven reservoir system. Standard whole-cell patch clamp methods were used record currents evoked by acetylcholine or by step depolarizations using an Axopatch 200B amplifier and Pulse Control/Igor Pro software. Patch pipettes were constructed out of 1.5 mm o.d. borosilicate glass (#TW150F-4; WPI, Sarasota, FL), coated with Sylgard elastomer (#24236–10, EMS, Hatfield, PA) and fire polished to resistances of 2.5–7 MΩ. The standard intracellular recording solution contained (in mM): 128 N-methyl-d-glucamine-Cl, 40 HEPES, 10 NaCl, 4 MgATP, 0.2 GTP, 0.1 Tris-EGTA, and pH adjusted to 7.2. IACh were induced by 10 s to 300 s applications of 100 μM ACh and recorded in physiological saline (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 25 mM glucose, pH 7.4). For recording of ICa, the superfusion solution was changed to a solution containing (in mM): 137 tetraethylammonium chloride, 5 CaCl2, 2 MgCl2, 10 HEPES, and 19 glucose, and pH adjusted to 7.2 with Tris. ICa current-voltage relationship was obtained in response to step depolarizations (30 ms) from a holding membrane potential of −90 mV. Pulses were applied in a randomized series to membrane potentials between −80 and +60 mV. All recordings were performed at room temperature. Cells with leak current greater than −80 pA or access resistance greater than 45 MΩ were excluded from our analysis.</p><!><p>The following materials were purchased and used without further purification: porcine brain L-α-phosphatidylcholine (bPC), porcine brain L- α-phosphatidylethanolamine (bPE), porcine brain L- α-phosphatidylserine (bPS), and L- α-phosphatidylinositol (liver, bovine) (PI), and porcine brain phosphatidylinositol 4,5bisphosphate (bPIP2) were from Avanti Polar Lipids; cholesterol, sodium cholate, EDTA, calcium, Opti-Prep Density Gradient Medium, sucrose, MOPS, glutamic acid potassium salt monohydrate, potassium acetate, and glycerol were from Sigma; CHAPS and DPC were from Anatrace; HEPES was from Research Products International; chloroform, ethanol, Contrad detergent, all inorganic acids and bases, and hydrogen peroxide were from Fisher Scientific. Water was purified first with deionizing and organic-free 3 filters (Virginia Water Systems) and then with a NANOpure system from Barnstead to achieve a resistivity of 18.2 MΩ/cm. Antibodies for synaptotagmin-1 (mouse monoclonal), synaptotagmin-7 (rabbit polyclonal) are from Synaptic Systems.</p><!><p>Pheochromocytoma cells (PC12), gifted from Tom Martin, with endogenous synaptotagmin-1 and −9 knocked down, described previously (Kreutzberger et al. 2017a), were cultured on 10 cm plastic cell culture plates at 37°C in 10% CO2 in 1x Dulbecco's modified Eagle's medium, high glucose (Gibco) supplemented with 10% horse serum (CellGro), 10% calf serum (Fe+) (HyClone), 1% penicillin/streptomycin mix, and 2 μg/ml of puromycin. Medium was changed every 2 to 3 days and cells were passed after reaching 90% confluency by incubating for 5 minutes in Hank's balanced salt solution and replating in fresh medium. Cells were passed no more than 30 times total. Cells were transfected by electroporation using ECM 830 Electro Square Porator (BTX). After harvesting and sedimentation, cells were suspended in a small volume of sterile cytomix electroporation buffer (120 mM KCl, 10 mM KH2PO4, 0.15 mM CaCl2, 2 mM EGTA, 20 mM HEPES-KOH, 5 mM MgCl2, 2 mM adenosine triphosphate, and 5 mM glutathione (pH 7.6), then diluted to ~14 × 106 cells/ml. Cell suspensions (~10 × 106 cells in ~700 μl volume) and 30 μg of NPY-mRuby DNA and 30 μg of synaptotagmin-1 or −7 DNA added and placed in an electroporator cuvette with a 4-mm gap. Then two 255-V, 8-ms electroporation pulses were applied. Cells were immediately transferred to a 10-cm cell culture dish with 10 ml of normal growth medium. Cells were cultured under normal conditions for 3 days prior to fractionation.</p><!><p>Dense core granules from PC12 cells were purified as described previously (Kreutzberger et al. 2017a). PC12 cells with shRNA mediated knockdowns of endogenous synaptotagmin-1 and −9 were transfected with NPY-mRuby (~20 10-cm plates) and plasmids for synaptotagmin-1 or −7 (Kreutzberger et al. 2017a; Kreutzberger et al. 2019). Cells were scraped into PBS and pelleted by centrifugation and then suspended and washed in homogenization medium (0.26 M sucrose, 5 mM MOPS, 0.2 mM EDTA) by pelleting and re-suspending. Following re-suspension in 3 ml of medium containing protease inhibitor (Roche Diagnostics), the cells were cracked open using a ball bearing homogenizer with a 0.2507-inch bore and 0.2496-inch diameter ball. The homogenate was spun at 4,000 rpm for 10 minutes at 4° C in fixedangle micro-centrifuge to pellet nuclei and larger debris. The post-nuclear supernatant was collected and spun at 11,000 rpm (8000 x g) for 15 min at 4° C to pellet mitochondria. The post-mitochondrial supernatant was then collected, adjusted to a final concentration of 5 mM EDTA, and incubated 10 min on ice. A working solution of 50% Optiprep (iodixanol) (5 vol 60% Optiprep: 1 vol 0.26M sucrose, 30 mM MOPS, 1 mM EDTA) and homogenization medium were mixed to prepare solutions for discontinuous gradients in Beckman SW55 tubes: 0.5 ml of 30% iodixanol on the bottom and 3.8 ml of 14.5% iodixanol, above which 1.2 ml EDTA-adjusted supernatant was layered. Samples were spun at 45,000 rpm (190,000 x g) for 5 hrs. A clear white band at the interface between the 30% iodixanol and the 14.5% iodixanol was collected as the dense core granule sample. The dense core granule sample was then extensively dialyzed in a cassette with 10,000 kDa molecular weight cutoff (24–48 hrs, 3 × 5L) into the fusion assay buffer (120 mM potassium glutamate, 20 mM potassium acetate, 20 mM HEPES, pH 7.4).</p><!><p>Syntaxin-1a (constructs of residues 1–288), SNAP-25, Munc18, and Munc13 (construct of residues 529–1407 containing the C1C2MUN region), and complexin-1 from Rattus norvegicus were expressed in Escherichia coli strain BL21(DE3) cells described previously (Zdanowicz et al. 2017; Kreutzberger et al. 2017b; Kreutzberger et al. 2017a).</p><!><p>Planar supported bilayers with reconstituted plasma membrane SNAREs were prepared by Langmuir-Blodgett/vesicle fusion technique as described in previous studies (Domanska et al. 2009; Kalb et al. 1992; Wagner & Tamm 2001). Quartz slides were cleaned by dipping in 3:1 sulfuric acid:hydrogen peroxide for 15 minutes using a Teflon holder. Slides were then rinsed in milli-Q water. The first leaflet of the bilayer was prepared by Langmuir-Blodgett transfer onto the quartz slide using a Nima 611 Langmuir-Blodgett trough (Nima, Conventry, UK) by applying the lipid mixture of 70:30:3 bPC:Chol:DPS from a chloroform solution. Solvent was allowed to evaporate for 10 minutes, the monolayer was compressed at a rate of 10 cm2/minute to reach a surface pressure of 32 mN/m. After equilibration for 5 minutes, a clean quartz slide was rapidly (68 mm/minute) dipped into the trough and slowly (5 mm/minute) withdrawn, while a computer maintained a constant surface pressure and monitored the transfer of lipids with head groups down onto the hydrophilic substrate.</p><p>Plasma membrane SNARE containing proteoliposomes with a lipid composition of bPC:bPE:bPS:Chol:PI:PI(4,5)P2 (25:25:15:30:4:1) were prepared by mixing the lipids and evaporating the organic solvents under a stream of N2 gas followed by vacuum desiccation for at least 1 hour. The dried lipid films were dissolved in 25 mM sodium cholate in a buffer (20 mM HEPES, 150 mM KCl, pH 7.4) followed by the addition of an appropriate volume of synatxin-1a and SNAP-25 in their respective detergents to reach a final lipid/protein ratio of 3,000 for each protein. After 1 hour of equilibration at room temperature, the mixture was diluted below the critical micellar concentration by the addition of more buffer to the desired final volume. The sample was then dialyzed overnight against 1 L of buffer, with one buffer change after ~4 hour with Biobeads included in the dialysis buffer. To complete formation of the SNARE containing supported bilayers, proteoliposomes were incubated with the Langmuir-Blodgett monolayer with the proteoliposome lipids forming the outer leaflet of the planar supported membrane and most SNAREs oriented with their cytoplasmic domains away from the substrate and facing the bulk aqueous region. A concentration of ~77 mM total lipid in 1.2 ml total volume was used. Proteoliposomes were incubated for 1 hour and excess proteoliposomes were removed by perfusion with 5 ml of buffer (120 mM potassium glutamate, 20 mM potassium acetate (20 mM potassium sulfate was used in buffers with acridine orange labeled granules), 20 mM HEPES, 100 mM EDTA, pH 7.4).</p><!><p>Dense core granule to planar supported bilayer fusion assay experiments were performed on a Zeiss Axio Observer 7 fluorescence microscope (Carl Zeiss), with a 63x water immersion objective (Zeiss, N.A. 0.95) and a prism-based TIRF illumination. Laser light at 514 nm from an argon ion laser (Innova 90C, Coherent), controlled through an acousto-optic modulator (Isomet), and at 640 nm from a diode laser (Cube 640, Coherent) were used as excitation sources. The characteristic penetration depths were between 90 and 130 nm. An OptoSplit (Andor Technology) was used to separate two spectral bands (540 nm – 610 nm, and 655 nm – 725 nm). Fluorescence signals were recorded by an EMCCD (iXon DV887ESC-BV, Andor Technology).</p><!><p>As previously described (Kreutzberger et al., 2017a), planar supported bilayers containing syntaxin-1a (1–288): dodecylated (d) SNAP-25 (bulk phase-facing leaflet lipid composition of 25:25:15:30:4:1 bPC:bPE:bPS:Chol:PI:bPIP2) were incubated with 0.5 μM Munc18 and 2 μM complexin-1. Secretory granules were then injected while keeping the concentrations of Munc18 and complexin-1 constant. Dense core granule docking was allowed to occur for ~20 minutes before the chamber was placed on the TIRF microscope and the microscope was focused on the planar supported membrane. Fluorescent images were recorded every 200 ms while buffer containing 100 μM calcium and a soluble Alexa647 dye to monitor the arrival of calcium at the observation site was injected.</p><p>Movies were analyzed as previously described (Domanska et al. 2010; Kreutzberger et al. 2017b; Kreutzberger et al. 2017a). Fusion efficiencies are reported as the percentage of granules in the field of view that fuse within 15 s. Fluorescent line shapes are presented as average from 20 single events as described previously (Kreutzberger et al. 2017b).</p><!><p>Graphpad Prism 7 was used for analysis of data collected from TIRF studies, immunocytochemistry, electrophysiological data and qPCR. Igor was used to fit and analyze data collected from single granule /supported membrane fusion assays. Data collected for studies utilizing primary chromaffin cell culture were collecting using at least 3 different cell preparations. Specifically, co-localization studies include the average and standard error of the mean (SEM). Imaging studies on the release of cargos were analyzed using non-linear regressions and Tukey's multiple comparisons tests (p<0.05) to determine if there were significant differences between groups. Fusion probability and release data were statistically analyzed using a nonparametric Mann-Whitney test, p<0.05. The choice of using a parametric or nonparametric test was determined by an F-test or a Brown-Forsythe test which determines if variances are significantly different; values are reported in results section if nonparametric tests were used. No sample calculation was performed to determine appropriate sample sizes. The ROUT method was used to identify outliers (Graphpad Prism).</p><!><p>Fluorescent labeling of endogenous Synaptotagmin-1 and −7 in bovine adrenal chromaffin cells revealed these proteins to exhibit a punctate intracellular distribution, with a low degree of co-localization (Rao et al. 2014). A similar pattern is observed in chromaffin cells harvested from the mouse adrenal medulla. As shown in Figure 1, endogenous Syt-1 and Syt-7 fluorescence is largely non-overlapping (in the range of 3 – 5%), punctate, and intracellular (Figure 1 A, B). It was previously reported that Syt-7 is sorted to the plasma membrane in PC12 cells and rat chromaffin cells (Sugita et al. 2001). However, the punctate pattern of staining observed here is most consistent with the protein being sorted to organelles. The co-localization of Syt-7 with plasminogen activator inhibitor-1 (PAI-1) (Figure 1 C, D) – a ubiquitous dense core marker in chromaffin cells (Bohannon et al. 2017; Abbineni et al. 2019) – shows that Syt-7 is present in secretory granules. Confocal imaging of immunolabeled Syt-7 demonstrates that it is co-localized with lysosome associated membrane protein-1 (LAMP-1), in the range of 14 – 18% (Figure 1 E, F). LAMP-1 and PAI-1 exhibited a roughly 10 – 11% co-localization frequency (Figure 1 G, H). LAMP-1 has been previously shown to label both granules and lysosomes in chromaffin cells (Hao et al. 2015).</p><p>To further characterize the subcellular distribution of Syt-7, bovine chromaffin cell lysates were layered on an iodixanol gradient and centrifuged to separate organelles by density (see Methods). Sequential fractions of 400 μl were collected and run on a polyacrylamide gel for western blotting. Immunoreactive bands corresponding to PAI-1, Syt-7, Syt-1, and LAMP-1 are evident in Figure S3. Given the intensity of the PAI-1 reactivity, fraction 13 likely contains dense core granule constituents. Less dense fractions (4–6) presumably contain lysosomes or endosomes. Syt-7 and Syt-1 also appear in these lighter fractions, in addition to those that contain PAI-1.</p><!><p>A major goal of this study was to delineate the ways in which the chromaffin cell secretory system depends on the presence of Syt-7. To avoid the potential ambiguities associated with overexpression of synaptotagmins in cells already expressing a full complement of endogenous proteins, most of the experiments in this study instead rely on cells harvested from Syt-7 KO mice. Figure S4A shows that compared to WT cells, expression of Syt-1 transcript remains unchanged in KO cells, while Syt-7 transcript is almost undetectable. Western Blot was also performed on adrenal gland lysates to verify the loss of Syt-7 protein in the KO. The alpha variant of Syt-7 (403 amino acids), targeted by Andrews and colleagues when generating the original Syt-7 KO mouse, has been reported to run at approximately 45 kDa (marked by arrow) (Martinez et al., 2000). That band is absent in the KO (Figure S4B).</p><p>Our first objective was to determine the release rate of NPY-pHl in Syt-7 KO cells compared to WT cells. Cells expressing fluorescent protein were identified by a brief exposure to 10 mM NH4Cl (Anantharam et al., 2010). Single cells were then depolarized by local perfusion of 100 mM KCl while exocytosis was imaged with a TIRF microscope. Figures 2A, B, and D show that the time necessary for NPY to be completely released from fused chromaffin granules is broadly distributed in WT cells (times ranged from 0.049 s to 8.10 s; the data are not normally distributed). In Syt-7 KO cells, NPY release durations were shifted to shorter times (ranging from 0.084 s to 1.17 s) (Figure 2C and D). A cumulative frequency distributions representing the time at which granules fused with respect to the start of stimulation, are plotted in Figure S5.</p><p>To assess if Syt-7 is responsible for slower rates of NPY release, myc-tagged Syt-7 and NPY-pHl (myc expression was verified post hoc) were co-expressed Syt-7 KO cells. Cells were stimulated with KCl and the rate of NPY release determined, as above. In cells expressing myc-Syt-7, a slow population of NPY release times was again observed (times ranged from 0.076 s to 8.05 s) (Figure 2D).</p><p>Certain lumenal cargo proteins, such as tissue Plasminogen Activator (tPA), exhibit intrinsically slower release or "discharge" times during exocytosis (Perrais et al., 2004; Weiss et al., 2014). In the case of tPA, this has been attributed to the protein's ability to stabilize the curvature of the fusion pore and thereby limit its rate of expansion (Bohannon et al., 2017). Here, the goal was to address whether tPA release from fused granules may be hastened by the abrogation of Syt-7 expression – a protein which imposes its own constraints on fusion pore expansion (Rao et al., 2014; Rao et al., 2017). As before, the rate of release of tPA-pHl was measured in both WT and Syt-7 KO cells depolarized with KCl. The average rate at which tPA is released is considerably slower than it is for NPY (tPA: 6.57 ± 0.59 s, NPY: 0.67 ± 0.13 s, Mann Whitney test, p<0.0001, Brown-Forsythe test, p<0.05) (Figure 2E). Transfection with tPA yielded secretion events from WT cells with a large distribution of release times ranging from 68 ms to 30.67 s (average time = 6.57 ± 0.59 s; n = 103 events). In Syt-7 KO cells, the distribution of the release time was somewhat narrower, and ranged from 84 ms to 17.44 s (average time = 3.16 ± 0.38 s; n = 120 events). The overexpression of myc-Syt-7 in the Syt-7 KO cells again restored the broader distribution of release times, which ranged from 100 ms to 33.4 s (average time = 7.00 ± 0.62 s; n = 89 events). * p<0.05, ** p<0.005 Tukey's multiple comparison test.</p><!><p>The number of NPY-GFP-labeled, docked granules that fused in response to 100 mM KCl depolarization in WT and KO cells was determined and used to calculate a granule fusion probability (Figure 3A; see also Supplementary Movie 1). The criterion for "docked" in this case, is that the granule is resident in the evanescent field at the start of KCl stimulation. The absence of Syt-7 severely disrupts the secretory response to KCl-based depolarization. This is evidenced by the fact that fusion probability of granules in KO cells is approximately 5-fold lower than that of granules in WT cells (Figure 3B).</p><p>In a previous study, we had reported that the frame-to-frame displacement (ΔR) of granules in bovine chromaffin cells harboring GFP-Syt-7 was, on average, lower than that of granules harboring GFP-Syt-1 (Rao et al., 2017). Thus, we also set out to determine whether granule mobility varies as a function of Syt-7 expression. NPY-GFP-labeled granules in WT and KO cells were tracked over a minimum of 200 frames (10 s in duration) (Figure S6A and B). From those tracks, ΔR of individual granules was calculated (Figure S6C) (Rao et al. 2017a). The distribution of ΔRs for granules from both cell-types, WT and KO, were best fit to a sum of two gaussians. The average ΔR from the slower WT population was 29.32 +/− 1.41 nm, and 48.39 +/− 1.13 nm for the faster population, with 53.58 +/− 30.67% of granules residing in the slower population. The differences between the slower and faster populations of KO granules were less pronounced, with a slow population ΔR of 27.82 +/− 0.59, a fast population ΔR of 39.97 +/− 3.76 nm, and 47.84 +/− 59.77% of granules residing in the slower population. Thus, the mobility of chromaffin granules labeled with NPY-GFP was not significantly different between WT and Syt-7 KO groups.</p><!><p>Chromaffin cell secretion, in situ, is triggered by activation of nicotinic receptors and subsequent Ca2+ influx (Douglas & Rubin 1963; Douglas 1968). Therefore, to understand how the lack of Syt-7 might disrupt secretion in a physiological setting, WT and KO cells were stimulated with ACh delivered locally via a perfusion pipet. ACh-triggered release kinetics of NPY-pHl was measured first (Figure 4A–D). As with elevated KCl stimulation, the range of NPY release times in response to ACh stimulation was broader in WT cells (0.06 s to 24.05 s) than in Syt-7 KO cells (0.07 s to 2.93 s). On average, the rate at which NPY is released was slower in WT cells compared to Syt-7 KO cells (WT: 1.47 ± 0.34 s, KO: 0.44 ± 0.1 s, Mann Whitney test, p < 0.05, Brown-Forsythe, p<0.05) (Figure 4D). Moreover, the overall fusion probability of NPY-GFP-labeled granules in Syt-7 KO cells was substantially lower than WT cells (Supplementary Movie 2) following cholinergic stimulation (3.2 ± 0.7% in KO versus 19.1 ± 3.4% in WT) (Figure 5A, B).</p><p>One potential explanation for the difference in granule fusion probability observed between the two cell types is that Ca2+ signaling is compromised in cells lacking Syt-7. Therefore, qualitative changes in intracellular Ca2+ in response to ACh stimulation were monitored using the fluorescent Ca2+ indicator, GCaMP5G (Akerboom et al. 2012). Neither the kinetics nor the peak amplitude of the GCaMP5G signal differed between WT and Syt-7 KO cells (Figure 5C, D). The possibility that differences in the magnitude of cholinergic or Ca2+ currents might underlie differences in the secretory phenotype of WT and KO cells was also tested. However, these were not different between the two groups (Figures S7A–D).</p><!><p>During the course of the experiments described in Figure 4, it became evident that the times at which NPY-pHl-labeled granules fused with respect to the start of stimulation varied depending on the genotype of the cell. To examine this issue more closely, WT and KO cells expressing NPY-pHl were stimulated for 2 minutes with 100 μM ACh while images were continuously acquired. The secretory response of a single WT and Syt-7 KO cell to prolonged ACh stimulation (beginning at 5 s after imaging begins) is shown in Figures 6A and B. Each of the vertical lines (blue for WT and red for KO) corresponds to the time of an individual NPY fusion event during the period of stimulation. In contrast to WT cells, the secretory response of KO cells rapidly wanes after an initial burst of fusion events.</p><p>The cumulative distribution of all fusion events in WT and Syt-7 KO cells (n = 7 cells for each group) occurring after ACh stimulation is plotted in Figure 6C. A distinctive feature of the cumulative time course for Syt-7 KO events is that the curve plateaus. Such a result would be expected if the absence of Syt-7 prevents a sustained response to cholinergic stimulation. The cumulative time course for WT cells stimulated with ACh does not plateau; secretion is observed for as long as the cell is stimulated although the frequency with which events occur does decline at later times. The curves were fit by the sum of two exponential (Figure 6C). Interestingly, over the first approximately 20 s of ACh stimulation, there is more secretory activity in the Syt-7 KO than in the WT cell (see inset, Figure 6C).</p><!><p>To better assign mechanistic roles to Syt-1 and Syt-7 in exocytosis, we utilized a previously characterized purified secretory granule to planar supported bilayer fusion assay (Figure 7A). PC12 cells lacking endogenous synaptotagmin (Kreutzberger et al. 2017a) were transfected to overexpress either Syt-1 or Syt-7 protein ((Kreutzberger et al. 2019) and Figure 7B). In the presence of SNARE regulatory proteins Munc18 and complexin-1, these granules will bind in an arrested state to planar supported bilayers (lipid composition of 70:30 bPC:Chol in the extracellular mimicking leaflet and 25:25:15:30:4:1 bPC:bPE:bPS:Chol:PI:PIP2) containing the plasma membrane SNARE proteins (syntaxin-1a and dSNAP-25). Injection of Ca2+ into this system readily stimulates fusion of dense core granules with the planar bilayer. Increasing the level of Ca2+ in the chamber containing docked dense core granules, triggers fusion with different efficiencies depending on the sensitivity to Ca2+ of the expressed synaptotagmin isoform (Figure 7C). Syt-7 containing dense core granules consistently fused at lower Ca2+ concentrations than those bearing Syt-1. The higher sensitivity of Syt-7 bearing granules to Ca2+ is consistent with the observation in cells that even mild depolarization selectively activates Syt-7 containing granules (Rao et al. 2014; Rao et al. 2017b; Bendahmane et al. 2018).</p><p>The intensity time course of NPY-mRuby fluorescence during release has noteworthy characteristics. Initially, a decrease in fluorescence is observed as NPY-mRuby begins to diffuse out of the early fusion pore and away from the fusion site (note dip in fluorescence in Figures 7D and 7E). After a brief delay, a sharp increase in fluorescence intensity is observed as the fusion pore expands and the granule membrane collapses (Kreutzberger et al. 2017b).</p><p>Previously, the delay time from the onset of NPY-mRuby release was shown to be sensitive to the presence of lipids with geometries that promote or inhibit fusion pore stability (Kreutzberger et al. 2017b). Here, we show this feature is also sensitive to the synaptotagmin isoform expressed. A close examination of the fluorescence intensity profile of Syt-1 granules shows that there is approximately a 0.4 s delay time from the initial decrease in fluorescence until the collapse of the granule into the supported bilayer (Figure 7D). In Syt-7 granules, the delay is lengthened to approximately 1 s (Figure 7E). An overlay of averaged release profiles of NPY from Syt-1 and Syt-7 granules is shown in Figure 7F. Images of individual NPY-mRuby release events from Syt-1 and Syt-7-bearing granules are shown in Figure S8.</p><!><p>Despite the broad interest in Syt-7 as a regulator of exocytosis (MacDougall et al. 2018), surprisingly basic aspects of its function remain unresolved, including its intracellular localization and role in regulating fusion pore expansion. Using a combination of subcellular fractionation and immunolabeling methods, we report that endogenous Syt-7 is co-localized with markers of the lysosome and dense core granule (LAMP-1 and PAI-1, respectively). The marker used in this study to identify granules, PAI-1, is ubiquitously expressed in chromaffin cells (Bohannon et al. 2017) and exhibits a high degree of co-localization with other markers of the dense core, tPA and dopamine β-hydroxylase (Bohannon et al. 2017; Abbineni et al. 2019). The conclusion that fraction 13 (Fr-13, Figure S3) contained the majority of dense core granule constituents is based on the strong PAI-1 signal. Syt-7 was most abundant in fractions 13 and 4–6. Approximately 20% of the total amount of Syt-7 in the cell can be found in fraction 13 (i.e., granules) – a number that is also consistent with the immunocytochemical data (Figure 1). Fractions 4–6 represent less dense organelles, including lysosomes. Immunoreactivity for LAMP-1 is greatest in fractions 4–6, but is also detected in fraction 13. This suggests that dense core granules house LAMP1 in addition to Syts 1 and 7 – a conclusion which is supported by immuncytochemical data (Figure 1) and published reports (Hao et al. 2015).</p><p>We presume that membrane proteins, such as Syt-7, are turned over more slowly than secreted proteins (Winkler 1971). The recycling of synaptotagmin isoforms (e.g., after fusion) would account for their higher abundance in lighter organelles which may form part of the endolysosomal system. Interestingly, Syt-1 is more abundant in fraction 13, as a percentage of total protein, than is Syt-7. This may reflect differences in the steady-state turnover of granules to which Syt-1 and Syt-7 are sorted.</p><p>The basic finding that Syt-7 is sorted to chromaffin granules corroborates what has been previously published in PC12 cells and chromaffin cells (Fukuda et al. 2004; Tsuboi & Fukuda 2007; Zhang et al. 2011; Rao et al. 2014). In contrast, neuronal Syt-7 has been reported to exist primarily as a plasma membrane bound protein (Weber et al. 2014; Jackman et al. 2016; Sugita et al. 2001) (Jackman et al., 2016; Sugita et al., 2001; Weber et al., 2014). The reason for differences in Syt-7 localization between neurons and neuroendocrine cells is unclear at this time.</p><p>A second key unresolved issue concerning the function of Syt-7 in chromaffin cells is whether it constrains (Rao et al. 2014), or alternatively, promotes (Zhang et al. 2019) fusion pore expansion. Here, two distinct experimental preparations were used to address this issue: 1) primary mouse chromaffin cells lacking Syt-7; and, 2) a reconstituted single granule fusion assay employing PC12 granules only expressing either Syt-1 or Syt-7. Data gathered from both experimental preparations provide support for the principle that Syt-7 imposes limits on the rate at which cargos are discharged during exocytosis. In the case of cargos that exhibit an intrinsically slow release profile (e.g., tPA), the effect of Syt-7 is additive. The findings are also consistent with TIRF-based measurements of secretion in other systems, including PC12 cells (Zhang et al. 2011) and mouse embryonic fibroblasts (Jaiswal et al. 2004), where it has been shown that Syt-7 restricts post-fusion soluble content release and diffusion of granule membrane proteins into the plasma membrane.</p><p>Although the mechanism by which Syt-7 acts at the pore to slow discharge is not resolved, the protein's high affinity for anionic phospholipids likely plays a key role. According to this model, the tight binding of the Syt-7 C2 domains to lipids constituting the fusion pore is a determining factor of pore stabilization (Bendahmane et al. 2018; Voleti et al. 2017; Tran et al. 2019). In support of this hypothesis, a mutant of Syt-1, whose C2B domain Ca2+/phospholipid-binding loops were exchanged for those of Syt-7, exhibited a higher intrinsic affinity for phospholipids and a slower dissociation from phospholipids in the presence of Ca2+, than the WT Syt-1 protein (Bendahmane et al. 2018). When expressed in cells, this Syt-1 mutant also stabilized membrane curvature associated with the fused granule/plasma membrane domain, thereby slowing the discharge of NPY-pHl (Bendahmane et al. 2018). It should be noted here that fluorescent peptide hormone release observed by TIRF microscopy is likely to represent a slower process than the transition from pre-spike foot to burst of neurotransmitter release captured by amperometry recordings, which occurs in a matter of milliseconds (Albillos et al. 1997; Lindau & Alvarez de Toledo 2003; Ales et al. 1999; Chow et al. 1992; Wightman et al. 1991; Segovia et al. 2010). Although both phenomena have been interpreted as expansion of the fusion pore, they are likely to represent temporally distinct kinetic steps in the process of exocytosis. Therefore, a clear distinction between fusion pore dynamics as described by imaging and electrochemical methods should be made.</p><p>This study shows that Syt-7 and Syt-1 are both necessary for a robust secretory response to native stimuli that elevate intracellular Ca2+ level (Figure 6). The number of fusion events, and the rate at which those fusion events occur, is significantly lower in Syt-7 KO cells than in WT cells. The decreased likelihood of observing fusion events in cells that do not express Syt-7 is not due to impaired Ca2+ "handling" or excitability. There is also no difference in the magnitude of Ca2+ or cholinergic current in Syt-7 KO cells compared to WT cells (Figure S7). Based on the data shown in Figures 6 and 7, a mechanism is proposed to explain why the secretory response to prolonged cholinergic stimulation is disrupted in Syt-7 KO cells. Over the course of a two-minute exposure to ACh, nicotinic receptors (nAChRs) undergo desensitization (Figure S7A). Free cytosolic Ca2+, initially elevated as a result of Ca2+ influx through nAChRs, and also possibly Cavs, is rapidly sequestered, buffered, or extruded (Neher & Augustine 1992). Despite the collapse of the Ca2+ gradient, fusion persists in WT cells throughout the period of ACh perfusion (Figure 8B, C). On the other hand, in Syt-7 KO cells, secretory activity quickly ceases after an initial burst of fusion. Thus, expression of Syt-7 is necessary for sustained activity in the setting of rapidly declining levels of intracellular free Ca2+ following nAChR desensitization. Presumably, the residual Ca2+ that remains is effective at triggering fusion only when Syt-7 is present (Figure 8C). The stark differences in Ca2+ affinities between Syt-1 and Syt-7 are likely sufficient to account for this phenomenon (Bhalla et al. 2005; Sugita et al. 2002). Dose-response curves (Figure 7C) show that there is at least a 6-fold difference in the [Ca2+]1/2 for fusion of purified dense core granules bearing Syt-1 (63 μM) versus Syt-7 (10 μM). In fact, at 10 μM Ca2+, there is no appreciable fusion of Syt-1 granules.</p><p>Interestingly, the time course of the cumulative frequency distribution of fusion events in Syt-7 KO cells stimulated by ACh and KCl is different (Figure S5 compare to Figure 6). This can be explained by the fact that Ca2+ elevations evoked by ACh stimulation (Figure 5) decay more rapidly in the subplasmalemma than those evoked by KCl secretion (Rao et al. 2014; Fulop & Smith 2007) which, in the absence of Syt-7, results in a more rapid inhibition of the secretory response. The rate at which NPY was released as a result of fusion in WT cells differed depending on whether KCl or ACh was used to trigger release (compare Figure 2 and Figure 4). It may be that ACh-based stimulation relies to a greater extent on activation of granules bearing Syt-7 than does KCl-based stimulation, or such variability may simply reflect the fact that experiments were performed in different groups of cells at different times. Irrespective of the mode of stimulation, the absence of Syt-7 has the consistent effect of speeding NPY cargo release, irrespective of the stimulus.</p><p>We note that no significant difference in the mobility of NPY-GFP-labeled chromaffin granules in WT and Syt-7 KO cells was observed in this study. A previous study, in which fluorescently-tagged Syts were overexpressed in cells, revealed that the frame-to-frame movement of granules harboring Syt-1 was greater than those harboring Syt-7 (Rao et al. 2017a). Therefore, the degree to which the molecular identity of a chromaffin granule regulates its mobility and whether this depends at all on the expression of a particular Syt isoform, all remain open questions for future work. We also note that the absence of Syt-7 inhibits the secretory response to a greater extent in these TIRF-based studies than what had been previously reported in studies that measured secretion by capacitance or amperometry (Schonn et al. 2008; Segovia et al. 2010). One can speculate as to the reasons why. What is reported here is the probability of fusion, which requires overexpression of NPY-GFP in order to identify docked granules. It is only the fusion of these labeled granules, which likely constitute a minority of the total fusion-competent population of granules within the cell, which is documented. The manner in which secretion was elicited in these studies, and previous ones in which capacitance was measured, is also different. Here, cells were stimulated via local perfusion of elevated KCl or ACh. In studies employing capacitance measurements, cells were stimulated by either directly depolarizing the membrane potential via the patch pipet, or by uncaging Ca2+ to concentrations in the range of tens of micromolar (Schonn et al. 2008). Secretion elicited by a strong stimulus, and associated with a greater increase in intracellular Ca2+, may be less likely to rely on fusion mediated by Syt-7.</p><p>To conclude, the experiments in this study demonstrate that the absence of Syt-7 has a profound impact on Ca2+-triggering of exocytosis and the discharge rate of peptide hormones. A key result is that the delayed kinetic component of the secretory response to cholinergic stimulation, during which subplasmalemmal Ca2+ elevations have collapsed to near baseline levels, is impaired in Syt-7 KO cells. This is shown in cumulative frequency histograms of the time course of fusion events, which can be fit by the sum of two exponentials representing the fast and slow/sustained phases of secretion. The fast phase, in WT cells, is a minor component of the curve (0.66%), whereas the slow phase dominates. In the Syt-7 KO, the fast phase of release constitutes a greater proportion of the span of the curve (58.08%) than the slow phase. The idea that the initial and later phases of exocytosis have different Ca2+ sensitivities is supported by previous work performed in permeabilized bovine chromaffin cells (Bittner & Holz 1992). By measuring [3H] norepinephrine in the medium after cells were exposed to different levels of Ca2+, Bittner and Holz (1992) concluded that an early phase of release (i.e., occurring with 5 s of stimulation) must correspond to a lower affinity process ([Ca2+]1/2 ≈ 100 μM) than a later phase of release (i.e., occurring between 5 −10 s ([Ca2+]1/2 ≈ 10 μM)). In this study, we propose a molecular mechanism to account for these observations – a mechanism that relies on the sequential activation of specialized low and high affinity Ca2+ sensors.</p>

PubMed Author Manuscript

pH induced reversible assembly of DNA wrapped carbon nanotubes

BackgroundReversible assembly and disassembly of nanostructures has important function in controllable construction of nanodevices. There are several methods to achieve reversible assembly/disassembly, such as pH, temperature, DNA hybridization and so on. Among these methods, pH driven reversible assembly presents superiority due to its ease-of-use and no waste produced. Herein we report a novel design that use two single-stranded (ss) DNAs wrapped single walled carbon nanotubes (SWCNTs) for the pH controlled assembly of SWCNTs without generation of waste.ResultsBoth of the two DNAs with a same wrapping sequence of d(GT)20 and different free terminals showed a very high tendency to wrap around carbon nanotubes. The assembly was driven by the hybridization between the two free terminals of wrapped DNAs on the neighboring SWCNTs: i-motif (four-stranded C-quadruplex) and its complemental stranded G-quadruplex which would form tight tetraplexes and break the hybridization under slightly acidic conditions. Thus the assembly and disassembly are reversibly controlled by pH. And this assembly/disassembly process can be easily distinguished by naked eyes. Gel electrophoresis and Atomic Force Microscope are used to demonstrate the assembly and disassembly of SWCNTs at different pH.ConclusionsA novel pH induced reversible assembly and disassembly of SWCNTs was realized which may have potential applications in the area of controlled assembly of nanostructures.

ph_induced_reversible_assembly_of_dna_wrapped_carbon_nanotubes

Background<!>The strategy<!><!>SWCNTs at slightly basic solution and slightly acid solution<!><!>Reversible assembly of SWCNT by adjusting pH<!><!>Gel electrophoresis and atomic force microscopy (AFM) illustration<!><!>Gel electrophoresis and atomic force microscopy (AFM) illustration<!><!>The kinatic of aggregation<!><!>Synthesis of DNA wrapped SWCNT<!>pH driven reversible SWCNT-DNA assembly<!>Kinetic of the assembly of two SWCNTs<!>Agarose gel electrophoresis<!>AFM analyses<!>Conclusion<!>Abbreviations<!>Competing interests<!>Authors’ contribution

<p>Nanostructure has potential applications in future fabricating and nanodevices [1-8]. "Bottom up" construction of exquisite nanostructure based on different kinds of nanomaterials has attracted numerous attentions [9-13]. Several studies are particularly devoted to nanostructure based on DNA and other nanomaterials such as gold nanoparticles [14,15], carbon nanotubes [16,17], graphene [18] and so on [19,20]. Nanostructures formed by single-walled carbon nanotubes (SWCNTs) and DNA combine both the size-dependent properties of the SWCNT and the molecular recognition and biological function of DNA, which have potential applications in molecular electronics [21,22] and biomedical engineering [23-25]. Both covalent [26] and noncovalent [27] associations are used to construct SWCNT–DNA complexes. However, self-assembly strategies based on the biorecognition capability of single-stranded DNA (ssDNA) have been proposed to be a promising one [16,25].</p><p>Reversible assembly and disassembly of nanostructures has important function in controllable construction of nanodevices [28-30]. There are several methods to achieve reversible assembly/disassembly, such as pH, temperature, DNA hybridization and so on. Deng and co-workers demonstrated reversible assembly/disassembly of DNA–SWCNT conjugates switched by DNA hybridization [31]. However, changes in pH offered a simple but versatile way to control the assembly of materials. Qu et al. reported a duplex-based DNA-SWCNT self-assembled nanostructure based on a four-stranded DNA structure, G-quadruplex, and i-motif DNA [32]. This driven mechanism is reversibly controlled by pH, but still need complex covalent modification of SWCNT.</p><p>Herein we report a pH driven reversible assembly of DNA wrapped SWCNTs. SWCNTs were wrapped by two single strand DNAs which differed at their terminal: one had an i-motif sequence and the other had its complemental sequence G-quadruplex. Both of the DNAs with a wrapping sequence of d(GT)20 had a very high tendency to wrap around carbon nanotubes and made the SWCNTs stably water dispersed. However, The SWCNTs formed conjugates as the two DNA terminals hybridized to each other under slightly basic conditions, and they were disassembled under slightly acid conditions as the tight G-quadruplex and i-motif DNA structure formed. Thus the design of a reversible SWCNTs nanostructure driven by pH changes realized.</p><!><p>SWCNTs were wrapped by two single-strand DNAs which had a same wrapping sequence of d(GT)20 and different free terminals respectively (Figure 1). One of the free terminals contains G-quadruplex (G4), and the other contains i-motif (C4) in their sequences. A single-base mutation was added to the G-quadruplex domain to prevent the nonspecific SWCNTs assembly caused by the formation of the interparticle G-quadruplex. As mentioned in literatures, this sequences were proved to be useful in making other DNA based nanodevices [33,34]. Under basic conditions, C4 hybridized with its complementary G4, therefore SWCNTs formed a net-shape conjugates. When the pH was decreased to 5.0, C4 and G4 formed intramolecular i-motif and G-quadruplex tetraplex respectively, so the SWCNTs do not aggregate. If the pH was adjusted to 8.0 again, the net-shape conjugate reformed. The assembly and disassembly process was reversible as the pH reversed. As a result, the pH actuated reversible SWCNTs assembly was formed.</p><!><p>Strategy of pH induced reversible assemble of DNA wrapped SWCNTs. The mixture of Two SWCNT-DNA conjugates (SWCNT-G4 and SWCNT-C4) formed 3D aggregates at pH 8.0 through hybridization of G4 and C4. While the pH of buffer solution was adjusted to 5.0, G4 and C4 are formed and SWCNTs were dispersed. This assembly and disassembly process was reversible by adjusting the pH. Sequence of C4 DNA:(GT)20 –TTT TTT TTT TCC CAA TCC CAA TCC CAA TCC C. Sequence of G4 DNA: (GT)20 –TTT TTT TTT TGT GAT TGT GAT TGT GAT TGT G.</p><!><p>The mixtures of SWCNT-G4 and SWCNT-C4 in stoichiometric equivalents were adjusted to pH 8.0 (Figure 2a) and pH 5.0 (Figure 2b) respectively. After a 25-min incubation and a short centrifugation at 2000 g for 10 s, these two vials were observed by naked eye. Precipitation was seen at pH 8.0 which was respected to the conjugation of SWCNTs, while the solution was homogeneous at pH 5.0 showing that SWCNTs were dispersed well. As a contrast, two other vials of SWCNT-G4 only were adjusted to pH 8.0 (Figure 2c) and pH 5.0 (Figure 2d) respectively and observed after the same treatment. Interestingly, both of them were dispersed well. This phenomenon proved the specific SWCNTs conjugation was not affected by the change of pH condition.</p><!><p>Photograph of the two vials containing SWCNT-G4 and SWCNT-C4 in pH 8.0 (a) and pH 5.0 (b), and two vials containing only SWCNT-G4 in pH 8.0 (c) and pH 5.0 (d). After staying at room temperature for 30 min, Centrifugations at 2000 g for 10 s were applied to the two vials.</p><!><p>Two SWCNTs (SWCNT-G4 and SWCNT-C4) were mixed in stoichiometric equivalents in a vial. Then the pH was adjusted to 5.0 and 8.0 sequently and this process was cycled several times. As was seen in Figure 3, precipitate was seen when pH was changed to 8.0 while no precipitate was seen at pH 5.0. And the deposition and re-dispersion of the precipitate was reversible when the pH reversed.</p><!><p>Reversible assembly and disassembly of SWCNTs by adjusting pH between 5.0 and 8.0. Solutions in tubes a, c, e, and g are at pH 5.0, and tubes b, d, and f are at pH 8.0. Three cycles of assembly and disassembly of SWCNTs by pH switching are demonstrated here. Centrifugations at 2000 g for 10 s were applied after each pH changing to make the aggregates more visually discernible.</p><!><p>Agarose gel electrophoresis and AFM were employed to interrogate the assembly and disassembly extent of SWCNT–C4/SWCNT–G4 at both acidic and basic conditions.</p><p>Gel electrophoresis was employed to characterize the migration status of SWCNTs at different conditions (Figure 4). SWCNT-G4 (lane 1) and SWCNT-G4 (lane 2) could migrate out of the well as a single band. The mixture of two SWCNTs in pH 5.0 (lane 3) and 8.0 (lane 4) showed different migration status: When the pH was adjusted to 5.0, the mixture could also migrate out of the well and the speed was the same as SWCNT-G4 and SWCNT-C4, which suggeted that the i-motif and G-quadruplex were formed and SWCNTs dispered well. While the pH of the mixture was adjusted to 8.0, the aggregation of the SWCNTs could not penetrate into a 0.5% agarose gel but stayed in the well, which suggested that that SWCNT-C4 and SWCNT-G4 hybridized with each other and crosslinked SWCNTs was formed.</p><!><p>0.5% agarose gel electrophoresis of the SWCNT-G4 (lanes 1), SWCNT-C4 (lanes 2), asswbly of SWCNT-G4 and SWCNT-C4 aggregates in pH 5.0 (lanes 3) and pH 8.0(lanes 4).</p><!><p>The SWCNT-DNA aggregation controlled by pH was further checked by AFM. A centrifugation-assisted precipitation was employed to visually observe the dispersion and the precipitate of SWCNTs mixture. As seen in Figure 5, the mixture of SWCNTs despersed well and uniformly at pH 5.0 and formed a big area of aggregate at pH 8.0. This result further confirmed the result in gel electrophoresis which reveals the sharp contrast between the dispersed and aggregated states of the DNA–SWCNT conjugates.</p><!><p>AFM images of the conjugation of SWCNT-G4 and SWCNT-C4 at pH 5.0 (left) and pH 8.0 (right).</p><!><p>To investigate the kinetic of SWCNTs assembly, the pH of mixture of SWCNT-G4 and SWCNT-C4 was adjusted to 8.0. The solution was incubated at room temperature and was observed by a naked eye. A centrifugation-assisted precipitation was adoped at certain points and photographs were taken. The precipitation process was shown in Figure 6. There was little deposit after 5 min when the pH was adjusted to 8.0. Obvious conjugates were seen after 10 min and supernatant was clear after 30 min. it was suggested that the assembly of SWCNTs reached saturation point after 30 min.</p><!><p>Photograph of assembly of SWCNTs at different time.</p><!><p>DNA oligonucleotides were purchased from Sangon Inc. (Shanghai, China) with their sequences listed in Figure 1. A mutation of the G-quadruplex domain with a single base (from GGG to GTG) will disrupt the formation of the interparticle G-quadruplex and prevent the SWCNTs assembly. DNA wrapped SWCNT was prepared following a reported process. In briefly, 100 μL of aqueous solution containing about 0.1 mg of SWCNTs (Sigma Chemicals), 0.025 mg G4 or C4, and 0.1 M NaCl was sonicated at a power of about 4 W using a VC130PB probe-type sonicator (Sonics materials inc.). The whole sonication process was incubated in an ice-water bath. Then free DNA was removed using centrifugation. Before centrifugation, MgCl2 was added to the DNA and SWCNT mixture with the concentration of 30 mM to help precipitation of the SWCNT/DNA conjugates from the solution. A centrifugation at 2000 g was taken for 2 min. The supernatant solution was carefully removed using a pipette tip. The resulted DNA/SWCNT conjugate was redispersed in 0.5xTBE buffer (Tris, 44.5 mM; EDTA, 1 mM; and boric acid, 44.5 mM, pH 8.0) containing 30 mM NaCl plus 10 mM extra EDTA to complex with residual Mg2+ in the precipitate. This step was cycled several times.</p><!><p>SWCNT-G4 and SWCNT-C4 were mixed in stoichiometric equivalents in a vial. Then the pH was adjusted to 5.0 by adding 1 M HCl, and then the solution was incubated for 30 min at 25°C. The resulted mixture was centrifuged at 2000 g for 30 s and observed. Then the pH was adjusted to 8.0 by adding NaOH and the solution was treated same as pH 5.0. The process was repeated several times. Assembled and disassembly products were both checked by gel electrophoresis or AFM.</p><!><p>SWCNT-G4 and SWCNT-C4 were mixed in stoichiometric equivalents in a vial and the pH was adjusted to 8.0. Then the solution was incubated at 25°C and observed at different time.</p><!><p>SWCNT-DNA conjugates were loaded into 0.5% agarose gel and run in 0.5 × TBE at 10 V/cm. The DNA-hybridization leaded to the SWCNT aggregates which could not run into the 0.5% agarose gel and was held as black deposit in the gel-loading wells.</p><!><p>AFM images were recorded using a Nanoscope IIIa apparatus (Digital Instruments, USA) equipped with a J Scanner. A droplet of SWCNTs mixture sample was cast onto a freshly cleaved mica surface, followed by drying at room temperature.</p><!><p>In summary, we provided a pH controlled reversible assembly of SWCNTs. SWCNTs were wrapped by two ssDNAs which contained wrapped sequence and different free terminals. This assembly was driven by the hybridization between their free terminals: i-motif and G-quadruplex. A mutation of the G-quadruplex domain with a single base (from GGG to GTG) was used to disrupt the formation of the interparticle G-quadruplex and prevent the nonspecific SWCNTs assembly. As i-motif and G-quadruplex were both formed at slightly acidic conditions, the assembly was controlled by pH and reversible. This assembly/disassembly process was easy to control without generation of waste and accompanied by precipitation that was clearly visible to the naked eye. This system may develop into a fast, highly reversible pH-sensitive device that may have potential applications in the area of nanobiotechnology. For example, this pH induced controllable assembly of SWCNTs can offer a potential method to desired novel pH-sensitive multifunctional architectures and/or biosensing devices.</p><!><p>ssDNA: Single-stranded DNA; SWCNT: Single walled carbon nanotube; C4: Four-stranded C-quadruplex; G4: Four-stranded G-quadruplex.</p><!><p>The authors declare that they have no competing interests.</p><!><p>YW proposed the subject, designed the study, participated in the results discussion and carried out the synthesis of DNA wrapped SWCNT. GL carried out the agarose gel electrophoresis and Atomic Force Microscopy Analyses and helped to draft the manuscript. XZ and YS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.</p>

PubMed Open Access

Electron Dynamics with Explicit-Time Density Functional Theory of the [4+2] Diels–Alder Reaction

The prototype Diels–Alder (DA) reaction between butadiene and ethene (system 1) and the DA reaction involving 1-methoxy-butadiene and cyano-ethylene (system 2) are investigated with an explicit-time-dependent Density Functional Theory approach. Bond orders and atomic net charges obtained in the dynamics at the transition state geometry and along the reaction coordinate toward reactants are used to provide a picture of the process in terms of VB/Lewis resonance structures that contribute to a resonance hybrid. The entire dynamics can be divided into different domains (reactant-like, product-like, and transition state domains) where different Lewis resonance structures contribute with different weights. The relative importance of these three domains varies along the reaction coordinate. In addition to the usual reactant-like and product-like covalent Lewis structures, ionic Lewis structures have non-negligible weights. In system 2, the electron-donor OCH3 on the diene and the electron-acceptor CN on the dienophile make more important the contributions of ionic Lewis structures that stabilize the transition state and determine the decrease of the reaction barrier with respect to system 1.

electron_dynamics_with_explicit-time_density_functional_theory_of_the_[4+2]_diels–alder_reaction

Introduction<!>Computational Background<!>QM Calculations<!><!>QM Calculations<!>Wave Function Dynamics<!><!>Wave Function Dynamics<!>A Schematic Representation of the Possible Ionic Lewis Structures for the Prototype DA Reaction<!>Wave Function Dynamics<!><!>Wave Function Dynamics<!>A Schematic Representation of Relevant Lewis Structures for the DA Reaction Occurring in System 2<!>Wave Function Dynamics<!>Schematic Representation of Ionic Lewis Structures for the DA Reaction Occurring in System 2<!>Wave Function Dynamics<!><!>Wave Function Dynamics<!>Conclusions<!>

<p>Molecules, due to the ever-present electron delocalization, can be represented as resonance hybrids to which Lewis (resonance) structures contribute with different weights.</p><p>The hybrid mesomeric wave function Ψ is described as a linear combination of Lewis structures, ϕi:1where ci are variational coefficients that represent the weight of ϕi and minimize the electronic energy. Because the energy of a resonance hybrid is lower than the energy of any of the individual Lewis structures, much of the chemical stability and reactivity can be rationalized in terms of mesomerism. High energy states, such as transition states (TSs), can be stabilized by the presence of various resonance structures with a consequent lowering of the activation barrier. Therefore, mesomerism helps to explain why some reactions are fast and others are slow or do not proceed at all. "Arrow chemistry" is the pictorial representation of mesomerism. This qualitative popular approach to mesomerism was demonstrated to be a powerful tool capable of rationalizing organic reactivity. Arrows indicate the motion of individual or pairs of electrons connecting different Lewis structures and, in a natural extension, leading to products from reactants.</p><p>The contributions ci of each resonance (Lewis) structure ϕi can be, in principle, computed with the Valence Bond (VB) theory, ascribable to the pioneering work of Ingold and Pauling.1−4 The pictorial movement of electrons, described by the curly arrows, can be related to the oscillations of the electronic wave function, which even in the stationary case can be caused by vibrations or by the environment. This electron density reorganization occurs in a subfemtosecond time scale (where nuclei are frozen) and can be related to the contributions of the various resonance structures ϕi's.</p><p>The VB approach is seldom used because of the high complexity and computational costs required for VB calculations. Instead, Molecular Orbital (MO) theory is routinely employed to determine Ψ, but it can give only a rough estimate of the contribution of the various resonance (Lewis) structures. In this way, the familiar description of reactivity in terms of Lewis structures and curly arrows (beloved by organic chemists) is almost completely missed. Nevertheless, in a few papers, the language of mesomerism was used to interpret the results of MO computations for some prototype organic reactions.5−7 Bernardi and co-workers computed resonance energies defined in the theory of aromaticity using a VB Hamiltonian obtained from CASSCF wave functions. They applied this analysis to the transition structures of ethylene dimerization and Diels–Alder (DA) reaction between ethylene and butadiene.5 More recently, the prototype DA reaction was investigated with a new approach able to extract the movement of the electrons from static (no-time-dependent) Hartree–Fock calculations that were used to construct a reference configuration and "excited" configurations.6 For the Claisen rearrangement and other reactions, the bond reorganization expressed by curly arrows was directly observed in ab initio calculations as transformations of intrinsic bond orbitals along the reaction coordinate.7</p><p>Even if it is commonly accepted that there is no time-dependent oscillation between the resonance structures (ϕi partakes in Ψ, but not as a function of time), time is implicitly present in "arrow chemistry". The arrows indicate how electrons move and reorganize in the reactants–products transformation and are an example of time dependence.</p><p>To evaluate the contribution of the Lewis structures to the resonance hybrid and renovate the language of mesomerism in the context of MO computations, a time-dependent picture where electrons reorganize at the ultrafast time scale can be employed. In this Article, we use an explicit-time Density Functional Theory approach to obtain a "mesomeric" picture of one of the most popular pericyclic reactions: the [4+2] Diels–Alder (DA) cycloaddition. We investigate two different systems: (i) the prototype DA reaction between butadiene and ethylene (system 1), and (ii) the reaction between 1-methoxy-butadiene and cyano-ethylene, labeled as system 2. The RT-TDDFT (real-time-dependent Density Functional Theory) dynamics, carried out at fixed nuclei, describes the change in time of electron densities in TSs and intermediate points along the Intrinsic Reaction Coordinate (IRC). Within the present theoretical approach, the initial nonstationary wave function, describing the two unperturbed fragments at the TS (or IRC point) geometry, is perturbed by the adiabatic Hamiltonian, which represents the effect of the working/chemical environment upon the molecular fragments. The perturbation activates the electron flow between and within the fragments. During the dynamics, the total density matrix population shifts between diagonal and off-diagonal elements and provides information on the evolution of local atomic charges (Q's) and bond orders (BOs). Because our dynamics does not provide the values of experimental observables, bond orders and atomic charges, which are related to the evolution of the wave function, are useful quantities to discuss many experimental results for the DA reaction and not directly measured by the dynamics. In this picture, BOs and Q's vary in time, they identify the resonance structures ϕi's explored by the hybrid Ψ, and they are crucial to decode the nature of the wave function in terms of VB structures.</p><!><p>The explicit-time-dependent DFT simulations were performed by means of a numerically stable algorithm that we applied in previous studies to predict the nonlinear electronic response in systems under the effect of external perturbations:8−162where ψ(t) is the nonstationary wave function at time(t), H is the Hamiltonian, and Δt is the simulation time-step, here set to 0.0048 fs. The time dependence of the electronic wave function is calculated using a generalized Cayley algorithm,17 based on a Dyson-like expansion of the time-evolution operator,18 which conserves probability and preserves orthogonality. Numerically, the algorithm evolves the time-dependent Schrödinger equation by means of the Crank–Nicolson method.19,20</p><p>Wave function coefficients and energies of the critical and intermediate geometries computed on the ground-state PES of systems 1 and 2, as reported in the QM section of the Supporting Information, were used to set up the wavepacket dynamics, in a vacuum. Initially, the wave function, ψ(t = 0), is built in terms of block-localized MOs for each fragment, that is, the diene (DN) and dienophile (DP). The ground-state electronic structures of both fragments, distinctly obtained on their frozen geometries at different PES points, were calculated at the M06-2X/6-31+G(d) level of theory:3</p><p>In a sense, the initial wave function is constrained on the electronic structures of the unperturbed fragments along the nuclear geometries of the reaction path. The ψDN0 and ψDP0 wave functions were orthonormalized by a Löwdin transformation.</p><p>The coupling between fragments is introduced by means of the electronic Hamiltonian calculated at the same level of theory on the Löwdin orthonormalized wave functions of the total system at each PES geometry.</p><p>To quantify the electron density redistribution activated by coupling between interacting fragments, bond orders and atomic effective charges were calculated in time for different PES points.</p><p>In particular, the time-dependent inter- and intramolecular bond order between atoms A and B was calculated from the total density matrix P(t), according to the Wiberg definition:21−234</p><p>The time-dependent atomic effective charge on atom A, for an atom centered basis set, was estimated by the Lödwin population analysis:5</p><p>The orbital occupation numbers, determined by projecting the time-dependent density matrix onto the initial orbitals, were also calculated:6</p><p>We consider short-time coherent electron dynamics, of about 5 fs time length, before vibrational relaxation and interactions with the environment occur.</p><p>Importantly, the wave function ψ(t + Δt) of eq 2 is nonvariational. If ψ(t = 0) is the eigenfunction of H, the dynamics is trivial and does not require numerical integration. In the present context, either the Hamiltonian or the wave function can be adiabatic:78where9</p><p>However, Hinteraction and ψcorrection are not related by an eigenvalue problem. In a perturbative scheme, ψcorrection can include contributions from electronic excitations from the ψ(t = 0) wave function. These excitations effectively make ψ(t) multiconfigurational. Analogously, Hinteraction includes perturbative contributions at all orders of perturbation.</p><p>In the present calculations, Hadiabatic operates on ψfragments.</p><!><p>All calculations were performed in vacuo with the M06-2X functional24 with the 6-31+G(d) basis set. A schematic representation of the critical points obtained for system 1 and system 2 is reported in Figures 1 and 2.</p><!><p>Prototype Diels–Alder reaction between butadiene and ethylene. Energies (kcal mol–1) relative to reactants Rx1 are reported in parentheses and include zero-point corrections. Bond lengths are in angströms.</p><p>Diels–Alder reaction between 1-methoxy-butadiene and cyano-ethylene leading to 3-methoxy-4-cyano-cyclohexene. Energies (kcal mol–1) relative to reactants Rx2 are reported in parentheses and include zero-point corrections. Bond lengths are in angströms.</p><!><p>Following the usual interpretation of a [4π+2σ] pericyclic reaction, the prototype DA reaction between butadiene and ethylene (system 1) is a concerted process (as confirmed by previous studies25). The transition state (TS1) is a cyclic symmetric aromatic-like structure, where two new σ C–C bonds between C1(C4) and C6(C5) (2.27 Å) form simultaneously to the elongation of the C1–C2(C3–C4) π-bonds (1.38 Å). The C2–C3 bond shortens from 1.47 Å in Rx1 to 1.41 Å in TS1. The energy barrier from Rx1 is 18.5 kcal mol–1. The formation of product Pd1 is highly exothermic (−41.8 kcal mol–1).</p><p>The DA reaction mechanism computed for the formation of 3-methoxy-4-cyano-cyclohexene from 1-methoxy-butadiene and cyano-ethylene (system 2) entails the passage through transition state TS2 where the new C–C bonds form asymmetrically. TS2 corresponds to the most favorable relative orientation of the two reactant molecules. All other possible orientations were discarded because they require a significantly higher activation energy (see Table S1). The σ-bond C4–C5 (2.05 Å) is almost formed when C1 and C6 are rather distant (2.57 Å). The energy of TS2 with respect to reactants Rx2 is 11.2 kcal mol–1, and the exothermicity of the reaction is 43.0 kcal mol–1.</p><!><p>As previously underlined, the results of our theoretical approach cannot be directly compared to the experiment. Thus, to interpret in terms of VB/Lewis structures the experimental evidence available for DA reactions and the results of MO computations, we used bond orders (BOs) and atomic net charges (Q's) obtained during the dynamics. In system 1, bond orders, BOs, and charges, Q's, are weakly periodic, as shown in Figure S1 and Scheme S1. The period of their oscillations is less than a femtosecond. In the following discussion, we refer to the BOs and Q's of the reactants (products) and transition state reported in Table 1 and in Tables S2–S5. The values for Rx1 (Pd1) were obtained from the DFT static computations, while the results reported for TS1 refer to 5 fs of dynamics carried out at the transition state geometry. The butadiene double bonds C1–C2 and C3–C4 have a BO value of 2.11 (1.15) in Rx1 (Pd1). During the dynamics, it oscillates between 2.03 and 1.31, with an average value of 1.60. The butadiene single bond C2–C3 has a BO value of 1.23 (2.04) and oscillates between 1.80 and 1.14 (with an average value of 1.42) in the simulation. The ethene bond C5–C6 has a BO value of 2.30 (1.15) in reactants (products) and oscillates between 2.26 and 1.21, with an average value of 1.65. The incipient bond has a BO value of 0.0 (1.13) and oscillates between 0.0 and 0.83 during the dynamics, with an average value of 0.42. These values are collected in Table S2.</p><p>If we choose the average value as a reference and consider a deviation of ±20% of the difference between the maximum and minimum values of each BO during the dynamics (see Table S2), we find that for 72% of the dynamics the C2–C3 BO ranges from 1.25 to 1.55 and the incipient C4–C5(C1–C6) BO is in the range from 0.25 to 0.55. These results are collected in Table 1.</p><p>Thus, assuming the BOs of C2–C3 and C4–C5(C1–C6) as meaningful indicators of the time evolution of electron motion, the electron distribution observed in the major part of the dynamics (72%) is represented by a resonance hybrid where the inner butadiene bond is becoming single and the incipient bond is in an advanced state of formation. This hybrid is a linear combination of various Lewis structures that must include covalent structures I and II of Scheme 1, with significant and comparable weights.</p><p>The limiting values of the oscillations reach close to the values of the reactants and product, suggesting a different contribution of I and II in various intervals of the dynamics. When in the oscillations, a large value of the BO of the incipient bond is associated with a large value of the inner C2–C3 butadiene bond (this occurs for 20% of dynamics, with BOs of C4–C5(C1–C6) > 0.50 and C2–C3 > 1.50), the Lewis structure II is dominant, and the corresponding resonance hybrid is product-like.</p><p>The contribution of I and II is reversed (I becomes dominant) when the BOs of C2–C3 and incipient bonds significantly decrease (BO of C2–C3 ≤ 1.25 and BO of C4–C5(C1–C6) ≤ 0.25). This occurs for 8% of dynamics, and the resonance hybrid becomes reactant-like. Thus, the entire dynamics can be thought of as divided into different domains: reactant-like, product-like, and transition state domains. We can name the central domain as a benzene-like domain because of the complete electron delocalization. A graphical representation of these three domains is given in Figure 3, reporting the time-dependency of C4–C5(C1–C6) and C2–C3 BOs (left and right sides of the diagram, respectively). The transition state domain corresponds to the central horizontal region of the diagrams, where the C4–C5(C1–C6) BO ranges from 0.25 to 0.55 and the C2–C3 BO from 1.25 to 1.55. The upper and lower regions represent the product-like and reactant-like domains. The transition state domain is marked by a benzene-like structure showing the concerted, synchronous character of the mechanism.</p><!><p>Time-dependency of (a) C4–C5(C1–C6) BO and (b) C2–C3 BO. The transition state (benzene-like) domain corresponds to the central horizontal zone.</p><!><p>The 5 fs dynamics were carried out for various points along the Intrinsic Reaction Coordinate (IRC) in the reactant direction (see Table 1). Energy values along the IRC are reported in Table S3. Importantly, as the system approaches the reactants, the percentage of dynamics characterized by a BO of the incipient bond C4–C5(C1–C6) ≤ 0.25 and of the inner C2–C3 bond ≤ 1.25 increases significantly (from 8% to 99%); that is, the reactant-like domain becomes rapidly more important. Simultaneously, we observed a decrease of the percentage corresponding to C4–C5(C1–C6) BO ≥ 0.25 and C2–C3 BO ≥ 1.25. This trend perfectly agrees with the gradual increase of the weight of Lewis structure I and the concurrent decrease of the importance of structure II.</p><p>The analysis of the atomic charges computed in the dynamics (see Table S4) can identify qualitatively the contribution of other Lewis resonance structures bearing formal negative and positive Q's on the atoms (see Scheme 2).</p><!><p>In a molecular orbital approach, some structures are multiconfigurational.</p><!><p>The terminal carbon atom of butadiene is characterized by a negative charge that is on average −0.47, more negative than the charge in reactants (products), which is −0.42 (−0.41). The increase of negative charge with respect to reactants (the maximum charge on C1(C4) is −0.32, while the minimum value is −0.68) suggests that almost one electron occasionally becomes localized on these atoms and indicates that ionic Lewis structures such as III and VII provide a non-negligible contribution to the resonance hybrid describing the transition state.</p><p>The charge of the inner carbon has an average value of −0.28, which is similar to that in reactants (products) that is −0.27 (−0.28). It ranges between −0.03 and −0.51, consistent with contributions of structures IV, V, VII, and VIII. The charge of the ethene carbons C5(C6) is on average −0.42, similar to that of the reactants (products) that is −0.44 (−0.42). In the simulation, this charge varies between −0.16 and −0.67, which is compatible with contributions of structures V and VI.</p><p>Thus, during the simulation, both C1(C4) and C5(C6) become either more positive or more negative with respect to reactants, suggesting that butadiene and ethylene can behave either as a donor (nucleophile) or as an acceptor (electrophile).</p><p>However, the increase of positive charge on C5(C6) is larger with respect to C1(C4) (−0.16 and −0.32 are the less negative values, respectively, and −0.42 and −0.47 are the corresponding average charges). Thus, the simulation shows that electrons have a stronger propensity to move from ethene to butadiene than in the opposite direction, suggesting that, at the transition state geometry, ethene is playing a more important role as a donor than butadiene. The trend of occupancies of the frontier orbitals can help to understand the difference between diene and dienophile. During 5 fs of dynamics (see Figure 4), the occupancies of the dienophile HOMO and diene LUMO show a regular and opposite oscillating trend in the ranges 0.8–0.2 and 1.2–1.8, respectively.</p><!><p>Electronic occupancies at the TS1 geometry over the 5 fs dynamics of the dienophile HOMO and diene LUMO and of the diene HOMO (left side), and of the dienophile LUMO and the C2–C3σ* orbital (right side).</p><!><p>The sum of the two occupancies is approximately 2. Thus, the two orbitals "are talking directly to each other", without involvement of other orbitals. A different behavior was observed for the occupancies of the diene HOMO and dienophile LUMO. During the first 2 fs of dynamics, the trend is similar to that previously discussed. After this time interval, the curves become flatter, and the involvement in the charge transfer of the σ* orbital of the inner (C2–C3) butadiene bond is evident. Thus, electrons are also moving within the butadiene fragment from the HOMO to the σ* orbital. These orbitals, at the transition state geometry, have the correct symmetry to interact because they are both antisymmetric with respect to the symmetry plane that characterizes the TS1 structure. The effect of this intrafragment charge transfer is that during the simulation butadiene appears as a worse donor than ethylene.</p><p>Additional information on the nature of the transition state hybrid can be obtained from the percentages of the dynamics corresponding to different ranges of Q's at the transition state TS1 and for various points along the IRC. These data are reported in Table S5 and discussed in detail in the Supporting Information. They demonstrate that structures such as V, VI, and VII must be taken into account to provide an accurate description of the resonance hybrid corresponding to the transition state.</p><p>Bond orders for reactants (product) and transition state for system 2 are collected in Table S6. Also, for system 2, bond orders and charges are weakly periodic (Figure S2 and Scheme S2). The BO of the stronger incipient bond C4–C5 oscillates between 0.0 and 0.90. Its average value, 0.55, is greater than that (0.42) calculated for the symmetric pathway of system 1. For the weaker incipient bond C1–C6, BO oscillates in the range 0.0–0.39, with an average value of 0.20, much lower with respect to that of system 1, reflecting the asynchronous character of TS2.</p><p>The butadiene double bond C1–C2 in reactants has a BO of 1.89, lower with respect to system 1 (2.11). This is consistent with the expected effect of the electron-donating group OCH3 and the consequent contribution of Lewis structures such as III′ in Scheme 3.</p><!><p>X = OCH3, Y = CN.</p><!><p>The effect of this group and the influence of structure III′ on the transition state resonance hybrid are evident during the dynamics because the C1–C2 BO varies in the range 1.74–1.22 with an average value of 1.47 (it was 1.60 in system 1). The BO value of the butadiene single bond C2–C3 in reactants (products) is 1.21 (2.01) and during the dynamics oscillates between 1.71 and 1.18, with an average value of 1.44, larger than that found in system 1 (1.42). Even in this case, the influence of structure III′ is evident. Finally, the ethene bond C5–C6 has a BO value of 2.10 (1.09) in reactants (products) and during the dynamics oscillates between 2.05 and 1.18 (average value of 1.49). The decrease of bond order with respect to system 1 (average value of 1.65) is coherent with the presence of the electron-withdrawing group CN and a non-negligible contribution of III′ and IV′ (Scheme 3).</p><p>In Table 2, we report the percentages of the dynamics corresponding to different ranges of the BOs for C2–C3 and C4–C5. Deviations of ±20% of the difference between maximum and minimum were used again to define the different domains.</p><p>For 49%, the C2–C3 BO and the C4–C5 BO vary in the intervals 1.25–1.55 and 0.40–0.70, respectively. Thus, for a significant percentage of the dynamics, electrons are "exploring" a resonance hybrid where the contributions of Lewis structures of Scheme 3 (in particular, II′, III′, and IV′) are significant. Also, the contribution of structure IV′ (a five-centers product-like structure) is consistent with the fact that the incipient C1–C6 bond is nearly absent in the transition state resonance hybrid. Importantly, Lewis structures such as III′ and IV′ are not present in system 1. In system 2, these structures contribute to stabilize further the transition state and lower the activation barrier. For 20% of dynamics, the C2–C3 BO is smaller than 1.25 and the C4–C5 BO is smaller than 0.40. This domain corresponds to a reactant-like hybrid structure, where Lewis structures such as I′ and III′ are dominant. 31% of dynamics is spent by electrons exploring a product-like resonance hybrid where the major contribution is represented by II′. The time-dependency of C2–C3 and C4–C5 BOs used as indicators of the time evolution of the electron flow is shown in Figure S3. Transition state, reactant-like, and product-like domains are evidenced.</p><p>The results of dynamics carried out along the IRC toward the reactants (Table 2) are again informative. In approaching the reactants, the time spent by electrons in exploring a reactant-like hybrid structure (C2–C3 BO < 0.40 and C4–C5 BO < 1.25) rapidly increases: there is a larger contribution of I′ and III′ to the hybrid resonance with a simultaneous decrease of Lewis structures II′ and IV′.</p><p>A more complete picture appears when local charges are considered (see Table S7). The terminal carbon atom of butadiene (C4) that forms the stronger incipient bond has a negative charge that is on average −0.44 (it oscillates between −0.24 and −0.66), identical to the charge in reactants. The terminal carbon atom of butadiene that forms the weaker incipient bond (C1) is often nearly neutral (it oscillates between 0.08 and −0.29), its average charge being −0.09. The charges of the two atoms oscillate out-of-phase (see Figure S3, right panel). The charge on the ethene carbon C5, involved in the stronger new bond, is on average −0.41 (it oscillates between −0.23 and −0.56), more negative with respect to reactants (−0.34). Because during the simulation both C4 and C5 can become either more positive or more negative with respect to reactants and the atomic charges oscillate out-of-phase, these two atoms can behave either as a nucleophilic or as an electrophilic center. All of the above evidence enforces the idea that Lewis structures such as III′ and IV′ strongly participate in the resonance hybrid that represents the transition state, but additional contributions, even though less important, arise from Lewis structures such as V′, VI′, and VII′ (Scheme 4). However, because the increase of negative charge with respect to reactants is larger on C5 (ethene) than on C4 (butadiene) (previously reported average charges are −0.41 and −0.44, respectively), the dynamics indicates that, at the transition state geometry, electrons spend more time on ethene than on butadiene. In other words, and contrary to what was found for system 1, butadiene becomes a better donor than ethene, in agreement with the presence on butadiene of the electron-donating group OCH3.</p><!><p>X = OCH3, Y = CN.</p><!><p>Further evidence of the effect of substituents on diene (OCH3) and dienophile (CN) is provided by the trend of occupancies of the frontier orbitals during the dynamics (Figure 5) and comparison with system 1. In system 1, the oscillating trends of the dienophile HOMO and diene HOMO are similar: at the beginning of the dynamics, the corresponding occupancies vary from 2 to ∼1.2, which indicates the transfer of ∼0.8 of an electron. In system 2, these two orbitals show a rather different behavior: the occupancy of the diene HOMO (right side of Figure 5) changes from 2 to 1.1, while that of the dienophile HOMO (left side) changes from 2 to 1.4. Thus, the charge transfer originating from the dienophile is significantly lower than that coming from the diene, and this in turn is slightly higher with respect to system 1. This trend is consistent with the presence of the electron-donating group OCH3 on the diene and the electron-withdrawing group CN on the dienophile, which partly "adsorbs" the moving electrons.</p><!><p>Electronic occupancies at the TS2 geometry over the 5 fs dynamics of the dienophile HOMO and diene LUMO (left side) and of the diene HOMO, the dienophile LUMO, and the C2–C3σ* orbital (right side).</p><!><p>Even if the results of our dynamics are not directly connected to the experiment, we have shown that bond orders and charges can be used to interpret experimental evidence in terms of VB structures. As a further example, we use the VB picture derived by BOs and Q's to elucidate some interesting experimental results obtained by Bartlett26 at the end of the 1960s and concerning the mechanism (concerted or two-step) of the DA reaction. This author demonstrated that in the case of few alkenes (for instance, fluorinated alkenes) the reaction with butadiene leads exclusively to four-membered rings (1,2 cycloaddition). In other cases, a mixing of four-membered and six-membered (1,4-cycloaddition) rings was observed. Furthermore, a careful reinvestigation of the prototype DA reaction (ethylene + butadiene) revealed, besides the dominant cyclohexene product, 0.02% of vinylcyclobutane. Also, the reaction is usually stereospecific, suggesting a concerted mechanism. However, such stereospecificity often disappears in the formation of four-membered adducts. These results are consistent with the existence of two possible reaction paths, a concerted path and a nonconcerted one involving a biradical or dipolar ion intermediate. The lifetime of this intermediate can be long enough to allow the internal rotation to compete with ring closure with loss of stereospecificity. The existence of a potentially competitive nonconcerted path involving a dipolar ion and leading to four-membered adducts is evidenced by VB structures VI and VIII in Scheme 2 and structures V′ and VII′ in Scheme 4. These structures are usually high in energy (for instance, in the prototype DA reaction). They can stabilize in the presence of appropriate electron-withdrawing and electron-donating substituents, making the 1,2 cycloaddition path more favorable. In some cases, the two reaction paths coexist (mix of 1,4 and 1,2 adducts), while in other cases (for instance, fluorinated ethylenes) the 1,2 reaction channel can become dominant.</p><!><p>Explicit-time-dependent Density Functional Theory was used to investigate the prototype [4+2] DA reaction between butadiene and ethylene (system 1) and the DA reaction between 1-methoxy-butadiene and cyano-ethylene (system 2). Analysis of bond orders (BOs) and atomic net charges (Q's) during the dynamics allows one to interpret the results of MO computations in terms of VB/Lewis structures.</p><p>The entire dynamics obtained for system 1 can be divided into three domains: reactant-like, product-like, and transition state domains. The transition state domain is characterized by a complete electron delocalization (benzene-like domain). It corresponds to a resonance hybrid that can be represented as a linear combination of various Lewis structures. Covalent reactant-like and product-like structures of Scheme 1 are dominant and contribute with comparable weights. Besides conventional covalent Lewis structures, other (ionic) Lewis structures (Scheme 2) give non-negligible contributions.</p><p>The relative importance of all VB structures varies along the reaction coordinate. The transition state domain is dominant at the transition state geometry, but its importance rapidly decreases when we move from transition state to reactant geometry. Simultaneously, the importance of the reactant-like domain (a resonance hybrid dominated by Lewis covalent structure II) increases.</p><p>In system 2, additional Lewis structures involving the electron-donor OCH3 on diene and the electron-acceptor CN on dienophile give important stabilizing contributions to the transition state resonance hybrid (Scheme 3) and determine a decrease of the reaction barrier with respect to the prototype case.</p><p>Our results indicate that during the dynamics both diene and dienophile can behave either as an electron donor or as an electron acceptor. For the prototype reaction, because of an internal electron transfer mechanism involving the diene HOMO and the σ* orbital of the inner (C2–C3) diene bond, ethylene appears as a better donor than butadiene. The situation is reversed in system 2 because of the presence of the two substituents on diene (OCH3) and dienophile (CN).</p><p>The VB picture derived by BOs and Q's helps to elucidate some unusual experimental results26 showing that four-membered rings can be obtained in the presence of specific substituents (F, Cl, CN) on the dienophile. VB structures such as VI and VIII in Scheme 2 (system 1) and V′ and VII′ in Scheme 4 (system 2) are consistent with the existence of a competitive nonconcerted path involving a dipolar ion intermediate and leading to four-membered adducts with loss of stereospecificity.</p><p>The result presented here are rather promising for possible future applications of explicit-time Density Functional Theory to chemical reactivity and its interpretation in terms of VB language.</p><!><p>Computational details for QM calculations and RT-TDDFT wavepacket dynamics; activation energies computed for eight different approaches of 1-methoxy-butadiene and cyano-ethylene; maximum, minimum, and average bond orders and Löwdin charges, over 5 fs of dynamics, for systems 1 and 2; and Cartesian coordinates for reactants, products, transition states, and IRC points for systems 1 and 2 (PDF)</p><p>ct9b00690_si_001.pdf</p><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Application of Novel Amino-Functionalized NZVI@SiO2 Nanoparticles to Enhance Anaerobic Granular Sludge Removal of 2,4,6-Trichlorophenol

A novel amino-functionalized silica-coated nanoscale zerovalent iron (NZVI@SiO2-NH2) was successfully synthesized by using one-step liquid-phase method with the surface functionalization of nanoscale zerovalent iron (NZVI) to enhance degradation of chlorinated organic contaminants from anaerobic microbial system. NZVI@SiO2-NH2 nanoparticles were synthesized under optimal conditions with the uniform core-shell structure (80–100 nm), high loading of amino functionality (~0.9 wt%), and relatively large specific surface area (126.3 m2/g). The result demonstrated that well-dispersed NZVI@SiO2-NH2 nanoparticle with nFe0-core and amino-functional silicon shell can effectively remove 2,4,6-trichlorophenol (2,4,6-TCP) in the neutral condition, much higher than that of NZVI. Besides, the surface-modified nanoparticles (NZVI@SiO2-NH2) in anaerobic granule sludge system also showed a positive effect to promote anaerobic biodechlorination system. More than 94.6% of 2,4,6-TCP was removed from the combined NZVI@SiO2-NH2-anaerobic granular sludge system during the anaerobic dechlorination processes. Moreover, adding the appropriate concentration of NZVI@SiO2-NH2 in anaerobic granular sludge treatment system can decrease the toxicity of 2,4,6-TCP to anaerobic microorganisms and improved the cumulative amount of methane production and electron transport system activity. The results from this study clearly demonstrated that the NZVI@SiO2-NH2/anaerobic granular sludge system could become an effective and promising technology for the removal of chlorophenols in industrial wastewater.

application_of_novel_amino-functionalized_nzvi@sio2_nanoparticles_to_enhance_anaerobic_granular_slud

1. Introduction<!>2.1. Materials<!>2.2. Synthesis of NZVI@SiO2-NH2<!>2.3.1. Removal of 2,4,6-TCP by Iron Nanoparticles in Aqueous System<!>2.3.2. Removal of 2,4,6-TCP by the Integrated NZVI@SiO2-NH2-Anaerobic Microorganism System<!>2.4. Analytical Methods<!>3.1.1. The Morphology and Composition<!>3.1.2. X-Ray Powder Diffraction and FT-IR Spectroscopy<!>3.2. TCP Dechlorination by the NZVI@SiO2-NH2 in Aqueous System<!>3.3.1. Influence of NZVI@SiO2-NH2 on the Anaerobic Biodegradation of 2,4,6-TCP<!>3.3.2. Influence of NZVI@SiO2-NH2 on the Anaerobic Microbial Activity<!>3.4. Effect of NZVI@SiO2-NH2 Dosage<!>3.5. Effect of the Initial Concentration of Fe2+<!>4. Conclusion

<p>Chlorophenols (CPs), a group of toxic and suspected carcinogenic pollutants, have been widely identified in chemical industrial wastewater. CPs are considered resistant to biodegradation and may cause adverse effects on human health and the receiving environment [1–3]. The development of technologies for the remediation of CPs from industrial wastewater has attracted a great deal of attention in recent years, such as activated carbon absorption, photocatalytic degradation, electrochemical oxidation, and biodegradation [4–7]. Among these methods, anaerobic biological treatment technology is widely applied in toxic industrial wastewater treatment process due to convenient usage and economy [8, 9]. However, anaerobic microorganisms for treating industrial wastewater containing high concentration of CPs have technical limitations such as low degradation rates, long cleanup times, and inefficient activity of biological system, due to the unfavorable environmental conditions, properties of CPs, and their sterilized effects on microorganisms [10, 11]. Therefore, it is necessary to develop the advanced anaerobic technologies with high biodegradation rates and suitable microbial system for removal and mineralization of CPs into harmless end products from industrial wastewater.</p><p>During the last decade, nanoscale zerovalent iron (NZVI) has been widely applied as a promising tool for the treatment of contaminated groundwater and soil [12]. NZVI can remove many kinds of recalcitrant pollutants such as chlorinated organic compounds, azo dyes, nitroaromatic pollutants, and heavy metals through reductive reaction mechanism [13–16]. Besides, NZVI-microorganisms system has also been conducted to investigate the codegradation pattern of chlorinated organics since they maintain a high removal rate and sufficient anaerobic microbial activity [17–19]. The role of NZVI is expected to help create an enhanced microbial environment combining all advantages of physical absorption, chemical reaction, and biodegradation. During the NZVI corrosion/hydrogen reduction reaction, the produced hydrogen gas has been considered as an electron-donor source for anaerobic microorganisms, such as methanogenic, homoacetogenic, sulfate-reducing bacteria and denitrifying bacteria [20–22]. Moreover, the removal of perchlorate, trichloroethylene, and p-chloronitrobenzene can be improved through the combination of NZVI reduction with anaerobic microorganisms [23, 24]. The removal of COD in integrated microbial-Fe0 treatment process also increased when compared with a control anaerobic reactor [17]. Thus, it is obviously worthy of enhancement of anaerobic biodegradation by adding iron-based nanoparticles. However, under the natural environment, using NZVI is highly controlled by its unique physical/chemical properties for treating chlorinated organics [25, 26]. For example, the decreasing degradation rate of the chlorinated contaminants in practice process is mainly due to the particle devitalization and aggregation, and the high surface energy leads to the oxidation of iron nanoparticle in the atmosphere [27].</p><p>To overcome these problems, surface modification is recommended for synthesizing more stable and efficient NZVI. The surface modifying agents have been reported including carboxymethyl cellulose, chitosan, silica, and polymeric electrolyte membrane [28], which can be coated onto the surface of the nanoparticles to provide electrostatic repulsion and steric or electrosteric stabilization [14]. Compared with the organic coating materials, SiO2 has characteristics of water solubility, nontoxicity, and biocompatibility features. Additionally, the SiO2 coating shell has an abundance of surface hydroxyl groups, which makes it easy for surface modification and grafting functional groups [29, 30]. Silica-coated core-shell magnetite nanoparticles, that is, Fe3O4@SiO2, have recently been prepared for potential biomedical applications [31]. Besides, amino-functionalized Fe3O4@SiO2 core-shell magnetic nanomaterial has been prepared as a novel adsorbent for aqueous heavy metals removal [30]. Nevertheless, there are only a few researches about the synthesis and application of amino-functionalized silica-nanoscale zerovalent iron technology to improve the antioxidation abilities in the atmosphere and reduce degradation capacity in the actual water treatment process.</p><p>The objectives of this study were to develop a novel amino-functionalized silica-coated nanoscale zerovalent iron (NZVI@SiO2-NH2) by using a one-step Stöber method with the surface functionalization of NZVI. Batch microcosm experiments were executed to investigate the degradation efficiency of 2,4,6-TCP in the NZVI@SiO2-NH2/anaerobic granular sludge system and to assess nanoparticles influence on the anaerobic microbial activity. In addition, the effect of NZVI@SiO2-NH2 dosage and the role of Fe2+ on the removal of 2,4,6-TCP were also evaluated. The obtained results in this study will be useful to better understand the feasibility of using NZVI@SiO2-NH2-anaerobic granular sludge system for the remediation of industrial wastewater with CPs contamination.</p><!><p>Chemicals ferrous sulfate heptahydrate (FeSO4·7H2O), ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), isopropanol, tetraethyl orthosilicate (TEOS, ≥98.0%), and methyl alcohol (99.93%, HPLC grade) and other chemical reagents were purchased from the Jinke Chemical Reagent Co., Ltd. (Guangzhou, China). 3-Aminopropyl trimethoxysilane (APTMS, ≥97.0%), poly(ethylene glycol) (PEG20000, ≥96.0%), and 2,4,6-trichlorophenol (2,4,6-TCP, 98%) were purchased from Aladdin Chemistry Co., Ltd. (China). All chemical solutions and reagents used in the experiments were of analytical grade without further purification.</p><p>The tested sludge used as the inoculum for batch experiment was taken from an industrial wastewater treatment plant of pulp and paper mill in Guangzhou. The ratio of volatile suspended sludge to total suspended sludge (VSS/TSS) of this sludge was 0.79. In order to avoid the interference of the residual contaminants in the growth environment for the removal experiments, the anaerobic granular sludge was washed with N2-sparged deionized water.</p><!><p>Nanoscale zerovalent iron (NZVI) was firstly prepared using conventional liquid-phase reduction by reducing FeSO4 with an excess of NaBH4 [14, 32]. The coating iron nanoparticle with silica (NZVI@SiO2) was prepared based on a Stöber method using silicon alkoxide as a silica source. In brief, three grams of FeSO4·7H2O was diluted in the 150 mL ultrapure water/isopropanol (1 : 2, v : v) solution and stirred in a three-neck flask at room temperature. Then, 30 mL of freshly prepared NaBH4 solution (1 mol/L) was added dropwise under nitrogen protection, resulting in a suspension of iron nanoparticles. Finally, 4 mL of TEOS and 1 mL NaOH solution were added to the above iron nanoparticle solution to generate the NZVI@SiO2.</p><p>Amino-functionalized silica-nanoscale zerovalent iron (NZVI@SiO2-NH2) was conducted on-site by APTMS as the modified agent. After the above coating process for 4 h, 0.8 mL of APTMS was then added to the suspension, and the mixture was preserved at room temperature with continuous stirring (150 rmp/min) for 12 h under nitrogen flow (40 mL/min). After the reaction, the fresh NZVI@SiO2-NH2 nanoparticles were separated and washed several times with deionized water and ethanol to remove associated polysiloxane and dried overnight at 60°C under vacuum.</p><!><p>The removal experiments of 2,4,6-TCP by NZVI, NZVI@SiO2, and NZVI@SiO2-NH2 were conducted in 250 mL saline bottles. 2,4,6-TCP (50 mg/L) was firstly spiked into the N2-sparged deionized water, and then iron samples (0.5 g/L) including NZVI, NZVI@SiO2, and NZVI@SiO2-NH2 were added to these TCP solutions, respectively. The reaction solution was deoxygenated by N2 stream for another 15 min and kept sealed with a rubber stopper during the reaction. The final volume of reaction solution was 200 mL at pH value of 7.0 which was adjusted by HCl or NaOH. The serum bottles were performed in a thermostatic incubator at 35°C with a rotation speed of 150 rpm. At given time intervals, an aliquot of reaction solution was sampled for the measurement of residual 2,4,6-TCP concentration and pH value.</p><!><p>Three parallel nanobiodegradation hybrid systems for 2,4,6-TCP removal were performed by using 250 mL saline bottles with rubber stoppers as anaerobic microcosm reactors: the combination of anaerobic granular sludge system/NZVI  (AGS + NZVI), the combination of anaerobic granular sludge/NZVI@SiO2 system (AGS + NZVI@SiO2), and the combination of anaerobic granular sludge/NZVI@SiO2-NH2 system (AGS + NZVI@SiO2-NH2). The synthetic nutrient solution in each system was deoxygenated at pH 7.0 including a certain amount of 2,4,6-TCP, 10 g VSS anaerobic granular sludge, and the same dose of nanoparticles of 0.5 L−1. Two control experiments were also conducted to clarify the intimal activity of anaerobic microorganism and the toxicity of TCP on the biodegradation: a single anaerobic granular sludge system (AGS) was the same as the mixed system, but without any iron nanoparticle; another common anaerobic biodegradation system (AGS only) was the same as AGS, except without the 2,4,6-TCP. The composition of synthetic nutrient solution in biodegradation experiment included 3000 mg/L glucose, 286 mg/L ammonium chloride, and 65 mg/L monopotassium phosphate as nutrient and energy source to facilitate growth of the biomass, with C/N/P ratio of 200 : 5 : 1. In addition, 1 mL stock solution liquor of necessary trace elements was added to the above solution containing the following composition (mg/L): CaCl2·2H2O, 330; EDTA, 5000; NiCl2·6H2O, 190; H3BO4, 14; ZnCl2, 205; MnSO4, 500; CuSO4·5H2O, 250; CoCl2·6H2O, 240; MnCl2·4H2O, 205; (NH4)6MoO4·4H2O, 9. The saline bottles were maintained in a constant temperature shaker at 35°C with a rotation speed of 150 rpm. At various time intervals, 2 mL of suspensions was withdrawn from each reactor and control bottle for the analysis of degradation rate of 2,4,6-TCP. Besides, the removal rate, methanogenic activity, and electron transport system (ETS) activity were measured in duplicate.</p><!><p>The prepared iron nanoparticles were characterized as follows. The surface morphology and size distribution were determined with an S-3700N scanning electron microscopy (SEM) characterization (Hitachi, Japan). The elemental composition was performed by energy-dispersive spectrometry (EDS, Bruker Quantax, Germany), with energy resolution 123 eV. The crystallographic structures of these nanoparticles and oxides were determined by a D8 Advance X-ray Diffraction system and Bruker AXS with a Cu target and Kα radiation (λ = 0.15418 nm) at 40 kV and 40 mA at 20°C. The scan rate was set at 1.2°/min, and the range was set from 10 to 80 (2θ). FT-IR spectra were measured by a Spectrum One-B FT-IR spectrophotometer (Nicolet Magna 550) under dry air at 20°C by a KBr pellets method. Each sample was scanned from 4000 to 400 cm−1 with a resolution of 4 cm−1. The sampled 2,4,6-TCP reaction solution was firstly centrifuged for 10 min at 10000 g, and the supernatant was filtered through a 0.45 μm hydrophilic polyethersulfone (PES) syringe filter for high-performance liquid chromatography (HPLC) analysis. 2,4,6-TCP was analyzed on a Shimadzu LC-2010A HPLC equipped with a UV detector. A C18 column (250 × 4.6 mm, 15 μm) was used for the separation. The column temperature was set at 30°C. Methyl alcohol and ultrapure water (80 : 20, v/v) were used as the mobile phase. The flow rate was set at 0.7 mL/min. The UV wavelength for 2,4,6-TCP detection was 290 nm. Electron transport system (ETS) activity was measured by an INT method for assessing metal influence on anaerobic sludge [33, 34]. A Shi's fermentation tube was used to measure biogas production. The gas composition of biogas was investigated using gas chromatography (A90, Echrom, China). Methane gas was analyzed with a 2 m × 8 mm stainless column packed with Porapak T (80/100 mesh) and the operational temperatures of detector, injection port, and column were set at 250°C, 200°C, and 100°C, respectively. Argon was used as a carrier gas with a flow rate of 30 mL/min.</p><!><p>The morphology, nanoparticle distribution, and element content of freshly prepared NZVI and NZVI@SiO2-NH2 were presented in Figure 1. All these freshly synthesized NZVI particles were tightly touching each other in roughly spherical forms (Figure 1(b)), possibly due to their aggregation properties in the aqueous solution and their tendency to remain in the most thermodynamically favorable state. On the contrary, the obtained NZVI@SiO2-NH2 nanoparticles showed a smoothly spherical shape (Figure 1(a)) and were uniformly covered by an amorphous outer layer of SiO2 (Figure 1(c)). SEM image of NZVI@SiO2-NH2 indicated an average diameter range of about 80–100 nm, while the NZVI particles ranged from 30 to 60 nm. The diameter of iron nanoparticles increased with the coating and surface modification.</p><p>The chemical composition of NZVI and NZVI@SiO2-NH2 was also determined by EDS in Figure 2, which can be used to confirm the relation between the changes of particle morphology and the size with the appearing SiO2 layer. The chemical composition of NZVI was 0.14 wt% of C, 8.88 wt% of O, and 91.03 wt% of Fe, while the NZVI@SiO2-NH2 was of 5.9 wt% of Si, 19.45 wt% of O, and 74.65 wt% of Fe, reflecting the coating of SiO2 on the NZVI surface. Moreover, the successful amino-functionalization of SiO2 layer was also indicated by CHN elemental analysis. The nitrogen content was in a proper range from 0.6 to 0.9% wt. on the NZVI@SiO2-NH2, while it was not detected on the surface of NZVI. The result confirmed the structure of a SiO2 shell on the nFe0-core surface and the aminopropyl modification of the SiO2 shell.</p><!><p>To better understand the primary characterization of the chemical and physical properties of the modified iron nanoparticle, XRD and FT-IR measurements were also made on NZVI and NZVI@SiO2-NH2 in Figure 3(a). Evidently, the same characteristic peaks were observed for NZVI@SiO2-NH2 and NZVI with a strong peak at 2θ = 44.76°and two weak peaks at 2θ = 65.16°and 82.48°, indicative of the body-centered cubic α-Fe0 in the internals of the modified iron nanoparticle. The crystalline phase of iron nanoparticles was stable during silica coating and surface amino-functionalization process [35]. In contrast to the other characteristic peaks of the NZVI, the NZVI@SiO2-NH2 showed an extra-large shoulder centered around 2θ = 23°, which can be ascribed to the existence of amorphous silica component; and no obvious peaks have shown the existence of the iron oxide as α-Fe2O3 at 2θ = 36°. Thus, it proved the presence of the silica shells on the surface of nFe0-core after the modified process. The FT-IR spectra for SiO2 coating layer and NH2-functional group were obviously discerned on the surface of NZVI@SiO2-NH2 in Figure 3(b). Compared to NZVI, the characteristic peaks of 1057 cm−1 in NZVI@SiO2 and NZVI@SiO2-NH2 were attributed to the Si–O–Si asymmetric stretching vibration which indicated the formation of silica shells on the surface of nFe0-core. In addition, the typical peak at 2934 cm−1 corresponding to –CH2– group of aminopropyl from APTMS molecules was attributed to the characteristic peaks of the amine groups, indicating the success of the aminopropyl functionalization onto the surface of NZVI@SiO2-NH2 particles during the sequential sol-gel process [30].</p><!><p>The reaction reactivity of the iron nanoparticles on 2,4,6-TCP dechlorination could depend on the physical-chemical characteristic of nanoparticles. The effect of modified process on reaction reactivity was evaluated by the degradation efficiency of 2,4,6-TCP in aqueous phase. The results of removal of 2,4,6-TCP by NZVI, NZVI@SiO2, and NZVI@SiO2-NH2 were displayed in Figure 4(b). In the control experiment, the concentrations of 2,4,6-TCP existed steadily over 120 h in anoxic aqueous solution without any iron nanoparticles. The concentration of 2,4,6-TCP decreased obviously with the increasing reaction time by different styles of iron nanoparticles in anoxic conditions. However, for NZVI, only about 14% of the 2,4,6-TCP was degraded within 120 h. On the contrary, NZVI@SiO2 and NZVI@SiO2-NH2 exhibited high reactivity toward 2,4,6-TCP degradation, with the removal rates of 38.9% and 52.3% within 120 h, respectively. On the one hand, the reduction capacity of iron nanoparticles was very sensitive to the water/oxygen in the surrounding media [36]. The freshly prepared NZVI was rapidly corroded and formed an oxidation film, resulting in the loss of reactivity, which may decline and delay the reactivity of further reaction with 2,4,6-TCP. Meanwhile, the higher removal rates of 2,4,6-TCP by NZVI@SiO2-NH2 and NZVI@SiO2 may come from the increased antioxidant capacity by SiO2 coating [37]. On the other hand, SiO2 cladding layer and NH2-functional groups of the modified iron nanoparticles could be used to enlarge the specific surface area and kept more surface active points, leading to the higher surface reactivity and faster contaminants removal rate. The specific surface area analysis showed that NZVI@SiO2-NH2 and NZVI@SiO2 had higher BET surface areas of 126.3 m2/g and 107.4 m2/g than that of NZVI (67.3 m2/g). Besides, the hydrophobic surface properties of NZVI@SiO2-NH2 were also improved by the introduced functional groups, resulting in good dispersibility, which could effectively improve the chance to contact with the active sites in surface of the nanoparticles and contaminants, thereby increasing the removal efficiency. At the initial reaction, removal of 2,4,6-TCP by NZVI, NZVI@SiO2, and NZVI@SiO2-NH2 under different reaction time could be described by first-order rate equation in different reaction time. The result was presented in Figure 4(b). The obtained k obs value was only 0.011 h−1 for the NZVI reaction with 2,4,6-TCP. The degradation efficiencies of modified NZVI@SiO2 and NZVI@SiO2-NH2 were 0.031 h−1 and 0.043 h−1, much higher than that of NZVI, indicating that introducing SiO2 cladding layer and NH2-functional groups could enhance their ability of reductive dechlorination.</p><!><p>Application of anaerobic microbial processes for the treatment of chlorinated organic compounds had drawn considerable attention in recent decade [38, 39]. Under unfavorable environmental conditions, maintaining a stable microbial activity during degradation of toxic organic pollutants was one of the challenges in anaerobic treatment process which could lead to irreversible reactor failure. In this study, batch microcosm experiments were used to investigate the microbial activity profiles of the function of NZVI@SiO2-NH2 on anaerobic granular sludge treating CPs. The influences of NZVI, NZVI@SiO2, and NZVI@SiO2-NH2 on the removal of 2,4,6-TCP in the anaerobic biochlorination system were compared in Figure 5. For the AGS system, anaerobic microorganism alone could biologically degrade 2,4,6-TCP slowly with the removal rate less than 70% during the 120 h experimental period. It was difficult to achieve complete degradation by only microbial action [40]. When NZVI@SiO2-NH2 was added to anaerobic microbial system, more than 90% of 2,4,6-TCP was removed from the system in 120 h. Consistent with the earlier studies, adding NZVI in anaerobic system was a promising approach to promote anaerobic microbial biochlorination [13]. It is noteworthy that adding the surface-modified nanoparticle (NZVI@SiO2-NH2 and NZVI@SiO2) to anaerobic granule sludge system was also capable of providing positive effect to promote anaerobic biochlorination processes. At the same time, the degradation efficiency of anaerobic microbial system for adding NZVI@SiO2-NH2, 94.6% of 2,4,6-TCP removal, was substantially higher than that from adding NZVI@SiO2 and NZVI, 88.1% and 78.3% of 2,4,6-TCP removal, respectively. The experimental results showed that surface-modified and amino-functionalized NZVI@SiO2-NH2 could effectively maintain more particle surface activity and improve the dispersibility, thereby improving the ability of the combination system to remove 2,4,6-TCP.</p><p>In order to explore the effect of NZVI@SiO2-NH2 further in anaerobic granule sludge system, the concentration of Fe2+ and pH were analyzed. Free ferrous iron were stared as the important iron reagents in practical application of environment. It can be found in Figure 6(a) that the concentration of Fe2+ was very low in NZVI/AGS system. In contrast, the concentration of Fe2+ went up sharply from 0 to 38.7 mg/L and 32.4 mg/L in NZVI@SiO2-NH2/AGS and NZVI@SiO2/AGS system, respectively. The concentration of Fe2+ was released from the Fe0 reaction, which could reflect the activity of the iron nanoparticle. However, if the freshly prepared NZVI was exposed in the atmosphere, air and water would trigger a rapid reaction with a large amount of iron corrosion product which formed the oxide layer on the surface of NZVI, identified by the earlier reports as magnetite, maghemite, and lepidocrocite [41]. Once the surface of NZVI was coated by corrosion product, only negligible Fe0 reduction reaction would occur, leading to little of Fe2+ being released into the reaction medium. Thus, the aminopropyl modification of SiO2 shell could effectively slow down the surface passivation of nanoparticles, and ferrous ion could migrate almost freely in the functionalization of layer.</p><p>Meanwhile, it has been widely reported that pH was one of the significance factors for the growth of anaerobic microbial activity and the degradation efficiency of chlorinated organic pollutants in anaerobic microbial system. As shown in Figure 6(b), the pH of AGS decreased to 5.6 when adding 2,4,6-TCP to the reaction medium, while the AGS only system was stable at 6.8. This may be due to the inhibition of anaerobic microbial growth and methanogens activity from the toxic chlorophenols, which would produce the accumulation of organic acids. With NZVI@SiO2-NH2 added, a gradual pH rise was observed in the AGS/NZVI@SiO2-NH2 system, which could be explained by the fact that H+ was required for the dissolution of iron and the iron hydroxide was positively charged by adsorbing H+ [42]. Adding NZVI@SiO2-NH2 could stabilize the reaction environment and maintain the activity of microorganism in the nFe0-microbial system. Considering that the reaction medium was unbuffered when the anaerobic granule sludge was exposed to 2,4,6-TCP, the relative change of pH in the nanobiosystem was directly affected by the reaction activity of nanoparticle: the NZVI@SiO2-NH2 induced increase in pH and was in excess of 0.21 and 0.74 pH units compared to NZVI@SiO2 and NZVI at the 120 h of experiment process. Thus, these results proved that NZVI@SiO2-NH2 had a higher activity than NZVI and NZVI@SiO2. Consequently, it is confirmed that the NZVI@SiO2-NH2 would contribute to the higher removal rate of 2,4,6-TCP, the plenty of electron donors, and the stable environment in the anaerobic granule, which further augmented the function of NZVI in the removal of chlorinated organic compounds.</p><!><p>To estimate the actual effect of NZVI@SiO2-NH2 particles on methane production, the yield of biogas and the content of methane on the combination of AGS/NZVI@SiO2-NH2, AGS/NZVI@SiO2, and AGS/NZVI system and the AGS system were shown in Figure 7 during the operation. The accumulative production of biogas was 273.5 mL, 242.7 mL, and 204.6 mL in the anaerobic system enhanced by NZVI@SiO2-NH2, NZVI@SiO2, and NZVI, respectively, in contrast with 168.3 mL in the control system. In all cases, the yield of biogas was the highest in AGS/NZVI@SiO2-NH2 system, increasing 12.5% and 33.7% when NZVI@SiO2 and NZVI were supplied, respectively. Apparently, the higher activity of NZVI@SiO2-NH2 available led to plenty of electron donors and a stale and low toxic environment in anaerobic dechlorination process, which could stimulate methanogenesis dramatically. Moreover, the subdued period of methane production was shortened in the anaerobic system enhanced by NZVI@SiO2-NH2 than AGS/NZVI@SiO2, AGS/NZVI, and the AGS system. The highest methane production increased gradually to a daily maximum of 42.2 mL/g VSS d on the 20th hour during the experiment, with methane content increasing 65.4%. Therefore, the present study provided a clear demonstration that the AGS-NZVI@SiO2-NH2 exhibits better performance in terms of higher methane production and shorter subdued period.</p><p>Electron transport system (ETS) activity of the combined AGS/NZVI@SiO2-NH2 system was also further analyzed by the INT method to describe the influence of NZVI@SiO2-NH2 on dehydrogenase activity of anaerobic sludge, as shown in Figure 8. The experimental results illustrated that long-term exposure on the 2,4,6-TCP could decrease the activity of anaerobic granule sludge. For example, the ETS activity of the exposed anaerobic granular sludge was 29.7% lower than that of the control granular sludge at all-time points. Meanwhile, the ETS activity of anaerobic granular sludge was stabled at 87.4%, 94.0%, and 113.2% of the control granular sludge after 120 h of experiment process in the combination of AGS/NZVI, AGS/NZVI@SiO2, and AGS/NZVI@SiO2-NH2 system, respectively. NZVI@SiO2-NH2 did not exert much stimulation and inactivation on the ETS activity of the anaerobic bacteria at the initial process of the experiment, because the core nFe0 was avoided directly contacting with microorganism by the surface-modified SiO2 shell. It can be observed that the addition of NZVI@SiO2-NH2 to anaerobic biodegradation system could enhance the activity of sludge and reduce the adverse influence of 2,4,6-TCP.</p><!><p>Four identical batch microcosm experiments were operated at different dosages of 0.1, 0.2, 0.5, and 1 g/L in parallel for 120 hours to evaluate the removal rate of 2,4,6-TCP. The removal rate of 2,4,6-TCP as a function of time was presented in Figure 9. It can be observed that high removal rate of 2,4,6-TCP occurred when the NZVI@SiO2-NH2 dosage increased in the anaerobic granular sludge system. For example, the removal rate of 2,4,6-TCP was found to be 80.7%, 87.4%, 95.4%, and 96.6%, respectively, when the addition dosage of NZVI@SiO2-NH2 to the anaerobic system was 0.1, 0.2, 0.5, and 1 g/L. In addition, the concentration of 2,4,6-TCP in combined system was obviously deceased in the first 10–12 h of the experiment, which indicated that 2,4,6-TCP can be directly chemically reduced and/or be adsorbed on the surface layer of NZVI@SiO2-NH2. The adsorptive and active sites on the surface of NZVI@SiO2-NH2 increased when the amount of NZVI@SiO2-NH2 increased. Moreover, adding NZVI@SiO2-NH2 dosage to anaerobic granule sludge system is capable of providing more electron donors to promote anaerobic metabolic processes, and the corrosion process of NZVI@SiO2-NH2 can produce Fe2+/Fe3+ and hydrogen which can be used as minerals for the anaerobic microorganisms; thereby, the remnants of pollutants and toxic intermediate products could be further removed biologically by the attached microorganism in the combined system.</p><p>Methanogenic activity test was used to determine the influence of NZVI@SiO2-NH2 dosage on the anaerobic sludge activity in various anaerobic processes. Figure 10 showed the summary of the cumulative biogas productions with the function of NZVI@SiO2-NH2 in anaerobic methanogenic process. The accumulative production of biogas of the seed granular sludge at 35°C was 268.1 mL in the AGS only system. When the anaerobic system was exposed to 50 mg/L of 2,4,6-TCP, the cumulative production of biogas decreased to 157.7 mL, far lower than the AGS only. The reason may be that chemical 2,4,6-TCP had a significant inhibitory effect on methanogenic activity of anaerobic microorganism, which affected the 2,4,6-TCP biological dechlorination and final mineralization by anaerobic microorganism. Maintaining a sufficient microbial activity was essential for a stable anaerobic treatment system on unfavorite condition. With increasing the dosage of NZVI@SiO2-NH2 from 0.1 g/L, 0.2 g/L, and 0.51 g/L to 1 g/L, obvious increase of the cumulative production of biogas in the integrated AGS/NZVI@SiO2-NH2 system during the operation was observed. And among these dosages of NZVI@SiO2-NH2, the cumulative production of biogas (0.2 g/L and 0.5 g/L) increased by 31.7% and 62.4%, respectively, more than that of the AGS system at 120 h. That is to say, the methanogenic activity of the seed sludge increased with the increasing dosage of NZVI@SiO2-NH2. The results implied that the activity of anaerobic microorganisms was significantly influenced by the function of the added iron nanoparticles. However, the results of cumulative biogas productions in anaerobic dechlorination processes with the different dosage of NZVI@SiO2-NH2 were largely different from the removal rate of 2,4,6-TCP (Figure 9). The methanogenic activity of 1 g/L of NZVI@SiO2-NH2 was considerably lower than that of 0.5 g/L, which means that the anaerobic system should be operated with appropriate concentration of iron nanoparticles.</p><!><p>The effect of Fe2+ on the removal of 2,4,6-TCP in combined anaerobic granule sludge/NZVI@SiO2-NH2 system was investigated at initial neutral pH, as shown in Figure 11. When only 100 mg/L of Fe2+ was presented in the reaction solution, the concentration of 2,4,6-TCP kept near 48 ± 0.1 mg/L within 120 h. However, the existence of Fe2+ may involve the reactions (Fe2+ → Fe3+ + e−) which might be attributed to increasing Fe2+ reducing ability. The result obviously indicated that the ferrous ion was not the major source on the removal of 2,4,6-TCP. When 50 mg/L to 200 mg/L Fe2+ was added to the AGS/NZVI@SiO2-NH2 system, the removal rate of 2,4,6-TCP decreased from 87.8% to 75.2%, indicating that the ferrous ion in the mixed system of NZVI@SiO2-NH2 and anaerobic sludge did not enhance the 2,4,6-TCP degradation. The higher content of Fe2+ had a negative effect on the 2,4,6-TCP degradation in the integrated AGS/NZVI@SiO2-NH2 system. Besides, Table 1 has shown that the added Fe2+ had a significant inhibitory effect on methanogenic activity of AGS/NZVI@SiO2-NH2 system. The specific methanogenic activity decreased from 48.7 mL/g VSS d to 37.5 mL/g VSS d at 200 mg/L of Fe2+. This may be because higher concentration of Fe2+ (200 mg/L) decreased very quickly in the reaction system with a large amount of amorphous colloidal Fe(OH)3. These iron precipitates can be attached to the surface of iron nanoparticles and anaerobic granular sludge, which would decrease the reduction ability of iron nanoparticles and the activity of anaerobic microorganisms. Thus, this would further affect the degradation efficiency and methanogenic activity of anaerobic microorganisms. Besides, the concentration of Fe2+ gradually raised and remained at a certain concentration in this combined system (Figure 6(a)), suggesting that the added NZVI@SiO2-NH2 could continue to release Fe2+ as the supply of electronics and elements for NZVI@SiO2-NH2-anaerobic granule sludge system.</p><!><p>NZVI@SiO2-NH2 was successfully synthesized by the surface functionalization of NZVI using TEOS and APTMS. The obtained NZVI@SiO2-NH2 had a good dispersibility and antioxidant capacity and can be stored in the air for long time. Compared to the NZVI, NZVI@SiO2-NH2 showed an appreciable reactivity with 2,4,6-TCP. The determined K obs was 0.043 h−1 at the neutral condition, much higher than that of NZVI. The combined anaerobic granule sludge system/NZVI@SiO2-NH2 had significant synergistic effects on the removal of 2,4,6-TCP. More than 94.6% of 2,4,6-TCP was removed from the combined AGS/NZVI@SiO2-NH2 system during the operation processes. The added NZVI@SiO2-NH2 to microbial system can decrease the toxic inhibition of 2,4,6-TCP, resulting in improved cumulative amount of methane production and ETS activity. Moreover, the combination of AGS/NZVI@SiO2-NH2 system should be operated with appropriate concentration of NZVI@SiO2-NH2. The 2,4,6-TCP degradation and methane production with extra Fe2+ (>50 mg/L) in the combined AGS/NZVI@SiO2-NH2 were remarkably adverse on the performance and methanogenic activity of AGS-NZVI@SiO2-NH2. The novel of modified nanoparticle could be an effective and promising material in the anaerobic treatment system for removal of CPs from industrial wastewater. However, further study should be carried out to control the efficiency and activity of microbial system in application of the novel NZVI@SiO2-NH2 in situ remediation of industrial wastewater.</p>

PubMed Open Access

Ginseng berry extract increases nitric oxide level in vascular endothelial cells and improves cGMP expression and blood circulation in muscle cells

[Purpose]The purpose of this study was to determine whether ginseng berry extract improves blood circulation by regulating vasodilator expression in exposed to tumor necrosis factor alpha (TNF-α)-exposed endothelial cells and muscle cells.[Methods]Nitric oxide (NO) and cGMP levels in human umbilical vein endothelial cells (HUVECs) and A7r5 cells exposed to ginseng berry extract were investigated. Based on the in vitro results, healthy participants were treated with ginseng berry extract for 4 weeks and then a non-invasive vascular screening device was used to confirm the improvement of blood circulation.[Results]When TNF-α-treated cells were exposed to the ginseng berry extract, the expression levels of endothelial nitric oxide synthase (eNOS), NO, and cGMP were recovered to almost normal levels. In addition, TNF-ɑ-induced overexpression of vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), e-selectin, and p-selectin was lowered by ginseng berry extract. The ginseng berry extract significantly increased ankle brachial pressure index compared to placebo (p < 0.05).[Conclusion]This study confirmed that the intake of ginseng berry extract improved blood circulation and therefore, its intake would be helpful for people having problems with blood circulation.

ginseng_berry_extract_increases_nitric_oxide_level_in_vascular_endothelial_cells_and_improves_cgmp_e

INTRODUCTION<!>Preparation of ginseng berry extract<!>Cell culture<!>A7r5 and HUVEC co-culture<!>Cell viability<!>Total RNA extraction and cDNA synthesis<!>Intracellular nitric oxide production in HUVECs<!>cGMP quantification in A7r5 cells<!>Reverse transcription-polymerase chain reaction<!><!>Subject characteristics and ginseng berry extract administration<!>Evaluation of improvement of blood circulation<!>Data analysis<!>Effects of ginseng berry extract on HUVEC viability<!>Ginseng berry extract contents and cell viability in HUVEC.Component of Ginseng Berry and Cell Viability in HUVECs.<!>Ginseng berry extract induces eNOS expression, which increases intracellular NO levels in HUVECs<!>Ginseng berry Extract induces eNOS expression which leads increase of intracellular NO in HUVEC.<!>Ginseng berry extract regulates adhesion molecules through the NO-cGMP pathway in A7r5 muscle cells<!>Ginseng berry Extract reduce TNF-a-induced adhesion molecules through NO-cGMP pathway in A7r5. RT-PCR results of cGMP and Adhesion Molecules Expression level by Ginseng berry extract in A7r5.<!>Ginseng berry extract reduces TNF-α-induced adhesion molecule and ET-1 expression in HUVECs<!>Ginseng berry Extract reduce TNF-a-induced adhesion molecules and ET-1 expression in HUVEC. RT-PCR results of Adhesion Molecules and ET-1 Expression level by Ginseng berry extract in HUVEXs.<!>Blood circulation changes following the intake of ginseng berry extract<!>DISCUSSION<!>CONCLUSION

<p>Blood via blood vessels delivers oxygen and nutrients to each tissue in the body and removes the waste produced by cells1,2, transports hormones, defends cells from external harmful substances, and maintains proper body temperature and homeostasis3. Therefore, a normal blood circulation is very important for maintenance of body function4.</p><p>The vascular endothelium is a layer of endothelial cells on the inner surface of the vascular layer5. It regulates fluidity and flow of blood and blood vessel tension via the synthesis and secretion of various kinds of substances having biological activity6. It also plays an important role in maintaining and controlling arterial blood vessel function and health by participating in platelet aggregation, thrombogenesis, and dissolution7. In other words, vascular endothelial cells primarily serve as an anatomical barrier to prevent blood circulation in the vessel wall, as well as to control blood vessel tension, partially control cell growth in the blood vessel wall, and regulate extracellular matrix deposition8. These cells are also involved in blood vessel protection from substances and cells, blood clotting in response to partial injury and inflammation, and homeostasis of blood vessels by repair responses9.</p><p>Continued inflammatory stimuli (e.g., oxidative stress, inflammatory cytokines, exposure to pathogenic agents) cause endothelial dysfunction, with endothelial cells becoming overactive or chronically active10. In this condition, damage to the vascular endothelium leads to excessive inflammation, resulting in excessive secretion of soluble adhesion molecules such as intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, e-selectin, and p-selectin from endothelial cells11. Oxidized cholesterol accumulates in the injured area, forming a plaque, and the inner wall of the blood vessel swells up, resulting in narrowing of the blood vessels and decreased blood circulation11.</p><p>Nitric oxide (NO), which plays a key role in maintaining endothelial homeostasis, is produced by endothelial nitric oxide synthase (eNOS) from the amino acid L-arginine in endothelial cells12. A typical function of NO is vasodilation13. The NO produced diffuses into the smooth muscle cells of the blood vessels and activates the soluble guanylate cyclase to induce cyclic guanosine-5'-monophosphate (cGMP)-mediated vascular relaxation14. cGMP acts as a second messenger to induce many biological effects of NO, such as relaxation of smooth muscle or inhibition of platelet aggregation15. NO inhibits the expression of proinflammatory cytokines, chemokines, and adhesion molecules in addition to vascular relaxation16. Therefore, it plays a key role in regulating vascular endothelial function, inhibiting vascular recruitment of white blood cells, proliferation of vascular smooth muscle cells, platelet aggregation, and inhibiting the production of tissue factor involved in thrombus formation17.</p><p>However, when functional and structural changes of vascular endothelial cells occur due to risk factors for vascular diseases such as hypertension, diabetes, hyperlipidemia, and smoking, endothelial cells cannot play the protective role mentioned above, but cause atherosclerosis18. NO production cannot be achieved by eNOS; NO is oxidized by reactive oxygen species to ONOO-, thus reducing the bioavailability of NO19. The resulting ONOO-oxidizes BH4, the coenzyme of eNOS, or increases the activity of arginase20. Therefore, eNOS uncoupling phenomenon (excessive O2 · - and H2O2 formation due to abnormal action of eNOS) is induced by decreasing L-arginine or insufficient BH4, which is the substrate of eNOS21. As a result, NO-mediated endothelium-dependent vascular dilation leads to impairment and negative effects on blood flow22.</p><p>Ginseng and its associated ingredients have been used in food or herbal medicines for thousands of years to treat many diseases23. It exhibits anti-aging, anti-diabetic, anticancer, and anti-fatigue effects through promotion of DNA, RNA, and protein synthesis24. In addition, ginseng contains many ingredients, such as ginsenosides, polysaccharides, polyacetylenes, fatty acids, mineral oil, peptides, and amino acids25. The bioactivity of ginseng is attributable to the presence of ginsenosides in the root26. Thus far, studies have focused on the effects of ginseng roots26. However, these ingredients were also distributed in other parts of ginseng, such as berries and leaves26. Ginseng berry was reported to have a higher ginsenoside content than roots, and its anti-hyperglycemia and anti-obesity activities were studied27,28. Despite its efficacy, ginseng berry has not received much attention29. No studies have been reported on the effects of ginseng berry extract in improving blood circulation. Therefore, the purpose of this study was to determine whether tumor necrosis factor (TNF)-□-induced endothelial cells and muscle cells would be recovered using ginseng berry extract and to investigate the effect of the extract intake on blood circulation. NO is expected to be produced in vascular endothelial cells owing to treatment with the extract, and the produced NO is expected to increase cGMP level and decrease the level of adhesion molecules in vascular muscle cells.</p><!><p>Raw ginseng berries were harvested from Hongcheon, Gangwon-do, South Korea, and the seeds were separated and removed. Ginseng berries were dried and refluxed with 100% distilled water and concentrated under reduced pressure at 45˚C (the patent number 10-2015-0101209).</p><!><p>Human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (A7r5) were both purchased from ATCC (Manassas, VA, USA). HUVECs were cultured in complete microvascular endothelial cell growth medium (EGM-2, Lonza Walkersville, Inc.), in humidified 5% CO2 incubator at 37˚C. A7r5 cells were cultured in complete medium (DMEM, Hyclone, GE Healthcare Life Sciences, South Logan, Utah, USA), in humidified 5% CO2 incubator at 37˚C.</p><!><p>Co-culture plates, SPLINsertTM Hanging plates were purchased from SPL (SPL Life Sciences, Pochun, Gyeonggi-do, South Korea). A7r5 cells were seeded at a density of 1 × 105 cells per well in complete DMEM, and HUVECs were seeded at a density of 5 × 104 cells per cassette in complete EGM-2. Cassettes with HUVECs were placed on each well and incubated in humidified 5% CO2 incubator at 37˚C.</p><!><p>HUVECs were seeded at a density of 1 × 104 cells per well in a 96-well plate. Cells were incubated for 24 h with EGM-2, in humidified 5% CO2 incubator at 37˚C. After 24 h, the medium was removed and cells were washed with 1× DPBS (Hyclone). Fresh medium was added and ginseng berry extract at various concentrations (0, 15. 63, 31. 25, 62. 5, 125, 250 and 500 μg/ml) was added. TNF-α (10 ng/ml) was added after 30 min and the cells were incubated in humidified 5% CO2 incubator at 37˚C. A Cell counting Kit-8 (Dojindo, Rockville, MD, USA) was used to determine the viability of HUVECs. CCK-8 solution (10 μl) was added in each well and incubated for 4 h in humidified 5% CO2 incubator at 37˚C. The absorbance was measured at 450 nm with a plate reader (Varioskan Lux, Thermofisher).</p><!><p>HUVECs were seeded at a density of 2 × 105 cells per well and incubated for 24 h. Culture medium without FBS was added, and ginseng berry extract at various concentrations (20, 100, and 500 μg/ml) was added. TNF-α (10 ng/ml) was added to each group (except normal cell group) and incubated for 24 h. Cells were washed with 1× DPBS and treated with easy-BLUETM (1 ml) of the Total RNA Extraction Kit (Intron Biotechnology, Sungnam, Gyeonggi-do, South Korea). Cell lysates were collected, 200 μl of chloroform was added, and centrifuged at 10,000 ×g (4˚C, 10 min). Supernatants were collected, iso-propanol (500 μl) was added, and the mixture was kept for standing for 10 min at room temperature. The mixed solution was centrifuged at 7,500 ×g (4˚C, 10 min), and the supernatants were discarded. Pellets were washed with 75% ethanol and centrifuged at 7,500 ×g (4˚C, 5 min) twice. Pellets were dissolved with distilled water and treated with DEPC. Extraction of total RNA from A7r5 cells, which were co-cultured with HUVECs, was performed following the same procedure.</p><p>cDNA synthesis was performed with Power cDNA Synthesis Kit (Intron Biotechnology), according to the manual in the kit. Total RNA (1 μg) was used to synthesize cDNA, which was used to perform reverse transcription-polymerase chain reaction.</p><!><p>HUVECs were seeded at a density of 2 × 104 cells per well in a 96-well black plate (SPL Life Sciences, Pochun, Gyeonggi-do, South Korea) and incubated in humidified 5% CO2 incubator at 37˚C. The culture medium was changed to FBS-free culture medium after 24 h, and ginseng berry extract at various concentrations (20, 100, and 500 μg/ml) was added to each well. TNF-α (10 ng/ml) was added after 30 min. Detection of intracellular NO was performed according to the kit, OxiSelectTM Intracellular Nitric Oxide (NO) Assay Kit (Cell Biolabs, San Diego, CA, USA) manual and fluorescence was measured with a fluorometric plate reader at 480 nm/530 nm.</p><!><p>A colorimetric cGMP ELISA Kit (Cell Biolabs) was used to determine the cGMP level in A7r5 smooth muscle cells. A7r5 cells were co-cultured with HUVECs and the samples were treated with or without with TNF-α for 24 h in humidified 5% CO2 incubator at 37˚C. Cells were lysed and the cGMP level was determined in the lysates following the kit manual. At the end of the experiment, the absorbance was read at 450 nm using a plate reader.</p><!><p>Primers for HUVECs, β-actin, eNOS, ET-1, VCAM-1, ICAM-1, e-selectin, and p-selectin mRNA specific primers were synthesized and purchased from Macrogen (Seoul, South Korea). Primers for A7r5, GAPDH, VCAM-1, ICAM-3, e-selectin, and p-selectin were also synthesized and purchased from Macrogen (Table 1). Maxime PCR PreMix Kit (i-Taq) from Intron was used to perform PCR amplification. PCR products were loaded on 1% agarose gel to perform electrophoresis.</p><!><p>Mean ± Standard deviation (SD)</p><p>GBE; ginseng berry extract supplements group, PG; placebo supplements group, RPWV; right brachial ankle pulse wave velocity, LPWV; left brachial ankle pulse wave velocity, RABPI; right ankle brachial pressure index, LABPI; left ankle brachial pressure index</p><!><p>In this study, 30 males (over 20 years of age) were included and divided into the following groups: Ginseng berry extract group (n=15; age, 34. 73 ± 6. 97 years; height, 172. 33 ± 7. 20 cm; weight, 73. 33 ± 12. 19 kg) and placebo group (n=13; age, 35. 07 ± 10. 25 years; height, 170. 79 ± 9. 19 cm; weight, 65. 43 ± 14. 67 kg). Two subjects in the placebo group voluntarily discontinued after 1 week of initiating the administration of ginseng berry extract and were excluded from the study. Thus, 28 patients orally took ginseng berry extract for 4 weeks, and ginseng berry extract distributed to the subjects completed an average intake of 66%. Ginseng berry extract was administered in the form of hard capsules containing ginseng fruit extract powder (500 mg strength); the capsules were administered twice daily to ensure a dose of 1000 mg per day. The intake setting was based on a significant increase in serum NO based on the previous animal study result9.</p><!><p>To evaluate the improvement of blood circulation, brachial ankle pulse wave velocity and ankle brachial pressure index were measured before and after ingestion of ginseng berry extract. All subjects randomly assigned to the ginseng berry extract group visited the laboratory before and 4 weeks after ingestion and took a rest for 10-15 minutes while they were in stable condition. During the resting period, the heart rate reached 60 bpm, and the stable heart rate was maintained for more than 2 min. All measurements were performed using a non-invasive vascular screening device (VP-1000 plus, Omron Healthcare Co., Ltd., Japan).</p><!><p>All data were analyzed using SPSS 21. 0 statistical package, and the mean and standard deviation were calculated. The changes in NO and cGMP levels were tested by independent t-tests. In addition, independent t-test was used to determine the difference among subject characteristics and baseline blood circulation values before the administration of ginseng berry extract, and 2-way mixed ANOVA was used to test the difference between groups before and after ingestion of ginseng berry extract. The significance test level was set at p < 0.05.</p><!><p>No cytotoxicity or adverse effects were observed in HUVECs even after treatment with the highest concentration of the ginseng berry extract (500 μg/ml; Figure 1). Since no cytotoxicity was observed at up to 500 μg/ml, we used the extract at a concentration up to 100 μg/ml in subsequent experiments.</p><!><p>(A) Percentage of Ginsenoside Re, Rg1, Rf, Rg2, 20R_Rg2, Rh1 and Rh4 in Ginseng Berry Extract. (B) Cell Viability in HUVECs treated with Ginseng Berry extract 0, 31.25, 62.5, 125, 250 and 500 ug/ml were measured by CCK-8 kit.</p><!><p>TNF-α-induced cells showed downregulated eNOS expression and decreased NO production compared to normal cells. Treatment with the ginseng berry extract recovered the expression of eNOS and thereby the production NO. This result confirms that the ginseng berry extract affects the production of NO (Figure 2).</p><!><p>eNOS Expression and Intacellular NO level in HUVEC. (A) eNOS exprssion level and (B) intracellular NO level in HUVECs, treated with 0, 20, 100 and 500 ug/ml. (mean ± s.d., *P<0.05, **P<0.01 as compared to TNF-a-induced Ginseng berry 0 ug/ml treated group).</p><!><p>HUVECs and A7r5 cells were co-cultured. Treatment with TNF-α decreased cGMP level, which was recovered by the ginseng berry extract in a dose-dependent manner. This result indicates that the ginseng berry extract has a significant effect on the NO-cGMP pathway.</p><p>The expression of the adhesion molecules VCAM-1, ICAM-1, e-selectin, and p-selectin increases in endothelial cells in response to excessive diet or stress. The results show that the expression of adhesion molecules increased in TNF-α-induced A7r5 cells and was reduced in cells treated with the ginseng berry extract (Figure 3).</p><!><p>(A) Reletive expression of cGMP and (B) adhesion molecules in A7r5, treated by 0, 20, 100 and 500 ug/ml Ginseng berry extract and (C) RT-PCR results, treated with 0, 20, 100 and 500 ug/ml. (mean ± s.d.,P *<0.05, **P<0.01 as compared toT NF-a-induced Ginseng berry 0 ug/ml treated group).</p><!><p>The increased expression of adhesion molecules and ET-1 in TNF-α-induced HUVECs was decreased by treatment with the ginseng berry extract (Figure 4).</p><!><p>(A) Reletive expression of adhesion molecules and ET-1 in HUVEXs, treated by 0, 20, 100 and 500 ug/ml Ginseng berry extract and (B) RT-PCR results, treated with 0, 20, 100 and 500 ug/ml. (mean ± s.d., *P<0.05, **P<0.01 as compared to TNF-a-induced Ginseng berry 0 ug/ml treated group).</p><!><p>This study confirmed the relaxation of smooth muscle cells by the ginseng berry extract through in vitro experiments and clinical trials. There was no significant difference in age and physical characteristics among the participants in this study. In addition, there was no significant difference between the groups in blood circulation factors (RPWV, LPWV, RABPI, LABPI) at baseline (p>0. 05). There was no interaction effect among RPWV, LPWV, and LABPI in the ginseng berry extract supplement group, but RABPI decreased in the ginseng berry extract supplement group and significantly increased in the placebo group (p=0. 016; Table 1).</p><!><p>TNF-α decreases eNOS expression and intracellular NO level and markedly increases the expression of adhesion molecules and ET-1 in endothelial cells and muscle cells, resulting in endothelium dysfunction. The results showed that these abnormalities prevented endothelium dysfunction. Many ginsenosides in ginseng berry, including ginsenoside Re, are reported to have effect on penile erection in in vivo studies. Erectile dysfunction is also associated with blood circulation and muscle contraction and relaxation30. The problem of blood circulation is caused by endothelial dysfunction. Endothelial dysfunction has been reported to result from a reduction in NO level, which is also referred to as impaired vasodilation31. The decrease in NO synthesis is due to a decrease in the expression of eNOS, a deficiency in substrate or cofactor required for eNOS activity, a change in cell signal transduction, impaired eNOS activation, and reactive oxygen species (ROS)32-35.</p><p>As expected, the ginseng berry extract first increased the expression of eNOS and decreased the expression of ET-1 in endothelial cells. Increased expression of eNOS in endothelial cells eventually leads to NO production, which leads to the expression of cGMP in smooth muscle cells. Increased expression of cGMP causes smooth muscle cell relaxation and ultimately induces vasodilation. In addition, expression of VCAM-1, ICAM-1, p-selectin, and e-selectin increased by high-fat diet was reduced by ginseng berry extract, resulting in smooth muscle cell relaxation. It is known that ET-1 is strongly expressed in obese conditions in which muscle cells shrink to interfere with blood flow; however, the mechanism is not clear (Figure 5).</p><p>A decrease in blood pressure and PWV levels was observed after ingesting ginseng berry extract for a certain period of time. Increasing the diameter of the blood vessel or decreasing the thickness of the vessel wall should occur to confirm PWV reduction. In vitro experiments using vascular endothelial cells and smooth muscle cells showed that ginseng berry relaxed muscles. When the muscles relax, the thickness of the vessel wall decreases and the blood vessels expand. Based on the results of our in vitro experiments and clinical trials, we found that the ginseng berry extract helps to improve blood circulation.</p><!><p>This study confirmed the effect of ginseng berry on blood circulation. Because of the difficulty in elucidating the clinical outcome, we constructed an in vitro system with two different cell lines. The change in the NO-cGMP pathway induced by ginseng berry extract was confirmed. Moreover, ginseng berry extract also decreased the expression of cell adhesion molecules causing inflammation in the blood vessel. As a result, this study suggests that the intake of ginseng berry extract improves blood circulation and therefore, ginseng berry extract intake would be helpful for people having problems with blood vessel function.</p>

PubMed Open Access

Chemically Orthogonal Three-Patch Microparticles**

Compared to two-dimensional substrates, only a few methodologies exist for the spatially controlled decoration of three-dimensional objects, such as microparticles. Combining electrohydrodynamic co-jetting with synthetic polymer chemistry, we were able to create two- and three-patch microparticles displaying chemically orthogonal anchor groups on three distinct surface patches of the same particle. This approach takes advantage of a combination of novel chemically orthogonal polylactide-based polymers and their processing by electrohydrodynamic co-jetting to yield unprecedented multifunctional microparticles. Several micropatterned particles were fabricated displaying orthogonal click functionalities. Specifically, we demonstrate novel two- and three-patch particles. Multi-patch particles are highly sought after for their potential to present multiple distinct ligands in a directional manner. This work clearly establishes a viable route towards orthogonal reaction strategies on multivalent micropatterned particles.

chemically_orthogonal_three-patch_microparticles**

<p>The directionally controlled presentation of chemical ligands on the surface of particles defines critical materials processes, such as three-dimensional gelation,[1] directed self-assembly,[2] or the controlled interaction of particles with biological cells.[3] While the spatially controlled presentation of chemical and biological ligands is well established for two-dimensional substrates,[4] very few methodologies exist for the spatially controlled decoration of three-dimensional objects, such as microparticles.[2f,5] Many of the patterning methods for two-dimensional substrates including photolithography, microcontact printing, dip-pen nanolithography, or blockcopolymer micelle nanolithography,[4d,6] are not easily extendable to the three-dimensional surfaces of microparticles. Thus, there is a clear need for efficient microstructuring methodologies for meso-scale particles and some of the more promising strategies include microfluidic and microforming techniques[5c,7] template-assisted polymerizations,[8] electrohydrodynamic co-jetting,[9] or the use of pickering emulsions.[10] With the requirement for spatially controlled immobilization of ligands also comes an increased need for the parallel immobilization of multiple, chemically distinct ligands on defined and independent interfacial patches of the same object. While most multi-ligand attachment strategies have relied on statistical distributions of ligand mixtures,[11] several studies have emphasized the need for the independent attachment of two or more ligands on the same surface by orthogonal immobilization strategies.[12] In this case, an important prerequisite is the compatibility with the biological "reaction" environment. Bertozzi et al. coined the term bio-orthogonal ligation to express the need for chemical selectivity relative to 1) the biological environment and 2) the different types of ligands to be co-presented on different surface patches.[13] Typically, different orthogonal immobilization schemes take advantage of click-type reactions and, in case of two-dimensional substrates, have been well-established,[14] including "double-click" strategies.[15] Recent work has revealed the potential of particles with multiple surface patches, however, the orthogonal functionalization of different surface locations still poses significant challenges.[16] Herein, we now report the spatially controlled immobilization of three chemically distinct patches on the same microparticle by orthogonal surface reactions. Combining electrohydrodynamic (EHD) co-jetting with synthetic polymer chemistry, we were able to create two- and three-patch microparticles displaying chemically orthogonal anchor groups on three distinct surface patches of the same particle.</p><p>As depicted in Figure 1, the preparation of multi-patch microparticles was achieved by EHD co-jetting of up to three polymer solutions in parallel. The EHD co-jetting process yielded well-defined microfibers that were subsequently sectioned into multi-patch particles.[17] In this study, we employed disk-shaped microparticles with an average diameter of 10–15 μm and an average aspect ratio of 1:2 (height:diameter). Size and shape of the microparticles can be controlled within a broad range,[17] but were ultimately selected to be in the 10–20 μm range for practical reasons, such as optimal imaging of the individual chemical surface reactions by fluorescence and Raman confocal microspectroscopy. The polymer used throughout this study was a biodegradable poly(lactide-co-glycolide) (PLGA) polymer. To impart chemical orthogonality, several different chemically functionalized polylactide (PLA) polymers were synthesized. The chemically orthogonal PLA derivatives can be added to the different PLGA compartments to provide chemical patches for directionally controlled surface modification. In our case, a set of five different PLA derivatives was selected for their ability to support orthogonal surface modification without cross-reactions (Figure 1).</p><p>The first step of the synthesis of the functionalized PLA derivatives 2–5 involved the ring-opening co-polymerization of monomer 1 and L-lactide in the melt, and subsequent palladium-catalyzed hydrogenation of the benzyl ether bonds to yield the hydroxy-modified PLA derivative 2 (Supporting Information Section 2.1). Polymer 2 was then used as the starting point for further diversification of the functionalities using a number of post-polymerization modifications: 1) Reaction with 3-(diphenylphosphino)-4-(methoxycarbonyl) benzoic acid with N,N′-dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP) yielded the PLA derivative 3 for subsequent Staudinger Reaction. 2) Alternatively, polymer 2 was converted with 2-(4-benzoylphenyl) acetic acid and DCC/DMAP under dry conditions to yield the photoreactive PLA derivative 4. 3) Polymer 5, a cyclooctyne-modified polylactide, was derived from polymer 2 by straightforward conversion with 2-(cyclooct-2-yn-l-yloxy) acetic acid and DCC/DMAP (Supporting Information Section 2.2).</p><p>In general, we found a satisfying compatibility of the PLA derivatives with the PLGA base polymer: For concentrations of up to 50 % of the PLA additives, no adverse effect on the electrohydrodynamic co-jetting process was observed—independent of the chemical nature of the PLA derivative that was added to the jetting solutions. On the other hand, preliminary immobilization experiments demonstrated that PLA concentrations above 20% were adequate to ensure effective surface coupling of model ligands (Supporting Information Section 2.3).</p><p>We then created a group of bicompartmental microparticles, which presented a single hemispheric patch selectively displaying only one of the functional anchor groups. In this case, the second hemisphere was composed of the PLGA base polymer only, and served as an internal reference for the surface reactions. Figure 2 depicts three different particle architectures. These architectures allowed for spatially controlled modification of one hemisphere only. The reference patch showed only very low levels of non-specific ligand adsorption. In Figure 2A, PLGA particles with one hemispheric surface patch containing polymer 3 (with green dye) was treated with azide-PEG-Biotin (PEG: polyethylene glycol) by Staudinger ligation. The biotin was then labeled with TRITC-Streptavidin (red dye) for imaging purposes. Coexistence of the red and green fluorescence in the CLSM images (Figure 2A3) confirms the spatially controlled surface modification of the microparticles. Similarly, Figure 2B displays particles with polymer 4 in the blue hemisphere only. As found for all PLA derivatives, the functional polymer is restricted to only one hemisphere of the microparticles (Figure 2B1) underpinning the high degree of patchiness obtained by the EHD co-jetting process. The microparticles were subsequently incubated with a protein, Bovine Serum Albumin (BSA) tetramethylrhodamine (red dye), and exposed to UV light at 365 nm to initiate the photoimmobilization of the BSA to the reactive surface patch (Figure 2B2). The high degree of selectivity of the photoimmobilization reaction is confirmed by the spatially controlled surface binding of the protein, as depicted in the overlay images of Figure 2B3. Similarly, the copper-free click chemistry of bicompartmental microparticles using polymer 5 was successfully carried out, as verified by CLSM analysis (Figure 2C). In this case, the unreactive PLGA hemisphere was labeled with a green fluorescence dye loaded in the bulk of the compartment, whereas the azide-reactive compartment that contained polymer 5 was non-fluorescent (Figure 2C1). The spatially controlled surface modification of these patchy microparticles with azide-PEG-biotin and Alexa Fluor 647 Streptavidin (magenta dye) was confirmed by selective surface binding, as shown in the overlay images of Figure 2C3. We further conducted spatially controlled surface modification of two-patch particles with one functionalized patch using both polymers 1 and 2 with analogue results (Supporting Information 2.3–4). In summary, this initial immobilization studies indicated two important findings: 1) The addition of functionalized PLA derivatives into one jetting stream resulted in well-defined patchy microparticles and 2) the selected surface chemistries are fully orthogonal to the base polymer (PLGA), as shown by the fact that only negligibly low levels of non-specific binding were observed on the reference hemisphere.</p><p>Encouraged by these initial data, we conducted a second immobilization study that included two different functionalized PLA derivatives in two separate hemispheres of the same particle. For example, Figure 3 displays the CLSM, Raman characterization, and surface functionalization of these twopatch particles containing the two functional polymers 1 and 4 in separate compartments. To confirm that polymers 1 and 4 were indeed localized in different hemispheres, rather than mixed throughout the entire particle, Raman confocal microspectroscopy was employed. This method can be used to gain chemical information of two- and three-dimensional substrates and has been used to characterize Janus particles.[9b] In this case, microparticles containing a low-molecular-weight PLGA polymer with a higher number of free carboxylic acid end groups in one hemisphere and a mixture of PLGA and polymer 4 in the second hemisphere were imaged by Raman microspectroscopy (Figure 3A). In Figure 3A1, the two Raman spectra that were obtained from the two different hemispheres are shown. The spectrum obtained on the PLGA hemisphere is shown in red, while the spectrum obtained on the hemisphere displaying polymer 4 is indicated by a yellow color. In addition to the expected PLGA bands, the yellow spectrum further revealed characteristic bands indicative of polymer 4. Specifically, two additional bands at 1610 and 1660 cm−1 signify the presence of benzophenone groups (Supporting Information Section 2.5). Therefore, the Raman maps shown in Figure 3 A3–A5 unambiguously confirm that the PLGA base polymer is present in both hemispheres (red), whereas the PLA derivative 4 is restricted to one side only (yellow). The two hemispheres were allowed to react with BSA tetramethylrhodamine (red dye) in the presence of UV light (first step) and amine-PEG-FITC (green dye) and EDC/sulfo-NHS (second step). The hemisphere containing polymer 4 autofluoresces in the blue channel (Figure 3B3) to further distinguish the two compartments. Subsequent analysis with confocal laser scanning microscopy (CLSM) confirmed successful, yet highly selective surface modification of both surface patches (Figure 3B2–B3). Taken together, the Raman microspectroscopic analysis and CLSM support the successful orthogonal surface reactions on these two-patch microparticles.</p><p>Beyond this particular combination of functionalized PLA derivatives, a number of different orthogonally substituted microparticles were prepared and selectively surface-modified, further elevating the potential scope of this novel approach. As depicted in Supporting Information Section 2.6, two-patch microparticles containing polymer 2 (with blue dye) and 5 (black, no dyes) in different hemispheres were prepared and the selective surface functionalization was demonstrated as well. Further expanding on the complexity of possible multifunctional particles, we adapted our EHD cojetting strategy to create three-patch microparticles. These particles, shown in Figure 4, contained patches displaying three different functionalized PLA derivatives, that is, polymers 1, 4, and 5. As noted above, it is important to ensure that the functionalized polymers are indeed localized in specific surface patches prior to conducting orthogonal surface modification. In fact, the defined localization of the reactive polymer species is an essential prerequisite for spatially controlled surface modification. Figure 4A displays the results of the Raman microspectroscopic analysis of three-patch particles. The Raman spectra obtained from the three patches displayed characteristic bands of the PLGA base polymer (blue). In addition, characteristic bands of polymer 4 at 1610 and 1660 cm−1 for benzophenone (black), and at 2200 cm−1 for the cyclooctyne of polymer 5 (red) were located in different surface regions, as shown in Figure 4A1 (Supporting Information 2.7.) The two-dimensional reconstruction of the Raman spectra displays three distinct patches on these microparticles. The PLGA polymer is detected in the entire particle (blue, Figure 4A3), while polymer 4 (white, Figure 4A4) and polymer 5 (red, Figure 4A5) are each restricted to two different surface hemispheres. We further conducted a series of orthogonal surface reactions on these microparticles and analyzed them by fluorescence confocal microscopy. The schematic for the sequential chemistries and the results obtained by CLSM imaging are shown in Figure 4B1 and 4B2, respectively. In this case, the blue patch, containing polymer 4, was functionalized by photochemical attachment of BSA tetramethylrhodamine (red dye), while the selective conjugation of azide-PEG-FITC (green dye) was restricted to the second surface patch that contained polymer 5. Efficient binding was achieved through click chemistry with the cyclooctyne groups of polymer 5. The third compartment, containing polymer 1, was treated with amine-PEG-Biotin through EDC/sulfo-NHS chemistry and labeled with Alexa Fluor 647 Streptavidin (magenta dye) for CLSM imaging.</p><p>Figure 4B3 shows the three-dimensional reconstruction of the microparticles on the basis of the fluorescence images collected in the z-direction. The particles were imaged in the xy-plane with a step size of 250 nm and were then reconstructed to display the threedimensional structure and characteristics of the patchy microparticles. We note that these results correspond well with the cross-sectional view (Figure 4B3, right image).</p><p>In conclusion, we have demonstrated an approach towards microparticles with fully orthogonal surface patches that takes advantage of a combination of novel chemically orthogonal polylactide-based polymers and their processing by electrohydrodynamic co-jetting to yield unprecedented multifunctional microparticles. These and other microstructured particles are highly sought after for their potential to present multiple distinct ligands in a directional manner. Applications may range from novel gels and particle self-assembly to use as carriers for cancer therapies with synergistic targeting effects from complementary ligands. For many of the above-mentioned applications, smaller particles are required. While designed as a proof-of-concept study to establish the feasibility of orthogonal reaction strategies on multivalent particles, future work will need to focus on refining critical particle characteristics, such as size or shape, while maintaining the same precision with respect to compartmentalization and spatially controlled surface modification. However, we have already shown that simpler versions of multi-patch particles can be made as small as 200 nm.[18] It is thus plausible that future work can access similar size ranges with the type of orthogonal multi-patch particles described in this study.</p><p>We acknowledge funding from the Multidisciplinary University Research Initiative of the Department of Defense and the Army Research Office (W911NF-10-1-0518), the DOD through an idea award (W81XWH-11-1-0111), the Tissue Engineering and Regenerative Medicine Training Grant (DE00007057-36), and the Scientific and Technical Research Council of Turkey (TUBITAK, 2219-International Postdoctoral Research Scholarship Programme). We also thank the Helmholtz Association for funding of this work through the BIF program and the Helmholtz-Portfolio Topic "Technology and Medicine".</p><p>Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201310727</p><p>c) Ref. [8e].</p><p>Fabrication of microparticles with orthogonally functionalized interfacial patches by EHD co-jetting. Functionalized polylactide derivatives (1–5) are incorporated into different jetting solutions, leading to compartmentalized fibers, which can be sectioned into the corresponding microdisks. Each compartment surface establishes a unique surface patch that is then selectively modified by orthogonal reactions.</p><p>Selective surface modification of microparticles containing three different orthogonally functionalized PLA derivatives exclusively present in one hemisphere. In (A)–(C), microparticles displaying polymers 3 to 5 in one hemisphere only are selectively surface functionalized through Staudinger ligation (A), photo-immobilization (B), and alkyne/azide click chemistry (C). CLSM images show the spatioselective nature of the surface modifications. See text for details.</p><p>Selective surface modification of two-patch microparticles. Microparticles with chemically orthogonal polymers 1 and 4 in different hemispheres were characterized through Raman microspectroscopy (Figure 3A), selectively surface-modified, and characterized using CLSM imaging (Figure 3B). See text for details.</p><p>Characterization and selective surface functionalization of three-patch microparticles. Particles containing polymers 1, 4, and 5 in separate surface patches were characterized using Raman microspectroscopy (A). The surface of each patch was then selectively modified using orthogonal chemistries and analyzed through CLSM imaging (B). Unless noted, all scale bars are 5 μm, See text for details.</p>

PubMed Author Manuscript

Engineering Ascorbate Peroxidase Activity into Cytochrome c Peroxidase

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibit each others activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struc. Biol. 10, 303\xe2\x80\x93307). The corresponding region in CCP has a much longer surface loop and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of \xe2\x89\x88 12 min\xe2\x88\x921 indicating that the engineered ascorbate binding loop can bind ascorbate.

engineering_ascorbate_peroxidase_activity_into_cytochrome_c_peroxidase

<!>Site-directed Mutagenesis<!><!>Site-directed Mutagenesis<!>Protein Expression and Purification<!>Steady-State Activity Assays<!>Transient-State Kinetic Studies<!>Electron Paramagnetic Resonance (EPR) Spectroscopy<!>X-ray Crystallography and Structure Refinement<!>Molecular Dynamics<!>Mutant Design<!>Crystal Structures and Molecular Dynamics<!>Spectroscopy<!>Stopped Flow Studies<!>Steady State Activity<!>Discussion

<p>Peroxidases are a large ubiquitous family of detoxifying enzymes that catalyze the reduction of H2O2 at the expense of various reducing substrates. Most peroxidases are heme-containing enzymes that use hydrogen peroxide to catalyze a number of oxidative reactions. The general reaction scheme of heme peroxidases is as follows: Fe3+R+H2O2→Fe4+−OR•+H2OCompoundI Fe4+−OR•+S→Fe4+=OR+S•CompoundII Fe4+=OR+S→Fe3+R+S•RestingStateEnzyme The enzyme reacts with H2O2 to form compound I. In compound I, the heme iron is oxidized from Fe3+ to Fe4+ and the porphyrin ring to a π-cationic radical (1). Yeast cytochrome c peroxidase (CCP) is an exception since Trp 191 in the proximal pocket just below the heme and adjacent to the His 175 heme ligand is the site of radical formation (2) and not the porphyrin ring. Compound I is then subsequently reduced back to the resting state in two successive one electron transfer reactions involving two molecules of substrate (S in the above scheme) via another enzyme intermediate called compound II.</p><p>In general, peroxidases can oxidize a wide variety of substrates including aromatic amines, indoles, phenols, lignin, halides, and manganese (3). When the first few crystal structures of peroxidases became available it appeared that the most likely region for small aromatic substrates to bind is at the one edge of the heme exposed to solvent which thus allows substrates to directly contact the heme for short and fast electron transfer to the porphyrin radical. Indeed, the crystal structure of horse radish peroxidase complexed with ferulic acid shows binding at the heme edge as expected (4). CCP, however, is poor at oxidizing small aromatic dyes but instead is specialized to oxidize cytochrome c. This is accomplished by providing a surface unique to forming a complex with cytochrome c (5) and by locating the radical in compound I on Trp 191 (2) rather than the porphyrin macrocycle. As more crystal structures became available, however, this simple view that, with the exception of CCP, substrates bind at the heme edge came into question. For example, manganese peroxidase has the substrate, Mn(II), coordinated to one heme propionate (6) thus providing a direct route of electron transfer to heme along the heme propionate group. Chemical modification (7), mutagenesis studies (8), and crystallographic studies (9) showed that ascorbate peroxidase (APX) also uses a similar site near the heme propionates.</p><p>A long standing goal in enzyme engineering is to introduce novel activities into enzymes by altering or introducing new substrate binding sites. Given the close similarity between the APX and CCP structures with the exception of ascorbate binding site, it appeared to us that it should be possible to introduce the APX substrate binding site into CCP and convert CCP into an APX. If successful, this would provide an important step in developing strategies of tailoring peroxidases to bind other types of substrates that could prove useful in a variety of practical applications such as bioremediation. Here we report our initial attempts at engineering CCP into an APX.</p><!><p>Oligonucleotide-directed mutagenesis experiments were performed on pT7-7 vector that contained the wild-type CCP. Residues 30–42 (LRED DEYDNYIGY) of WTCCP were replaced with residues 27–32 (IAEKKC) of APX in order to introduce the ascorbate-binding loop. The following 69 base oligonucleotide & its reverse complement was obtained from QIAGEN's Operon Technologies (Alameda, CA) GTG TAC AAT GCG ATT GCA CTC AAG ATT GCG GAA AAG AAG TGT GGG CCC GTA TTA GTC CGT CTT GCT TGG.</p><!><p>ACC CAC TTG AAG CGC TCT GGA TAC GAA</p><!><p>Site-directed mutagenesis was carried out by overlap extension method by PCR (10) using Thermal Ace Polymerase (Invitrogen) with outside primers "T7 " TAATACGACTCACTATAGG and "Lac-80" CAGTCACGACGTTGTAAAAC, which were also purchased from QIAGEN's Operon Technologies and the requisite internal primers. These mutations were introduced iteratively in stages to follow properties of the protein as a consequence of mutations. First, the ascorbate-binding loop was introduced into CCP followed by the N184R point mutation. The resultant mutant was called CCP2APX. Although the mutant has fewer amino acids than wild type CCP, the wild type amino acid numbering will be used. In order to enable this mutant to form a porphyrin π-cation radical during catalysis Trp 191 was converted to Phe using CCP2APX as template to give CCP2APX/F191. The mutant clones were analyzed and confirmed by restriction analysis and automated DNA sequencing at the sequencing facility of Biotech Diagnostic, Suite 372 23974, Aliso Creek Road, Laguna Niguel CA 92677-3908.</p><!><p>Expression plasmids were transformed into Escherichia coli BL21(DE3) STAR competent cells and the transformants were grown in 7 L of terrific broth with vigorous shaking at 37°C until the cultures reached an A600 of 0.8. At this point, protein production was induced from the T7 promoter by adding 750 µM of IPTG. Cells were allowed to express protein overnight at a reduced temperature of 25°C and reduced shaking to 100 rpm. Cells were harvested, lysed and chromatographed on 2 L Sephadex G-75 column followed by heme incorporation according to published protocols (11, 12). After heme incorporation and dialysis proteins were further purified using DEAE-Sephacel anion exchange chromatography. Fractions were collected, pooled, and concentrated by ultrafiltration using YM30 membrane and the protein was stored in 50 mM potassium phosphate, pH 6.0 after extensive dialysis against the same buffer. Protein concentrations were determined spectrophotometrically using an extinction coefficient ε408 = 96 mM−1 cm−1.</p><!><p>Spectrophotometric assays were carried out at room temperature using a Cary 3E UV-visible spectrophotometer. Steady-state activity of wild-type and mutant proteins was measured using the native substrate of CCP, yeast cyt. c (ferrocytochrome c), as well as L-ascorbic acid. The steady state oxidation of cytochrome c was measured at room temperature using a Δε550 = 19.6 mM−1 cm−1 using 25 µM sodium dithionite reduced cyt. c, 100 mM Tris-phosphate pH 6.0, and 100 µM H2O2. Peroxidation of L-ascorbic acid was measured by following the decrease in optical density at 290 nm using a ε290 = 2.8 mM−1 cm−1 in 50 mM potassium phosphate pH 7.5, 250 µM H2O2, and varying concentrations of ascorbate. Hydrogen peroxide concentrations were standardized with KMnO4 using the method of Fowler and Bright (13).</p><!><p>The rate of compound I formation and decay was determined using an Applied Photophysics SX.18MV-R stopped-flow spectrophotometer at room temperature. Wild-type and mutant proteins at a concentration of 6 µM were mixed with H2O2 at concentrations ranging from 6 to 20 µM. The transient-state reaction was examined using a diode array attached to the SX.18MV-R stopped-flow spectrophotometer and was used to determine the maximal change in absorbance for each of the proteins. Buffers used were 50 mM potassium phosphate, pH 7.0 with 0.1 mM EDTA for all proteins except CCP2APX/F191 for which in addition to the previous buffer, 100 mM sodium citrate buffer, pH 5.0 was also used. The formation of compound I was examined for 20 ms and data were fit to a single-exponential curve using Applied Photophysics software. In APX and the mutant where Trp 191 is converted to Phe, there is an initial rapid decrease in absorbance owing to formation of a porphyrin π-cation radical followed by a slower increase in optical density owing to the spontaneous reduction of the porphyrin radical. The kinetics of compound I decay was estimated from the rate of increase in absorbance after the initial formation of the compound I porphyrin π-cation radical.</p><!><p>Quartz EPR tubes (715-PQ-250m) were purchased from WILMAD. EPR spectra were recorded on a Bruker ESP300 spectrophotometer equipped with an Air Products LTR3 liquid helium cryostat. To observe the radical in compound I, 200 µL of 300 µM wild-type or mutant CCP protein was combined with an equal volume of 360 µM H2O2 in 50 mM potassium phosphate, pH 6.5. The solution was mixed and transferred to a quartz EPR tube and flash-frozen in a nhexanes-LN2(1) slurry, a process which took between 18 and 27 s. The EPR spectra were obtained at 10 K as an average of 10 scans using the following instrument parameters: microwave frequency, 9.387 GHz; modulation amplitude, 0.40 G; modulation frequency, 100 kHz; field sweep rate, 23.84 G/s; 0.638mW microwave power, 1.0 ×104 receiver gain, and 5.120ms time constant.</p><!><p>CCP2APX failed to produce decent quality diffracting crystals, while crystals CCP2APX/F191 and CCP/R184 were of high quality. Only peak fractions obtained during purification were used for crystallization. Crystallization conditions were optimized starting with conditions established for wild-type CCP. Sitting-drop vapor-diffusion with a well solution of 30% 2-methyl-2,4-pentanediol (MPD) in 50 mM Tris-phosphate, pH 6.0 was used initially. The best crystals grew in drops consisting of 400 µM protein, 22% MPD, and 50 mM Trisphosphate, pH 6.0 at 4°C. The following day, one round of touch seeding using wild type CCP crystals was done to initiate crystal growth. Immediately after crystal growth mutant crystals were flash-frozen in liquid nitrogen and stored for data collection later.</p><p>High-resolution data collection for CCP2APX/F191 and near atomic resolution data for CCP/R184 was collected at SSRL beamline 9-1. Data were reduced using HKL2000 and SCALEPACK (14). CCP2APX/F191 and CCP/R184 crystals were isomorphous with WTCCP crystals and belong to space group P212121. As a result, the structure of WTCCP was used as the starting model for refinement in CNS version 1.1 (15) after molecular replacement. Rigid body refinement was followed by slow-cool simulated annealing starting at 3000 K as implemented in CNS version 1.1 (15) and the remaining cycles that followed consisted of a few cycles of conjugate gradient minimization and water picking. The program O (16) was used for further adjustment and modeling of protein atoms, ligands, and water molecules. The final refinement was carried out using SHELXL (17) and anisotropic B-factors were used for main chain atoms. Refinement of the 1.06 Å structure of the CCP/R184 mutant followed a similar protocol. Data collection and refinement statistics are summarized in Table 1.</p><!><p>Molecular dynamics simulations were carried out with Amber 9.0. Charges and optimal geometry for ascorbate were obtained using the antechamber routine in Amber and AM1-bcc charges with ascorbate assigned a net charge of −1.0. Heme parameters were provided by Dr. Dan Harris (18). The APX-ascorbate structure used was taken from the known crystal structure (9). Ascorbate was modeled into the crystal structure of engineered version of CCP, CCP2APX/F191, assuming that ascorbate binds the same way as in APX. Since we were focusing only on the substrate binding site, a full periodic boundary simulation was not carried out. Instead a sphere of TIP3 water molecules was placed within a 20 Å sphere of the ascorbate, which is sufficient to properly model the interaction of ascorbate and nearby protein groups with solvent. Prior to MD simulations, structures were energy minimized for 1,000 cycles with all H atoms and water molecules allowed to move. Next, all atoms except the ascorbate were allowed to move for 1,000 cycles followed by a final 2,000 cycles of minimization with all atoms allowed to move. For MD runs, atoms greater than 18 Å from the ascorbate were constrained to the starting position using a force constant of 2 kcal/Å. This was done to avoid "boiling off" of solvent since a periodic boundary was not used. MD Simulations were run for 10 ns with structures saved every 10 ps.</p><!><p>Fig. 1 shows a comparison between CCP (red) and APX-ascorbate complex (green) in the vicinity of the ascorbate binding site identified in the crystal structure of the APX-ascorbate complex (9). A loop on the surface between the A and B helices provides the ascorbate binding pocket. In CCP this loop is 7 amino acids longer than in APX. Based on structural superposition of WTCCP and APX as shown in Fig.1 and after subsequent sequence analysis the ascorbate binding site was introduced into WTCCP. This was achieved by replacing wild type residues 30–42 (LREDDEYDNYIGY) with residues 27–32 (IAEKKC) from APX. In the APX-ascorbate complex, Arg 172 (Fig. 1) H-bonds with the substrate and is known to be critical for activity in APX (8), whereas the corresponding residue in CCP is Asn 184, and thus was replaced with Arg.</p><!><p>Crystals of CCP2APX/F191 diffracted to 1.3 Å resolution which provided sufficient data to justify the use of anisotropic B-factor refinement for main chain atoms. The fit of the engineered ascorbate binding site to the electron density map including Arg 184 is shown in Fig. 2A. The engineered ascorbate binding loop superimposes well on APX (Fig. 2B).</p><p>We made several unsuccessful attempts to obtain crystals of the CCP2APX/F191-ascorbate complex. Soaking crystals in ascorbate resulted in crystal cracking which could be due to ascorbate induced structural changes incompatible with crystal lattice. Alternatively, the increase in ionic strength upon addition of sodium ascorbate could be a problem since crystals are grown at low ionic strength. We also attempted to co-crystallize the CCP2APX-ascorbate complex but this, too, failed to generate useful crystals. We therefore used MD simulations to obtain some insight on the structure, stability, and energetics of the engineered ascorbate binding site. The results of MD simulations are summarized in Fig. 3 and Table 2. Structures shown in Fig. 3 were averaged over the last 5 ns of the 10 ns simulation followed by energy minimization. As shown in Fig. 3A and Table 2, the H-bonding interaction between ascorbate, Arg 172, and heme in APX remain intact during the simulation. However, the H-bond between Lys 30 and ascorbate found in the crystal structure of the APX-ascorbate complex (9) breaks early on in the simulation resulting in Lys 30 forming a stable ion pair with Glu 29 indicating that Lys 30 does not play an important role in ascorbate binding. This agrees well with the experimental data since replacing Lys 30 with Ala actually increases kcat (19). In sharp contrast Arg 172 in APX is essential for catalysis (8, 19) and the Arg 172-ascorbate interaction remains intact over the course of the 10 ns simulation (Fig. 3A and Table 2). For CCP2APX/F191 structure the ascorbate rotates up toward Asn 87 (Ile 76 in APX) in order to form a new H-bond with Asn 87 (Fig. 3B). This movement requires breaking of the ascorbate-heme H-bond. In addition the rms fluctuation of ascorbate is higher in CCP2APX/F191 than APX (Table 2). The main difference in the substrate binding pocket and the reason ascorbate moves so much in CCP2APX/F191 is that Asn 80 (Asn 87 in WTCCP numbering) is Ile 76 in APX. In APX Ile 76 lies on top of the ascorbate to assist the ascorbate to remain in position for H-bonding with Arg 172. We therefore carried out an in silico mutagenesis experiment and converted Asn 87 to Ile in CCP2APX/F191 followed by a 10 ns simulation. As expected CCP2APX/F191/N87I more closely resembled APX than CCP2APX/F191. As shown in Fig. 3C and Table 2 ascorbate remains in position for H-bonding with Arg and heme and the rms fluctuation is slightly lower than in CCP2APX/F191 (Table 2). A priori one might conclude that the overall ΔG of binding is about the same for all 3 structures since all 3 have 2 H-bonds between the substrate and neighboring groups. The main difference is the more extensive van der Waals contacts between Ile and ascorbate in APX and CCP2APX/F191/N87I.</p><p>We also solved two other structures: the first with just the ascorbate-binding loop introduced into CCP and the second with just Asn 184 converted to Arg which we call CCP/R184. With respect to engineering CCP into an APX neither structure provides any further insights than CCP2APX/F191 structure. However, crystals of CCP/R184 provided an unexpected benefit of diffracting to atomic resolution which enabled the structure to be refined to a nominal resolution of 1.02 Å (Table 1). Applying a I/σI>2.0 cutoff, the resolution would decrease to 1.06 Å. Although outside the scope of the present paper, it is worth a slight diversion to describe some interesting insights provided by the CCP/R184 structure. Fig. 4 shows the 2Fo−Fc maps in the active site region. The main change induced by the N184R mutation is in Arg 48, a critical active site residue. In WTCCP Arg 48 occupies multiple positions (20) but in CCP/R184 Arg 48 occupies only one position. Both Arg 48 and the mutant Arg 184 side chains interact with the same heme propionate via a network of H-bonded solvent molecules. This additional ordering perhaps accounts for the decrease in flexibility of Arg 48. Such subtle changes apparently have little effect on enzyme activity since the CCP/R184 mutant exhibits about 50% of WTCCP activity.</p><p>One last feature to be highlighted is the level of detail provided for key active site H-bonding residues. Asp 235 (Fig. 4C) is an invariant residue that H-bonds with the His 175 heme ligand. At atomic resolution it is possible to discern the difference between C-O single and double bonds in well ordered carboxylate side chains. To obtain an objective picture on the precise bond distances in Asp 235, 10 cycles of SHELXL refinement were carried out with no distance or angle restraints imposed on Asp 235. The resulting 2Fo−Fc electron density map is shown in Fig. 4D. Note that the electron density contoured at 5σ is continuous along the CG-OD2 bond but broken along the CG-OD1 bond. In addition, the CG-OD2 unrestrained bond distance is 1.228 Å compared to 1.290 Å for the CG-OD1 bond. In order to assess the significance of this difference we carried out a round of full matrix least squares refinement. Standard uncertainties on bond lengths can be calculated by inversion of the normal matrix using SHELXL. All restraints on positional parameters were excluded for the calculation. The matrix included all of the positional parameters and none of the thermal parameters. The resulting bond distances and standard deviations are CG-OD1 1.290±0.017 Å and CG-OD2 1.228±0.014 Å. Thus the difference in bond lengths is more than 3 standard deviations above the error and hence is significant. Both the map and distances indicate that the CG-OD1 bond has less double bond character with more electron density localized on OD1 than OD2. Therefore, OD1 should be a particularly strong H-bond acceptor from His175. Indeed, the H-bond angle between Asp235 and His175 is a near ideal 119° while the H-bond angle between Asp 235 and Trp 191 is 127°. The strong His 175-Asp 235 H-bond found in many heme peroxidase structures is generally consideredto be an important factor in the low heme redox potential of peroxidases compared to other heme proteins with His ligands such as the globins. However, there is nothing unusual about the Asp-His H-bond other than ideal geometry and distance which thus precludes an unprotonated His ligand or low barrier H-bond.</p><!><p>Fig. 5a shows the absorption spectrum of CCP2APX before, immediately after, and one hour after the addition of one equivalent of H2O2. Because the Trp 191 radical in compound I does not contribute significantly to the absorption spectrum, the spectrum of compounds I and II are very similar and thus are characteristic of the Fe4+=O center. As with WTCCP, the compound I Soret band red shifts relative to the Fe3+ resting enzyme with new bands at 530 nm and near 560 nm. The spectrum is quite stable and slowly relaxes back toward the Fe3+ spectrum. However, there is a decrease in absorption of the main Soret band immediately after addition of peroxide and remains below the starting level. This indicates that a small fraction of heme may be destroyed during the decay of compounds I and II. Fig. 5b shows the EPR spectrum of CCP2APX immediately after the addition of peroxide. The compound I signature of WTCCP is typically axially symmetric with a broad envelope due to a Trp 191 π-cation radical, which is in magnetic exchange with S = 1.0 oxyferryl heme iron center with a g = 2.01–2.04. The spectrum for CCP2APX is very similar to WTCCP indicating that engineering the ascorbate-binding loop into CCP does not effect formation of Trp 191 cation radical. Taken together the EPR and UV-vis data indicate that compounds I and II of CCP2APX are long lived with very similar spectral properties of WTCCP although there does appear to be a small amount of heme lost during the decay of compounds I and II.</p><!><p>As shown in Table 3 the rate of compound I formation in the mutants is very similar to wild type CCP. With CCP2APX/F191 we expected the initial reaction with H2O2 to generate a porphyrin π-cation radical. In order to capture this reaction intermediate we employed diode array stopped flow. As shown in Fig. 6a the initial product formed 6 ms after mixing with H2O2 exhibits decreased absorption in the Soret band and a red shift in the maximum to longer wavelengths. In addition, there are distinct changes in the 500–580 nm region. These changes are very similar to what was observed for the porphyrin π-cation radical in the W191F CCP mutant (21) but with clear differences. With CCP containing the W191F mutation the first spectrum captured immediately after mixing with peroxide exhibits a decrease in the Soret maximum of about 35% (taken from Fig. 4 in ref. (21)) while CCP2APX/F191 exhibits only a 10% decrease. This together with the less well defined α and β bands in the 500–580 nm region indicates that CCP2APX/F191 forms a porphyrin π-cation radical to less of an extent than the single W191F mutant. It could be that one full peroxide oxidizing equivalent resides on the porphyrin immediately after mixing but rapidly migrates elsewhere. APX compound I spontaneously converts to compound II and the rate of compound I decay can be estimated from the compound I to II spectral changes (22). CCP2APX/F191 compound I decays about 160 fold faster than APX compound I (Table 3). Fig. 6b shows the spectrum of CCP2APX/F191 before, immediately after, and about 30 seconds after the addition of one equivalent of H2O2 which should generate the compound II spectrum. However, the spectrum closely resembles the starting Fe3+ enzyme with a diminished Soret maximum although there is the band near 560 nm is indicative of compound II. This indicates that CCP2APX/F191 forms a short-lived porphyrin radical which does not decay to a clearly defined and stable compound II species.</p><!><p>CCP2APX exhibits about ~ 2% WTCCP activity which is not too surprising since the loop engineered out in the mutant provides direct contact with cyt. c in the CCP-cyt. c complex (5). Under the assay conditions employed in this study WTCCP exhibits no ascorbate peroxidase activity. However, as shown in Fig. 7, CCP2APX exhibits reasonably good ascorbate peroxidase activity. From where the curve in Fig. 7 plateaus, we can estimate a kcat ≈ 12 min−1. CCP2APX/F191 exhibited activity as well but only about half the level of CCP2APX.</p><p>The MD work suggested that the Ile in APX which replaces Asn 87 in CCP helps to hold the ascorbate in place for proper H-bonding with the heme propionate (Fig. 3). We therefore replaced Asn 87 with Ile in CCP2APX/F191 and found no improvement in enzyme activity.</p><!><p>Our results show that it is relatively straight forward to engineer the ascorbate binding site found in APX into CCP. While it is true that CCP and APX are structurally very similar, it may seem somewhat surprising that the removal of a 7 residue surface loop in CCP required to form the ascorbate site that has very little effect on structure, stability, or the ability to form well ordered crystals. However, loop insertions or deletions between stable elements of secondary structure is a common strategy employed by nature to alter function. Loop swapping also has been successfully employed in the redesign of other metalloproteins (23). The fact that engineered ascorbate-binding site in CCP2APX/F191 is the same as in APX and the mutants exhibit ascorbate peroxidase activity strongly suggests that ascorbate is binding to the engineered site and is responsible for the observed ascorbate peroxidase activity in the mutants.</p><p>The rate of ascorbate peroxidation for CCP2APX is 12 min−1, while WTCCP exhibits no activity under the same experimental conditions clearly demonstrating that we were successful in engineering a new activity. Even so this value is well below the kcat of 40–100 sec−1 observed for plant APX. One possible reason is that the engineered site may bind ascorbate but not very well. The MD simulations showed that Ile 76 in APX helps to hold the substrate in place such that it H-bonds with both Arg 172 and one heme propionate. This provides a direct connection between the substrate and heme. The MD work also indicates that in CCP2APX/F191 the substrate fluctuates more and rotates up toward Asn 87 and no longer maintains a stable short contact with the heme propionate. Even so the substrate maintains a close interaction with the engineered Arg residue (Table 2) and it is doubtful that a slight reorientation of the substrate in CCP2APX compared to APX is responsible for the lower activity in CCP2APX. Moreover, replacing Asn 87 with Ile resulted in no further improvement in activity.</p><p>A second possibility is that in CCP2APX the radical site in compound I is stably located on Trp 191 while in APX the radical is located on porphyrin macrocycle. Thus the electron transfer distance is much longer in CCP2APX, about 9 Å, and does not follow a direct route to the radical site as in APX. However, a rough estimate of the expected electron transfer rate at a distance of 9.0Å can be made using the web-based electron transfer calculator (http://www.uphs.upenn.edu/biocbiop/local_pages/dutton_lab/golden.html). To make such an estimate the overall thermodynamic driving force of the reaction and reorganization energy must be known. The typical values used for reorganization energy are 07.-1.0V. The redox potential of the ascorbate/ascorbate radical couple is +282mV (24) while the Trp 191/Trp 191 radical couple is in the range of 1V (25). Although there are uncertainties in the actual redox potential of Trp 191 in the protein the overall driving force for the oxidation of ascorbate is large and in the range of 400–700mV. Even assuming that the driving force is 0V and large errors in reorganization energy, the rate of electron transfer over a distance of 9.0Å is orders of magnitude more than the observed turnover so it is doubtful that the distance between the ascorbate and Trp 191 radical is limiting. Even so anticipating this problem is the reason we also prepared the CCP2APX/F191 mutant since this mutant should form a porphyrin radical. However, based on the magnitude of spectral changes obtained in our diode array experiments, the extent of porphyrin radical formation is much less in CCP2APX/F191 compared to plant APX and the decay of porphyrin radical is about 160 times faster in CCP2APX/F191 than in APX. Thus CCP2APX/F191 forms neither a stable porphyrin radical, an amino acid radical, nor a stable compound II which can account for its lower ascorbate peroxidase activity in comparison to CCP2APX mutant.</p><p>A third possibility relates to the reactivity of compound II. The reduction of compound II is often the rate limiting step in peroxidases and thus is the obvious place to focus for improving activity. Here the efforts of Yeung et al. are highly relevant (26). In this work the manganese peroxidase Mn(II) site was introduced into CCP. The initial design effort resulted in a mutant CCP that oxidizes Mn(II) at 15 min−1 compared to 14,500 min−1 for authentic manganese peroxidase. In subsequent studies the activity was improved to about 240 min−1 by converting both Trp 191 and the distal pocket Trp 51 to Phe (27, 28). These mutations have nothing to do with improving Mn(II) binding but do affect the reactivity of compounds I and II. Pfister et al. (29) argued that the improved activity in the Trp 191Phe/Trp51Phe double mutant is due to an increased reactivity of compound II which is consistent with other studies (30). Trp 51 directly contacts the Fe-linked oxygen atom in compound I (20) so it is not surprising that the conversion of Trp 51 to Phe dramatically alters reactivity. Similar arguments could hold for our present work. The spectral data suggest that Fe4+=O center in CCP2APX is stable and thus may lack the reactivity required for fast ascorbate oxidation. Indeed, CCP compounds I and II are unusually stable among heme peroxidases. This could be biologically advantageous since more reactive compounds I and II would be subject to non-specific reduction by small molecule reductants. Thus CCP had to evolve a somewhat more elaborate electron transfer process to ensure only cyt. c is oxidized. One novel feature that helps to control selectivity in CCP is both electrons from cyt. c required to reduce compound I back to the resting Fe(III) enzyme must pass through Trp 191 (31). This means that after reduction of the Trp 191 radical in compound I, there is an internal electron transfer from Trp 191 to Fe4+=O to give Trp 191.+/Fe3+-OH and it is this species that accepts the second electron from cyt. c. The cyt. c heme directly contacts Ala 193 (5) in CCP and thus there is a direct polypeptide electron transfer path to Trp 191. The binding of cyt. c also could effect the energetics of Trp 191-to-Fe4+ internal electron transfer process and thus the energetics of compound II reduction. High resolution structures (20) show that the Ala193 section of polypeptide is disordered in resting Fe3+ CCP but becomes well ordered in compound I such that the Trp 191 cationic radical is more effectively stabilized by the surrounding protein. The crystal structure of the covalent CCP-cyt. c complex (32) shows that this region is well ordered in CCP indicating that the binding of cyt. c can affect the local Trp 191 environment and could promote the formation of compound II. None of these effects associated with cyt. c binding is possible in the CCP2APX mutant alone. As a result, the reactivity of compound II is too low to support rapid oxidation of ascorbate. This appears to be the most reasonable explanation and is consistent with previous studies (27).</p><p>A low compound II reactivity at first may seem puzzling since Fe4+=O is a powerful oxidant and there is no thermodynamic barrier to electron transfer from ascorbate. However, the reduction of Fe4+=O to Fe3+-OH is a proton coupled electron transfer process which can present a substantial kinetic barrier to electron transfer. Here again the internal electron transfer from Trp 191 to Fe4+=O is the key to understanding compound II reactivity since once Trp 191.+/Fe3+-OH is formed there is very little thermodynamic or kinetic barrier to reducing the Trp cation radical. The rate limiting and least understood step is the coupling of electron transfer from Trp 191 with protonation of Fe4+=O oxygen atom and how this process may be affected by cyt. c binding. The success in substantially increasing the Mn(II) peroxidation activity of engineered versions of CCP with Trp 51 converted to Phe could have directly affected the kinetic barrier to protonation of Fe4+=O resulting in increased Mn(II) peroxidation rates. There thus appears to be a path to further improving CCP2APX activity by making additional distal pocket mutants that increase the reactivity of compound II. In conclusion, it appears that engineering novel peroxidase activities is a two part problem. First, introducing the proper substrate binding site and second, altering the activity of enzyme oxidant, usually compound II, whose reduction is rate limiting. The first goal is relatively straight forward since loop swapping is sufficient to provide a novel binding pocket without altering the core structure. The second goal is more difficult since this is less of a structural problem and more of a chemical reactivity issue where the effects of mutagenesis are not so simple to interpret. Nevertheless, engineered peroxidases with altered activity may provide a window into understanding the more challenging problem of proton coupled electron transfer.</p>

PubMed Author Manuscript

Ultraviolet photochemistry of ethane: implications for the atmospheric chemistry of the gas giants†

Chemical processing in the stratospheres of the gas giants is driven by incident vacuum ultraviolet (VUV) light. Ethane is an important constituent in the atmospheres of the gas giants in our solar system. The present work describes translational spectroscopy studies of the VUV photochemistry of ethane using tuneable radiation in the wavelength range 112 ≤ λ ≤ 126 nm from a free electron laser and event-triggered, fast-framing, multi-mass imaging detection methods. Contributions from at least five primary photofragmentation pathways yielding CH2, CH3 and/or H atom products are demonstrated and interpreted in terms of unimolecular decay following rapid non-adiabatic coupling to the ground state potential energy surface. These data serve to highlight parallels with methane photochemistry and limitations in contemporary models of the photoinduced stratospheric chemistry of the gas giants. The work identifies additional photochemical reactions that require incorporation into next generation extraterrestrial atmospheric chemistry models which should help rationalise hitherto unexplained aspects of the atmospheric ethane/acetylene ratios revealed by the Cassini–Huygens fly-by of Jupiter.

ultraviolet_photochemistry_of_ethane:_implications_for_the_atmospheric_chemistry_of_the_gas_giants†

Introduction<!>(a) Ethane absorption and the energetics of its various dissociation channels<!>(b) Ion imaging studies<!>(c) H atom photofragment time-of-flight (TOF) spectra<!>(d) Active photofragmentation channels<!>CH2 radical formation<!>C–C bond fission<!>C–H bond fission<!>(e) Implications for modelling the atmospheres of the gas giants<!>Conclusions<!>Data and materials availability<!>Conflicts of interest

<p>Understanding, and perhaps one day exploiting, the environment of extraterrestrial bodies is a central objective of planetary science. The gas giants in our solar system (Jupiter, Saturn, Uranus and Neptune) are rich in molecular chemistry and remain targets of intense scientific study. Like Earth, each of these planets orbits the sun with its own eccentricity and obliquity leading to seasonal variations in incident solar radiation and thus a cycling chemical composition with latitudinal and altitudinal variations in the abundances of the various molecular constituents.1 Absorption of near-infrared solar radiation by methane (CH4) makes important contributions to heating the upper atmospheres (stratospheres) of these planets.1–3 Methane contributes less to stratospheric cooling, however, which is more reliant on emission from ethane (C2H6) and acetylene (C2H2).1 Understanding the balance and interplay between CH4 and C2H6/C2H2 is central to understanding the atmospheric dynamics of the gas giants.</p><p>Chemical processing in the stratospheres of the gas giants is driven by incident vacuum ultraviolet (VUV) light,4 even in the distant, gas-poor giants Uranus and Neptune.5 Numerous possible reactions merit consideration, but common photochemical models for these planetary atmospheres necessarily employ a reduced set pruned from a much larger library of reactions, along with their corresponding rates/branching fractions. These models describe many aspects of the atmospheres of Saturn and Jupiter reasonably well1–3 but have recognised shortcomings. For example, the dominant C2H6 and C2H2 generation mechanisms are assumed to involve secondary reactions following photolysis of CH4.6–8 But both the Cassini–Huygens fly-by of Jupiter and terrestrial measurements reveal very different meridional and latitudinal distributions for C2H6 and C2H2. Such would be surprising if both species are tightly coupled to methane photolysis.3,9,10 Neglect of ion-molecule chemistry has been suggested as one possible explanation for this discrepancy,3,11,12 but it is also appropriate to question the inputs to the commonly used photochemical schemes. These draw on data8 from a range of (often indirect) sources, including predictions, wherein chemical pathways have been included or removed on the basis of how well the model fits the measurements. Ethane is an important participant in these models and, whilst VUV photolysis is accepted as its main destruction route,13,14 the dominant fragmentation pathways and photoproducts are not well determined.</p><p>Early laboratory studies of C2H6 photolysis at the resonance wavelengths emitted by a xenon lamp (λ = 147.0 and 129.5 nm) deduced the involvement of (at least) three fragmentation pathways. Two involve loss of H2 or two H atoms, the other yields CH4 + CH2 products.15 Subsequent studies using Kr and Ar resonance lamps (λ = 123.6 and 106.7/104.8 nm, respectively) suggested additional primary fragmentation channels, to CH3 + CH3 and, particularly, H + C2H5 products.16–18 These studies all involved careful end-product analysis but could not distinguish primary photofragmentation processes from secondary reactions following photolysis, nor yield any dynamical information. More recent imaging studies showed formation of H atoms following C2H6 photolysis at the Lyman-α wavelength (λ = 121.6 nm, the most intense VUV wavelength in the solar spectrum), with an isotropic velocity distribution peaking at low kinetic energies and a weak tail extending to higher energies. The form of this distribution was attributed to initial C–H bond fission, yielding a fast H atom and an electronically excited fragment, followed by a second (slow) H atom from unimolecular decay of the latter.19</p><p>The present study employs two cutting-edge technologies – the intense, pulsed VUV free electron laser (FEL) at the Dalian Coherent Light Source (DCLS)20 and an event-triggered, fast framing, Pixel Imaging Mass Spectrometry (PImMS2) sensor21 – to advance understanding of C2H6 photochemistry and to identify similarities and differences with the photochemistry of both lighter (i.e. CH4) and heavier (e.g. propane (C3H8)) alkanes. The reported data derive from two sets of collision-free experiments: (i) multi-mass velocity-map ion imaging (PImMS2 detected)22 studies following one-color VUV photolysis of ethane and 'universal' (i.e. not quantum state selected) photoionisation of CH2 and CH3 photoproducts at four (FEL-produced) wavelengths in the range 112.0 ≤ λ ≤ 125.6 nm, and (ii) VUV photolysis at λ = 121.6 nm (using photons generated by four wave mixing outputs from a tabletop pulsed laser) and subsequent detection of H atom products using the high resolution H-atom Rydberg tagging technique.23,24 The experimental procedures have all been described previously and are thus confined to the ESI.†</p><!><p>Fig. 1 shows the chosen photolysis wavelengths superimposed on the electronic absorption spectrum of ethane.25,26 As with the other alkanes, the absorption of C2H6 lies in the VUV region but, uniquely amongst the alkanes, its room temperature absorption spectrum displays resolved vibronic structure. This structure is attributed to transitions from the near degenerate highest occupied 3a1g and 1eg valence orbitals to orbitals with dominant 3p Rydberg character. One or more of these are suggested to have significant antibonding valence σ* character also.27 Excitations to the 3s Rydberg orbital in C2H6 are predicted at lower energies, but to be weak – as a result of the molecular center of symmetry – thus distinguishing the 3s Rydberg excitations of ethane from those in CH4 and the heavier alkanes which all show large absorption cross-sections. This seemingly simple description hides a wealth of potential complexity, however. The degeneracy of the ground (X̃2Eg) state of the C2H6+ cation is lifted by Jahn–Teller distortion, and the structure and dynamics of the resulting cation states are further complicated by the energetic proximity of the low lying Ã2A1g excited state – with the result that even a full interpretation of the threshold photoelectron spectrum of C2H6 remains elusive.28 Such interactions must also affect the Rydberg states of current interest – since they share the same ion core(s) – and thus affect the absorption spectrum shown in Fig. 1.</p><p>Contemporary computational chemistry methods have enabled global investigations of the ground (S0) state potential energy surfaces (PESs) for species involved in the early stages of the pyrolysis of ethane and other C1–C3 hydrocarbons29 but have yet to be directed at the excited state photochemistry of any but the very simplest alkanes. Fig. 2 shows the lower-lying dissociation limits of C2H6. The predicted minimum energy conical intersections were located using the global reaction route mapping (GRRM) method and are discussed later. The S0 state correlates adiabatically with the ground state products from either C–C or C–H bond fission (i.e. ground state CH3 + CH3 and H + C2H5 fragments). The former is the weaker bond, and the formation of 1CH2 + CH4 products is attributed to an (essentially barrierless) H atom transfer between the incipient CH3 radicals.29 The energetic thresholds for these three processes are all lower than the calculated barrier to H2 elimination on the S0 PES (∼5.1 eV).29 As Fig. 2 also shows, many more spin-allowed fragmentation channels are energetically accessible following electronic excitation of ethane. Table 1 lists no fewer than 17 chemically intuitive channels that require less than the 10.2 eV of energy provided by a Lyman-α photon. Of these, 8, 7, 6 and 5 of the channels yield, respectively, H atoms, H2 molecules, CH2 and CH3 radicals amongst the dissociation products. Such commonalities provide a major challenge for quantitative studies of the primary photochemistry of ethane (and larger alkanes). Of particular relevance to the present study, the reduced models currently used to describe the atmospheric chemistry of Jupiter and Saturn1–3 recognise just reactions (1)–(5) in Table 1.</p><!><p>Fig. 3 shows a representative time-of-flight mass spectrum (TOF-MS) of the ions formed following FEL excitation (at λ = 121.6 nm) of a jet-cooled sample of C2H6 in helium. The spectrum is dominated by a peak associated with H+ ions. This is unsurprising, given that this wavelength is resonant with the Lyman-α transition from the ground (n = 1) state of the H atom. The remainder of the TOF-MS, displayed on a 5× expanded vertical scale, reveals two clumps of partially-resolved peaks corresponding to CHx+ (x = 2, 3) and C2Hy+ (y = 3–6) ions. The most intense features in the latter are associated with C2H3+ and C2H5+ ions. Tables S1 and S2 in the ESI† list relevant adiabatic ionisation and dissociative ionisation thresholds, respectively, and show that four of the neutral products of particular interest (i.e. CH2, CH3, C2H3 and C2H5) are amenable to single photon ionisation at 10.2 eV, with dissociative ionisation only a (potential) concern if any of these species carry high levels of internal excitation. Note, however, that the observation of some parent C2H6+ ion signal highlights the difficulty of completely excluding multiphoton processes even when operating at threshold FEL pulse intensities (<100 nJ).</p><p>The inset to Fig. 3 shows that the relative intensity of the CH2+ signal increases as the excitation wavelength is decreased. Note that the data shown in the inset were recorded with the detector sensitivity raised for just the relevant narrow range of mass/charge (m/z) ratios, thus allowing averaging over many more acquisitions and improved signal-to-noise ratios. The λ-dependent trend in the CH2+ signal is also recognisable in spectra recorded using higher FEL pulse energies but, as shown in Fig. S1,† the relative peak intensities are also pulse energy dependent. Such variations are not unexpected, given the inevitable differences in the wavelength and internal energy dependent photoionisation cross-sections for CH3, 1CH2 and 3CH2 radicals.</p><p>Use of the PImMS2 sensor affords not just TOF mass spectra such as those presented in Fig. 3, but also an ion image for each mass channel, in a single acquisition. This provides velocity distributions for each ion peak in Fig. 3. Since C–C bond rupture processes are likely to be pivotal in the cycling of ethane and methane and thus to have a major effect on the atmospheric dynamics, we first present kinetic energy distributions of CH2 and CH3 fragments (monitored via the corresponding ions) from the photofragmentation of ethane.</p><p>Fig. 4 presents the total kinetic energy release P(TKER) distributions (calculated on the basis that the partner to the observed fragment carries all of the remaining mass) and TKER-dependent best-fit recoil anisotropy (β) parameters30 obtained from analysing the ion images retrieved from the central time slice of the TOF-MS peaks corresponding to (A, B) CH2+ and (C, D) CH3+ ions recorded at FEL wavelengths λ = 125.6 nm (9.87 eV), 121.6 nm (10.19 eV), 118.2 nm (10.49 eV) and 112.0 nm (11.07 eV). Note that the signal intensities at TKER > 35 000 cm−1 are too low for recoil anisotropy parameters to be fitted satisfactorily. Fig. 4A and C also show the corresponding [P(TKER)]1/2 plots (dotted lines) to allow better visualisation of the high TKER data. The raw ion images are shown in Fig. S2 of the ESI.†</p><p>The distributions derived from the CH2+ ion images (Fig. 4A) assume that the co-fragments are CH4 (i.e. that the CH2 fragments derive from reaction (4) in Table 1). This assumption must be correct for the more translationally excited CH2 products, which display an anisotropic velocity distribution characterised by a positive recoil anisotropy parameter, β ∼ +0.5–0.7 (Fig. 4B), i.e. the CH2 fragments recoil preferentially along the axis parallel to the polarisation vector ε of the photolysis laser photons. But the P(TKER) distributions extend to TKER ∼0 – implying substantial internal excitation of some of the CH2 and/or CH4 fragments. Indeed, as Table 1 shows, the chosen photon energies exceed the thresholds for several three-body fragmentation processes that yield CH2 products. Some or all of channels (6), (13), (14) and (17) in Table 1 could contribute to the increased low-TKER product yield observed at the two shortest excitation wavelengths – a point to which we return later. Thus the precise form of the P(TKER) distribution at low TKER is ill-defined, since the momentum conservation arguments used to convert a measured CH2 fragment velocity (derived from the image radius) into a TKER value are likely not to apply in a three-body dissociation. But this does not negate the conclusions that (i) the relative yield of slow fragments in the CH2+ images increases with increasing photon energy and (ii) the slower fragments, which display minimal recoil anisotropy (β ∼ 0), likely arise via one or more of the three-body fragmentation processes.</p><p>The distributions derived from the CH3+ ion images (Fig. 4C) peak at TKER ∼0 and show a tail extending to higher TKER that becomes more anisotropic (to positive β) and relatively more intense as the photolysis wavelength is reduced. As can be deduced from Table 1, the maximum possible TKER of CH3 fragments formed via reaction (5) following excitation at λ = 121.6 nm (Fig. 4C) would be ∼6.38 eV (∼51 500 cm−1); the high-TKER tails of the P(TKER) distributions shown in Fig. 4C (derived assuming C–C bond fission) extend to values for which the direct C–C bond fission channel (5) is the only possible one photon induced CH3 fragment formation pathway. Most of the imaged CH3 fragments appear with much lower TKER, however. Table 1 shows several potential sources of slow CH3 radicals, including three-body dissociations (6), (12) and (14) and the production of an electronically excited CH3 partner (channel (16)), the relative likelihoods of which are discussed below.</p><!><p>H atom TOF spectra were recorded following photolysis of a jet-cooled ethane sample in He at λ = 121.6 nm with ε aligned at, respectively, ϕ = 0, 54.7 and 90° to the detection axis and converted to the corresponding P(TKER) and β(TKER) distributions, shown in Fig. 5A and B, by assuming C2H5 as the co-fragment. The fastest products have TKER ∼35 000 cm−1 (∼4.3 eV). This TKER value is greater than that reported in the earlier imaging study at this wavelength19 but still well below the maximum allowed by energy conservation assuming single C–H bond fission in ethane (channel (7), for which TKERmax ∼ 6.5 eV). In contrast to the case of CH4, however, the H atom recoil velocity distribution is essentially isotropic.</p><!><p>The present work identifies fragments formed by VUV photolysis of C2H6, assures that these arise via collision-free unimolecular dissociation and affords insights into the fragmentation dynamics. The translational spectroscopy data for the CH2, CH3 and H atom products hint at similarities in the fragmentation mechanisms following VUV photoexcitation of C2H6 and CH4 and it is useful to summarise current knowledge of the photofragmentation dynamics of CH4 to provide context for the discussion that follows.</p><p>Only the ground (S0) state and a repulsive triplet excited state of CH4 correlate with the lowest energy C–H bond fission limit (associated with H + CH3 products). The first excited singlet (S1) state of CH4 correlates adiabatically with products; the electronically excited fragments predissociate rapidly to H + 1CH2(ã) products.31 (Here and henceforth, we use superscript * and # symbols to indicate, respectively, electronically and rovibrationally excited products). Nonetheless, experiments find a substantial quantum yield of ground state C–H bond fission products following VUV photoexcitation of CH4, and the H atom products display anisotropic recoil velocity distributions – implying that the photoexcited molecules dissociate on a time scale that is much shorter than the rotational period of the parent molecule (which is estimated to be a few picoseconds).30,32–34 These findings highlight the importance of non-adiabatic couplings via conical intersection (CIs) between the S1 and S0 PESs.35,36 Theory shows that, to form H + CH3 products, one C–H bond in the photoexcited CH4 must start stretching and sweep through the plane defined by the other atoms to access the S0 PES and dissociate. Angular momentum conservation requires that the resulting CH3 products are highly rotationally excited; indeed, some of these CH3(X̃)# fragments are formed with so much internal energy that they dissociate further – to H + CH2 and/or H2 + CH products. Rival distortions have also been identified, whereby photoexcited CH4 molecules dissociate by eliminating H2. Theory suggests that the partner CH2 fragments in this case are formed in the ã1A1 state (for dissociations occurring after non-adiabatic coupling to the parent S0 PES) and the b̃1B1 state (if dissociation occurs adiabatically on the excited state PES).36</p><p>Quantitative simulations of the early time nuclear motions following photoexcitation of C2H6 remain challenging but global reaction route mapping (GRRM)37,38 calculations (summarised in the ESI†) can offer important insights by predicting low-lying conical intersections (CIs) between the PESs for the S0 and S1 states. The present VUV photoexcitations will populate one or more Sn (n > 1) states of ethane, but we henceforth assume that molecules excited to these higher Sn states undergo efficient non-radiative coupling to the S1 state. As Fig. 2 showed, the S1 state of ethane correlates with and products (channels (15) and (16) in Table 1). The and species are both unstable and dissociate to give, respectively, H + C2H4 (ref. 39–41) and H + 1CH2(ã) (ref. 31) products. As in CH4, C2H6(S1) molecules can also decompose after non-adiabatic coupling to the S0 PES. The nuclear distortions required to access the predicted CIs between the S1 and S0 PESs (shown in Fig. 2) correlate well with 1CH2 elimination once an H atom has inserted between the two C atoms, with C–C or C–H bond fissions and with loss of H2. The present study is sensitive to the first three fragmentation processes, which are considered in turn. Given the photon energies involved and the multi-dimensional nature of many of these distortions, we can anticipate that (as in the case of CH4 (ref. 30, 33 and 34)) many of the polyatomic products will be formed with sufficient internal energy that they will fragment further.</p><!><p>The imaging studies reveal CH2 fragments, with non-zero β values, implying that these are again formed on a timescale shorter than the parent rotational period. The P(TKER) distributions extend to values where the partner fragment can only be CH4, but not to sufficiently high TKER values to allow unambiguous determination of the electronic state of the CH2. Spin-conservation arguments suggest that CH2 radicals formed in tandem with CH4 will be in their ã1A1 state (for dissociations that occur following non-adiabatic coupling at a CI with the S0 PES) and/or b̃1B1 state (for dissociation on the S1 PES). But the distributions also extend to TKER ∼0, showing that one or other or both fragments are formed with a broad spread of internal energies. The photoexcitation energies are sufficient to induce three-body fragmentations and, simply on energetic grounds, any of channels (6), (13), (14) and (17) in Table 1 could contribute to signal in the CH2+ ion images. Of these, unimolecular decay of any sufficiently internally excited CH#4 partner would be expected to contribute to the yield of (slow) H and CH3 products, i.e. the net reaction (14) in Table 1.</p><p>Fig. 4A shows an additional feature at low TKER in the distributions derived from the CH2+ images measured at the two shorter wavelengths. This might signify the opening of a new (three-body) route to 1CH2 products, but this feature more likely indicates the presence of 3CH2 photoproducts: The ground states of the 3CH2 radical and the CH2+ cation have very similar geometries. Photoionisation of 3CH2 thus tends to be vibrationally adiabatic (i.e. to favour Δv = 0 transitions)42–44 and, from Table S1,† should only be expected at Ephot >10.39 eV (i.e. at λ < 119.3 nm). Note that the feature at low TKER in the P(TKER) distributions shown in Fig. 4A appears to 'turn on' and become more prominent as the photon energy is tuned above this threshold. Several possible routes to forming 3CH2 products can be envisaged, including the unimolecular decay of highly internally excited CH#3 fragments (from initial C–C bond fission) or of C2H#5 fragments (following primary C–H bond fission) after non-adiabatic coupling to the S0 state – as discussed below. Both would contribute to net process (6) in Table 1, though not necessarily exhibit similar energy disposals.</p><!><p>The tails of the P(TKER) distributions derived from the CH3+ images extend to TKER values that can only be accommodated by assuming C–C bond fission and formation of two CH3 radicals (i.e. reaction (5) in Table 1). Most of the measured CH3 fragment velocities imply TKER values far below the maximum allowed by energy conservation, however. Focussing first on the high TKER region in Fig. 4C, the CH3 fragment yield is clearly rising with decreasing TKER, indicating a preference that one (or both) CH3 fragments from reaction (5) are formed internally excited. Such energy disposal would likely be a consequence of the nuclear motions that enable non-adiabatic coupling to the S0 PES. Again, the non-zero β parameter revealed by the CH3 images imply that these nuclear motions and the ensuing C–C bond fission on the S0 PES also occur on a timescale shorter than the parent rotational period.</p><p>In principle, the entire P(TKER) distribution derived from the CH3 image could be attributed to channel (5) if the fragmentation dynamics were heavily biased towards forming very highly internally excited CH#3 products. The unimolecular decay of these CH#3 fragments would be a source of the 3CH2 products inferred above (reaction (6)), and these 3CH2 products would be expected to display a similar translational energy distribution to that of the CH#3 products (since the light H atom partner would take the bulk of any excess energy released in the secondary fragmentation). Such expectations are consistent with the experimental data and, as noted above, the non-observation of a peak attributable to 3CH2 products at longer wavelengths (e.g. at λ = 121.6 nm) need not imply that CH#3 fragments are not formed but simply that the 3CH2 products from their decay are not amenable to photoionisation at the longer wavelengths.</p><p>The dominance of translationally 'cold' (i.e. internally 'hot') CH3 products in the P(TKER) distributions is striking, however. Table 1 suggests several other potential sources of slow CH3 products. Adiabatic dissociation on the S1 PES to products is an interesting contender. This process is exoergic at all wavelengths studied, though the adiabatic S1 PES will likely exhibit a barrier at short RC–C bond extensions as the Rydberg function acquires increasing σ* antibonding valence character.6 The radicals would be unstable with respect to H + 1CH2(ã) products.31 Again, the H atoms would carry most of any kinetic energy release, so the translational energy distributions of any 1CH2(ã) fragments formed in this way should broadly mirror that of their precursor. 1CH2(ã) fragments are amenable to photoionisation at all wavelengths investigated in the present work, but the TKER distributions derived from the CH2+ images measured at the longer excitation wavelengths show no 'spike' at low TKER – suggesting that any contribution to the 1CH2(ã) yield from adiabatic dissociation to products on the S1 PES must be small compared to that from reaction (4).</p><!><p>The P(TKER) distribution derived from the H atom TOF measurements (Fig. 5A) extends to TKER values that can only be attributed to prompt C–H bond fission following VUV photoexcitation of C2H6, i.e. to reaction (7) in Table 1. The C2H5 co-fragments are formed with a very broad spread of internal energies. Analogy with CH4 suggests that this energy disposal is a consequence of the nuclear motions that promote C–H bond fission by non-adiabatic coupling to the S0 PES.6,35 Most of the 'C2H5' products assumed in deriving the P(TKER) distribution have sufficient internal energy to dissociate further – by loss of another H atom (net reaction (2)), or H2 (net process (9)), or both (net channel (10)), or to two CHx species (e.g. via net channels (6) or (11) in Table 1).42 However, the smoothly varying P(TKER) distributions shown in Fig. 5A suggest that such overall three- (or more-) body dissociations occur sequentially, i.e. via a prompt C–H bond fission and subsequent unimolecular decay of the resulting C2H#5 radicals. The low-TKER peak in Fig. 5A could also indicate an adiabatic contribution to the overall dissociation, yielding as primary products – as suggested in the earlier imaging study at λ = 121.6 nm.19 Any fragments would dissociate, yielding H + C2H4 products with a spread of translational energies39,45 (i.e. net reaction (2)). Many of the C2H4 products formed by decay of C2H#5 or species may well be formed with sufficient internal energy to decay yet further, to H + C2H3 (vinyl) radical products, or by eliminating H2 to yield C2H2. The former products are observed in the present study, via the C2H3+ peak in the TOF-MS in Fig. 3 and corresponding small ion image shown in Fig. S3 of the ESI,† but the current work is blind to C2H2 products – which were identified by end-product analysis in the early VUV photolysis studies of C2H6 under collisional conditions.16–18 For completeness, we note that C2H2 products could also arise via sequential H2 eliminations from, first, C2H#6 (formed by non-adiabatic coupling to the S0 state) and then from the resulting C2H#4 intermediates (i.e. net reaction (3) in Table 1). C2H2 formation by loss of four H atoms from C2H6 is energetically forbidden at the VUV wavelengths of current interest.</p><p>The present study does not return quantum yields and, as noted above, is silent regarding some molecular elimination channels. But it certainly identifies several active fragmentation channels and provides new insights into the likely fragmentation dynamics. The present analysis finds no compelling evidence for adiabatic dissociation on an excited state PES – implying efficient non-adiabatic coupling between excited states of C2H6 and to the S0 PES. Many of the present interpretations align with the results of recent quasi-classical trajectory surface hopping calculations for the next larger alkane, propane (C3H8), following excitation at λ = 157 nm, wherein it was concluded that most dissociations occur after internal conversion to the S0 PES, that the energy disposal in the resulting fragments is governed by dynamical rather than statistical factors, and that the three-body fragmentation processes occur sequentially.46</p><!><p>This work provides detailed new insights into the VUV photochemistry of ethane. The results discussed in detail in the above subsections are summarised below in terms of their implications for modelling the atmospheres of the gas giants. These new results must influence future models:</p><p>(i) The HRA-PTS studies reveal kinetic energy distributions extending to values that, on energetic grounds, can only be attributed to prompt C–H bond fission, confirming primary C–H bond fission yielding H + C2H5 products (reaction (7) in Table 1). This reaction does not feature in current models used to describe the chemical processing in the stratospheres of the gas giants. Most of the C2H5 species are formed with enough internal energy to decay further, almost certainly yielding some H + C2H4 products. The present study thus supports inclusion of reaction (2) in the modelling and implies that the two H atoms in that case are lost sequentially.</p><p>(ii) The kinetic energy distributions derived from the CH3+ ion images extend to TKER values that can only be attributed to C–C bond fission yielding two CH3 radicals, confirming that the C–C bond fission channel (reaction (5)) is active and supporting its inclusion in the modelling. The finding that the P(TKER) distributions peak at TKER ∼0 implies that one of the CH3 fragments is generally formed with sufficient internal energy to decay further. If C–C bond fission completes after non-adiabatic coupling to the S0 PES, the resulting CH#3 fragments most likely decay to H + 3CH2(X̃) products (i.e. net reaction (6)). This reaction is not included in the current model and, according to the present analysis, will have significantly higher quantum yield than reaction (5).</p><p>(iii) The imaging studies confirm formation of CH2 fragments, with a smooth P(TKER) distribution that extends to TKER values such that the partner fragment can only be CH4. Spin-conservation arguments and the deduced efficiency of non-adiabatic coupling to the S0 PES suggest that these faster CH2 fragments are formed in the ã1A1 state. The inclusion of reaction (4) in the photochemical modelling is vindicated.</p><p>(iv) The primary fragmentations and resulting product energy disposals following VUV photoexcitation of ethane are shown to be governed by dynamical rather than statistical factors; three-body dissociations are commonplace and occur sequentially. Clearly, quantitative branching ratios for the various active channels are still needed, but the present work offers several clear pointers. Reaction (7) and, particularly, the three-body fragmentation (6) are active and require incorporation in future modelling. The yield of (currently neglected) reaction (6) is deduced to be larger than that of reaction (5). The processes revealed in this study all involve relatively 'prompt' C–H or C–C bond fission, after non-adiabatic coupling to the S0 PES. As Fig. 2 shows, the respective bond energies are lower than the energy barriers to C2H4 formation by H2 elimination on the S0 PES. Analogy with CH4 suggests that any H2 and C2H4 products formed via process (1) will be both translationally and vibrationally excited. The likelihood that the C2H#4 species would have sufficient internal energy to surmount the barrier to eliminating a further H2 (to yield C2H2 or H2CC) is unclear. We further note that the substantial (∼60%) branching into C2Hx species following VUV photoexcitation of C2H6 assumed in the current planetary atmospheric photochemistry models derives from indirect measurements made more than half a century ago, and is predicated on an assumption that the decomposition of the internally excited C2H#4 species formed via reaction (1) would mimic that deduced following VUV photoexcitation of strategically deuterated ethene (CH2CD2) molecules.16,17 Such an assumption must be questionable, given the differences in available energy and the recognised importance of dynamics (i.e. the topographies of, and non-adiabatic couplings between, the PESs sampled in the two cases) in determining the product branching and energy disposal. It seems likely that the current models overestimate the relative yield of C2Hx (particularly C2H2) photoproducts.</p><!><p>Translational spectroscopy methods employing two cutting-edge technologies – the Dalian Coherent Light Source (DCLS) Free Electron Laser (FEL) and a fast-framing PImMS2 camera – have revealed many new insights into the rich (and hitherto largely impenetrable) VUV photochemistry of ethane. The present findings should serve to stimulate ab initio molecular dynamics simulations of this prototypical alkane following photoexcitation at VUV wavelengths and substantial refinements of the models currently used to describe the atmospheric photochemistry of the gas giants. This study (i) concludes that, as in CH4, the VUV photochemistry of ethane is driven by efficient non-adiabatic coupling to, and subsequent direct (and sequential) dissociations on the S0 PES, (ii) highlights the need to revise current photochemical models of the stratospheric photochemistry of the gas giants – by incorporating the hitherto neglected C–H bond fission channel (7) and the three-body decomposition (6) to CH3 + 3CH2 + H products and down-grading the relative yield of primary C2Hx photoproducts – and (iii) emphasises the pressing need for quantitative product branching fractions. Stratospheric C2H6 production in the gas giants is driven by VUV photodissociation of CH4, but the present analysis implies that the subsequent photochemical coupling between C2H6 and C2H2 is likely to be weaker than currently assumed.</p><!><p>The raw ion event lists, H-atom TOF spectra and calculation log files are available from the authors upon reasonable request.</p><!><p>There are no conflicts to declare.</p>

PubMed Open Access

Immunogenicity of In Vitro-Transcribed RNA

CONSPECTUS: In vitro-transcribed RNAs are emerging as new biologics for therapeutic innovation, as exemplified by their application recently in SARS-CoV-2 vaccinations. RNAs prepared by in vitro transcription (IVT) allow transient expression of proteins of interest, conferring safety over DNA- or virus-mediated gene delivery systems. However, in vitro-transcribed RNAs should be used with caution because of their immunogenicity, which is in part triggered by double-stranded RNA (dsRNA) byproducts during IVT. Cellular innate immune response to dsRNA byproducts can lead to undesirable consequences, including suppression of protein synthesis and cell death, which in turn can detrimentally impact the efficacy of mRNA therapy. Thus, it is critical to understand the nature of IVT byproducts and the mechanisms by which they trigger innate immune responses. Our lab has been investigating the mechanisms by which the innate immune system discriminates between \xe2\x80\x9cself\xe2\x80\x9d and \xe2\x80\x9cnonself\xe2\x80\x9d RNA, with the focus on the cytoplasmic dsRNA receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated 5 (MDA5). We have biochemically and structurally characterized critical events involving RNA discrimination and signal transduction by RIG-I or MDA5. We have used in vitro-transcribed RNAs as tools to investigate RNA specificity of RIG-I and MDA5, which required optimization of the IVT reaction and purification processes to eliminate the effect of IVT byproducts. In this Account, we summarize our current understanding of RIG-I and MDA5 and IVT reactions and propose future directions for improving IVT as a method to generate both research tools and therapeutics. Other critical proteins in cellular innate immune response to dsRNAs are also discussed. We arrange the contents in the following order: (i) innate immunity sensors for nonself RNA, including the RIG-I-like receptors (RLRs) in the cytosol and the toll-like receptors (TLRs) in the endosome, as well as cytoplasmic dsRNA-responding proteins, including protein kinase R (PKR) and 2\xe2\x80\xb2,5\xe2\x80\xb2-oligoadenylate synthetases (OASes), illustrating the feature of protein\xe2\x80\x93RNA binding and its consequences; (ii) the immunogenicity of IVT byproducts, specifically the generation of dsRNA molecules during IVT; and (iii) methods to reduce IVT RNA immunogenicity, including optimizations of RNA polymerases, reagents, and experimental conditions during IVT and subsequent purification.

immunogenicity_of_in_vitro-transcribed_rna

INTRODUCTION<!>CELLULAR INNATE IMMUNITY<!>RLRs<!>TLRs<!>Other dsRNA-Binding Proteins<!>RNA IMMUNOGENICITY DUE TO IVT REACTION<!>METHODS TO REDUCE IMMUNOGENICITY<!>RNA Polymerases<!>Chemically Modified Nucleotides<!>Transcription Conditions<!>Template Sequences<!>Purification of In Vitro-Transcribed RNAs<!>OUTLOOK

<p>Messenger RNA (mRNA)-based therapy can be used to replace endogenous malfunctioning genes or deliver antigens either from pathogens or tumors to the immune system.5 As our understanding of RNA biology and technology increases, mRNA-based therapy has become a promising direction for treatment. mRNA vaccines against SARS-CoV-2 are the most well-known example of its application to date.6</p><p>Therapeutic mRNAs are produced through in vitro transcription (IVT) catalyzed by DNA-dependent RNA polymerases derived from bacteriophages, such as T3, T7, or SP6.7 Of these, T7 RNA polymerase is the most extensively studied and widely used.8 These RNA polymerases selectively recognize the promoter region in DNA templates to synthesize RNA transcripts based on downstream template sequences. The resultant transcripts are naked RNA molecules—uncapped at the 5′-end and non-polyadenylated [poly(A)] at the 3′-end. To mimic naturally occurring mRNAs, extra steps are taken during and after IVT to cap the 5′-end (linking of N7-methylguanosine to the first nucleotide through a 5′–5′ triphosphate bond), optimize the sequences in the 5′- and 3′-untranslated regions (UTRs) and the coding sequence (CDS), and add the poly(A) tail.9</p><p>Although these modifications improve the stability and translational efficiency of in vitro-transcribed RNAs, they are challenged by the host innate immune system, which recognizes nonself molecules frequently found in invading pathogens or under pathological conditions. The innate immune response impedes therapeutic efficacy, as it not only affects treatment safety because of aberrant immune activation but also reduces the translation efficiency as part of cellular stress responses.10 Advances in innate immunity and IVT research have led us to believe that the purity and nucleotide composition of in vitro-transcribed RNA contribute to its immunogenicity. In this Account, we focus on our progress in our understanding of innate immune response to nonself RNA and IVT RNA.</p><!><p>Nonself molecules are sensed by host-encoded pattern recognition receptors (PRRs). PRRs generally do not recognize specific sequences from DNA, RNA, or protein but rather the molecular structures or kinds. Ligand-bound PRRs activate type-I interferon (IFN-I, such as IFNα or IFNβ) and inflammatory nuclear factor kappa B (NF-κB) signaling pathways to direct and modulate innate and adaptive immunity.11,12 Aberrant activation elicited by, for example, mutations in critical genes results in inflammation and immune diseases.13 Cellular functions, including gene expression, metabolism, proliferation, and differentiation, are under stress in cells involved in the innate immune response.14,15 In such cases, the translation of in vitro-transcribed mRNAs is negatively affected.</p><p>We will discuss PRRs that recognize nonself RNAs, most notably cytosolic RIG-I-like receptors (RLRs) and endosomal toll-like receptors (TLRs). Other double-stranded RNA (dsRNA)-binding proteins that shape cellular stress responses to dsRNA will also be discussed.</p><!><p>Upon RNA virus infection, dsRNAs are formed intracellularly and recognized by RLRs, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated 5 (MDA5).16,17 They have similar amino acid sequences and domain architectures, with two tandem N-terminal caspase activation and recruitment domain (CARD) motifs, a central DExD/H box helicase domain, and a C-terminal domain (CTD) (Figure 1a).16 RIG-I and MDA5 use the same downstream adaptors, kinases, and transcription factors to activate IFN signaling.16 Thus, the study of one receptor provides insights into the other.</p><p>We determined the structure of MDA5 bound to dsRNA and found that MDA5 binds dsRNA in a sequence-independent manner by interacting with the backbone of the RNA duplex (Figure 1b).1 Upon dsRNA binding through the helicase domain and CTD, proteins oligomerize along the length of the dsRNA and form a filamentous structure.18 The two CARD motifs (2CARDs) oligomerize upon filament formation,19 activating the downstream adaptor mitochondrial antiviral signaling protein (MAVS) in the mitochondria. We used RIG-I 2CARD oligomers to study their interaction with MAVS and found that they interact with the CARD motif of MAVS, triggering MAVS to form prion-like aggregates.2 It was shown that this MAVS–MAVS interaction is mediated through its CARD–CARD binding2 in the way that 2CARD oligomers from the RIG-I filament nucleate MAVS assembly (Figure 1c).2 This work uncovered how signals are transduced from RLR–dsRNA to MAVS. Activated MAVS then recruits tumor necrosis factor (TNF) receptor-associated factors (TRAFs), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3) to activate IFN signaling (Figure 1d).2,20</p><p>Despite sharing similarities, RIG-I and MDA5 have different substrate preferences and modes of filament formation. We and others have shown that RIG-I senses 5′-triphosphate (5′-ppp) or 5′-diphosphate (5′-pp) dsRNA with minimal lengths of ~20 base pairs (bp) and 40–150 bp for optimal signal transduction efficiency.21–24 First, it binds to the 5′-end of dsRNA and translocates to the inner side of dsRNA by hydrolyzing adenosine triphosphates (ATPs). A second RIG-I molecule then binds to the exposed dsRNA end and processes it in the same manner.19 MDA5 prefers longer dsRNA molecules (normally >1000 bp), independent of cap structure. It binds to the inside of dsRNA to nucleate protein association for filament formation.2,18 It was found that nucleation is a rate-limiting step compared with filament elongation. Hydrolyzing ATP leads to dissociation of MDA5 from the filament end and new MDA5s then assemble to fill the gap. Long dsRNA is preferred because a certain length of filament can be maintained under such assembly-and-disassembly conditions.18,22 The ATPase activities of both RIG-I and MDA5 play important roles in discrimination of nonself from self RNA substrates.18,25,26 The sensitivity of RLRs needs to be balanced with the specificity. The gain-of-function mutations of MDA5 showed more IFN-activating capacity at the cost of self-recognition,3 leading to the onset of autoimmune diseases. RLRs were found to be involved in in vitro-transcribed RNA-mediated activation of IFN signaling.27,28</p><!><p>Viruses invading cells through endocytosis enter the endosomes for uncoating and release of their genomic materials into the cytosol. Several types of TLRs localize and face toward the inside of the endosome.29 Recognition of dsRNAs with a minimal length of ~40 bp by TLR330 leads to TLR3 dimerization and subsequently activates the downstream adaptor protein Toll or interleukin-1 receptor (TIR) domain-containing adapter-inducing interferon-beta (TRIF). TRIF then recruits TRAF3 and TRAF6 for transcriptional activation of IFN and NF-κB activation.29,31 Structural studies have revealed that TLR3 binds to the ribose–phosphate backbone and not individual bases of dsRNA.32</p><p>Single-stranded RNAs (ssRNAs) are also recognized by TLR7 or TLR8 in the endosome. TLR7 dimerizes upon RNA binding, whereas TLR8 exists as a dimer before RNA recognition.33,34 Once bound to their ligands, TLR7 and TLR8 recruit the downstream adaptor myeloid differentiation primary response 88 (MyD88), which in turn associates with interleukin-1 receptor-associated kinase 4 (IRAK4) and IRAK1/IRAK2 to form the Myddosome. The Myddosome recruits TRAFs to activate IFN and NF-κB signaling.35–38 Human TLR8 prefers GU-rich ssRNAs.39 The immunogenicity of U-rich stretches was demonstrated using mouse and human cells in a study showing that uridine stretches are strong ligands for TLR7.40 Consistently, structural analysis revealed that both TLR7 and TLR8 bind to uridine in a uracil- and ribose-dependent manner (Figure 2a for TLR7 and Figure 2b for TLR8).33,34 It is noteworthy that the binding sites on the uracil base are still present in chemically modified versions of uridine, such as pseudouridine (ψ) (Figure 2c), which will be discussed in more detail later in this Account. Several studies have confirmed that in vitro-transcribed RNAs stimulate TLR3, TLR7, and TLR8.41–43</p><!><p>When protein kinase R (PKR) binds to dsRNAs with a minimal length of ~33 bp, it dimerizes and gets activated by autophosphorylation,17,44 which leads to the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylated eIF2α blocks global translation initiation and subsequently leads to activation of NF-κB-mediated apoptosis.45,46 Similar to RLRs, PKR binds to dsRNA in a sequence-independent manner.47,48 In vitro-transcribed RNAs have been reported to trigger PKR activation in vitro and in cells.49,50</p><p>2′,5′-Oligoadenylate synthetase (OAS) binds to dsRNA to produce 2′,5′-oligoadenylate (2–5A), which serves as a secondary messenger to activate RNase L by inducing its dimerization or oligomerization.51 Activated RNase L then globally degrades cellular RNAs52 and actively arrests global translation.53–55 Interestingly, defense mRNAs such as IFNβ are not cleaved and translate during this process. All of these reprogram the cellular environment toward making IFNs.53–55 In humans, the OAS family comprises OAS1, OAS2, OAS3, and OAS-like protein (OASL); OASL is enzymatically inactive.56 OAS3 is more potent in responding to dsRNAs than OAS1 and OAS2 and is the primary activator of RNase L.57 In vitro-transcribed RNAs activate the OAS–RNase L pathway and inhibit their own translation.58</p><p>A schematic view of cellular innate immunity is shown in Figure 3.</p><p>It is noteworthy that theoretically a potential consequence of in vitro-transcribed RNAs applied in vivo is the raising of anti-RNA antibodies. These RNA-recognizing antibodies are evident in the case of autoimmune diseases like systemic lupus erythematosus.59,60 Studies have shown that TLR7 in B cells contributes to such antibody production and that RNA antigens can be RNA epitopes on small nuclear RNAs (snRNAs), 28S rRNA (rRNA), certain tRNAs (tRNAs), and even some mRNAs.59–62 Although reports on anti-IVT RNA antibodies are still lacking, one might consider examining mRNAs for therapeutics, as they themselves do not induce anti-RNA antibodies.</p><!><p>IVT RNAs are often used to investigate the RNA specificity of RNA sensors in the innate immune system. IVT can be easily performed using commercial kits or homemade T7 RNA polymerase. However, IVT yields not only the desired full-length ssRNA but also several types of byproducts, including short transcripts due to abortive transcription63 and dsRNAs.4,64,65 Such complexity can be exemplified by the observation that RNA produced by IVT activated OAS while that with the same sequence produced by chemical synthesis did not.66 Understanding how dsRNA byproducts are formed during IVT will help reduce unwanted immunogenicity.</p><p>dsRNA byproducts are mainly produced through the 3′-extension of the ssRNA transcript, caused by annealing of the 3′-end to complementary sequences in cis or trans and continued transcription (Figure 4a).64,65 Additionally, we identified that dsRNA byproducts that were resistant to ssRNA-specific RNase I digestion but sensitive to dsRNA-targeting RNase III treatment (Figure 4b) were composed of two separate reverse complementary RNA strands (Figure 4c). The sense strand in the dsRNA corresponds to the desired ssRNA product, whereas the antisense strand was a full-length RNA molecule transcribed from the 3′-end of the template to the promoter region (Figure 4c), indicating that the antisense strand is transcribed in a promoter-independent, 3′-end extension-free manner and anneals to the sense strand to form a duplex (Figure 4d). This full-length dsRNA byproduct activates both RIG-I and MDA5 for IFN signaling (Figure 4e).4 The promoter-free transcription from the 3′-end of the DNA template is dependent on the sequence of the 3′-end in the DNA template.4,67</p><p>Double-stranded structures can also be formed through annealing of complementary sequences intra- or intermolecularly.68 Therefore, checking in vitro-transcribed RNA for the presence of dsRNA byproducts even after RNA purification is critical. Traditional purification methods, including phenol/chloroform extraction, lithium chloride precipitation, and column-based purification methods, cannot distinguish dsRNA from ssRNAs, and short abortive RNAs are not always removed. We used acridine orange (AO) to stain RNAs on native PAGE because AO helps distinguish between ssRNA and dsRNA (Figure 4b). Moreover, analysis of RNA products using different types of RNases is helpful (Figure 4b).</p><!><p>Many groups, including ours, have attempted to eliminate the dsRNA byproduct during transcription or purification. We have arranged this section based on operational order, from during IVT to after IVT. During IVT, optimizations are performed on RNA polymerases, chemically modified NTPs, reaction conditions, and template sequences. For the subsequent purification steps, chromatography-based methods are effective.</p><!><p>IVT can be performed at higher temperatures (51–55 °C) by using an engineered thermostable T7 RNA polymerase to yield ssRNA products lacking 3′-extension-derived dsRNA byproducts. Higher temperatures are believed to decrease the efficiency of either polymerase binding to the 3′-end of RNA or 3′-end priming for antisense synthesis.67 Interestingly, this high-temperature condition could not reduce the formation of the dsRNA byproduct formed by the annealing of sense and antisense RNAs. However, introduction of the poly(A) tail encoded by the template reduced antisense byproduct formation at higher temperatures. Thus, a combination of high temperature and poly(A) tailing was proposed to reduce immunogenicity67</p><p>RNA polymerases from other bacteriophages have also been studied. An RNA polymerase from the psychrophilic phage VSW-3 was shown to be more effective than T7 RNA polymerase in maintaining the yield of ssRNA products and decreasing the yield of dsRNA byproducts. Reactions can be performed at ≤25 °C, which is reportedly beneficial for the stability of RNA products.69</p><!><p>DNA molecules undergo modifications such as cytosine methylation on nucleosides, not to affect base paring but to influence protein binding.70 Similarly, mammalian RNAs, including mRNAs, are modified post-transcriptionally. Chemically modified nucleosides include pseudouridine (ψ), N6-methyladenosine (m6A), 5-methylcytidine (m5C), etc.71 Other common modified nucleosides include 2-thiouridine (s2U) and N1-methylpseudouridine (m1ψ) (Figure 2c). Such modifications are suggested to mark the RNAs as self and protect them from innate immune recognition.42 Extensive efforts have been made to investigate the protective role of modified nucleosides toward in vitro-transcribed mRNAs. Studies in dendritic cells (DCs) from peripheral blood have found that m5C and m6A present immunostimulatory effects comparable to those with unmodified bases, whereas ψ, s2U, and ψ/m6A are nonstimulatory.42 This suggests a dominant role of ψ (over m6A) in protecting RNAs from recognition and that mRNAs containing m1ψ are less immunogenic.72,73 Consistently, in mice, modified nucleosides such as s2U/m5C, ψ/m5C, and ψ reduce the immunogenicity of in vitro-transcribed mRNA.43,74,75</p><p>These chemically modified nucleotides containing in vitro-transcribed RNAs may lose efficient binding affinity to innate immune receptors. We reported that the presence of ψ, s2U, or m6A in the RNA duplex suppresses RIG-I from forming filaments on dsRNA.19 Consistently, Gehrke et al. observed that m6A protects in vitro-transcribed RNAs from binding to RIG-I, whereas ψ, m1ψ, and m5C prevent RIG-I conformational change after binding.28 A combination of m5C and s2U protects mRNAs from binding to TLR3, TLR7, TLR8, and RIG-I.43</p><p>Studies have shown that for IVT mRNA, translation efficacy is higher with modified nucleotides than with normal nucleotides.43,72,73,75 For example, in vitro-transcribed mRNAs containing ψ or m5C exhibited a dramatic increase in translation, which was immunogenicity-independent for in vitro-transcribed mRNAs containing ψ.75 Comparison of in vitro-transcribed yeast tRNA and natively isolated tRNA showed that the IVT product is a strong agonist of PKR activation and that nucleoside modifications, including ψ, m5C, and s2U, abolished this activation.76 Consistently, tests with in vitro-transcribed mRNAs have shown that ψ, m1ψ, and s2U are responsible for protection against PKR recognition.49,50,77 Moreover, replacement of U with ψ protects the RNA molecule from activating OAS and the downstream RNase L-mediated RNA cleavage event.58 Mounting evidence from mice studies supports the beneficial role of modified nucleosides (ψ and m1ψ) in mRNA translation.58,72,73,75,78–80 However, it should be noted that exactly why certain modifications are more effective than others in evading translational suppression is unclear. Not all modifications are beneficial to translation, even if they contribute to reducing PKR binding. Additionally, different cells may exhibit different sensitivities to modified nucleosides. For example, in vitro-transcribed mRNAs containing s2U and m6A in HEK293 cells (an immortal human embryonic kidney cell line) and murine monocyte-derived dendritic cells (MDDCs) are not translated.75 Furthermore, ψ and m1ψ could not improve translation of in vitro-transcribed mRNAs in HeLa cells (an immortal human cervical cancer cell line) or keratinocytes (an epidermal cell line) but could enhance the translation of in vitro-transcribed mRNA in C2C12 cells (an immortalized mouse myoblast cell line).72</p><p>These observations suggest that the effect of RNA modification on immunogenicity and translation suppression may not be entirely due to the direct effect of modified nucleosides on the RNA sensors. In fact, our study found that modified nucleotides in the IVT reaction also alter the behavior of the RNA polymerase and consequently the synthesized RNA products. We tested selected modified nucleotides to replace original nucleotides for IVT and found that antisense-mediated dsRNA byproducts were reduced when ψ, m1ψ, or m5C was used but not when m6A was used (Figure 5a, 512B transcript).4 A similar observation was made by Karikó et al. when ψ was used in place of U.78 Intriguingly, the MDA5-mediated IFN signaling efficacy was unaffected by gel-purified dsRNA containing modified nucleotides (Figure 5b, purified 512B:c512B), consistent with the notion that MDA5 binds to the dsRNA backbone rather than interacting with bases, where modification often occurs. Our data suggest that modified nucleotides in part reduce the immunogenicity of in vitro-transcribed RNA by inhibiting the production of dsRNA byproducts, thus improving RNA purity. It is also possible that modified nucleotides in the IVT reaction also suppress other immunogenic byproducts besides the dsRNA byproduct as described above, which would be an interesting area of future research.</p><!><p>We tested different IVT conditions, such as the concentrations of RNA polymerase, template, NTPs, and NaCl, to decrease dsRNA formation and found that lowering the Mg2+ concentration (from 30 to 5 mM) reduced the amount of dsRNA byproducts generated by the promoter-free antisense RNA synthesis mechanism (Figure 5c) independent of template length.4 Although the reason for this improvement is unclear, we speculate that the T7 RNA polymerase in the promoter-free antisense RNA synthesis maintains its conformation in the elongation phase to stay transcriptionally active and that a high concentration of Mg2+ (30 mM) promotes maintenance of such a conformation.4</p><!><p>Sequence manipulation can reduce the immunogenicity of in vitro-transcribed mRNA. By using different restriction sites to change the 3′-end sequence in the IVT template and digestion of these sites to create different structures, we noticed that the 3′-end sequence and structures impact the promoter-free antisense transcription (Figure 5d).4 Other studies have shown that using uridine-depleted sequences lowers the immunogenicity of in vitro-transcribed Cas9 mRNA containing modified nucleosides by an unknown mechanism,81 supporting the notion that TLR7 and TLR8 bind U-containing ssRNAs.</p><!><p>On the basis of RNA size and structure, native PAGE or agarose gel electrophoresis can identify the correct ssRNA products on a small scale.82 For larger amounts of in vitro-transcribed RNAs, chromatographic purification effectively separates IVT byproducts, such as short abortive transcripts, nucleotides, and dsRNAs with distinct conformations.83–85 High-performance liquid chromatography (HPLC) was reported to effectively remove dsRNA contaminants; however, the resultant in vitro-transcribed mRNA, composed of unmodified nucleotides, remained immunostimulatory—DCs maintained high levels of TNF-α and IFN-α. By contrast, HPLC-purified ψ-, m5C- or ψ/m5C- modified mRNAs did not induce cytokine release.85 Findings from a study using m1ψ were consistent.86 Thus, using a combination of modified nucleotides for IVT and HPLC purification can produce non-immunostimulatory mRNAs.</p><p>Another approach to remove dsRNA contaminants takes advantage of the fact that dsRNA prefers to bind to cellulose in ethanol-containing buffer78 and is utilized in the study of microorganisms.87,88 Cellulose-bound dsRNA is separated in the spin column from the ssRNA flowthrough. Nucleoside-modified RNAs (m1ψ) can be effectively purified using this method. Results from in vivo assays suggested that mRNAs purified using this method are comparable with HPLC-purified mRNAs in terms of translation efficiency.78 Consistent with results from HPLC purification, cellulose-purified in vitro-transcribed mRNAs containing unmodified nucleosides were immunostimulatory, whereas m1ψ-containing in vitro-transcribed mRNAs were not, further highlighting the protective role of modified nucleosides.</p><p>A summary of methods to reduce immunogenicity is illustrated in Figure 6. Purification of in vitro-transcribed mRNAs to discard dsRNA contaminants or abortive short transcripts is not effective enough to keep the in vitro-transcribed mRNAs from being immunogenic. For now, a combination of modified nucleotide usage for transcription and chromatographic purification appears to be promising for in vitro-transcribed mRNA preparation.</p><!><p>Progress in innate immunity has increased our understanding of the immunogenicity of in vitro-transcribed mRNAs. Mechanistic studies of the interactions between host-cell-encoded receptors and their RNA ligands have revealed details of innate immune activation, and investigations of IVT reactions have shed light on the origin of such immunogenicity. Although immunogenicity can be potentially beneficial in the case of vaccination,89 a well-controlled in vitro-transcribed mRNA product is crucial for therapeutic applications, as the goal of mRNA therapy is to express proteins of interest in target cells. The use of chemically modified nucleotides for IVT and chromatographic purification steps makes in vitro-transcribed mRNAs competent in protein translation and immunosilencing.78,85,86 New RNA polymerases, including the optimized T7 RNA polymerase, are also worth exploring.67,69</p><p>The use of chemically modified nucleotides in IVT contributes not only to reducing the immunogenicity of purified ssRNAs but also to decreasing dsRNA production.4,78 We speculate that modified nucleotides may benefit IVT by increasing transcript purity and decreasing transcript immunogenicity. Because of the complex nature of in vitro transcription, using AO staining can be a straightforward way to check the presence of dsRNA byproducts, thus reducing uncertainties about the IVT samples.</p><p>How modified nucleotides suppress dsRNA production or RNA immunogenicity is still unclear. Considering immune evasion as an example, the dsRNA-binding proteins discussed here sense RNA in a sequence-independent manner. For the ssRNA-sensing receptors TLR7 and TLR8, structural studies have shown that the receptor-bound atoms in uridine include oxygen atoms linked to C2 and C4 and N3 in uracil (Figure 2a,b), all of which are equally present in ψ or m1ψ (Figure 2c). Precisely how these modified nucleotides affect immunogenicity and translational efficiency, whether it is directly or indirectly mediated by RNA sensors, and how much of these effects are due to their impact on the IVT reaction itself remain to be investigated. We believe that studying the molecular mechanism ofn this process will improve our understanding of how innate immunity distinguishes "nonself" from "self" RNAs.</p>

PubMed Author Manuscript

Absolute Free Energy of Binding Calculations for Macrophage Migration Inhibitory Factor in Complex with a Drug-like Inhibitor

Calculation of the absolute free energy of binding (\xce\x94Gbind) for a complex in solution is challenging owing to the need for adequate configurational sampling and an accurate energetic description, typically with a force field (FF). In this study, Monte Carlo (MC) simulations with improved side-chain and backbone sampling are used to assess \xce\x94Gbind for the complex of a drug-like inhibitor (MIF180) with the protein macrophage migration inhibitory factor (MIF) using free energy perturbation (FEP) calculations. For comparison, molecular dynamics (MD) simulations were employed as an alternative sampling method for the same system. With the OPLS-AA/M FF and CM5 atomic charges for the inhibitor, the \xce\x94Gbind results from the MC/FEP and MD/FEP simulations, \xe2\x88\x928.80 \xc2\xb1 0.74 and \xe2\x88\x928.46 \xc2\xb1 0.85 kcal/mol, agree well with each other and with the experimental value of \xe2\x88\x928.98 \xc2\xb1 0.28 kcal/mol. The convergence of the results and analysis of the trajectories indicate that sufficient sampling was achieved for both approaches. Repeating the MD/FEP calculations using current versions of the CHARMM and AMBER FFs led to a 6-kcal/mol range of computed \xce\x94Gbind. These results show that calculation of accurate \xce\x94Gbind for large ligands is both feasible and numerically equivalent, within error limits, using either methodology.

absolute_free_energy_of_binding_calculations_for_macrophage_migration_inhibitory_factor_in_complex_w

INTRODUCTION<!>Absolute Binding Free Energy Calculations.<!>System Preparation.<!>Monte Carlo Simulations.<!>Molecular Dynamics Simulations.<!>Calculations for the Unbound Ligand; Comparison of Water Models.<!>Calculations for the Complex.<!>MD Calculations for the Unbound Ligand; Comparison of Small-Molecule Force Fields.<!>Conformation of the Unbound Ligand.<!>MD Results for the Bound Complex with the OPLS/CM5 Force Field.<!>MD Results for the Four Force Fields and the Protonation State for Proline 1.<!>Structural Analyses.<!>CONCLUSION

<p>Alchemical binding free energy calculations have been rapidly developing and are now being widely applied in structure-based drug design (SBDD).1–6 Different statistical mechanics approaches have been explored to try to achieve accurate binding affinity predictions.1,7 Perturbative free energy methods such as thermodynamic integration (TI), free-energy perturbation (FEP) and Bennett's acceptance ratio (BAR) are based on the assumption that the configurational space of two different states is similar enough to obtain valid evaluations of the difference in free energies. To ensure this condition, the stratification technique splits the transformation path into a number of intermediate steps or "λ-windows" that yield adequate overlap of the configurational spaces. Relative binding free energy (ΔΔGbind) calculations, where the initial and final molecules are very similar, have been dominant in structure-based drug design (SBDD) studies.5,6 In contrast, absolute binding free energy calculations decouple energetically the ligand entirely from its environment, either the surrounding solvent molecules or a protein binding site.8,9 As the removal of the entire ligand molecule is performed, such calculations are computationally demanding and potentially sensitive to sampling and numerous setup issues for the protein. On the other hand, they do address the fundamental thermodynamic gauge of molecular recognition and the results can be directly compared to experimental binding data, after corrections for standard states are introduced.10–12 The calculations when performed in a prospective manner provide a rigorous test of current methodologies and force fields.13 However, such calculations are still far from routine and, as considered here, further examination of methodological issues and the impact of alternative force fields is needed.</p><p>Much work in the area has been done with molecular dynamics (MD) methods using software packages such as GROMACS,14 AMBER,15 NAMD,16 CHARMM,17 and OpenMM.18 Much less work has used Monte Carlo statistical mechanics (MC), though it can be very efficient compared to MD for liquid simulations.19 Unlike MD, where a new configuration is generated by integrating equations of motion for all atoms, MC explores the configurational space by localized random moves of solvent and solute molecules.19 It also permits enhanced sampling of conformational changes and of local regions of interest, e. g., near the protein binding pocket. Moreover, NVT and NPT ensembles are readily implemented through the Metropolis sampling without the need to apply thermostats and barostats. Recent improvements in the MC-based software package MCPRO have resulted in enhanced sampling of protein side chains and backbone atoms.20 Similar sampling and absolute free energies of binding were obtained for complexes of benzene and analogs with T4 lysozyme L99A using MD or MC.20 It is of interest, then, to extend this study to a more drug-relevant biomolecular system and further assess the sampling performance of MC and MD. For a common force field, MC and MD are expected to converge to the same ΔGbind results, once sufficient configurational sampling is achieved.</p><p>Macrophage migration inhibitory factor (MIF) is both a keto-enol tautomerase and a cytokine associated with inflammatory diseases and cancer.21,22 It was selected as the subject of this study since it has many characteristics that make it a suitable benchmark system: (1) the trimeric protein has moderate size with 342 residues; (2) multiple high-resolution crystal structures of complexes of MIF with tautomerase inhibitors are available; (3) the crystal structures for many inhibitors show modest conformational changes for binding-site residues; and, (4) experimental binding data, Ki and Kd values, are available from inhibition and fluorescence polarization assays. In particular, for this work, we have chosen to study the complex of the inhibitor MIF180 with human MIF, as illustrated in Figure 1 from the crystal structure obtained in our laboratory.22 As indicated, the complex features a combination of hydrogen bonding, van der Waals, and aryl-aryl interactions, which is typical for protein-drug complexes. In contrast, the widely used L99A T4-lysozyme system binds benzene analogs primarily through the hydrophobic effect.20</p><p>Once adequate sampling is achieved, the effects of the accuracy of the force field and other methodological factors can be evaluated for the benchmark system via ΔGbind calculations. In our laboratory, much effort has been devoted to steady improvements of the OPLS force fields. Recently, the OPLS-AA force field for proteins and nucleic acids has been improved through extensive reoptimization of the torsional parameters using high-level quantum mechanical calculations and MC and MD simulations of series of peptides, proteins, nucleotides and polynucleotides to yield OPLS-AA/M.23,24 In addition, OPLS parameters for general small-molecule ligands are now available with atomic charges from QM calculations, after optimization through studies of properties of pure liquids and free energies of hydration.25 CHARMM26 and AMBER27 are two other popular force fields initially parameterized for proteins and later extended to nucleic acids, lipids, and small molecules (CGenFF28 and GAFF29). In the present work, four combinations of protein-ligand force fields are utilized, namely OPLS-AA/M with OPLS-AA/CM5, OPLS-AA/M with OPLS-AA/CM1A, CHARMM 36 with CGenFF, and AMBER ff14sb with GAFF. These will be referred to as OPLS/CM5, OPLS/CM1A, CHARMM/CGenFF, and AMBER/GAFF.</p><p>In this work, ΔGbind results for the MIF180/MIF complex have been obtained from Monte Carlo free energy perturbation (MC/FEP) and MD/FEP calculations using the OPLS/CM5 force field for comparison with each other and with the Kd measurement from a florescence polarization assay (ΔGbind = RT ln Kd).30 Six-degree-of-freedom (6DoF) restraints were adopted for all simulations of the complex.12,13,31 In addition, the remaining force field combinations, OPLS/CM1A, CHARMM/CGenFF and AMBER/GAFF, have been applied using the same MD/FEP protocol to evaluate the sensitivity of the ΔGbind results to these alternative choices.</p><!><p>Absolute binding free energy calculations were conducted via the double decoupling method (DDM) following the thermodynamic cycle depicted in Figure 2 and using eqs 1 and 2.8–12 The difference between the two sides of the cycle, which effectively transfers the ligand from aqueous solution to the binding site, represents the binding affinity of the protein-ligand complex. The ligand intermolecular interactions are turned off (decoupled) from the water solvent in the unbound simulation to yield ΔGunbound. All calculations of ΔGbound and ΔGunbound were done in two stages with scaling of the atomic charges (1)ΔGbind=ΔGunbound−ΔGbound+ΔGrestr−ΔGvb (2)ΔGrestr=−kTln[8π2V(KrKθAKθBKφAKφBKφC)1/2ra,A,02sinθA,0sinθB,0(2πkT)3] to zero followed by removal of the intermolecular Lennard-Jones interactions. In the bound simulations, the decoupling from the solvent and the protein is done with the use of geometric restraints. They are introduced to keep the disappearing ligand in the observed position and orientation in the binding site (Figure 1) yielding ΔGvb, and their effect on the free energy is corrected analytically via ΔGrestr. The latter term is calculated using eq 14 from the paper by Boresh et al.13 The equation is reproduced here as eq 2 and reflects imposition of restraints for six degrees of freedom (6DoF) that keep the ligand translationally and rotationally stable in the binding site.32–34 The specific algorithm from Wang et al.35 is implemented in the colvars module of the utilized MD program NAMD36 to control the six variables.31,37 The same algorithm is also used in our MC program, MCPRO. In the MC implementation, the restraints are turned on simultaneously with the removal of the atomic charges so ΔGvb is included in the electrostatic portion of ΔGbound, but it requires a separate simulation in the MD calculations using NAMD.</p><p>The sum of terms in eq 1 is sufficient to calculate ΔGbind of most ligands that are either conformationally rigid or freely interconverting. The ligand in this study, MIF180, occurs in two different, non-interconverting conformations during the simulations bound in the protein and in the unbound aqueous phase. Therefore a correction term, ΔGconf, needs to be added to the result of eq 1 as a penalty for conversion of the ligand from the most stable conformation in aqueous solution or in the gas phase with the triazole and quinoline nitrogen atoms anti to the syn conformer observed in the complex (Scheme 1). This term was estimated via potential of mean force (PMF) calculations for rotation about the bond connecting the rings. The decoupling was only for interactions with the environment; intramolecular energy terms did not contribute to the FEP results, so the conformational change required separate assessment.</p><!><p>All structures were initially built starting from crystal structures of the complexes for MIF180 and a close analog (PDB IDs: 4WR8 and 4WRB)22 using the MCPRO clu utility. The full structure with 342 residues was retained and relaxed via a conjugate-gradient optimization using MCPRO with a dielectric constant of 2.0. For the ligand, two different OPLS-AA charge models, OPLS-AA/CM1A and OPLS-AA/CM5 were considered with the usual scaling factors for neutral-molecule partial charges of 1.14 for CM1A and 1.20 for CM5.38 In addition, CHARMM general force field (CGenFF) parameters were obtained from its webserver.28 It should be noted that the output included warnings about low quality for torsional parameters for several dihedral angles. Similarly, the ligand parameters were assigned for the general AMBER force field (GAFF)29 with the Antechamber package including AM1-BCC atomic charges.39 The protonation states for the protein residues were determined by H++40,41 and PropKa3.42,43 In both cases, the N-terminal proline was predicted to be neutral. Pro1 is the putative catalytic base for the tautomerase reaction.21 However, no neutral N-terminal proline parameters were available for the CHARMM3626 and the AMBER ff14SB27 force fields. In order to obtain the necessary parameters, the C-terminal-capped PRO-MET dipeptide was processed using CGenFF and GAFF. The parameters thus obtained were then mapped into the CHARMM and AMBER protein force fields for the neutral terminal proline. Complexes for MIF with both the neutral and protonated Pro1 were prepared for all four force field combinations.</p><!><p>The OPLS-AA/M force field23 was used for the MIF protein, while OPLS-AA with the 1.20*CM5 charge model38 was used to represent the inhibitor, MIF180. The calculations of absolute free energies were carried out following the double-decoupling scheme (Figure 2). The ligand electrostatic and Lennard-Jones (LJ) interactions were decoupled consecutively with simple overlap sampling (SOS).44 The charges were first scaled to zero linearly with the λ parameter, then the intermolecular LJ interactions were turned off using 1-1-6 softcore potentials.45 The charge and LJ removals were split into 15 and 18 windows for the unbound state and 15 and 41 windows in for the bound state respectively. Each window comprised 80 million (80M) configurations of equilibration and 180M configurations of averaging (80M/180M) for the unbound state; the bound-state calculations used 80M/240M configurations for the electrostatic and 320M/240M for the LJ calculations. The PMF calculations for the rotation around the bond connecting the triazole and quinoline rings were conducted using the same system as prepared for the unbound simulations using 1M configurations of equilibration and 5M averaging at 10° intervals.</p><p>During the bound state annihilation, the ligand was restrained to its initial position relative to the protein using the six degree-of-freedom (6DoF) restraints.13,33 The required coordinate system was constructed by choosing three sites from the ligand and three from nearby protein residues. Specifically, the hydroxyl oxygen atom, the midpoint of N2 and N3 in the triazole ring, and the quinoline N atom were selected as the three groups on the ligand; and the geometric center of the heavy atoms for the Tyr36C side-chain, the Ile64A backbone and the Lys32A side-chain were used for the protein. Analytical corrections for the restraints to the fully interacting ligand and standard state were included to obtain the absolute binding free energy.13,35,46,47 The force constants for the six restraints were 10 kcal/mol-Å2 and 0.1 kcal/mol-deg2 for distances and angles, respectively. All restraint terms were gradually increased starting from zero during the electrostatic decoupling and then kept constant.</p><p>For MC/FEP simulations, the unbound ligand was solvated in a 40-Å periodic cube containing 2100 TIP4P water molecules. For the complexes, a 25-Å radius cap with ca. 2000 TIP4P water molecules centered on the ligand was used. In all cases, a residue-based cutoff of 10 Å was applied to maintain consistency with the MD simulations.</p><!><p>All molecular dynamics (MD) simulations were performed using the NAMD program version 2.11.16 The ligand was solvated in a 40-Å periodic cube of TIP3P water,48 the default model for NAMD, and the complex was solvated in TIP3P water with 12-Å padding in all dimensions. The systems with unprotonated Pro1 were electrically neutral, and the ones with the three copies of the proline protonated were neutralized by addition of three chloride ions.</p><p>Langevin dynamics49 was applied to enforce a temperature of 300 K and a pressure of 1 atm. The time step was set at 2 fs using the SHAKE algorithm50 to constrain all bonds to hydrogen atoms. Coulombic interactions were truncated at 10 Å, and the Particle-Mesh-Ewald (PME) method was used to include long-range electrostatic interactions.51,52 The LJ interactions were smoothly switched off between 8 and 10 Å. For all of the MD simulations, the equilibration protocol consisted of 50,000 steps of conjugate-gradient minimization, followed by 2 ns of isothermal-isobaric dynamics for equilibration. The free energy rotational profiles were calculated from the unbound ligand setup using 1 ns equilibration and 5 ns averaging at 1° intervals.</p><p>The production FEP calculations were performed bi-directionally using 43 lambda windows. Each λ-window featured another 1 ns of equilibration and 5 ns of averaging for both the unbound and bound state, for a total 215 ns in each direction. The 6DoF restraints were applied to the ligand in binding site in the same manner as with MCPRO. The calculations of the absolute free energy of binding with protonated Pro1 using the AMBER/GAFF force field required an additional conformational restraint to maintain the bound syn conformation. All of the MD simulations were run in triplicate with small initial changes to generate independent trajectories.</p><!><p>Since the MC simulations used TIP4P water, while the MD simulations used TIP3P, the effect of the choice was first examined for the unbound state by evaluating free energies of hydration (ΔGhyd) of the ligand. For this purpose, full annihilations of the ligand in aqueous solution and in the gas phase were carried out with sequential removal of the Coulombic and LJ interactions. The net MC results for ΔGhyd were similar, specifically, −14.92 ± 0.10 kcal/mol for TIP4P and −14.23 ± 0.05 kcal/mol for TIP3P water. The results for the aqueous FEP calculations were taken as ΔGunbound.</p><!><p>Bound-state MC/FEP calculations were carried out following similar protocols as in the lysozyme study.20 However, a few methodological adjustments were necessary due to the larger size and asymmetry of the MIF180 ligand. First, the hard wall (HW) restraint was replaced by the 6DoF restraints. While the HW restraint was sufficient for benzene and analogs as ligands, it resulted in very slow convergence and large numerical fluctuations in initial studies with MIF180. With the 6DoF restraints, the bound-state results showed good numerical stability.</p><p>Next, 41 λ-windows were used to ensure sufficient configuration-space overlap for the FEP calculations. Simple overlap sampling (SOS) was used, where the midpoint of the window λM is the end-point for each perturbation. The bound-state free energy changes for each window and the corresponding fluctuations are plotted in Figures 3 and 4 from the 80M/240M run lengths. While the electrostatic free-energy fluctuations were all below 0.13 kcal/mol, the LJ free-energy changes exhibit two behaviors. Low fluctuations are exhibited in the first two-thirds of the calculations, up to window 26 (0.00 ≤ λ ≤ 0.67), and then higher fluctuations occurred for the remaining windows (0.67 ≤ λ ≤ 1.00). In the latter region, all the electrostatic interactions and a large portion of the Lennard-Jones ones have been eliminated. Thus, the protein backbone and side chains should be free to relax, and water molecules migrate into the emptying binding site. These changes can be expected to be accompanied by larger energy fluctuations. As a result, increase of the equilibration stage for the LJ decoupling was explored yielding the results in Figures 5 and 6. The electrostatic decoupling reached convergence after the 80M configurations of equilibration and ca. 200M of averaging (Figure 5A). The LJ decoupling, however, still showed a slight downward drift after 240M MC steps of averaging following the 80M equilibration (Figure 6A); the corresponding total free-energy change for 80M/240M is shown in Figure 6B. Restarting the averaging at this point corresponds to an equilibration of 320M configurations. This was done and followed by another 240M configurations of averaging to yield the results in Figure 6C, which show good convergence after ca. 100M configurations of averaging. The total free-energy change as a function of λ for the LJ component is then shown in Figure 6D for the 320M/240M run lengths and reflects a change of about 2 kcal/mol from Figure 6B. All the LJ windows are well converged with the 320M/240M protocol, as shown in Supplementary Figure S4.</p><p>The free energy changes for the unbound inhibitor were 27.56 ± 0.03 kcal/mol for the Coulombic term and 1.59 ± 0.10 kcal/mol for the LJ term, while the corresponding values for the bound state were 34.09 ± 0.44 kcal/mol and 16.53 ± 0.60 kcal/mol with 320M/240M. The 6DoF restraints correction, ΔGrestr, was 12.01 kcal/mol and the free energy penalty for the ligand conformational change in TIP4P water was 0.66 kcal/mol (see below). Thus, the absolute free energy of binding for the MIF180 complex via eq 1 is −8.80 ± 0.74 kcal/mol. The statistical uncertainty mainly arises from the bound-state calculations and places significant limits on the precision. The accord with the experimental value of −8.98 ± 0.28 kcal/mol30 is notable; it is in the 1-kcal/mol error range reported for relative free-energy results.6,53 However, many more examples are needed with different ligands and methodological variations before general conclusions can be reached. The following results provide some insights along these lines.</p><!><p>It is informative to compare results with the small-molecule force fields for computation of the absolute free energy of hydration of the MIF180 ligand. MD simulations in the CHARMM version of TIP3P water and in gas phase were used to calculate ΔGhyd with the OPLS-AA/CM5, OPLS-AA/CM1A, CGenFF and GAFF force fields using NAMD. The resulting values for the electrostatic (ΔGQ) and van der Waals (ΔGLJ) components of the free energy of hydration are listed in Table 1. The aqueous-phase results were also used to calculate ΔGbind. From Table 1, the predictions for ΔGhyd range over 2.7 kcal/mol, with ranges of 1.1 and 3.4 kcal/mol for the LJ and Coulombic components, respectively. Thus, the major differences likely arise from variations in the partial charges for the ligand. This is documented for the atoms with the largest differences in Figure 7 and Table 2. It can be seen that the largest variations are concentrated in the central quinolinyltriazole fragment, especially for the nitrogen atoms and C2 of the quinoline ring (C0I). The differences are substantial and show that current fixed-charge force fields are far from agreement on this important item, which has obvious implications for interactions with any surrounding water or biomolecules. In the present case, N2 and N3 of the triazole and the quinoline nitrogen all participate in hydrogen bonds with MIF (Figure 1). The magnitudes of the partial charges with CGenFF are particularly large, though this does not translate into a lower ΔGhyd in Table 1. The complete list of atomic charges for the four force fields can be found in Supplementary Table S1 as well as a graphical representation of the electrostatic potentials in figure S5.</p><!><p>To calculate the ΔGbind with the present decoupling methodology, it is necessary to include a penalty for the conformational change of the ligand from anti in water to syn upon binding as reflected in Scheme 1. Thus, potential of mean force (PMF) calculations were run with NAMD using TIP3P water for each force field; the dihedral angle for the bond connecting the quinoline and triazole rings (N0F-C0E-C0I-N0O) was driven from 0° in the syn conformation to 180° in the anti conformation in both the aqueous and gas phases. The calculated PMFs are depicted in Figure 8.</p><p>In TIP3P water, the OPLS/CM5 force field gives the smallest free energy difference from anti to syn with a ΔGconf of 0.44 kcal/mol. While OPLS/CM1A (1.00 kcal/mol) and CGenFF (0.71 kcal/mol) gave similar free-energy curves, GAFF showed a stronger preference for the anti conformer, 1.60 kcal/mol. An analogous MC/PMF calculation was done for MIF180 in TIP4P water with the OPLS/CM5 force field, which yielded a ΔGconf of 0.66 kcal/mol. It may also be noted in Figure 8 that the GAFF force field gives a significantly lower syn to anti barrier. A consequence seemed to arise in the case with protonated Pro1 for which the syn conformation was not maintained in the protein binding site during initial MD runs using the GAFF force field. So, in this case, an additional restraint was applied to fix the syn preference for the complex.</p><!><p>As noted above, the simulations for the bound complex were run in triplicate using 43 lambda windows and averaging for 5 ns. With Pro1 unprotonated, the absolute free energies of binding (ΔGbind) from the three runs were −8.35, −7.49 and −9.55 kcal/mol, to give an average of −8.46 ± 0.85 kcal/mol. The ΔGbind obtained by MD/FEP matches well with the MC/FEP result, −8.80 ± 0.74 kcal/mol, and the experimental value at −8.98 ± 0.28 kcal/mol,30 given the range of the uncertainties and the methodological differences. The bound MC/FEP simulations used a TIP4P water cap without electrostatic long-range corrections, whereas the MD/FEP runs used TIP3P water in a periodic truncated octahedron with Ewald corrections for long-range electrostatic interactions. Both simulation methods demonstrated a statistical uncertainty near 1 kcal/mol, which is in line with their difference of 0.34 kcal/mol. The evolutions of the free energies of binding are compared for MD/FEP and MC/FEP in Figure 9. On this basis, the results for both methods appear to be converged to within ca. 0.3 kcal/mol using the 240M configurations of averaging with MC and 3 × 5 = 15 ns of averaging per window with MD.</p><p>The simulations described here were designed to probe the convergence of each sampling method and are therefore longer than typical production runs would be. Hence, the comparison of timings and efficiency between these methods is at best qualitative at this point, and should be used as such. In the bound calculations, a single MC run of 100M configurations required 120 core-hrs in a Xeon E5–2660 as compared to 375 core-hrs for 5 ns of MD. Since NAMD is well parallelized, the wall-clock timing of a MD run can be easily reduced by using multiple cores. A single complete calculation for the bound MIF-MIF180 complex utilized 33,300 and 38,700 cpu-hrs in MC and MD simulations, respectively.</p><p>Concerning the migration of water into the binding site as the ligand disappears, Figure 10 shows the final configuration of the last λ window in the forward direction of the MD/FEP calculation. Only water molecules within 5 Å of the ghost of the ligand are shown. It is clear that water molecules penetrated well into the binding pocket during the decoupling of the bound ligand. The average numbers of water molecules within 8 Å of N06 at the center of the ligand were 3 and 18 and the beginning and end of the MC/FEP simulations, and 6 and 11 for MD/FEP.</p><!><p>The MD simulations for ΔGb were performed in the same way with the other three force fields. Furthermore, in order to investigate the preferred protonation state for Pro1 of MIF in the complex, the FEP calculations were also performed with Pro1 protonated in all three MIF monomers. The results of the ΔGb calculations with the four force fields are summarized in Table 3 for MIF180/MIF with and without the protonated N-terminal proline. As mentioned above, additional conformational restraint of the ligand to the syn conformation was needed for the simulations with Pro1 protonated using the AMBER/GAFF force field. The results for ΔGbind are clearly very sensitive to both the choice of force field and protonation state for Pro1. In all cases, ΔGbind is more favorable with neutral Pro1 and closer to the experimental result of −8.98 ± 0.28 kcal/mol.30 The OPLS/CM5, OPLS/CM1A, and AMBER/GAFF results strongly support the assignment of Pro1 as neutral. The 2.3-kcal/mol spread of ΔGbind results with neutral Pro1 from these three alternative force fields is probably a reasonable reflection of the current state of the art. Expecting 1 kcal/mol accuracy from any given fixed-charge force field on a specific complex of a protein with a drug-like ligand is overly optimistic. The results with CHARMM/CGenFF are an outlier. It is difficult to trace the origin of the problem, but it may reflect the strong variation in partial atomic charges shown in Table 2 or uncertainties about the quality of some of the torsional parameters noted by the on-line server.28 In any event, it would be premature to draw general conclusions about the force fields. For the OPLS force fields, there is no particular reason to favor the use of 1.20*CM5 over 1.14*CM1A charges based on results for pure organic liquids and free energies of hydration,38 so the results with CM5 may just be fortunate and unique in this case.</p><!><p>The crystal structure of the complex features multiple aryl-aryl interactions and hydrogen bonds as indicated in Figures 1 and 11. The phenolic hydroxyl group is hydrogen bonded with Asn97C (r(OO) = 2.52 Å), and the triazole N2 with Ile64A (r(NN) = 2.90 Å). Also, the quinoline N, triazole N3 and backbone O of Ile64A (r(NN) = 3.33 Å, r(NN) = 2.95 Å, r(NO) = 2.81 Å) are hydrogen-bonded to the ammonium nitrogen of Lys32A.22 For comparison with the MD results in TIP3P water, the five hydrogen-bond distances were averaged over the final 10 ns of the MD trajectories for the fully formed complexes. The average intermolecular hydrogen-bond distances designated in Figure 11 are compared in Table 4 from the simulations with Pro1 unprotonated. Histograms for the hydrogen-bond distances showing the sampled ranges with the four force fields are provided in Supplementary Figure S2.</p><p>The shortest average protein-ligand contacts are found for OPLS/CM5 and AMBER/GAFF, which is consistent with their most favorable ΔGbind results (Table 3). All of the force fields yield a hydrogen bond between the phenolic hydroxyl group and Asn97C, and a short contact for the Lys32A ammonium group and the oxygen atom of Ile64A, in agreement with the crystal structure. However, the predictions for the three N…N hydrogen bonds are varied. While AMBER/GAFF retains the three hydrogen bonds, the other three force fields do not to different degrees. The most separated structure is found for CHARMM/GenFF, which is consistent with its overly weak ΔGbind in Table 3. OPLS/CM5 retains the hydrogen bond between the triazole N2 and the backbone NH of Ile64A, while the coordination of the Lys32A ammonium group with N3 of the triazole and the quinoline nitrogen atom is weakened. This may be reasonable since Lys32A is at the entrance to the binding site and is largely solvent-exposed. There are also interprotein contacts in this region in the crystal structures,22 which provide for some exclusion of water and may lead to differences in structure for the crystal and dilute aqueous solution.</p><p>The corresponding results with Pro1 protonated are provided in Supplementary Table S3 and Figure S2. In this case, the ligand is much more separated from the protein. Basically, all of the hydrogen bonds are broken except for the one with the phenolic oxygen atom with OPLS/CM1A, CHARMM/CGenFF, and AMBER/GAFF.</p><!><p>Computation of the absolute free energy of binding (ΔGbind) for the complex of a drug-like ligand, MIF180, and human MIF has been investigated with both Monte Carlo statistical mechanics and molecular dynamics using double decoupling and four current fixed-charge force fields. Both MC and MD protocols were devised that yielded well converged ΔGbind values, though more efficient protocols using fewer λ-windows may be possible.54 The MC/FEP protocol with improved sampling techniques20 and the OPLS/CM5 force field performed well and gave an accurate estimate for ΔGbind in comparison to the experimental data. It was confirmed that a very similar result is obtained using the same force field in molecular dynamics simulations with the NAMD program. The MD/FEP calculations were then carried out for three additional force fields OPLS/CM1A, CHARMM 36 with CGenFF, and AMBER ff14sb with GAFF. The results for ΔGbind notably cover a 6 kcal/mol range, though three of the results are within 2.2 kcal/mol (Table 3). Significant differences in the computed structures for the complexes are also found with the general observation that shorter average protein-ligand contacts do correlate with more negative ΔGbind values. Many additional studies of this type are needed to make such computations more routine, to identify optimal protocols, and to reveal unambiguously any problematic issues for current force fields and sampling methods. It is proposed that human MIF is a good test system for such work owing to its moderate size and to the availability of multiple high-resolution crystal structures as well as accurate binding data for numerous, diverse inhibitors.21,22,30</p>

PubMed Author Manuscript

Synthesis of a Highly Aromatic and Planar [10]Annulene

As the next neutral structure following Hückels (4n+2)erule, a planar and aromatic [10]annulene is the ideal framework to study the link between ring size and aromaticity. However, the puckered geometry of the parent [10]annulene (1) suggests that the aromatic stabilization energy (ASE) is not sufficient to overcome the ring strain that exists when the system is forced into planarity. 1 It has been shown computationally that this ring strain can be alleviated through the addition of two or more cyclopropane rings to the periphery (5-6), 2 thereby creating theoretically aromatic structures. An alternative strategy to eliminating the issue of ring strain was demonstrated experimentally with the successful preparation of the highly aromatic 1,6-didehydro[10]annulene (2). 3 However, the system rapidly cyclizes at -40 °C to a naphthalene diradical due to the close proximity of the in-plane p-orbitals present in the system. Here we show that cyclopropanating one side of the unstable annulene (2) successfully prevents the destabilizing cross-ring interaction while maintaining a highly aromatic structure. Remarkably, the formed [10]annulene ( 7) is bench stable and can be stored for extended periods of time.

synthesis_of_a_highly_aromatic_and_planar_[10]annulene

<!>Associated Content

<p>Aromaticity is a fundamental concept that permeates many facets of chemistry. 4 Identifying (anti)aromatic motifs in various forms has helped explain the (in)stability of both transition state 5 and ground state structures. 6 Our working understanding and ability to predict its occurrence has also informed the preparation of materials. 7 However, despite these advances, we still lack much in the way of a fundamental understanding.</p><p>The traditional criteria for aromaticity proposed by Hückel require that a compound be cyclic, planar, conjugated, and contain (4n+2) electrons. 8,9,10 Any compound that can satisfy these criteria is predicted by Hückel molecular orbital (HMO) theory to have some degree of aromatic stabilization energy. 11 Besides the benzenoid archetypes, charged Hückel systems of various ring sizes have also been prepared to explore the effect of ring size on aromaticity. 12 Unfortunately, the systematic study of these charged species is often quite challenging when compared to neutral annulenes. The larger [18]annulene has been prepared and has been shown to be aromatic, 13 though its low-frequency out-of-plane vibrations and flexibility in solution make it difficult to probe its aromaticity experimentally. 14,15 There has also been a considerable amount of interest in very large aromatic systems with nanoscale structures, such as those prepared by Anderson et al. 16 Figure 1: a. The parent [10]annulene (1) has been found to be non-aromatic due to its non-planarity; b. The cyclization of 1,6-didehydro [10]annulene (2) to a biradical (3); c. The structure of 1,6-methano [10]annulene (4) and the unusual anisotropy of the induced current density (AICD) isosurface; d. The expansion of CCC bond angles upon cyclopropanation has been shown to generate theoretically planar [10]annulenes (5-6); e. Cyclopropanation of the thermodynamically stable 2 may afford a kinetically stable and planar annulene (7) that retains its aromaticity.</p><p>As the next highest Hückel aromatic neutral hydrocarbon, a rigid and planar [10]annulene would provide a desirable framework to study the link between aromaticity and ring size. Because the parent [10]annulene (1) is non-aromatic 1, 17,18 (Figure 1a) it must be constrained to force it to adopt a planar conformation that allows for electron delocalization. Myers and coworkers found success in this regard through the preparation of a 1,6-didehydro [10]annulene derivative (2), which has shown evidence of aromaticity. 3 However, the -bonds orthogonal to the conjugated aromatic system readily interact across the ring, resulting in a rapid intramolecular cyclization above -40 o C (Figure 1b). The first reported alternant aromatic [10]annulene is the bridged 1,6-methano [10]annulene (4). It was prepared by Vogel in 1964 19 and has recently shown potential as an organic material. 7b The structure is stable and aromatic with a slight deviation from planarity (4, Figure 1c). Despite this, it has been shown that the electronic structure of 4 more closely resembles a homonaphthalene due to cross-ring interactions. 20,21 Additionally, the presence of trans double bonds as well as the unusual and non-homogenous magnetically induced current also suggests it is not an ideal material to study the aromaticity of expanded benzene-like planar [10]annulenes. 22 In an effort to identify potential planar [10]annulene frameworks for further study Schleyer et al. explored cyclopropanated derivatives (Figure 1d). 2 It was shown computationally that cyclopropanated all-cis [10]annulenes (5 and 6) could adopt a planar structure due to the increased exo bond angles of cyclopropenes (Figure 1d, left). 2 However, despite their significant predicted aromatic stabilization energy no preparation of a cyclopropanated [10]annulene has been reported to date. Consequently, no experimental information is available regarding their thermodynamic and kinetic stability.</p><p>In order to definitively study the role of ring size on aromaticity we sought to identify a bench stable all-cis [10]annulene with a sigma framework more reminiscent of an expanded benzenoid. As the Myers annulene was found to be a thermodynamic minimum, it was believed that cyclopropanation of one of the in-plane −bonds of 2 (Figure 1e) would prevent the transannular orbital interaction, thereby creating a kinetically stable structure (7, Figure 1e). This would generate a structure reminiscent of those predicted by Schleyer and one that closely resembles an all-cis and planar [10]annulene. The essential strategy employed involved constructing a suitably oxidized but strain-free 10-membered ring that could be rapidly aromatized (Figure 2a, 11). A slow and stepwise introduction of strain was rejected in favour of the use of a late-stage rapid sequence of highly reliable strain inducing transformations, bypassing the need to carry unstable and non-aromatic intermediates through the synthesis. As kinetic stability of the final product was a concern, the possibility for a mild aromatization procedure had to be considered. A dichlorocyclopropane group was thought to be beneficial for this purpose as it would improve substrate versatility. Elimination of chloride would generate a transient cyclopropene that would isomerize to an exomethylene cyclopropane, irrespective of an aromatic or conjugative driving force (Figure 2b). This aromatization step was shown to work for the preparation of cyclopropabenzene (see SI). 23 The preparation of diene 19 closely followed literature procedures 24 with some minor modifications. 25 The synthesis began with the preparation of 14 via a Diels-Alder reaction between sulfolene (12) and maleic anhydride (13). The adduct (14) could easily be converted to 16 via an ethanolysis and cyclopropanation. LiAlH4 reduction afforded diol 17, which was readily mesylated to provide 18. Elimination to the diene 19 proceeded with a high yield when it was first converted to the corresponding diiodide (S1, see SI). Exposing the diene 19 to tetrachlorocyclopropene (21) in THF for 3 days generated 22 as a 3:1 ratio of cis and trans diastereomers. A subsequent ozonolysis with a standard reductive quench afforded the corresponding diketone 23, which could be isolated on smaller scales. However, this diketone proved very sensitive and a transannular aldol product (24) was typically obtained when the reaction was scaled up, regardless of the quenching reagent and temperature. Fortunately, a sodium borohydride quench of the ozonolysis reaction mixture at -78 °C was found to generate diol 25 as a single diastereomer in 70% yield (Figure 4). Note that typically only cis-22 underwent ozonolysis and all attempts to induce an ozonolysis of trans-22 by prolonging the reaction time or increasing the temperature resulted in the formation of an intramolecular aldol product like 24. All attempts to dehydrate diol 25 by elimination of the corresponding dimesylate only afforded the mono-alkene and decomposition products (not shown). Hoping that the other diastereomer of the diol would yield more favourable results, it was decided to attempt a double Mitsunobu inversion. Unexpectedly and to our delight, the desired diene 26 was directly obtained in high yield. We speculate that the nucleophile could not approach the concave face of the rigid system, resulting in the more favourable elimination pathway instead. The conditions strongly favoured the symmetric diene 26, presumably due to the strong electron withdrawing nature of the adjacent tetrachlorocyclopropane moiety. 26 Careful exposure of 26 to MeLi at 0 °C generated a dichlorocyclopropene (not shown) by a 1,2-didechlorination without noticeable formation of Skattebøl rearrangement products. 27 The intermediate dichlorocyclopropene could be hydrolyzed in situ to generate the bench stable cyclopropenone 27.</p><p>Decarbonylation of 27 by exposure to 300 nm UV light in dilute dichloromethane proceeded smoothly to yield the dienyne 28. Initial attempts to oxidize 28 or to carry out a double elimination/isomerization sequence with tBuOK at room temperature led to material degradation. Decreasing the temperature eventually produced observable aromatic signals (> 7 ppm). These were obtained alongside a major product in the 1 H NMR spectrum that was identified as 29. It was presumed that the major product 29 was obtained by deprotonation of the allylic proton in 28 with a subsequent cyclopropane ring opening. Interestingly, the crude reaction mixture remained intact over several days and the aromatic signals that were produced persisted despite storage in CDCl3. Unfortunately however, only minute amounts of material could ever be obtained. Purification attempts were further complicated due to the unexpected extreme volatility of 7 and the surprisingly similar behaviour of the two products (7 and 29). The materials were observed to coelute in both normal and reverse phase chromatography and even co-distilled. 28 Reverse phase HPLC showed peak resolution, but the small amounts of material and its high volatility would have rendered material recovery impractical. Eventually it was discovered that doping a PTLC plate with AgNO3 prior to development resulted in a separation that was sufficient to purify the materials. 29 As the alkyne in 7 is heavily delocalized in the aromatic system, it presumably has a weaker affinity to the Ag + dopant than the alkyne in the non-aromatic 29. Using this technique, the compound could be purified and fully characterized. Interestingly, despite the presence of only trace amounts of material it had a very distinct and pleasant smell. It is also notable that the product is remarkably stable, persisting in CDCl₃ for months at -20 °C and weeks at room temperature without noticeable degradation. The experimental 1 H NMR spectrum of the purified [10]annulene 7 displayed four distinct signals, three of which appeared in the aromatic region between 7.6 and 8.2 ppm (Figure 5, top). 30 The signals displayed the expected multiplicities with minor broadening. Second order effects and couplings rendered experimental coupling constant extraction challenging, although a spectrum obtained with computed chemical shifts and coupling constants very closely matched the signals observed in the aromatic region (Figure 5, bottom). 31 The close matching computed 1 H-NMR spectrum obtained in the absence of rovibrational considerations suggests it is a highly rigid structure that maintains both planarity and aromaticity. 32 This is in contrast to other larger aromatic structures such as [18]annulene, whose computed 1 H-NMR spectrum can only be obtained after considering dynamic motion. 14b Nucleus-independent chemical shift (NICS) 33 computations show a highly aromatic system with NICS(1)iso = -12.0 and NICS(1)zz = -31.9 (benzene NICSiso = -10.6 and NICS(1)zz= -30.09). The uniformity of the magnetically induced current isosurface as calculated by the anisotropy of the induced ring current (AICD) method is also quite apparent (Figure 6). 34 Additionally, all computed sp 2 -sp 2 bond lengths fall between 1.37 Å and 1.41 Å, further corroborating the high degree of delocalization.</p><p>Additional minima corresponding to a potential cumulenic structure could not be located, suggesting that the annulene exists as one fully delocalized minimum rather than two interconverting species. Indeed, natural resonance theory 35 analysis shows dominant contributions from the cumulenic and alkynic valence bond structures with a slight preference for the latter. 36 Interestingly, the presence of the cyclopropane ring appears to have no effect on the aromatic system, 37 and induces only a limited amount of strain (see SI). 38 This is consistent with the literature data on benzocyclopropenes where it has been shown repeatedly that such Mills-Nixon effects do not exist. 39 The quest to prepare an aromatic [10]annulene has been a long standing challenge for synthetic chemists. Here we show that it is possible to synthesize the first kinetically and thermodynamically stable all-cis [10]annulene structure by formally cyclopropanating the Myers 1,6-didehydro [10]annulene (2), thus confirming our hypothesis. The planar [10]annulene 7 by all measures is highly aromatic and remains remarkably conjugated despite the presence of both an alkyne and cyclopropane ring. The successful synthesis also supports the indicated mechanism for endo-to-exo cyclopropene isomerization (Figure 2b) 40 and shows the largely untapped potential of this transformation for generating strained systems. The transformation appears to have no dependence on aromaticity and may ultimately prove to be useful in the preparation of other annulenes.</p><!><p>Experimental procedures and characterization data are provided in the supporting information (SI).</p>

ChemRxiv

DIA-SIFT: A precursor and product ion filter for accurate stable isotope data-independent acquisition proteomics

Quantitative mass spectrometry-based protein profiling is widely used to measure protein levels across different treatments or disease states, yet current mass spectrometry acquisition methods present distinct limitations. While data-independent acquisition (DIA) bypasses the stochastic nature of data-dependent acquisition (DDA), fragment spectra derived from DIA are often complex and challenging to deconvolve. In-line ion mobility separation (IMS) adds an additional dimension to increase peak capacity for more efficient product ion assignment. As a similar strategy to sequential window acquisition methods (SWATH), IMS-enabled DIA methods rival DDA methods for protein annotation. Here we evaluate IMS-DIA quantitative accuracy using stable isotope labeling by amino acids in cell culture (SILAC). Since SILAC analysis doubles the sample complexity, we find that IMS-DIA analysis is not sufficiently accurate for sensitive quantitation. However, SILAC precursor pairs share common retention and drift times, and both species co-fragment to yield multiple quantifiable isotopic y-ion peak pairs. Since y-ion SILAC ratios are intrinsic for each quantified precursor, combined MS1 and y-ion ratio analysis significantly increases the total number of measurements. With increased sampling, we present DIA-SIFT (SILAC Intrinsic Filtering Tool), a simple statistical algorithm to identify and eliminate poorly quantified MS1 and/or MS2 events. DIA-SIFT combines both MS1 and y-ion ratios, removes outliers, and provides more accurate and precise quantitation (<15% CV) without removing any proteins from the final analysis. Overall, pooled MS1 and MS2 quantitation increases sampling in IMS-DIA SILAC analyses for accurate and precise quantitation.

dia-sift:_a_precursor_and_product_ion_filter_for_accurate_stable_isotope_data-independent_acquisitio

<p>Standard shotgun proteomics acquisition methods rely on repeating cycles initialized by a precursor scan and followed by dynamic selection, isolation, and fragmentation of a fixed number of abundant ions for sequence annotation1. In regions of high chromatographic complexity, the rate of the data-dependent acquisition (DDA) duty cycle limits isolation and fragmentation to only the most intense precursor ions. This under under-sampling fundamentally ties the number of analyzed peptides to the instrument scan speed, limiting proteome coverage2. Additionally, low mass resolution of multipole ion selection often results in undesirable ion interference, yielding chimeric fragmentation spectra across >50% of MS2 spectra3. The resulting composite fragment spectra incur search penalties that limit confident peptide annotations. These variables diminish reproducible analysis, prompting the need for new approaches to enhance ion discrimination and deliver high-quality fragmentation spectra.</p><p>To address this gap, data-independent acquisition (DIA) methods have been developed that bypass abundance-based ion isolation. Instead of iteratively selecting the most abundant ions for isolation and fragmentation, all-ion fragmentation methods, such as MSE, use alternating MS scans collected at low and high collision energies, generating precursor (MS1) and product (MS2) ion spectra across the entire mass range4. Post-acquisition alignment of peptide elution profiles for MS1 and MS2 spectra allow accurate assignment of precursors to their corresponding product ions. Sequential window acquisition of all theoretical mass spectra, or SWATH, further segregates the precursor mass range into limited m/z windows for confined fragmentation, resulting in reduced ion interference while preserving reproducibility5,6.</p><p>Recent commercial mass spectrometers incorporating ion mobility separation (IMS) provide an orthogonal approach to reduce ion interference for DIA workflows. In general terms, IMS describes a gas-phase electrophoresis separation, separating ions based on their size, shape, and charge on a millisecond timescale. These inherent physical properties impart distinct IMS drift times, providing an orthogonal analytical dimension that increases peak capacity, reduces ion interference, and delivers multi-parameter peptide analytics (retention time, drift time, precursor and product m/z) for high-definition analysis7,8. Furthermore, drift-time dependent collision energy assignment produces more efficient fragmentation, where higher collision energies are assigned to peptides with longer drift times9. Altogether, IMS-DIA methods yield a 2.3-fold higher annotation rate than the theoretical maximum for a first-generation quadrupole Orbitrap instrument using higher-energy collisional dissociation (HCD)9. This results in 1/3 more annotated proteins and twice as many annotated peptides. Based on these values, IMS separation improves both peak capacity and fragmentation efficiency to provide a unique platform for multi-dimensional DIA analysis8.</p><p>Label-free quantitation methods typically measure the 3 ions with the highest intensity (top-3 analysis), which largely correlate with protein abundance4. This procedure immediately triages the bulk of the peptide features from the final measurement, reducing statistical power often needed for confident cross-experiment analyses. SWATH acquisition quantified by summing the 5 most intense product ions from the 3 most intense peak groups generally yields intra-day CVs less than 20% across replicate runs, but variance increases significantly between different laboratories operating the same instrument10.</p><p>Incorporating internal standards for direct quantitation would correct many sources of additional variance.</p><p>We hypothesized that stable isotope labeling by amino acids in cell culture (SILAC) analysis might improve reproducible DIA quantitation across the proteome, especially since comparative ratios are internally quantified and eliminate many sources of experimental error. In this approach, control and experimental cells are grown separately in "Light" media or stable isotope-labeled "Heavy" media supplemented with L-arginine (13C6, 15N4) and L-lysine (13C6, 15N2). The samples are then mixed and digested with trypsin for comparative mass spectrometry analysis. Since trypsin digests proteins at arginine and lysine residues, the integrated intensity of each control ("Light") and experimental ("Heavy") peptides can be comparatively quantified across all proteolytic peptides. When SILAC is combined with IMS-DIA methods, even at the most complex elution time, IMS separation limits ion interference to only 5.5%11. Therefore, IMS enhances peak capacity to resolve much of the complexity introduced through binary SILAC analysis. Nevertheless, it remained unclear if this purity is sufficient for accurate quantitative analysis. Here we sought to benchmark stable isotope quantitation using IMS-DIA methods, particularly for accurate quantification of fractional abundance changes.</p><p>Since SILAC "Light" and "Heavy" peptide pairs share common retention and ion mobility drift times, the paired peptides co-fragment, and the resulting y-ion fragment spectra include "Light" and "Heavy" peak pairs derived from the C-terminal arginine or lysine labels. In current IMS-DIA proteomic analysis workflows, these product ion pairs are automatically binned together during drift and retention time alignment during peptide annotation (Figure 1A-B). To access these product ion ratios for quantitation, we developed a SILAC quantitation pipeline to process output files generated by the APEX algorithm provided in the PLGS analysis software (Waters)12,13. This post-processing algorithm retroactively extracts y-ion peak pair assignments, and since the DIA workflow samples peptide fragments across their chromatographic elution profile, the peak area can be quantified for all assigned y-ion pairs.</p><p>A series of human SILAC-labeled 293T cell tryptic digests of known Light / Heavy ratios were analyzed using a quadrupole IMS time-of-flight mass spectrometer (Synapt G2-S HDMS; Waters). Across different SILAC mixtures, MS2 y-ion ratio measurements nearly doubled the number of quantifiable observations, producing up to 60,000 quantifiable ratios summed across 3 replicates using a 105 min gradient (Figure 1C). Across this triplicate dilution series, 1254 ± 153 (SD) proteins were identified with an average of 19 precursor and 21 y-ion SILAC ratios per protein. Fragment ion intensities average 1-2 orders of magnitude lower than precursor ions, yet remain well within the dynamic range of the detector, allowing accurate quantitation (Figure 1D). By including these intrinsic y-ion SILAC ratios, on average, peptides are measured ~5 times, providing inherent validation of the quantified precursor ratio.</p><p>Label-free MS2 quantitation of IMS-DIA data reportedly increases accuracy of protein quantification, even when protein abundance spans a wide dynamic range14) In order to explore this further, we quantified the distribution of SILAC ratios across peptides from the different SILAC mixtures. In analyses of predefined mixtures spanning a 5-fold range, MS1 quantitation yielded a broad distribution of ratios with a 95% confidence interval for the coefficient of variation between 28-30% (Figure S1A), signifying extensive ion interference amplified by the increased sample complexity. In comparison, MS2 quantitation of y-ion ratios acquired with IMS-DIA methods yielded a broad distribution of measurements with a 95% confidence interval for the coefficient of variation between 76-80% (Figure S1B). However, the quantified ratios across individual proteins were largely Gaussian, with outliers amplifying the overall variance. These protein-level CVs are similar to retrospective analysis across several archived DDA experiments acquired on different generation Orbitrap instruments (Figure S2)15–17.</p><p>To address these observations, we introduced a simple statistical filter, termed DIA-SIFT, to eliminate outliers from pooled MS1 and MS2 ratios (Figure 2A). Since MS1 and MS2 ratios are both intrinsic to the actual ratio, precursor and y-ion measurements are weighted equally. The pooled measurements for each protein are then quantile filtered, removing any statistical outliers from the overall protein measurement. For example, the chaperone protein CCT4 reports a largely Gaussian distribution of both precursor and y-ion ratios in a 1:1 (Light : Heavy) analysis. DIA-SIFT filtering removes interfered outliers while retaining sufficient measurements for accurate quantitation (Figure 2B). Pooled protein measurements are only filtered when there are at least three observations, and no quantified proteins are outright eliminated from the analysis.</p><p>MS1-based SILAC measurements are typically reported as median values, which complicate later statistical analysis. Here, a sufficient number of measurements are retained to average any inconsistencies and more accurately reflect the true SILAC ratio. Applying a 0.2 inner quartile range (IQR) filter (see Supporting Experimental Methods) retains 67 ± 10 % (mean ± standard deviation) of the observations in the final protein quantitation (Figure 2C-D), while reducing the coefficient of variation >4-fold across the SILAC dilution series. The selected filter thresholds balance stringency with retention of quantified observations. Outlier ratios are equally distributed across the chromatographic gradient, and largely eliminated after quantile filtering (Figure 2E). Overall, DIA-SIFT restores accurate protein quantitation in SILAC IMS-DIA analysis without removing any protein identifications.</p><p>Across the tested dilution series, DIA-SIFT dramatically improves accurate quantitation of fractional changes across the proteome (Figures 3A, S3, and S4). For example, in a 1:1 mixture the mean protein-level coefficient of variation is reduced from 0.55 to 0.12, and the mean peptide-level variation is reduced from 0.24 to 0.06. The overall result is a reduction in outliers, which is essential for accurately measuring biologically significant changes. Weighting MS1 ratios more heavily than y-ion ratios had no effect on the overall accuracy, highlighting the broad dynamic range of TOF-based SILAC quantitation (Figure S5). Overall, quantile filtering reduces the mean coefficient of variation to less than 15% across all samples across the dilution series.</p><p>As further validation, human 293T cells initially grown in Heavy SILAC media (with arginine +10 and lysine +8) were switched to Light media and collected after different time intervals. The rate of Heavy to Light amino acid incorporation reports the ratio of old and newly synthesized proteins (Figure 3B). Unprocessed data is highly variable and cannot be reasonably interpreted. After processing the data with DIA-SIFT, individual proteins are more accurately quantified to determine their rate of synthesis and turnover.</p><p>Overall, DIA-SILAC methods offer several unique advantages for proteomics data analysis. Recent NeuCoDIA methods also quantify y-ion ratios, leveraging neutron-encoded SILAC labels to avoid increased sample complexity18, yet require extended acquisition times for high resolution analysis (120,000 resolution at 200 m/z) of the neutron-encoded labels. This limits analysis to only a narrow mass range (100 m/z) with four 27 m/z-wide SWATH windows. In comparison, IMS-DIA SILAC methods profile the entire mass range, collect more frequent MS1 and MS2 scans for better chromatographic peak definition and yield significantly more quantifiable observations than NeuCoDIA analysis. Nonetheless, both DDA and DIA SILAC analysis reduce the total number of annotated proteins by ~20%11,19. When accuracy is critical, this fractional loss in proteome coverage is less of a concern. In addition, the largest ratio changes are typically the most biologically interesting. Thus, implementing DIA-SIFT will reduce false positives and direct further biological validation to bona fide targets.</p><p>Here we demonstrate that DIA analysis yields y-ion ratio measurements that enhance SILAC quantitation. y-ions can be easily identified as fragments pairs with matched drift and retention times. Since the y-ion pairs are matched based on the drift and retention time peaks, they represent multiple measurements and not single scans typical of DDA spectra. Beyond quantitation, these y-ion pairs could also be leveraged to enhance confidence in peptide search algorithms, since they provide an orthogonal validation of accurate peptide fragmentation20. Implementing y-ion pair statistics in label-assisted de novo sequencing (LADS) algorithms could further enhance peptide annotation, quantitation, and reproducibility. Overall, DIA-SIFT algorithms can be adapted for SWATH analysis of SILAC or NeuCode-labeled peptides, providing a simple filter to leverage y-ion ratios for more accurate quantitation.</p>

PubMed Author Manuscript

Compact analytical flow system for the simultaneous determination of l-lactic and l-malic in red wines

During the malolactic fermentation of red wines, l-malic acid is mainly converted to l-lactic acid. Both acids should be precisely measured during the entire process to guarantee the quality of the final wine, thus making real-time monitoring approaches of great importance in the winemaking industry. Traditional analytical methods based on laboratory procedures are currently applied and cannot be deployed on-site. In this work, we report on the design and development of a bi-parametric compact analytical flow system integrating two electrochemical biosensors that could be potentially applied in this scenario. The developed flow-system will allow for the first time the simultaneous measurement of both acids in real scenarios at the real-time and in remote way. Miniaturized thin-film platinum four-electrode chips are fabricated on silicon substrates by standard photolithographic techniques and further implemented in a polymeric fluidic structure. This includes a 15 µL flow cell together with the required fluidic channels for sample and reagent fluid management. The four-electrode chip includes counter and pseudo-reference electrodes together with two working electrodes. These are sequentially modified with electropolymerized polypyrrole membranes that entrap the specific receptors for selectively detecting both target analytes. The analytical performance of both biosensors is studied by chronoamperometry, showing a linear range from 5 × 10 −6 to 1 × 10 −4 M (LOD of 3.2 ± 0.3 × 10 −6 M) and from 1 × 10 −7 to 1 × 10 −6 M (LOD of 6.7 ± 0.2 × 10 −8 M) for the l-lactate and the l-malate, respectively. Both biosensors show long-term stability, retaining more than the 90% of their initial sensitivity after more than 30 days, this being a prerequisite for monitoring the whole process of the malolactic fermentation of the red wines (time between 20 and 40 days). The flow system performance is assessed with several wine samples collected during the malolactic fermentation process of three red wines, showing an excellent agreement with the results obtained with the standard method.

compact_analytical_flow_system_for_the_simultaneous_determination_of_l-lactic_and_l-malic_in_red_win

<!>Experimental<!>Devices and equipment.<!>Electrochemical procedures.<!>Red wine samples from malolactic fermentation. Three different red wines provided by the Catalan<!>Results<!>Analytical characterization of the bi-parametric compact analytical flow-system.<!>Conclusions

<p>The malolactic fermentation (MLF) is a process in the winemaking industry in which bacteria convert l-malic acid into primarily l-lactic acid. The aroma and taste of many wines depend on this process, especially in red wines, but also in certain types of white wines. Besides, this process enables the stabilization of the wine colour, and it also allows its microbiological and bacterial control 1 . The control of the MLF during all the process (from 20 to 40 days) is crucial to obtain a highly qualified wine. Current standard methods 2 are applied in decentralized laboratories located far away from the wineries, meaning in long processes with associated high costs. They are based on chromatography and colorimetry and used bulky equipment which has to be used by highly skilled personnel. Enzymatic approaches based on absorbance detection of nicotinamide adenine dinucleotide (NADH) have been also proposed 3,4 , but they are also applied in external laboratories, meaning in more extra steps to carry out the process (sample uptake, sample stabilisation, sample transport and sample storage). This challenge should be solved for enabling on-time and in-situ corrective actions in the MLF process, to correct possible unpredictable problems.</p><p>The miniaturization of analytical methods could be of high interest for this type of applications because they enable the integration of multiplexed analysis in low-cost and fast-response portable devices by requiring very low volume of reagents 5 . Compact and portable flow-systems should have an associated manufacturing and maintenance low cost to be competitive in the winemaking industry. Besides, the materials used to fabricate OPEN 1 Instituto de Microelectrónica de Barcelona (IMB-CNM), CSIC, Campus UAB, 08193 Bellaterra, Spain. 2 Institut Català de La Vinya i el Vi (IRTA-INCAVI), Plaça Àgora 2, 08720 Vilafranca del Penedès, Spain. 3 CIBER de Bioingeniería, Biomateriales y Nonomedicina (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain. * email: pablo.gimenez@csic.es; cesar.fernandez@csic.es them have to be cheap and tough, but also easy to machine. First approaches done in research for these devices used glass, ceramic and silicon 6 because they are easy to manufacture and very reproducible, but the integration of sensors or other flow elements is very complicated. To solve this challenge, polymers have been proposed from several decades ago for flow-systems because they are very low cost 7 . The most common polymer used in fluidic systems is the polymethyl methacrylate (PMMA) 8 because it is rigid, hard and very easy to manufacture by fast-prototyping techniques (i.e. milling and laser ablation) [9][10][11][12][13][14] . Regarding the sensing part of the device, the electrochemical biosensors have been extensively reported as the best chance for the monitoring of analytical processes, including food control. They are also easy to integrate in portable analytical flow-systems for on-site analysis 15 .</p><p>We previously reported on the development of individual amperometric biosensors for the detection of l-lactate and l-malate in batch. Both biosensors showed long-term working stability of more than 37 days. This is the key factor that enabled the application of these biosensors to monitor the MLF process. The biosensors' architecture included a thin-film electrochemical transducer selectively electromodified with a bienzymatic membrane, based on a three-dimensional matrix of electrogenerated polypyrrole (PPy). For the l-lactate biosensor (Fig. S1a, in the Supplementary Information-SI), the PPy membrane entrapped lactate oxidase (LOX) and horseradish peroxidase (HRP) as enzymes, while ferrocyanide (Fe(CN) 6 4-) in solution was used as redox mediator 16 . In the case of the l-malate biosensor (Fig. S1b, in the SI), the PPy membrane entrapped malate dehydrogenase (MDH) and diaphorase (DP) as enzymes, together with hexaammineruthenium (III) (HAR) as redox mediator, while β-Nicotinamide adenine dinucleotide (NAD + ) in solution was used as co-factor 17 . In both cases, the selected redox media was oxidized or reduced on the surface of the electrochemical transducer, and the faradic signal resulted from this process was associated to the concentration of the analyte in the sample.</p><p>In this work, the production of a miniaturized flow-system integrating the above described biosensors is addressed for the very first time. The developed cost-effective flow-system could be readily used in field and in an automatic fashion, representing a significant advance in the precise monitoring of the malolactic fermentation in the winemaking industry. The simultaneous determination of l-lactic and l-malic in red wine samples with this flow-system is thoroughly assessed. A silicon chip comprising a platinum four-electrode electrochemical cell (counter, pseudo-reference and two working electrodes) was integrated in the compact flow-system. The working electrodes were selectively and sequentially modified with electrogenerated polypyrrole membranes to construct the corresponding on-chip biosensors. The resulting biochip was integrated in a multi-layered PMMA flow cell fabricated by a laser cutting process, which allowed the simple alignment of the chip with the fluidic reservoir and channels. Then, the bi-parametric fluidic system was applied to the monitoring of l-lactic and l-malic in samples collected along the MLF process of three different red wines and the results were compared with those recorded with the standard methods.</p><!><p>Reagents and solutions. High pure (or analytical grade) reagents from Sigma-Aldrich (Spain) were used in this work. Deionized water was used to prepare the solutions. For the mechanical cleaning of the electrodes, ethanol 96% and 6 M sulfuric acid (H 2 SO 4 ) were used. The distillation of the pyrrole (reagent grade, 98%) was carried out once per week and then it was preserved at − 20 °C in the freezer. Potassium phosphate monobasic (KH 2 PO 4 ) was used to prepare a 0.05 M phosphate buffer (PB) solution (pH 7), which was used for the sensors fabrication and their characterization.</p><p>The bienzymatic l-lactate biosensor was fabricated by using 10-µL aliquots of 1 U µL −1 Lactate oxidase (LOX, from Pediococcus sp., lyophilized powder, ≥ 20 U mg −1 solid), and then they were preserved at − 20 °C. The enzyme horseradish peroxidase (HRP, type VI-A, essentially salt-free, lyophilized powder, 250-330 U mg −1 solid) was preserved in a refrigerator at 4 °C as it was purchased. As redox mediator for the HRP enzyme, the potassium ferrocyanide (K 4 [Fe(CN) 6 ]) was employed. The bienzymatic l-malate biosensor was fabricated by using 15-µL aliquots of 5 U µL −1 Malate dehydrogenase (MDH, from porcine heart, freeze-dried material, ≥ 119 U mg −1 solid, Sorachim, S.A.) preserved at − 20 °C. β-Nicotinamide adenine dinucleotide hydrate (NAD + , ≥ 96.5% enzymatic, from yeast) and diaphorase (DP, from Clostridium kluyveri, lyophilized powder, 3-20 U mg −1 solid) were preserved in a freezer at − 20 °C as they were purchased. Every day, a 1-mL solution containing 0.1 M NAD + was prepared to be used as co-factor for the MDH. Moreover, the reagent hexaammineruthenium(III) chloride (Ru(NH 3 ) 6 Cl 3 , 98%) (HAR) was used as redox mediator for the DP.</p><!><p>A 11 × 9-mm 2 silicon chip formed by four in-parallel platinum (Pt) microelectrodes was designed and fabricated by using standard photolithographic techniques 18 (Fig. S2, in SI). The larger electrode (2 × 2.5 mm 2 ) was used as counter electrode (CE), the two internal ones (1 × 2.5 mm 2 ) were used as working electrodes (WE 1 and WE 2) and the last one (1 × 2.5 mm 2 ) was used as pseudo-reference electrode (p-RE). The separation between adjacent electrodes was 0.6 mm and the contact pads were located 2.9 mm far from the electrode areas.</p><p>The chip was inserted in a PMMA cell, designed by Corel Draw v.17 software and machined using a CO 2 -laser printer (Epilog Mini 24, Epilog Laser, USA). Two different cell architectures were mechanized. The first one, shown in Fig. 1a, was used for the batch sequential fabrication of both sensors, whereas the second one, shown in Fig. 1b, was used for the sensor analytical characterization under flow conditions. The chip can be used without any encapsulation process and was directly inserted in the cell where an integrated four spring-loaded connector (RS Components, Switzerland) was placed in contact with the electrode pads to enable contacting the chip with the measuring instrument. The distance from the electrodes to the contact pads enabled leaving enough room for the fluidic cell, providing an easy integration of the chip into the flow cell and a proper approach for connecting the cell to the measuring potentiostatic device. Both PMMA cells were formed by a 3-mm-thick PMMA bottom part fixed to a 0.5-mm-thick PMMA layer using double-sided PSA (175 μm thick) as adhesive. The 0.5-mm layer defined a 11 × 9 × 0.5-mm 3 well to host and align the silicon chip. The top PMMA part was 5-mm-thick. The one used for the sensor fabrication (Fig. 1a) defined a 50-µL chamber, which was aligned over the area of the electrodes. A 180-µm-thick PDMS layer was sandwiched between the bottom and top PMMA layers and the three layers were clamped together with four 1-mm diameter screws to avoid fluid leakage. During all the electropolymerization and activation steps, a 2-mm-diameter stainless-steel wire was used as CE and a 1.5-mmdiameter Ag/AgCl (3 M KCl) flexible Dri-Ref (World Precision Instruments, Sarasota, USA) was used as RE.</p><p>The top PMMA part of the flow cell used for the sensor characterization (Fig. 1b) included several layers, which were fixed using 175-µm-thick double-sided PSA as adhesive. It comprised a 15-µL cell, two fluidic channels (1 mm width, 7 mm length) showing a thickness of 175 μm and 1-mm-diameter holes to enable fluidic connection between consecutive layers. Two fitting threads for connecting the fluidic inlet and outlet with external Teflon tubes (1.0-mm inner diameter, Teknokroma, Barcelona, Spain) were also included. A 180-µm-thick PDMS layer was also sandwiched between the top and bottom PMMA parts and fixed with four 1-mm diameter screws to avoid the fluid leakage. Here, the electrochemical cell comprised both biosensors together with the integrated Pt on-chip CE and p-RE electrodes. An image of the assembled compact analytical flow-system is shown in Fig. 1c. A cross-section of the fabricated bi-parametric compact analytical flow-system is shown in Fig. 1d. Conditioned samples were flowed inside the device by using a peristaltic pump (403U/VM3, Watson Marlow, UK) in this first approach. www.nature.com/scientificreports/ An Autolab workstation (PGSTAT-100 potentiostat-galvanostat, Ecochemie, Uthecht, The Netherlands) was employed to carry out the electrochemical measurements. The potentiostat was controlled by using the software NOVA v2.0 (Metrohm Autolab, Utrecht, Netherlands).</p><!><p>Firstly, the electrodes were cleaned and activated as follows: 96% ethanol, 6 M H 2 SO 4 and deionized water were used to mechanically clean the surface of the electrodes, and subsequently they were electrochemically activated by cyclic voltammetry (20 scans from + 0.8 to − 2.2 V at 100 mV s −1 ) in a 0.1 M KNO 3 solution 18 . Then, the surface of both WEs was selectively electro-modified by applying the conditions optimized in our previous works, allowing the integration of the two biosensors on a single chip. A l-lactate biosensor was constructed in the WE 1 and a l-malate biosensor in the WE 2. In both cases, the PPy membranes were electrogenerated by fixing an overpotential of + 0.7 V (vs. Ag/AgCl) in a 50-μL 0.05 M PB solution at pH 7, which also contained 0.4 M pyrrole and 0.1 M KCl (named after generation solution). For the fabrication of the l-lactate biosensor, the generation solution also included 10 U of LOX and 200 U of HRP. The overpotential was applied until reach an accumulation charge of 500 mC cm −216 . For the electrosynthesis of the l-malate biosensor, a first membrane of PPy with an accumulated charge of 250-mC cm −2 was electrosynthesized. The generation solution used in this step also included 10 mM HAR(III) as redox mediator. Then, a second membrane of PPy with an accumulated charge of 500 mC cm −2 was generated by adding to the generation solution 45 U of MDH and 7.5 U of DP 17 . Afterward, the as-produced two-biosensor chips were rinsed with PB solution to remove the (bio)reagents physically adsorbed onto the PPy surface. Finally, they were preserved in a freezer at 4 °C in a PB solution when they were not in use. For ensuring a stable base line for both biosensors through all the measurements, the PPy membranes were overoxidized just after their electrosynthesis. This oversoxidation was carried out by cycling the potential from 0 to + 1 V 60 times at 100 mV s −1 in a PB solution 19 . The overoxidation process only had to be done once after the electrosynthesis of the biosensors, and it was not necessary to repeat the process during the life of the biosensors.</p><p>The biosensor responses were based on the cascade (bio) reactions depicted in Fig. S1 (in the SI). All analytical measurements were carried out in the electrochemical flow cell and under stop flow conditions. The p-RE was positioned upstream the biosensors to avoid the potential changes caused by the enzymatic reactions on the biosensors. Initially, cyclic voltammograms (CVs) were recorded at 20 mV s −1 by injecting 125 μL of a 0.05 M PB solution (pH 7) containing 0.5 M KCl and all the other reagents required to complete the bi-enzymatic reaction for both biosensors. For the l-lactate biosensor, 1 mM l-lactate and 2 mM K 4 [Fe(CN) 6 ] as redox mediator were added. Regarding the l-malate biosensor, 1 mM l-malate and 5 mM NAD + as co-factor were added to the characterization solution. The solution also included 0.5 M KCl to minimize the potential drop and the hysteresis effects of the electrochemical processes, which were observed when biosensors were measured in compact cells under flow conditions 20 . Once the optimum operational potential applied for chronoamperometric measurements was set, calibration curves were performed in triplicate for both target analytes. l-lactate was measured in a concentration range between 1 × 10 -7 and 1 × 10 -3 M, whereas l-malate was measured in a concentration range between 1 × 10 -7 and 1 × 10 -5 M.</p><p>The biosensors' performance was assessed in terms of sensitivity, linear range, limit of detection (LOD) and reproducibility of the fabrication procedure by using three biosensors fabricated under the same experimental conditions. Biosensor selectivity was evaluated in PB solutions containing the main interferences in wine samples (glycerol, glucose, gluconic acid, fructose, acetic acid, citric acid, ethanol, l-lactic, l-malic, tartaric acid and ascorbic acid) with a concentration of 5 × 10 -5 M or 5 × 10 -7 M for the l-lactate and the l-malate detection, respectively. A set potential of 0.35 V and − 0.4 V (vs Pt p-RE) was applied for the l-lactate and the l-malate detection, respectively. Concerning the working stability with time over long-times, both bio-chips were tested by calibrating them every 2 or 4 days in a concentration range of 1 × 10 -7 M-1 × 10 -5 M and 1 × 10 -6 M-1 × 10 -4 M for the l-malate and the l-lactate, respectively.</p><p>All solutions and samples used for the evaluation of the bi-parametric compact analytical flow-system were flowed continuously during 30 s at 0.25 mL min −1 , in order to ensure that the previous solution filling the electrochemical flow cell was flowed out and replaced by fresh solution for carrying out the following measurement.</p><!><p>Institute of Vineyard and Wine (IRTA-INCAVI) were selected to validate the developed flow-system for the simultaneous determination of l-malic and l-lactic. The wines were collected from the 2013 vintage and their vineyards were harvested in the region of Tarragona (Spain). The MLF process was induced in the samples after the alcoholic fermentation process by a strain of the specie Oenococcus oeni. A set of samples for each of the three wines were collected along the MLF process, in a concentration range from 0 to 8 × 10 -3 M (0-1.2 g L −1 ) for the l-malic, and from 0 to 6 × 10 -3 M (0-0.5 g L −1 ) for the l-lactic acid. The number of the collected samples was 10 of the Wine 1 (along 28 days), 5 of the Wine 2 (along 33 days) and 13 of the Wine 3 (along 45 days). All the samples were analysed with the bi-parametric compact analytical flow-system. The wine samples were diluted to adjust the l-malic acid and the l-lactic acid concentrations to the linear range of the individual biosensors obtained previously in our group (from 5 × 10 -6 to 1 × 10 -4 M and from 1 × 10 -7 to 1 × 10 -6 M, for the l-lactate and the l-malate, respectively) 14,15 . The wine samples were eventually diluted 1:10,000 by carrying out two intermediate dilutions of 1:100 in a 0.05 M PB solution (pH 7) containing 0.5 M KCl to adjust the l-malic acid concentration to the linear range of the biosensor. For the l-lactic determination, the dilution in a PB solution to adjust the concentration to the linear range of the biosensor for Wine 1 and Wine 2 was 1:50, meanwhile for Wine 3 was 1:20.</p><p>The results obtained with the bi-parametric compact analytical flow-system were compared with them obtained by the standard enzymatic method applied by the IRTA-INCAVI. The standard method is based on www.nature.com/scientificreports/ the enzymatically catalysed reaction between the l-lactate or l-malate and the NAD + to produce NADH, whose concentration is stoichiometrically related to the analyte concentration in the sample. The change of the NADH concentration is measured spectrophotometrically at 340 nm 21 .</p><p>Regarding the hydrodynamic performance of the bi-parametric compact analytical flow-system, the procedure used for the detection of both parameters is summarized as follows: Firstly, the wine sample was diluted in two 125-μL PB solutions for adjusting the concentration of the parameter to the linear range of each biosensor. Then, for the l-lactic detection, the ferrocyanide was added to one of these diluted solutions and was inoculated in the bi-parametric compact analytical flow-system during 30 s to totally clean the system from the last sample. The same procedure was repeated for the l-malic detection, but inoculating the diluted sample containing the NAD + .</p><!><p>Biosensor fabrication. The use of electropolymerized membranes to produce the biosensor recognition layers on top of the electrodes allowed for the on-chip integration of both l-lactate and l-malate biosensors. The electrosynthesis of both PPy membranes was sequentially carried out in the electrochemical flow cell by applying potentiostatic conditions, as was described in the experimental section. The electrogeneration method applied avoided the cross contamination between the four integrated electrodes of the silicon chip. As can be seen in Fig. 2, both biosensors were selectively electrodeposited in the WE 1 and WE 2. The current profiles recorded under potentiostatic conditions during the potentiostatic electropolymerization of the PPy films are depicted in Fig. S3 (in the SI). The chip architecture facilitates the integration in a flow cell and can easily be connected via the embedded spring-loaded connectors to the measuring instrument. Millimetre-sized electrodes show suitable dimensions to work in a compact flow system like the one shown in this work with the electrode distance and electrochemical cell layout being adequate to avoid any electrochemical cross-talk due to the reactions taking place at the counter electrode on the biosensor devices and Ohmic drop. The biosensor fabrication was carried out sequentially without observing any chemical interference. The pseudo-reference electrode was placed upstream the biosensors to avoid any potential changes caused by the enzymatic reaction. Also, the counter electrode was placed downstream to avoid possible interference on the biosensor responses, as pointed out above. No chemical cross-reactions were observed in the biosensor responses, convincingly demonstrating the suitability of the architecture used to develop the chip.</p><p>In both biosensors, the PPy membranes were overoxidized in order to obtain a stable base-line signal. This process consisted of carrying out 60 consecutive potential cycles between 0 and + 1 V at 100 mV s −1 in a 0.05 M PB solution (pH 7) 17 . The voltammetric signals (cycles 5, 30, 45 and 60) obtained during the overoxidation of the l-lactate and the l-malate biosensors are shown in Figs. S4a and S4b (in the SI), respectively. As can be observed, the non-faradaic current decreased with the increase in the number of CV until almost stabilization. This is associated with the electrochemical oxidative degradation of the conducting polymer 22 . Although the overoxidation of the polypyrrole membrane causes a loss of its conductivity or electroactivity, the moderate conditions applied in this work enabled to obtain a more stable background signal, which resulted in an improved analytical biosensor performance 23 .</p><!><p>The voltammetric responses recorded with the two on-chip biosensors in the electrochemical flow cell were used to fix the applied potential for the chronoamperometric characterisation of both biosensors. In the case of the l-lactate biosensor, after the addition of 1 mM l-lactate to a 0.05 M PB solution at pH 7 which also contained 0.5 KCl, an increase of the cathodic current at around − 0.35 V (vs Pt p-RE) occurred (Fig. S5a-in the SI). This increase was caused by the ferricyanide reduction, which was previously generated by the bienzymatic reaction involving the LOX and the HRP. For the l-malate biosensor, the addition of 1 mM l-malate and 5 mM NAD + to a 0.05 M PB solution (pH 7) containing 0.5 M KCl caused an increase of the anodic current (Fig. S5a, in the SI). This current increase was the consequence of the re-oxidation of the HAR(II) previously generated by the bienzymatic reac- From the obtained chronoamperograms in the electrochemical flow cell, the corresponding calibration curves of the two on-chip biosensors were constructed. Figure 3a shows that larger negative current densities were recorded along 120 s in solutions containing increasing concentrations of l-lactate. In all cases, the recorded currents for each analyte concentration became stabilized at around 90 s. Therefore, the mean value of the current density of the last 30 s was used as analytical signal for plotting the calibration curve for the l-lactate biosensor (Fig. 3b). Concerning the l-malate biosensor, similar response profiles were recorded but here higher anodic currents were recorded when the l-malate concentration increased in solution (Fig. 4a). The corresponding calibration curve was also plotted by using the mean current density value of the last 30 s vs. the l-malate concentration in solution (Fig. 4b). The l-lactate biosensor showed a sensitivity of (− 173 ± 8) × 10 2 µA M −1 cm −2 (r = 0.997, n = 7) in a linear range from 5 × 10 -6 to 1 × 10 -4 M and a LOD (3σ IUPAC criterion) of 3.2 ± 0.3 × 10 -6 M. A linear response for the l-malate biosensor was observed in a range from 1 × 10 -7 to 1 × 10 -6 M, with a sensitivity of (5.53 ± 0.6) × 10 2 mA M −1 cm −2 (r = 0.997, n = 5) and a LOD of 6.7 ± 0.2 × 10 -8 M. For concentrations above 1 × 10 -4 M of l-lactate and above 1 × 10 -6 M of l-malate, the respective enzymatic biosensors showed a saturation behaviour, this being in agreement with a process following a Michaelis-Menten kinetics. The reproducibility of the biosensors fabrication methodology was evaluated for each analyte by calibrating three different on-chip biosensors fabricated in three different silicon chips. The obtained relative standard deviation (RSD) of the biosensor sensitivity was lower than 8% and 6% for the l-lactate and the l-malate, respectively, meaning that the reproducibility of the fabrication method is very good.</p><p>The selectivity and the working stability of both biosensors were studied in detail and reported in our previous works as is detailed in the experimental section 16,17 . It can be assumed that this is going to be analogous in the bi-parametric compact analytical flow-system reported here. Regarding the selectivity, any of the checked interferences showed a signal that could affect the analyte detection at the applied set potential for both biosensors. www.nature.com/scientificreports/ Concerning the working stability, the biosensors maintained more than 90% of their initial response during 52 days and 37 days for the l-lactate and the l-malate detection, respectively, meaning that these integrated biosensors can be used for the monitoring of long analytical processes, such as the MLF.</p><p>Malolactic fermentation monitoring with the bi-parametric flow-system. Finally, the bi-parametric flow-system was applied to the monitoring of the malolactic fermentation of three different red wine samples collected along the MLF process. l-lactic and l-malic acids were determined consecutively in the red wine samples. Considering the required time for the electrochemical flow-cell filling (30 s) and the amperometric detection (120 s) carried out twice, the complete analysis of each sample took around 5 min. Figure 5 shows a comparison between the results obtained by the standard method and those obtained by the developed bi-parametric flow-system. There was an ideal agreement between both compared methods, with absolute errors below 0.15 g L -1 . Moreover, it is important to notice that all the experimental values obtained with the bi-parametric flow-system are within the 95% uncertainty range of the standard method. The evolution of both acid concentrations in the samples was as expected: at the beginning of the MLF process the l-malic acid concentration was high and there was not l-lactic acid. Along the process, the concentration of l-malic acid decreased and the l-lactic acid concentration increased. Finally, the MLF process finished when both concentrations were stable. The MLF process can take from a few days to a few weeks. The control of both parameters along the MLF process is essential to ensure the quality of the wine, because it is very important to determine the end-point of the process when the complete duck-out of the l-malic acid occurred. The appropriate control of the end-point avoids the organoleptic deviations caused by the growth of damaged microorganisms, implying a new inoculation of artificial strain from laboratories for restarting the process and consequently, it provokes additional cost and time for the process. Besides, the control of l-lactic acid concentration is also necessary because the maintenance of the ratio between both acids along the process determines that there are no unwanted parallel www.nature.com/scientificreports/ processes in the MLF. Only one bi-parametric fluidic system without re-generating the on-chip biosensors was employed during all the assays (including the calibrations of the system performed before and after the analysis of each wine), resulting in a total number of assays above 80. It was shown that both biosensors kept more than 91% and 93% of the initial sensitivity for the l-lactate and the l-malate, respectively. www.nature.com/scientificreports/</p><p>The proposed bi-parametric compact analytical flow-system reduces the number of reagents involved in the determination of both acids compared to the commercial enzymatic methods, because almost all the reagents are immobilized on the biosensor membrane, which only has to be re-generated after 37 days of intensive use. The volume of the reagents consumed is small, leading to less expense associated with the fermentation control. Moreover, the use of microfluidics enables to high dilute the wine samples, avoiding the potential matrix effects in the p-RE that could interfere in the determination of both acids. The flow-system also enables the fast and easy recalibration of the bio-chips before each analysis, meaning in a stable medium along all the assays. It is important to highlight that, in general, along the malolactic fermentation process, the l-malic acid concentration can decrease from around 3 g L −1 (2.2 × 10 -2 M) to 0 g L −1 (0 M). Nevertheless a concentration of l-malic below 0.3 g L −1 (2.2 × 10 -3 M) is considered as the end point of the MLF process in the winemaking industry. Therefore, the l-malic range can be actually considered from 2.2 × 10 -2 to 2.2 × 10 -3 M. Regarding the l-lactic acid, its concentration usually can increase from 0 g L −1 (0 M) up to 1.5 g L −1 (0.01 M). Thereupon, considering the general concentration ranges of these two biomarkers in red wines (from 2.2 × 10 -3 to 2.2 × 10 -2 M and from 0 to 1 × 10 -2 M, for the l-malic and the l-lactic, respectively) we could anticipate that the dilution factors would be between 1000 and 10,000 for all the possible acid concentrations in the winemaking industry. For the analyses of l-lactic, a sample dilution of 1:1000 will be carried out, whereas for the analyses of l-malic the sample will be diluted 1:10,000, in order to work within the biosensor linear concentration range. In this work, a different and more adjusted dilution ratio was used for both biosensors because the concentration range during the MLF process was previously determined by the standard method, but the general dilution factor could have been applied, too.</p><p>As summary, the results in this work demonstrate the high potential of the developed compact analytical flow-system for the monitoring of both l-lactic and l-malic acids in the field of the winemaking industry. The accuracy of the proposed flow-system is appropriate for the considered application: the on-line control of both parameters in fermentation barrels of wines, which allows applying corrective actions in real time if it were necessary. Besides, the compacted size of the system will allow its easy integration within barrels, which will enable the biosensors calibration and the l-lactic and l-malic detection in an automated mode including the sample collection and dilution, the reagents inoculation, the analysis and the data processing. It results in an easier and more accurate method for the MLF process monitoring in comparison to conventional current methods without the fluidic part. Regarding the calibration of the system and the analysis of the real samples in the field, it will be as follows: the fluidic inlet of the compact analytical flow system could be easily connected to an automated and remotely controlled system of multi-valves, which will enable the flow of the rest of the reagents involved in the determination in an automatic mode. A total of six valves should be connected to 5 reservoirs containing (1) PB solution to dilute the sample, (2) l-lactate and (3) l-malate to calibrate the system every day, and (4) ferrocyanide and ( 5) NAD + to complete the bi-enzymatic reactions. The total volume consumed for every assay is very low (125 μL) and it is used for enabling the perfect renovation of the fluidic microchannels with next solutions, which avoids the memory effect of the sensors from previous assays by removing any trace of l-lactic or l-malic coming from them. From these numbers, and considering one calibration and one assay per day to control the malolactic fermentation process, if 10 mL containers for each solution were connected to the automated system, the microanalytical flow-system could work during all the malolactic fermentation process without any user intervention. Therefore, the proposed analytical flow-system will enable the control of the l-lactic and the l-malic acids on-site, creating a new portable and automated flow-system for the MLF process monitoring with breakthrough attributes.</p><p>Comparison with other bi-parametric systems. Table 1 shows some characteristics of the developed bi-parametric compact analytical flow-system and other bi-parametric systems based on enzymes for simultaneous determination of l-lactic and l-malic in wines, previously reported. As can be seen, two systems use independent enzymatic reactors 24,25 , one for each parameter, two other systems are based on the immobilization of the enzymes in different membranes 26,27 and another one is based on the entrapment of the enzymes in a solid composite 28 . The proposed bi-parametric compact analytical flow-system in this work is the only one constructed by simultaneously entrapping both enzymes, and even the redox mediator, in an electrogenerated PPy membrane. This fabrication methodology enables the deposition of the required species on the same silicon chip without affecting the other integrated electrodes, meaning in an excellent approach for the fabrication of flow devices integrating microtransducers. As a result of this simplification, there is a reduction of volumes consumed of reagents and samples in comparison to the other works in the literature, which is especially interesting for reducing costs along the monitoring of long-term processes, as the MLF.</p><p>All the other works use common species in solution for carrying out the detection of both target analytes: fluorimetric detection of NADH generated for l-LDH and l-MDH, amperometric detection of O 2 consumed for LOX, HRP and MDH/DP and the chronoamperometric detection of the redox mediator ferricyanide. This is the reason why all of them are combined with a FIA system to manage the liquids, except one which is performed in batch 28 . In the bi-parametric compact analytical flow-system presented in this work, two specific redox mediators, ferrocyanide and HAR, were used for each analyte. In the case of the l-malate biosensor, the HAR is incorporated in the PPy membrane, allowing a continuous flow analysis.</p><p>The analysis time per sample is similar in all the cases, this being between 3 and 6 min. In the other hand, the system described in this work outperforms the other approaches in terms of limit of detection (LOD), especially in the case of l-malate. This may be partially related with the immobilization of the biochemical species in the conductive polypyrrole membrane synthesized under biocompatible conditions that may preserve the enzyme activity to a large extent. www.nature.com/scientificreports/ A system applied to the monitoring of the malolactic fermentation must show a long-term working stability under continuous use because the fermentation process takes around 30 days. Some of the systems in Table 1 show good operational stability values as long as 6 months. The system developed in this work maintains 92% of its initial sensitivity after more than 80 measurements in continuous use, with a lifetime of 37 days, being able to monitor the entire malolactic fermentation process. Finally, all the systems have been applied to the bi-parametric determination in finished wines samples, which are commercially available. However, the bi-parametric compact analytical flow-system presented in this work is the only one that has been assessed using real samples collected during the malolactic fermentation of red wines. This compact analytical flow-system integrates for the first time both sensors on a new automated flow platform, resulting in a simply system for the malolactic fermentation monitoring. It represents a significant advance in the precise monitoring of the process in the winemaking industry because this novel research will provide for the very first time a cost-effective and easy-to-use way to determine l-lactic and l-malic on-site and on-line based on this new concept of test for winemaking control with unique and unprecedented attributes. No other previous reports are focused on the on chip integration of both l-malate and l-lactate biosensors in a flow-system that could show the potential of these devices for monitoring the malolactic fermentation of red wines in field and in an automatic fashion.</p><!><p>A bi-parametric compact analytical flow system for the simultaneous determination of l-lactate and l-malate was designed, fabricated and optimized. The development of a bi-parametric compact system included the design and the fabrication of a 11 × 9-mm 2 silicon chip comprising two working electrodes, together with a counter and a pseudo-reference electrode, all made of platinum. These miniaturized electrochemical sensors are cheap, highly reproducible and robust. The working electrodes were modified with polypyrrole-based enzymatic membranes to produce the two on-chip biosensors for l-lactate and l-malate target analytes. The use of an electropolymerization approach enabled the strict controlled deposition of the required enzymes over the selected electrodes.</p><p>The biochip did not require encapsulation and wire-bonding and thus allowed for the simple integration in a robust miniaturized PMMA flow cell, mechanized by using rapid prototyping techniques. The resulting bi-parametric compact analytical flow-system showed superior analytical and operational features for the determination of l-lactate and l-malate and was eventually assessed in red wine samples collected during the malolactic fermentation process, showing a good agreement with the results obtained for both analytes with the standard colorimetric methods. As summary, the overall novelty of the proposed flow-system is twofold: (1) it has the ability to fully process the sample and the reagents required for the malolactic fermentation control, by using a low-cost and robust miniaturized PMMA flow cell. The design of the flow cell reduces drastically the complexity of current traditional tests, meaning in a method with a total analysis time of 5 min and a required sample volume of 125 μL per analysis. Besides, the automated flow cell avoids the human error and the potential contamination of barrels derived from the manipulation of the sample and reagents. (2) The flow-system integrates both l-lactate and l-malate sensors in the same chip, which allows the reduction of reagents and sample consumption. Moreover, the monitoring of both parameters using a compact system is proposed for the very first time in literature, meaning in a novel system for the fast and total control of the malolactic fermentation in field, in real-time and on-site in the winemaking industry. The proposed flow-system would set a new paradigm for integration of electrochemical sensing in field by solving the limitations of current methods applied in the winemaking industry. This approach would open new opportunities for in-situ and real-time control of processes by making accessible to untrained personnel an easy handling method for fermentation monitoring. It would also facilitate better management to winemakers and the application of corrective actions, if required, allowing unprecedented time and cost savings.</p>

Scientific Reports - Nature

Gold–carbonyl group interactions in the electrochemistry of anthraquinone thiols self-assembled on Au(111)-surfaces

New anthraquinone derivatives with either a single or two thiol groups (AQ1 and AQ2) were synthesized and immobilized in self-assembled monolayers (SAMs) on Au(111) electrodes via Au-S bonds. The resultant AQ1-and AQ2-SAMs were studied by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), which enabled mapping of the gold-carbonyl group interactions and other dynamics in the Au-S bound molecular framework. Understanding of these interactions is important for research on thiol-coated gold nanoclusters, since (I) anthraquinone derivatives are a major compound family for providing desired redox functionality in multifarious assays or devices, and (II) the gold-carbonyl interactions can strongly affect anthraquinone electrochemistry. Based on equivalent circuit analysis, it was found that there is a significant rise in polarization resistance (related to SAM structural reorganization) at potentials that can be attributed to the quinone/semi-quinone interconversion. The equivalent circuit model was validated by calculation of pseudocapacitance for quinone-to-hydroquinone interconversion, in good agreement with the values derived from CV. The EIS and CV patterns obtained provide consistent evidence for two different ECEC (i.e. protoncontrolled ET steps, PCET) pathways in AQ1-and AQ2-SAMs. Notably, it was found that the formal reorganization (free) energies obtained for the elementary PCET steps are unexpectedly small for both SAMs studied. This anomaly suggests high layer rigidity and recumbent molecular orientation on gold surfaces, especially for the AQ2-SAMs. The results strongly indicate that gold-carbonyl group interactions can be controlled by favorable structural organization of anthraquinone-based molecules on gold surfaces.

gold–carbonyl_group_interactions_in_the_electrochemistry_of_anthraquinone_thiols_self-assembled_on_a

Introduction<!>CV of the quinone group<!>Reductive desorption of the Au-thiol surface bond<!>AQ-SAMs surface imaging<!>Frequency response analysis<!>Electrochemical kinetics analysis<!>Conclusions<!>Chemicals<!>Au(111) electrodes and sample preparation procedures<!>Instrumentation and electrochemical measurements<!>Conflicts of interest

<p>There is presently a great interest in gold nanoclusters, i.e. gold in sub-nanoparticle size in a variety of shapes. 1 These span polyhedral structures containing from a few to hundreds of gold atoms. Gold nanoclusters are intensively investigated due to their potential exploitation e.g. as catalysts 2 and to singleelectron charging and other quantum effects. 3,4 One of the "traditional" methods of gold nanoparticle synthesis and stabilization is the use of coating compounds terminated with thiol groups, which is comprehensively exploited in the creation of gold nanoclusters. 1 The organization of dithiol-type compounds on gold nanoclusters in comparison to compounds with only a single thiol group is further important from fundamental points of view. The interaction and surface distribution of added functional groups is of particular interest, 1 which would be different for mono-and dithiol compounds. Additional functionalization provides other useful approaches for ne-tuning of gold nanocluster properties, and for controlled aggregation of the functionalized Au-clusters into new "smart" materials. 1,4 One feasible step towards assessing such interactions is the use of anthraquinone derivatives. Anthraquinones with two thiol linkers are attractive for molecular electronics, 5 due to their molecular redox functionalities, and ease of tuning properties by adding functional groups through well-established organic syntheses. [6][7][8] The putative application of anthraquinone thiol derivatives as molecular linkers or redox probes also relates to the great importance of quinone electrochemistry in bioenergetics. 9 Future devices based on hybrids of anthraquinone thiols with gold nanoclusters could therefore potentially be utilized e.g. in investigation of electron transfer (ET) kinetics in electrochemical systems based on redox enzymes. 10,11 The quinone-to-hydroquinone conversion process also depends strongly on pH, since each ET step is accompanied by proton transfer (PT). [12][13][14] Besides the proton concentration and the surrounding medium, the electrochemical conversion of carbonyl groups can also be affected by the nature of the gold surfaces. 15 In-depth understanding of these interactions in the context of anthraquinone-gold systems is thus highly important.</p><p>In this report we present a study of the electrochemical properties of self-assembled molecular monolayers (SAMs) of in-house synthesized mono-and dithiol anthraquinone derivatives (denoted as AQ1-and AQ2-SAMs, Scheme 1) assembled on Au(111) electrode surfaces. Deposition of the compounds on a single-crystal (i.e. atomically at) gold surface enabled rst the recording of interfacial faradaic processes of both the carbonyl group and the surface Au-S linking units.</p><p>Secondly, electrochemical features related to structural reorganization events in the whole AQ-SAMs could be recorded. The assignment of these features to specic reactions is based on cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Particularly, the EIS data combined with equivalent circuit analysis allowed us to assess at the same time changes in pseudocapacitance and polarization resistance (R p ). The calculated pseudocapacitances based on equivalent circuit analysis are consistent with those that correspond to faradaic process as obtained from CV, thus supporting the proposed model. A signicant change in R p is an indicator of structural reorganization in the layer subjected to electric elds. 16 An overall electrochemical assessment of gold-carbonyl group interactions framed within the quinone-to-hydroquinone interconversion process is provided. We particularly focus on the carbonyl group proximity to gold which triggers specic structural SAM reorganization, as well as solvent and intramolecular reorganization that accompanies the "elementary" PCET steps. Very notably the latter was found to be unexpectedly small and much smaller than reorganization free energies commonly encountered for electrochemical ET processes. As noted, the understanding of gold-carbonyl interactions is also more broadly important for future electrochemical investigations of anthraquinone compounds assembled on gold nanoclusters.</p><!><p>Cyclic voltammograms of AQ2 SAMs show two reversible electrochemical reactions (referred to as reactions ( 1) and ( 2)), Fig. 1A. Both reactions depend on pH, and their formal potentials shi with pH (ca. 60 mV pH À1 ) following the Nernst equation for a one-electron-one-proton (PCET) process, Fig. 1B. Since the overall quinone/hydroquinone conversion in aqueous buffer is a 2-proton/2-electron process, 13 the observed oneelectron-one-proton features must be part of a two-step process each step with a PCET-type mechanism. 17 Reaction ( 2) is therefore likely to be related to the formation of semiquinones, and reaction (1) to the fully reduced AQ2 hydroquinone state. The peak related to semi-quinone formation has in fact been observed for quinone lms at high pH, 18 or in CV of freely mobile quinones in unbuffered solutions. 14 On the other hand, reaction (2) for anthraquinone derivative SAMs in buffered solutions at moderate pH should not be observed. This is the case with our reference compound (AQ1-SAM), insert in Fig. 6. This feature therefore not only depends on the proton concentration, but is also likely to depend on the particular organization of the attached quinones at the surface. The latter is in turn controlled by molecular interactions within the layer Scheme 1 The reactions and resultant chemical structures of AQ2 (5) and AQ1 (6) compounds. and by specic interactions with the electrode surface. Signicantly different peak capacitances for reactions (1) and (2) are therefore observed, which we address further below using frequency response analysis.</p><!><p>Different structural organization of the AQ1-and AQ2-SAMs is expected, as AQ1 binds only via a single Au-S bond, while AQ2 can bind either by a single or two Au-S bonds. AQ2 is therefore expected to form a structurally more rigid surface layer than AQ1. Pronounced differences between the AQ1-and AQ2-SAMs on Au(111) electrodes is in fact substantiated by reductive desorption voltammograms recorded for 0.1 M NaOH, Fig. 2. AQ1-SAMs exhibit a single dominating sharp feature at ca. 0.15 V (actual desorption) and a satellite feature at ca. À0.10 V (arising due to electrode roughness), according with typical voltammograms for desorption of alkanethiol monolayers. 19 AQ2-SAMs exhibit less well-dened features, likely to originate from e.g. sequential desorption or more constrained angular degrees of freedom, both arising from the prevalence of two binding sites. The estimated surface coverages (mol cm À2 ) from reductive desorption of AQ1-and AQ2-SAMs are ca. 6.8 Â 10 À10 and 7.4 Â 10 À10 , respectively, assuming single-ET for AQ1 and two-ET for AQ2 in the desorption processes. This supports that there are in fact two binding sites for AQ2, but only a single binding site for AQ1. The estimated surface coverages are close to the expected coverage of a dense monolayer (with interchain spacing of z5 Å) of 7.76 Â 10 À10 mol cm À2 on a (111)-facet. 20</p><!><p>Scanning tunnelling microscopy (STM) in both the ex situ and the electrochemical in situ modes were undertaken. Highresolution images could be recorded, but their interpretation remains presently elusive. As can be seen from Fig. S1, † cluster formation was consistently observed, resulting in signicantly disordered adlayers. Such structures can strongly affect tunnelling current pathways in different ex situ and in situ STM tip/target molecule/gold surface systems. However, below 150-200 Å, the resolution was not sufficient to distinguish singlemolecule features or surface orientation of the anthraquinone thiols. This could be attributed to a persistent tendency of anthraquinone thiol derivatives for p-stacking competing with Au-S surface bonding. The synthetized molecules have relatively short side chains, and strong p-p interactions are to be expected. We therefore focused further attention on electrochemical kinetic and frequency response analysis in the assessment of gold-carbonyl group interactions.</p><!><p>EIS for AQ1-and AQ2-SAMs was recorded to obtain insight in the nature of reaction (2) and its relation to reaction (1). The EIS experiments were designed to complement optimally the CV data. An example of AQ2-SAM frequency response and equivalent circuit is shown in Fig. 3. The equivalent circuit is analogous to the typical circuit used to describe faradaic impedance of strongly adsorbed molecular entities. 12 The capacitive element related to the faradaic process was modied by utilizing a constant phase element (CPE). This small modication enabled a far more exible t to the wide potential range than utilization solely of a capacitor.</p><p>Additionally, the use of a CPE offers insight into the roughness of the surface. 21 The complex CPE admittance Y(u) can be described by the equation:</p><p>where j ¼ (À1) 1/2 , n is a phenomenological number describing the deviation of the CPE from an ideal capacitor, and Y 0 the magnitude of the admittance (S s n ). The phase angle of the CPE is independent of frequency and has a value of À(90 n) . Y 0 is thus an ideal capacitor if n ¼ 1, and an ideal resistor if n ¼ 0. In the context of surface roughness, the change in the value of n from 1 to 0.5 can be interpreted as the change from a perfectly at to a highly rough surface. 21 Y 0 can be converted to capacitance (C r ) using the following equation:</p><p>where f mf is the frequency of maximum phase angle. The conversion of Y 0 to C r using eqn (2), is based on the assumption that the imaginary part of the CPE impedance equals the impedance of tted capacitance for a given frequency range (eqn S1 †). Based on the applied model (inset in Fig. 3 The C r values (aer correction for C dl as noted) can be directly used for the validation of the applied equivalent circuit. As seen from Fig. 4, the calculated specic capacitances for reaction (1) are quite similar to the values derived from CV, thus justifying the application of the proposed equivalent circuit for the description of faradaic impedance of adsorbed anthraquinone thiols.</p><p>Fig. 5 shows potential induced changes in CPE admittance (and thus in C r ). There is a signicant admittance rise in reaction (1) for both AQ1-and AQ2-SAMs (0.15-0.20 V), and a much smaller change for reaction (2) (ca. 0.35 V). These changes are consistent with the CV features obtained (inset in Fig. 6). As noted, n gives a rough estimate of the adlayer deformation. The particular numerical values of this parameter are shown in the brackets for selected potentials, Fig. 5. The change in n with applied potential for AQ2-SAMs, is only signicant for reaction (2) suggesting a major structural reorganization in a narrow potential range. The n values for AQ1-SAMs are signicantly lower than for both bare and AQ2-coated Au(111) electrodes, making similar assessment challenging. This nding might be indicative of more complex intermolecular interactions in the AQ1 adlayers.</p><p>The plot of R p against applied potential implies that two different EC (ET/PT) reaction pathways specic for AQ1-and AQ2-SAMs operate, Fig. 6. The kinetics for freely mobile anthraquinone-type compounds was found to proceed by EECC (pH z 10), ECEC (at pH 7-4) and CECE (pH 1 and below) mechanisms (where E is ET and C is PT in the sequence). 13 We could not obtain sufficient electroactivity above pH 7 for the SAMs here, and the highest electroactivity was found at pH z 4.5. The latter nding is reected in the observed pH dependence of the current densities for both reactions (1) and ( 2), being similar at pH 7 and pH 1, but notably higher at pH 4.5. The apparent pK a of the rst protonation step is around 4 for the ECEC mechanism. 13 The maximum current density can therefore be associated with the maximum concentration of semiquinone at pH 4.5 compared to pH 7. In the case of pH 1, the apparent pK a of the rst protonation step is estimated to be below 1 for the CECE mechanism. 13 It can then be suggested that the gold-carbonyl group interactions may interfere more strongly with the rst CE step than with the corresponding EC step, resulting in the different current densities at pH 4.5 and pH 1.</p><p>Since the ECEC mechanism likely prevails at pH 4.5, and the relaxation between the ET and the PT steps is fast, the two distinct reorganization events in the AQ1-SAM structure (separated by ca. 0.15 V) can be assigned to two consecutive EC steps, resulting in a single broad CV redox wave. The assigned EC steps (i.e. reactions (1) and ( 2)) of AQ2-SAMs are separated by a larger potential difference of ca. 0.25 V. The change in R p associated with reaction (2) is signicantly higher than for reaction (1), although the change in admittance and pseudocapacitance for reaction (2) is still very small.</p><!><p>We attempted rst to estimate the interfacial electrochemical ET rate constants (k ox /k red ) for AQ1-and AQ2-SAMs by a Laviron analysis and the Butler-Volmer limit of the current/ overpotential (i/h) correlation, Fig. 7:</p><p>F, R and T have their usual meaning, a is the transfer coefficient, h the overpotential, and k 0 the standard rate constant at h ¼ 0 V. The apparent i/h correlations for the anodic and cathodic processes appear symmetric around h ¼ 0 V, but approach a quadratic form already at very small overpotentials |h| < 0.1 V.</p><p>In accordance with electrochemical molecular charge transfer concepts and theoretical concepts and formalism introduced by Marcus, Hush, Gerischer, and particularly by Levich, Dogonadze, Kuznetsov and associates, [25][26][27][28][29][30][31] the latter correlation can be represented as:</p><p>from which the formal reorganization (free) energy (l) of the PCET elementary steps can be estimated. l represents the change in low-frequency solvent and intramolecular structures, and is distinct from the structural reorganization in the SAM detected with EIS discussed above. The summary of this analysis is given in Fig. 7. Slightly asymmetric Tafel plots for AQ1-SAM were obtained, in contrast to AQ2-SAM, suggesting that the layer of the latter is less prone to molecular structural changes in the PCET steps. Notably, l was found to be only ca. 0.05 eV for the AQ1-SAM and 0.02 eV for the AQ2-SAM. l for reaction (2) could not be determined, due to difficulties in reaching the current plateau region, even at high scan rates.</p><p>The difference in apparent l could indicate that the carbonyl groups are closer to the electrode surface for AQ2-SAMs than for AQ1-SAMs, which is supported by the specic faradaic resistance (U cm 2 ) of ca. 6.1 Â 10 5 for the AQ1-SAM and 3.5 Â 10 4 for the AQ2-SAM (Fig. S3 †). The values of l obtained are, however, very small and correspond to almost step-like transition from the Butler-Volmer to the activationless overpotential region. More importantly, the emerging limiting slopes at small overpotentials which represent the electrochemical transfer coefficient, a are quite different from the input values in the Laviron forms (e.g. 1.6 vs. 0.7, Fig. S4 and S5 †). These observations prompt alternative considerations given below.</p><p>Consistent use of the Laviron and Butler-Volmer formalism rests on the notion of strong electronic-vibrational coupling and large reorganization free energies, l [ k B T. The observed current rise from thermal to activationless behavior is far too abrupt to be compatible with the broadly observed much smaller curvature in both simple electrochemical processes and ET processes in homogeneous solution. To account for step-like i/h behavior the notion of weak electronic-vibrational coupling can instead be proposed. In this limit the i/h correlation is dominated by the step-like Fermi function in the electrochemical rate constant rather than by the Gaussian molecular vibrational energy density form as in "normal" electrochemical ET processes. This difference can be illustrated by more detailed rate constant forms that incorporate contributions from all electronic levels of the metal electrode and not only from levels around the Fermi energy. The current density, here cathodic is: [27][28][29][30][31]</p><p>where f(3 À 3 F ) is the Fermi function of the metallic electronic energy spectrum 3, and 3 F the Fermi energy. A is a h-independent constant available from molecular charge transfer theory. 31 f(3 À 3 F ) and the vibrational distribution function g(3 À 3 F ; h) depend strongly on 3, and dominate the i/h correlations with contributions from all electronic levels of the metal electrode, at higher overpotentials:</p><p>f(3 À 3 F ) has a step-like functional form, changing from unity to exponentially small values over an energy range of a few k B T.</p><p>The vibrational distribution g(3 À 3 F ; h) is given the normal Gaussian form. The Gaussian width is D ¼ 2Olk B T z 0.2-0.3 eV for l ¼ 0.5-1.0 eV as in "normal" strong-coupling electrochemical ET. This limit implies that the current varies from quadratic to activationless overpotential dependence over a range of $0.5 V. Since the Gaussian width 2Olk B T signicantly exceeds the "width" of the Fermi function the current monitors essentially the Gaussian vibrational energy density up to overpotentials around l. In the opposite limit of weak coupling, the Gaussian width approaches the "width" of the Fermi function, say D ¼ 2Olk B T z 0.04-0.07 eV or (1-2) Â k B T for l ¼ 0.02-0.05 eV, Fig. 7. In the limit of very weak coupling g(3 À 3 F ; h) would assume a Lorentzian form. 31 Current is not recorded in these limits, until the overpotential has taken the maximum of the, now very narrow Gaussian or Lorentzian g(3 À 3 F ; h) function up to the Fermi level, with a very narrow hrange changing the current from "normal" to activationless behavior. What is recorded in the i/h correlations then, is essentially the Fermi function f(3 À 3 F ) and not the vibrational "bandshape" g(3 À 3 F ; h). Fig. 8 illustrates the difference between the strong-and weak-coupling limits.</p><p>Kuznetsov has provided a quantitative formalism in the weak-coupling limit both for ET in homogeneous solution and electrochemical ET processes. 32 A detailed formalism for analogous optical electronic transitions, for which the weakcoupling Lorentzian bandshape limit is much more common, is also available. 33 It thus appears that the i/h correlations obtained accord formally with weak electronic-vibrational coupling but poorly with the much more commonly encountered limit of strong electronic-vibrational coupling. The question regarding physical reasons, why the coupling should be weak particularly for the thiol-derived anthraquinones bound to the Au(111)-electrode surfaces via strong Au-S chemisorption then arises.</p><p>Based on the results from CV and EIS, it can be suggested that the signicant structural reorganization in AQ2-SAMs in reaction (2) can be attributed to the impeding effect of goldcarbonyl group interaction on the semi-quinone formation. This is different from the nuclear reorganization in the elementary faradaic processes and resembles autoinhibition in electrochemical systems where a mercury electrode surface is saturated with target adsorbate compounds. 34,35 Together with the unexpected voltammetric behavior in the i/h pattern for both AQ1-and AQ2-SAMs (Fig. 7), with a sharp ((1-2) Â k B T) transition between "normal" and activationless i/h behavior, the major ndings of our study can then be summarized as:</p><p>(I) The anthraquinone molecules in the AQ2-SAMs are in close proximity to the electrode surface leading to strong goldcarbonyl group interaction, in contrast to anthraquinone molecules in the AQ1 adlayers.</p><p>(II) Gold-carbonyl interactions create an energy barrier, leading to a split CV redox wave (denoted as reactions ( 1) and ( 2)) and a narrowing of the peak attributed to reaction (1) (inset in Fig. 6).</p><p>(III) The strong gold-carbonyl group interactions in the AQ2-SAMs are supported by the Nernstian pH dependence of both reactions ( 1) and ( 2) (Fig. 1), as well by the signicant difference Fig. 8 The dependence of normalized transition probability (with respect to h ¼ 0 V) and the Fermi function on the electronic energy (A), together with calculated normalized i/h relationship (B), for selected lvalues. The transition probability was calculated using E g 8, and the i/h relationships using a reformulation of eqn (6) (eqn S2 †).</p><p>in the R p -potential dependence between AQ1-and AQ2-SAMs (Fig. 6).</p><p>(IV) The clear R p changes can be interpreted as structural reorganization events in the AQ-SAMs.</p><p>(V) Structural reorganization in reaction ( 2) is signicantly more pronounced than in reaction (1) (Fig. 6).</p><p>(VI) Pseudocapacitance for reaction ( 2) is almost negligible compared to reaction (1) (Fig. 5).</p><p>(VII) A very small environmental reorganization (free) energy accompanies the PCET processes in both AQ1-and AQ2-SAMs (Fig. 7 and 8).</p><p>Regarding point (V), it might be speculated that prior to reaction (2), anthraquinone molecules in the AQ2-SAM interact with gold either via a single or both carbonyl groups. These binding modes could result in SAMs composed of specic differently organized domains (hypothetical State 1). The STM data offer some support for such a view, Fig. S1 † Aer completion of reaction ( 2), the resulting semi-quinones maintain interaction with gold via a single remaining carbonyl group (hypothetical State 2). The structural reorganization from State 2 into fully reduced AQ2-SAM might be lower in reaction (1) than in reaction (2), since the molecular orientation in State 1 is more random than in State 2.</p><p>Point VI suggests that the appearance of reaction (2) in the cyclic voltammograms of AQ2-SAMs is of complex nature. As a comparison, intermolecular interaction of sulfonated anthraquinones gives narrow spike-like CV features. 34 This is in contrast to the observed broad reaction (2) CV features of AQ2-SAMs, and probably associated with greater disorder of State 1 in the AQ2-SAM, than in layers composed of loosely adsorbed anthraquinones at mercury electrodes. 34 Correspondingly the transition from State 1 to State 2 could result in a decrease of AQ2-SAM compactness, which might rationalize that the observed apparent rate for reaction (2) is about twice higher than for reaction (1) (Fig. 7B).</p><p>The notable observation in point VII remains open. AQ2 layers are expected to be more rigid than AQ1 layers, due to single Au-S binding for AQ1 and binding by either a single or two Au-S bonds for AQ2, reected in around twofold higher apparent l and about an order of magnitude higher faradaic resistance for AQ1 than for AQ2. On the other hand, the apparent l-value for AQ1-SAMs is also "unexpectedly small". Furthermore, the ET distances for both AQ1 and AQ2 SAMs are small (i.e. below 1 nm), and the possibly of planar (or close to planar) molecular orientation could indeed result in small reorganization energies. It can therefore be proposed that overall AQ-SAMs rigidity and close proximity of anthraquinone molecules to the electrode surface would result in the small l observed.</p><p>"Small" apparent l-values for "simple" electrochemical ET processes are known for the mammalian heme redox protein cytochrome c 36,37 and the bacterial blue copper protein azurin. 38,39 These values are, however, still signicantly larger, z0.25 eV or so, than the apparent l-values presently observed. Although structurally "small", these proteins are also still complex molecules compared with AQ1 and AQ2, and offer options for more complex, multi-step electrochemical ET that involve e.g. structural gating, pre-organization in the protein conformational systems, or other overpotential independent elementary steps that could lower the apparent l-values in the overall process.</p><p>A second rationale for small l-values could be that quinoneto-hydroquinone interconversion involves both ET and PT in an overall PCET process. PCET processes can involve all degrees of coupling between the elementary ET and PT steps. 40,41 ET and PT can be independent, vibrationally fully relaxed events, each involving charge transfer and signicant environmental reorganization, but the steps are "coupled" in the sense that a given, say ET step affects the kinetic parameters of the subsequent PT step, or vice versa. In the opposite limit ET and PT are fully coupled invoking the character of the quinone-to-hydroquinone conversion as a hydrogen atom transfer process. An electrostatically neutral particle is then transferred, with little solvent reorganization. This expectation also applies when the time sequence between separate ET and PT steps is shorter than the solvent relaxation time (z10 À11 s) in the interfacial electrode surface region. All the limits can be considered in the AQ1 and AQ2 processes, but strong coupling between the ET and PT steps is needed to rationalize the small reorganization energies observed. Reorganization in the intramolecular nuclear modes would not be reected conspicuously in the i/h correlations, as the appropriate C-C, C-H and O-H modes involve high vibrational frequencies represented by nuclear tunnelling in the preexponential factor of the current density forms rather than in the h-dependent activation factors. 31</p><!><p>We have synthesized new thiol-derived anthraquinones with both a single (AQ1) and two (AQ2) thiol groups linking the molecules in SAMs to a single-crystal, atomically planar Au(111)-electrode surface via either a single or two Au-S bonds. We have explored the electrochemical SAM behavior using electrochemical techniques particularly CV and EIS. The electrochemical studies addressed voltammetry and EIS of both the quinone and the -SH moieties as well as crucial potential dependent structural reorganization events of the surface bound target molecular SAMs. Overarching objectives were, rst to introduce a class of challenging electrochemical probe molecules with prospects as building blocks in new "smart" materials as hybrids with Au-nanoparticles and in other ways. The anthraquinone thiol SAMs on Au(111) electrodes offer, secondly sensitive probes for fundamental structural reorganization studies arising from low single-crystal surface roughness and a direct dependence of redox center proximity to gold on the number of binding sites. The reorganization events can therefore also be probed by potential dependent polarization resistance.</p><p>Faradaic monolayer CV and EIS analysis based on interfacial capacitance and resistance, and interfacial electrochemical ET rate constants has led to a coherent view of the elementary electrochemical ET processes and other elementary reorganization steps that accompany the conversion between the fully oxidized and fully reduced AQ1 and AQ2 target molecules. It was found, notably that gold-carbonyl group interactions effectively impede formation of semi-quinones, which results in signicant reorganization events that can be attributed to specic EC steps. Unexpectedly and also notably, very small apparent lvalues were observed for both AQ1-and AQ2-SAMs, probably associated with the layer rigidity, close proximity of anthraquinone thiols to the electrode surface, as well as strong coupling between the ET and PT steps.</p><p>The outcome of the study has disclosed novel features of thiol-derived anthraquinones based on different electrochemical techniques targeting the interactions of both the molecular quinone and the thiol moieties with the single-crystal Au(111)-surfaces. The work offers other steps towards understanding of thiol-derived quinones also bound to gold nanoclusters which may have more direct impact in molecular scale electronics than planar electrode surfaces. The apparently weak electronic-vibrational coupling might here hold advantages by reduced thermal broadening and noise in the electronic functions to be targeted.</p><!><p>All reagents and materials were purchased from well-known chemical suppliers and used without further purication. Aqueous solutions were prepared with freshly deionized water (18.2 MU cm resistivity) obtained with the Sartorius ultrapure water system. Synthesis of 2,6-bis(3-hydroxyprop-1-yn-1-yl)anthracene-9,10dione (3)</p><p>The compound was prepared using Sonogashira coupling conditions between a terminal alkyne species and an arylbromide. The reaction was performed under inert conditions to prevent undesired homolytic coupling of the terminal alkyne. In an oven-dried, condenser-equipped and degassed round-bottomed ask, containing a magnetic stirring bar, a 1 : 1 solution of tetrahydrofuran (THF, 44 mL) and diisopropylamine (DIPA, 44 mL), the crystalline compounds 2,6dibromoanthracene-9,10-dione 1 (0.354 mg, 0.968 mmol, 1 equiv.), CuI (9.2 mg, 48.4 mmol, 0.05 equiv.), PdCl 2 (PPh 3 ) 2 (13.6 mg, 19.4 mmol, 0.02 equiv.) and PPh 3 (12.7 mg, 48.4 mmol, 0.05 equiv.) were dissolved, followed by 30 minutes of degassing. Propargyl alcohol (170.0 mL, 2.904 mmol, 3 equiv.) was added dropwise to the solution and the reaction mixture was set to stir for 24 h under reux. The reaction was quenched with H 2 O (50 mL) followed by three consecutive extractions with Et 2 O (3 Â 25 mL). The organic phase was collected and dried over Na 2 SO 4 , ltered and concentrated in vacuo. The remaining grey precipitate was recrystallized in EtOAc and ltered, isolating 3 (232.6 mg, 76%); 1 H NMR (400 MHz, DMSO-d 6 ) d 8.17 The compound was prepared using Sonogashira coupling conditions between a terminal alkyne specie and an arylbromide. The reaction was performed under inert conditions to prevent undesired homolytic coupling of the terminal alkyne. In an oven-dried, condenser-equipped and degassed roundbottomed ask, containing a magnetic stirring bar, a 1 : 1 solution of THF (36 mL) and DIPEA (36 mL), the crystalline compounds 2-bromoanthracene-9,10-dione 2 (294 mg, 1.03 mmol, 1 equiv.), CuI (9.79 mg, 51.4 mmol, 0.05 equiv.), PdCl 2 (PPh 3 ) 2 (14.4 mg, 20.6 mmol, 0.02 equiv.) and PPh 3 (13.48 mg, 51.4 mmol, 0.05 equiv.) were dissolved, followed by 30 minutes of degas. Propargyl alcohol (178.0 mL, 3.09 mmol, 3 equiv.) was added dropwise to the solution and the reaction mixture was set to stir for 48 h under reux. The reaction was quenched with H 2 O (50 mL) followed by three consecutive extractions with Et 2 O (3 Â 25 mL). The organic phase was collected and dried over Na 2 SO 4 , ltered and concentrated in vacuo. The remaining grey precipitate was recrystallized from EtOAc, washed with heptane and ltered, isolating 4 (179 mg, 66%); 1 The compound was prepared from 3, using Mitsunobu conditions to carry out a thioesterication of the primary alcohols. In an oven-dried and degassed round-bottomed ask, containing a magnetic stirring bar, 3 (200 mg, 0.63 mmol, 1 equiv.), dry THF (50.4 mL), diethyl azodicarboxylate (DEAD, 40 wt% in toluene, 379.7 mL, 1.26 mmol, 2 equiv.) and PPh 3 (328.0 mg, 1.26 mmol, 2 equiv), thioacetic acid (95.2 mL, 1.26 mmol, 2 equiv.) was added and the reaction was le for 48 h. The reaction mixture was concentrated in vacuo followed by purication by ash column chromatography (CH 2 Cl 2 ), isolating 5 (180 mg, 61%); 1 The compound was prepared from 4, using Mitsunobu conditions to carry out a thioesterication of the primary alcohol. In an oven-dried and degassed round-bottomed ask, containing a magnetic stirring bar, 4 (113.3 mg, 0.43 mmol, 1 equiv.), dry THF (34.4 mL), DEAD (40 wt% in toluene, 195. 9 mL, 0.65 mmol, 1.5 equiv.) and PPh 3 (169.2 mg, 0.65 mmol, 1.5 equiv.), thioacetic acid (49.1 mL, 0.65 mmol, 1.5 equiv.) was added and the reaction was le overnight. The reaction mixture was concentrated in vacuo followed by purication by ash column chromatography (CH 2 Cl 2 ), isolating 6 (31.3 mg, 23%); 1</p><!><p>In-house made Clavilier-type bead Au(111) electrodes (ca. 0.04 cm 2 ) were annealed at 850 C for 8 h. Compounds 5 or 6 were dissolved in 4 mL of isopropanol (0.1 mmol), mixed with 1 mL of 25% NH 3 (aq.), and kept in sealed container at 100 C for 8 h, using a microwave synthesizer (initiator, biotage), in order to remove acetyl groups. Au(111) electrodes were annealed in a hydrogen ame, quenched in ultrapure water saturated with dihydrogen, and further immersed in the solutions containing target compound for ca. 24 h. Finally, the samples were kept for 40 min overall in ethanol and subsequently in ultrapure water, prior to each electrochemical experiment.</p><!><p>Both CV and EIS studies were performed using three-electrode one-compartment glass cells and an Autolab potentiostat/ galvanostat (PGSTAT 12, Metrohm) controlled by the Nova 2.0 soware. All measurements were performed at room temperature (23 AE 2 C), using bead Au(111) working electrodes and Pt coiled-wire counter electrodes. The reference electrode was a reversible hydrogen electrode (RHE) (lled with the same supporting electrolyte as in the cell) prepared before each electrochemical experiment. The reported values of the applied potential are shown in Fig. 1 aer recalculation to standard hydrogen electrode (SHE). EIS studies were performed at different potentials (from 0.55 to 0 V vs. RHE, DE ¼ 0.05 V) in the frequency range of 100 kHz to 0.1 Hz (61 data points per measurement) by using an excitation amplitude of 0.01 V. Before each EIS measurement a constant potential, which corresponded to specic DE was applied for 2 min. The Nova 2.0 soware was used for the tting of the impedance spectra. Tafel plots were constructed from selected CV experiments at various scan rates, ranging from 0.005 to 40 V s À1 . Prior to each CV or EIS experiment the electrolyte solutions were degassed with Ar gas for 30 min and an Ar atmosphere maintained above the electrolytes for the whole duration of electrochemical experiments. The electrolytes used were the following: 0.1 HClO 4 (pH 1), 0.1 M KH 2 PO 4 (pH 4.5), 0.1 M phosphate buffer (pH 7), and 0.1 M NaOH (pH 13). All glassware was cleaned prior each experiment by boiling in 15% HNO 3 (aq.) for ca. 20 min.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.

calpain_activation_mediates_microgravity-induced_myocardial_abnormalities_in_mice_via_p38_and_erk1/2

<!>Deficiency of Capns1 inhibits NADPH oxidase activation and oxidative stress in tail-suspended mouse hearts<!><!>Calpain deficiency is associated with reduced phosphorylation of Ser-345 on p47phox in tail-suspended mouse hearts and cultured cardiomyocytes under microgravity<!><!>MAPK signaling mediates calpain-promoted phosphorylation of Ser-345 on p47phox during microgravity<!><!>Inhibition of calpain does not affect MuRF1 and atrogin-1 expression in cultured cardiomyocytes under microgravity<!>Discussion<!>Animals<!>Experimental protocol<!>Echocardiography<!>Histological analyses<!>Determination of protein and DNA concentrations<!>Determination of oxidative stress levels in hearts<!>Neonatal mouse cardiomyocyte cultures and simulated microgravity<!>Western blotting analysis<!>Quantification of mitochondrial DNA copies by real-time PCR<!>Calpain activity<!>NADPH oxidase activation<!>Xanthine oxidase activity<!>SOD activity, catalase activity, and GPx activity<!>Statistical analysis<!>Data availability<!>Supplementary Material<!>

<p>Edited by Roger J. Colbran</p><p>Spaceflight or microgravity conditions are associated with myocardial atrophy, which contributes to the functional depression of the heart (1, 2, 3). Depressed myocardial function is an important factor leading to post-flight orthostatic intolerance (4, 5). However, the exact mechanisms that govern the regulation of myocardial atrophy in microgravity are incompletely understood.</p><p>Protein degradation systems play important roles in muscle atrophy (6). There are three major protein degradation systems in the cardiovascular system, including the calpain system, autophagy, and the ubiquitin proteasome system (7). Previous studies investigated the muscle RING finger protein-1 (MuRF1), an E3 ubiquitin ligase, and atrogin-1, a striated muscle-specific ubiquitin ligase, in the development of cardiac muscle atrophy under certain conditions (8, 9, 10). It was reported that autophagy is induced during regression of cardiac hypertrophy by unloading of the heart (11, 12) and in myocardial atrophy induced by tail suspension (13), a condition simulating microgravity. Of note, inhibition of autophagy induction reduced myocardial atrophy and dysfunction in tail-suspended rats (13), indicating an important role of autophagy in promoting myocardial atrophy. However, little information is available on the role of calpain in myocardial atrophy under microgravity.</p><p>Calpains belong to a family of calcium-dependent proteases with 15 identified isoforms. Two major isoforms of calpain, calpain-1 and calpain-2, are heterodimers consisting of distinct large 80-kDa catalytic subunits encoded by Capn1 and Capn2, respectively, and a common small 28-kDa regulatory subunit encoded by Capns1. The regulatory subunit is indispensable for calpain-1 and calpain-2 activities, and thus, deficiency of Capns1 disrupts calpain-1 and calpain-2 (14). Calpains have been implicated in cardiac injury due to a variety of stressors and the progression of heart failure (15, 16, 17, 18, 19, 20, 21, 22, 23). A previous study demonstrated that mechanical unloading of the heart activated the calpain system and induced myocardial atrophy (24). Interestingly, calpain activation was observed in hearts of rats with tail suspension (25). These prior studies suggest a possible role of calpain in microgravity-induced myocardial atrophy.</p><p>NADPH oxidase-derived ROS contributes to muscle atrophy under various conditions (2, 26). In cardiomyocytes, NOX2-containing NADPH oxidase and NOX4 are two predominant isoforms of NADPH oxidase (27, 28). The NOX2-containing NADPH oxidase is composed of a membrane-bound complex (NOX2 or gp91phox and p22phox) and a cytosolic complex (p40phox, p47phox, p67phox, and a small G protein, Rac1). Activation of NADPH oxidase initiates multiple steps, mainly including phosphorylation of cytosolic subunits (e.g. p47phox), their translocation to the membranes, and the assembly of both cytosolic and membrane-bound complexes (27, 29). p47phox has multiple serine phosphorylation sites, among which Ser-345 is located in a mitogen-activated protein kinase (MAPK) consensus sequence that is phosphorylated by p38 and ERK1/2 of the MAPK family (29). Phosphorylation of Ser-345 on p47phox causes conformational changes of p47phox by binding the proline isomerase Pin1, thereby facilitating its phosphorylation on other sites, leading to full activation of the NADPH oxidase (29). We recently reported that selective inhibition of NADPH oxidase preserved cardiomyocyte size and heart mass and improved myocardial function in tail-suspended mice (30), underscoring an important role of NADPH oxidase in microgravity-induced myocardial abnormalities. However, it remains unknown how NADPH oxidase activation is modulated in response to microgravity, and it has never been shown whether calpain regulates NADPH oxidase activation under microgravity.</p><p>In this study, we investigated whether and how calpain promotes NADPH oxidase activation and oxidative damage, leading to myocardial abnormalities during simulated microgravity.</p><p>Tail suspension induces calpain activation, myocardial atrophy, and dysfunction in mice. Adult male mice were subjected to tail suspension for 14 or 28 days. A, hind limb muscle mass. Data are mean ± S.D. (error bars), n = 5–7 in each group. One-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (F = 59.01, p < 0.0001). *, p < 0.05 versus sham. B, calpain activities in heart tissues from Capns1-KO mice and their WT littermates. Data are mean ± S.D., n = 6 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (interaction, F = 19.27, p < 0.0001; row factor, F = 33.81, p < 0.0001; column factor, F = 56.78, p < 0.0001). *, p < 0.05 versus sham; †, p < 0.05 versus 14 days after tail suspension in WT; #, p < 0.05 versus 28 days after tail suspension in WT. C, the ratio of heart weight relative to tibia length. Data are mean ± S.D., n = 5–7 in each group. One-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (F = 75.13, p < 0.0001). *, p < 0.05 versus sham; †, p < 0.05 versus 14 days after tail suspension. D, fractional shortening (%) was analyzed by echocardiography. Data are mean ± S.D., n = 5–7 in each group. One-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (F = 32.44, p < 0.0001). *, p < 0.05 versus sham; †, p < 0.05 versus 14 days after tail suspension. E, top, a representative staining of wheat germ agglutinin in heart tissue sections; bottom, quantitation of cardiomyocyte cross-sectional area. Data are mean ± S.D., n = 5–6 in each group. One-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (F = 100.8, p < 0.0001). *, p < 0.05 versus sham; †, p < 0.05 versus 14 days after tail suspension.</p><p>Tail suspension induces myocardial atrophy and dysfunction in WT mice, which are attenuated in Capns1-knockout mice. Mice with cardiomyocyte-specific deletion of Capns1 (KO) and their WT littermates (WT) were subjected to tail suspension (TS) for 28 days. A, the heart weight/tibia length ratio was decreased in tail-suspended WT mice. Deletion of Capns1 prevented the decrease of the heart weight/tibia length ratio. Data are mean ± S.D. (error bars), n = 5–7 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (interaction, F = 5.749, p = 0.0264; row factor, F = 19.55, p = 0.0003; column factor, F = 1.819, p = 0.1925). *, p < 0.05 versus sham + WT; †, p < 0.05 versus TS + WT. B, a 28-day TS resulted in lower cardiomyocyte cross-sectional area in WT mice, which was prevented in KO mice. Data are mean ± S.D., n = 4–6 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (interaction, F = 8.137, p = 0.011; row factor, F = 23.48, p = 0.0002; column factor, F = 1.929, p = 0.1828). *, p < 0.05 versus sham + WT; †, p < 0.05 versus TS + WT. C, TS resulted in myocardial dysfunction in WT mice as determined by lower fractional shortening (%), which was prevented in KO mice. Data are mean ± S.D., n = 7 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (interaction, F = 18.76, p = 0.0002; row factor, F = 42.35, p < 0.0001; column factor, F = 8.51, p = 0.0075). *, p < 0.05 versus sham + WT; †, p < 0.05 versus TS + WT.</p><p>Tail suspension induces NADPH oxidase activation in WT mice, which is prevented in Capns1-knockout mice. Mice with cardiomyocyte-specific deletion of Capns1 (KO) and their WT littermates (WT) were subjected to TS for 14 or 28 days. Cell membranes were isolated from mouse hearts, and NADPH oxidase activation was determined by measuring p47phox, p67phox, and Rac1 in cell membrane fractions relative to Na+/K+-ATPase. A, representative Western blots from three different hearts for p47phox, p67phox, and Rac1 in cell membranes (top) and whole heart lysates (bottom). B, a representative Western blot from two of six different hearts for p47phox, p67phox, Rac1, and Na+/K+-ATPase in the membranes. C–E, quantitation for p67phox (C), p47phox (D), and Rac1 (E) relative to Na+/K+-ATPase. Data are mean ± S.D. (error bars), n = 6 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis. C, interaction, F = 22.28, p < 0.0001; row factor, F = 3.988, p = 0.052; column factor, F = 2.522, p = 0.1194. D, interaction, F = 12.62, p = 0.002; row factor, F = 6.15, p = 0.0222; column factor, F = 1.455, p = 0.2417. E, interaction, F = 6.97, p = 0.0134; row factor, F = 1.898, p = 0.1792; column factor, F = 7.08, p = 0.0128. *, p < 0.05 versus sham + WT; †, p < 0.05 versus TS + WT.</p><p>Deletion of Capns1 inhibits tail suspension-induced oxidative stress in mouse hearts. Mice with cardiomyocyte-specific deletion of Capns1 (KO) and their WT littermates (WT) were subjected to TS for 28 days. Oxidative stress was assessed by measuring ROS production (A), MDA production (B), and protein carbonyl content (C) in mouse hearts. Data are mean ± S.D. (error bars), n = 4–7 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis. A, interaction, F = 3.358, p = 0.0855; row factor, F = 5.995, p = 0.0263; column factor, F = 5.915, p = 0.0271. B, interaction, F = 9.633, p = 0.0073; row factor, F = 1.004, p = 0.3322; column factor, F = 12.56, p = 0.0029. C, interaction, F = 7.275, p = 0.0194; row factor, F = 2.682, p = 0.1274; column factor, F = 9.306, p = 0.0101. *, p < 0.05 versus sham + WT; †, p < 0.05 versus TS + WT.</p><!><p>Because cardiomyocytes predominantly express NOX2 and NOX4, we also analyzed NOX4 expression, as NOX4 is primarily regulated through transcriptional mechanisms (31). Tail suspension resulted in higher protein levels of NOX4 in tail-suspended mouse hearts (Fig. S2). However, deficiency of Capns1 did not change the higher protein levels of NOX4 due to tail suspension (Fig. S2). In addition, neither tail suspension nor deletion of Capns1 changed mitochondrial ROS generation and xanthine oxidase activity in tail-suspended mouse hearts (Fig. S3 (A–C)).</p><p>Increased ROS production and consequent oxidative damage may also result from a defect of antioxidant systems. Therefore, we measured the main antioxidant enzymes in hearts. As shown in Fig. S4 (A–C), neither tail suspension nor deletion of Capns1 changed the activities of SOD, GPx, and catalase in heart tissues. These results exclude the possibility that changes of antioxidant enzymes play a role in calpain activation-associated oxidative stress in tail-suspended mouse hearts.</p><!><p>Inhibition of calpain prevents phosphorylation of Ser-345 on p47phox in tail-suspended mouse hearts and cultured cardiomyocytes in response to simulated microgravity.A and B, mice with cardiomyocyte-specific deletion of Capns1 (KO) and their WT littermates (WT) were subjected to TS for 14 or 28 days. A, a representative Western blot from three different hearts in each group for phosphorylated p47phox (Ser-345) and total p47 phox. B, top, a representative Western blot from two of six different hearts in each group for phosphorylated p47phox (Ser-345) and total p47 phox; bottom, quantitation for phosphorylated p47phox (Ser-345) relative to total p47 phox. Data are mean ± S.D. (error bars), n = 6 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis (interaction, F = 14.2, p = 0.0008; row factor, F = 27.2, p < 0.0001; column factor, F = 9.388, p = 0.0048). *, p < 0.05 versus sham + WT or sham + KO. †, p < 0.05 versus TS + WT or sham + KO. C–E, neonatal mouse cardiomyocytes were subjected to a condition of simulated microgravity (SMG) in the presence of calpain inhibitor-III (CI-III) or vehicle for 24 h. C, calpain activities. D, total protein/DNA ratio in cardiomyocyte lysates. E, top, a representative Western blot from three different cell cultures with each in duplicate for phosphorylated p47phox (Ser-345) relative to total p47phox; bottom, quantitation for phosphorylated p47phox (Ser-345) relative to total p47phox. Data are mean ± S.D., n = 3–5 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis. C, interaction, F = 11.75, p = 0.0090; row factor, F = 18.26, p = 0.0027; column factor, F = 12.13, p = 0.0083. D, interaction, F = 7.366, p = 0.0153; row factor, F = 86.86, p < 0.0001; column factor, F = 9.917, p = 0.0062. E, interaction, F = 5.361, p = 0.0313; row factor, F = 31.8, p < 0.0001; column factor, F = 5.368, p = 0.0312. *, p < 0.05 versus sham + vehicle; †, p < 0.05 versus SMG + vehicle.</p><!><p>To provide further evidence supporting the role of calpain in promoting p47phox phosphorylation, we simulated microgravity in cultured cardiomyocytes using the rotary cell culture system. Simulated microgravity increased calpain activities (Fig. 5C), and this was correlated with a reduction of total protein/DNA ratio (Fig. 5D) and an increase in phosphorylation of Ser-345 on p47phox (Fig. 5E). Incubation with calpain inhibitor-III prevented calpain activation, increased total protein/DNA ratio, and attenuated microgravity-induced phosphorylation of Ser-345 on p47phox in cardiomyocytes (Fig. 5, C–E). These results recapitulated the inhibitory effect of calpain disruption on p47phox phosphorylation in tail-suspended mouse hearts.</p><!><p>Tail suspension induces activation of p38 and ERK1/2 in WT mice, which is attenuated in Capns1-knockout mice, and inhibition of calpain attenuates phosphorylation of ERK1/2 and p38 in cardiomyocytes under microgravity.A–C, mice with cardiomyocyte-specific deletion of Capns1 (KO) and their WT littermates (WT) were subjected to tail suspension (TS) for 14 or 28 days. A, a representative Western blot from three different hearts in each group for total and phosphorylated p38 and ERK1/2. B, top, a representative Western blot from two of six different hearts in each group for total and phosphorylated ERK1/2; bottom, quantitation for phosphorylated ERK1/2 (p-ERK1/2) relative to total ERK1/2. C, top, a representative Western blot from two of six different hearts in each group for total and phosphorylated p38; bottom, quantitation for phosphorylated p38 (p-p38) relative to total p38. Data are mean ± S.D. (error bars), n = 6 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis. B, interaction, F = 39.53, p < 0.0001; row factor, F = 0.1185, p = 0.7366; column factor, F = 19.48, p = 0.0008. C, interaction, F = 82.32, p < 0.0001; row factor, F = 14.01, p = 0.0028; column factor, F = 101.8, p < 0.0001. *, p < 0.05 versus sham + WT; †, p < 0.05 versus KO + vehicle. D–F, neonatal mouse cardiomyocytes were subjected to SMG in the presence of calpain inhibitor-III or vehicle for 6 and 24 h. D, a representative Western blot from three different cell cultures for total and phosphorylated p38 and ERK1/2. E, top, a representative Western blot from three different cell cultures with each in duplicate for total and phosphorylated ERK1/2; bottom, quantitation for phosphorylated ERK1/2 (p-ERK1/2) relative to total ERK1/2. F, top, a representative Western blot from three different cell cultures with each in duplicate for total and phosphorylated p38; bottom, quantitation for phosphorylated p38 (p-p38) relative to total p38. Data are mean ± S.D., n = 3 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis. E, interaction, F = 46.37, p < 0.0001; row factor, F = 5.254, p = 0.0329; column factor, F = 42.27, p < 0.0001. F, interaction, F = 7.9, p = 0.0108; row factor, F = 155.2, p < 0.0001; column factor, F = 5.059, p = 0.0359. *, p < 0.05 versus sham + vehicle; †, p < 0.05 versus SMG + vehicle.</p><!><p>To further address this in cultured cardiomyocytes, we showed that the protein levels of phosphorylated ERK1/2 were higher 6 h but not 24 h after simulated microgravity (Fig. 6D). Incubation with calpain inhibitor-III prevented ERK1/2 phosphorylation in cardiomyocytes 6 h after simulated microgravity (Fig. 6E).</p><p>The protein levels of phosphorylated p38 were higher 6 h and even higher 24 h after simulated microgravity (Fig. 6D). We then chose to determine the effect of calpain inhibition on p38 phosphorylation 24 h after simulated microgravity. Similarly, incubation with calpain inhibitor-III prevented p38 phosphorylation (Fig. 6F).</p><!><p>Inhibition of ERK1/2 and p38 prevents phosphorylation of Ser-345 on p47phox in cultured cardiomyocytes in response to simulated microgravity. Neonatal mouse cardiomyocytes were subjected to SMG in the presence of PD98059, SB203580, or vehicle for 24 h. A and B, top, a representative Western blot from three different cell cultures with each in duplicate for total and phosphorylated p47phox (Ser-345); Bottom, quantitation for phosphorylated p47phox relative to total p47phox. C, effect of ERK1/2 inhibition on p38 phosphorylation in cardiomyocytes after SMG. Top, a representative Western blot from three different cell cultures with each in duplicate for total and phosphorylated p38; bottom, quantitation for phosphorylated p38 (p-p38) relative to total p38. Data are mean ± S.D. (error bars), n = 3 in each group. Two-way ANOVA followed by Newman–Keuls test was performed for statistical analysis. A, interaction, F = 5.958, p = 0.0311; row factor, F = 15.48, p = 0.002; column factor, F = 3.788, p = 0.0754. B, interaction, F = 8.13, p = 0.0145; row factor, F = 3.085, p = 0.1045; column factor, F = 2.035, p = 0.1792. C, interaction, F = 0.1223, p = 0.7356; row factor, F = 48.81, p = 0.0001; column factor, F = 0.29, p = 0.6049. *, p < 0.05 versus sham + vehicle; †, p < 0.05 versus SMG + vehicle.</p><!><p>Because both MuRF1 (8, 9, 10) and atrogin-1 (32, 33) have been implicated in promoting cardiac muscle atrophy or inhibiting cardiac hypertrophy, we also analyzed the protein levels of MuRF1 and atrogin-1 in cardiomyocytes. Simulated microgravity resulted in higher levels of MuRF1 and atrogin-1 in cultured cardiomyocytes; however, inhibition of calpain did not change the protein levels of MuRF1 and atrogin-1 in cardiomyocytes under normal conditions or microgravity (Fig. S6).</p><!><p>The major findings of this study are that disruption of calpain preserves cardiomyocyte size, heart mass, and myocardial function in tail-suspended mice, indicating an important role of calpain in myocardial atrophy under microgravity. Furthermore, calpain activation mediates NADPH oxidase activation by facilitating p38 and ERK1/2 activation and subsequent phosphorylation of Ser-345 on p47phox in cardiomyocytes in response to microgravity. Thus, this study also reveals an unrecognized role of calpain in promoting NADPH oxidase activation via p38 and ERK1/2 signaling during microgravity.</p><p>Calpain activation was reported in heart tissues from tail-suspended rats (25) and unloading mouse and human hearts (24). In line with these prior observations, this study demonstrates that tail suspension induces calpain activation in mouse hearts. Importantly, deletion of Capns1 preserved cardiomyocyte size and heart mass and protected myocardial function in tail-suspended mice, underscoring a critical role of calpain in microgravity-induced myocardial atrophy and dysfunction. Our in vitro study using cultured cardiomyocytes further supports the role of calpain in myocardial atrophy, as inhibition of calpain prevented the reduction of protein/DNA ratio after simulated microgravity. Although a previous study reported that transgenic overexpression of calpastatin failed to reduce myocardial atrophy in a mouse model of heart transplantation (24), the transplanted heart does not have any loading of its left ventricular chamber, a condition totally different from the heart of a tail-suspended mouse. As further evidence in support of our finding, inhibition of calpain by transgenic overexpression of calpastatin slowed muscle waste during murine muscle disuse (34). Additionally, there is usually a compensatory increase in catecholamine release after returning from a long-term spaceflight (25). We have reported that incubation with the catecholamine, norepinephrine, induces calpain activation and apoptosis in cardiomyocytes (35). Along with this, a prior study showed that calpain activation was induced and contributed to myocardial apoptosis in rats during the recovery period after tail suspension (25). Thus, calpain may represent a useful target for prevention and therapy for myocardial disorders during and after microgravity.</p><p>Our recent study demonstrated that NADPH oxidase is activated and oxidative stress is induced in tail-suspended mouse hearts and that inhibition of NADPH oxidase with apocynin preserves cardiomyocyte size and heart weight and improves myocardial function in tail-suspended mice (30). This finding suggests an important role of NADPH oxidase in myocardial abnormalities under microgravity. Although activation of NADPH oxidase has been implicated in a variety of cardiac diseases (36) and modulation of NADPH oxidase activation has received intensive attention, the molecular mechanisms underlying NADPH oxidase activation remain poorly understood. An important finding of this study is that calpain activation promotes NADPH oxidase activation in tail-suspended mouse hearts. This was demonstrated by determining the translocation of its cytosolic subunits, including p47phox, p67phox, and Rac1, to the cell membranes and concomitant oxidative stress. Our study provides further evidence suggesting that calpain activation promotes phosphorylation of Ser-345 on p47phox in tail-suspended mouse hearts. Studies have implicated ERK1/2 and p38 in mediating phosphorylation of Ser-345 on p47phox (29). In line with this, we report that the protein levels of phosphorylated ERK1/2 and p38 are higher in cultured cardiomyocytes under microgravity and in tail-suspended mouse hearts. Of note, deletion of Capns1 or inhibition of calpain prevented the difference in phosphorylated ERK1/2 and p38 in tail-suspended mouse hearts or cultured cardiomyocytes under microgravity, respectively. Furthermore, pharmacological inhibition of ERK1/2 and p38 attenuated phosphorylation of Ser-345 on p47phox in cultured cardiomyocytes under microgravity. Thus, we argue that deletion of calpain inhibits phosphorylation of Ser-345 on p47phox by blocking ERK1/2 and p38 activation, thereby preventing translocation of cytosolic subunits of NADPH oxidase to the membrane, NADPH oxidase activation, and oxidative stress in tail-suspended mouse hearts. Nevertheless, future studies are needed to determine how calpain mediates ERK1/2 and p38 activation during microgravity. It is important to mention that simulated microgravity induces ERK1/2 and p38 activation 14 days but not 28 days after tail suspension, suggesting that ERK1/2 and p38 activation accounts for NADPH oxidase at the early phase of microgravity. This is indeed supported by in vitro studies using cultured cardiomyocytes. Moreover, inhibition of ERK1/2 or p38 attenuated but did not completely block p47phox phosphorylation in cardiomyocytes induced by simulated microgravity. It is therefore possible that additional mechanisms mediate NADPH oxidase activation and subsequent cardiomyocyte atrophy during the late phase of microgravity, which merits further investigation.</p><p>Interestingly, our data do not support any role of calpain in NOX4 expression in cultured cardiomyocytes and mouse hearts under microgravity. In addition to NOX2-containing NADPH oxidase and NOX4, other main sources of ROS generation are proposed in cardiomyocytes, including mitochondria and xanthine oxidase (37, 38, 39). However, deletion of Capns1 did not change mitochondrial ROS generation and xanthine oxidase activity during microgravity in tail-suspended mouse hearts. It is important to mention that excessive ROS production and subsequent oxidative stress is the consequence of an imbalance between ROS generation and antioxidant mechanisms (40, 41). By examining the main antioxidant enzymes, including SOD, catalase, and GPx, this study demonstrated that deletion of Capns1 had no impact on the activities of these antioxidant enzymes and, thus, rules out the possibility that defects of the main antioxidant enzymes contributed to calpain-mediated oxidative stress in tail-suspended mouse hearts.</p><p>In addition to calpain, autophagy and the ubiquitin proteasome system have been implicated in protein degradation in the cardiovascular system (7), which contributes to muscle atrophy. Indeed, the ubiquitin proteasome system has been reported to promote cardiac muscle atrophy. For example, both MuRF1 and atrogin-1 expression are implicated in promoting cardiac muscle atrophy or inhibiting cardiac muscle hypertrophy, and inhibition of either reduces cardiac muscle atrophy under certain conditions (8, 9, 10, 32, 33). In the present study, we also report that simulated microgravity induces an increase in MuRF1 and atrogin-1 protein levels in cultured cardiomyocytes. However, inhibition of calpain did not attenuate the higher protein levels of MuRF1 and atrogin-1 in cardiomyocytes under microgravity. Considering that calpain mediates NADPH oxidase activation and our recent study demonstrates that inhibition of NADPH oxidase did not affect MuRF1 expression in tail-suspended mouse hearts (30), the calpain/NADPH oxidase pathway may represent a new mechanism contributing to microgravity-induced myocardial atrophy, which is independent of MuRF-1 and atrogin-1.</p><p>Although myocardial atrophy weakens myocardial contractility, the underlying mechanisms remain to be determined. Our data show that tail suspension for 14 and 28 days reduced mitochondrial Mtnd1 DNA copies in mouse hearts. However, ATP production remains unchanged in heart tissues after tail suspension. This disparity between mitochondrial DNA copies and ATP production may be due to a reduction in cardiomyocyte size after tail suspension (by about 33% in Fig. 1E) as a smaller cardiomyocyte may have a lesser number of mitochondria, whereas ATP production remains the same per unit of cell volume. Thus, a reduction of mitochondrial DNA copies is not a potential mechanism for myocardial dysfunction induced by microgravity. Future studies are needed to determine the detailed mechanisms by which microgravity induces myocardial dysfunction.</p><p>In summary, we have demonstrated an important role of calpain in promoting myocardial abnormalities in tail-suspended mice. Thus, targeting calpain may be a useful strategy to protect the heart under conditions of microgravity. Given that pharmacological inhibitors of calpain are under clinical trials, our findings provide important preclinical evidence to support future translational studies.</p><!><p>This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (8th Edition, 2011). Breeding pairs of C57BL/6 mice were purchased from the Jackson Laboratory. Mice with cardiomyocyte-specific disruption of Capns1 (Capns1-KO) were generated by breeding mice bearing the targeted Capns1PZ allele containing loxP sites flanking essential coding exons and mice with cardiomyocyte-specific expression of Cre recombinase under the control of α-myosin heavy chain as we described recently (23). Deletion of Capns1 was confirmed by analyzing Capns1 mRNA expression in Capns1-KO mouse hearts as described previously (23) (Fig. S7). All mice used in this study, including controls, were littermates of the same generation. A breeding program was implemented at Soochow University's animal care facilities. All experimental protocols were approved by the Animal Use Subcommittee at Soochow University (Suzhou, China).</p><!><p>Tail suspension was performed in Capns1-KO mice and their WT littermates (males aged 2 months) to simulate microgravity using the methods described previously (30, 42). Briefly, a piece of tape was attached to both the tail and a swivel tied to a horizontal bar at the top of cage. Mice had free access to food and water during the tail suspension. 14 or 28 days later, mice were subjected to various experiments.</p><!><p>Animals were lightly anesthetized with inhalant isoflurane (0.5–1%) and imaged using a 40-MHz linear array transducer attached to a preclinical ultrasound system (Vevo 2100, FUJIFILM VisualSonics, Toronto, Canada) with nominal in-plane spatial resolution of 40 μm (axial) × 80 μm (lateral). M-mode and two-dimensional parasternal short-axis scans (133 frames/s) at the level of the papillary muscles were used to assess changes in left ventricular (LV) end-systolic inner diameter, LV end-diastolic inner diameter, LV posterior wall thickness in end-diastole and end-systole, fractional shortening (%), and ejection fraction.</p><!><p>Mice were euthanized by cervical dislocation under anesthesia using a mixture of ketamine (100 mg/kg)/xylazine (5 mg/kg, intraperitoneally). Heart tissues were collected, fixed, processed, and sectioned. For cardiomyocyte size measurement, several cross-sections of the whole heart (5 μm thick) were prepared and stained for membranes and nuclei with FITC-conjugated wheat germ agglutinin (Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific), respectively. Single cardiomyocytes were measured using an image quantitative digital analysis system (NIH Image version 1.6) as described previously (43). The outlines of at least 200 cardiomyocytes were traced in each section.</p><!><p>The protein levels were determined by DCTM Protein Assay (Bio-Rad). Genomic DNA was extracted from cultured cardiomyocytes using a Hiyield Genomic DNA Isolation Kit (cultured cells) (Cedarlane Laboratories Ltd., Burlington, Canada) and measured by a Nanodrop spectrophotometer (A260).</p><!><p>The formation of ROS in heart tissue lysates was measured using the Amplex® Red hydrogen peroxide/peroxidase assay kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Briefly, frozen heart tissues were homogenized in an assay buffer. The homogenates (50 μg of protein) were incubated with a fluorescent probe Amplex® Red and hydrogen peroxide/peroxidase at 37 °C. The fluorescent product formed was quantified using a spectrofluorometer measured at 485/525 nm. Changes in fluorescence were expressed as arbitrary units.</p><p>Protein carbonyl content was determined using a protein carbonyl colorimetric assay kit (Cayman Chemical Co.) following the manufacturer's instructions. A total of 700 μg of protein was used for each sample.</p><p>Lipid peroxidation in heart tissue lysates was assessed by measuring MDA production using a TBARS assay kit (Cayman Chemical) following the manufacturer's instructions. A total of 500 μg of protein was used for each sample.</p><!><p>Neonatal mice (born within 24 h) were euthanized by decapitation. Neonatal cardiomyocytes were prepared and cultured in Dulbecco's modified Eagle's medium with 10% newborn calf serum and penicillin-streptomycin according to methods described previously (44).</p><p>Right after isolation, cardiomyocytes (1.5 × 106) were cultured with Cytodex 3 microcarrier beads (175-μm particle size, spherical) for 24 h. To simulate microgravity, we dispersed cardiomyocytes on Cytodex 3 microcarrier beads in 5 ml of Dulbecco's modified Eagle's medium with 10% newborn calf serum and penicillin-streptomycin and then inoculated them through the syringe port inside the 5-ml high-aspect ratio vessels of a rotary cell culture system (RCCS-4, Synthecon Inc.). After being assembled, they were placed on their rotary base and maintained in a 37 °C incubator with a 5% CO2/air mixture and saturating humidity. Vessel rotation was set at 18 rpm according to our recent report (30). After 24 h, cells were collected for Western blotting analysis. For the treatment with various inhibitors (PD98059 (10 μm), SB23580 (10 μm), and calpain inhibitor-III (10 μm)), the inhibitors or vehicle were added to culture media at the time of inoculation into the rotary device. The cells were incubated under simulated microgravity for 24 h.</p><!><p>40 μg of protein from heart tissue or cell lysates or 10 μg of protein from isolated membrane lysates were loaded onto SDS-polyacrylamide gels. After electrophoresis, the separate proteins were transferred onto Bio-Rad polyvinylidene difluoride membranes. After blocking in 5% nonfat milk for 1 h, the membranes were incubated with antibodies against NOX4 (1:1000 dilution; Abcam), Rac1 (1:1000 dilution; Abcam), MuRF1 and atrogin-1 (1:1000 dilution; Abcam), Na+/K+-ATPase (1:1000 dilution; Abcam), p67phox (1:1000 dilution; Abcam), phosphorylation of Ser-345 and Ser-370 on p47phox and total p47phox (1:1000 dilution; Thermo Fisher Scientific Inc.), phosphorylated p38 and total p38 (1:1000 dilution; Cell Signaling Technology), phosphorylated ERK1/2 and total ERK1/2 (1:1000 dilution; Cell Signaling Technology), and GAPDH (1:5000 dilution; Cell Signaling Technology), respectively, followed by secondary relevant antibodies conjugated with horseradish peroxidase. The signals were then developed using an enhanced version of the chemiluminescence reaction.</p><p>The protein ladders were purchased from FroggaBio Inc. (Concord, Canada) for cultured cardiomyocytes (245, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, and 11 kDa) and Thermo Fisher Scientific China Co. Ltd. for heart tissue samples (170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kDa).</p><!><p>Total DNA was extracted using a DNA extraction kit (Qiagen), following the manufacturer's instructions. Real-time PCR was conducted using primers specific to the mitochondrial Mtnd1 region of the mitochondrial genome and β2-microglobulin (B2M) as a nuclear gene reference. The sequences of primers are as follows: Mtnd1, 5′-GAGGGAACCAAACTGAACGC-3′ and 5′-TGGATCCGTTCGTAGTTGGAG-3′; B2M, 5′-CAGACTCTGCGATGTTTCCA-3′ and 5′-GCCTGAGCACTTCCAGAAAC-3′. The mitochondrial DNA copies were expressed as the ratio of Mtnd1 to B2M.</p><!><p>Calpain activities were measured in tissue and cell lysates (15 μg of protein) using a fluorescence substrate, N-succinyl-LLVY-7-amido-4-methylcoumarin (Cedarlane Laboratories) as described previously (35).</p><!><p>NADPH oxidase activation was determined by measuring the translocation of Rac1, p47phox, and p67phox to cell membranes. Briefly, cell membranes were isolated from heart tissues using a commercial kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. The protein levels of Rac1, p47phox, and p67phox in cell membranes and whole-heart lysates were analyzed using Western blotting. Na+/K+-ATPase and GAPDH were used as loading controls for the membranes and whole lysates, respectively.</p><!><p>Xanthine oxidase activity was determined in heart tissue lysates using a commercial assay kit (Beyotime Biotechnology), following the manufacturer's instructions. A total of 100 μg of protein was used for each sample in the assay.</p><!><p>These activities were measured in heart tissue lysates using commercial assay kits (Beyotime Biotechnology), following the manufacturer's instructions. A total of 100 μg of protein was used for each sample in the assay.</p><!><p>All data are expressed as means ± S.D. Differences between two groups were compared by unpaired Student's t test. For multigroup comparisons, ANOVA followed by Newman–Keuls test was performed. A value of p < 0.05 was considered statistically significant.</p><!><p>All data presented are available upon request from Tianqing Peng (tpeng2@uwo.ca or tqpeng@suda.edu.cn).</p><!><p></p><!><p>Author contributions—L. L, H. L., L. Q., L. Z., G.-C. F., P. A. G., J. L., D. L. J., and T. P. conceptualization; L. L., H. L., L. Q., L. Z., and G.-C. F. data curation; L. L., H. L., T. C., L. Q., L. Z., G.-C. F., and T. P. formal analysis; L. L., L. Q., L. Z., G.-C. F., P. A. G., J. L., D. L. J., and T. P. supervision; L. L. and T. P. funding acquisition; L. L., T. C., L. Q., L. Z., D. L. J., and T. P. validation; L. L., H. L., T. C., L. Q., L. Z., G.-C. F., P. A. G., and J. L. investigation; L. L., H. L., T. C., L. Q., L. Z., G.-C. F., P. A. G., J. L., and D. L. J. methodology; L. L., H. L., L. Q., L. Z., G.-C. F., J. L., and D. L. J. writing-original draft; L. L., T. C., L. Q., L. Z., G.-C. F., P. A. G., J. L., D. L. J., and T. P. writing-review and editing; L. Z. project administration.</p><p>Funding and additional information—This study was supported by the opening foundation of the State Key Laboratory of Space Medicine Fundamentals and Application, Chinese Astronaut Research and Training Center (Grants SMFA15K02 and SMFA18K01), Natural Science Foundation of Jiangsu Province Grant BK20171216, Natural Science Foundation of Zhejiang Province Grant LY14H020005, and Priority Academic Program Development of Jiangsu Higher Education Institutions.</p><p>Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.</p><p>mitogen-activated protein kinase</p><p>glutathione peroxidase</p><p>left ventricular</p><p>malondialdehyde</p><p>reactive oxygen species</p><p>superoxide dismutase</p><p>tail suspension</p><p>knockout</p><p>simulated microgravity</p><p>analysis of variance</p><p>glyceraldehyde-3-phosphate dehydrogenase.</p>

PubMed Open Access

Highly Fluorinated Interphases Enable High-Voltage Li-Metal Batteries

Li metal is regarded as the ''Holy Grail'' electrode because of its highest specific capacity and lowest electrochemical potential. However, challenges arising from the low Coulombic efficiency (CE) and dendritic nature of Li metal in carbonate electrolytes remain to be resolved. Here, by increasing LiFSI salt concentration in the carbonate electrolyte, we successfully increased the CE to 99.3% while suppressing Li dendrite formation. An NMC622jjLi cell was paired and showed excellent cycling performance.

highly_fluorinated_interphases_enable_high-voltage_li-metal_batteries

INTRODUCTION<!>RESULTS AND DISCUSSION<!>X-ray photoelectron spectroscopy (XPS) analysis revealed the chemical compositions of the interphase formed on the Li-metal surface in different electrolytes (Figures 4E and S14<!>EXPERIMENTAL PROCEDURES Materials<!>Material Characterization<!>Electrochemical Measurements<!>DFT Calculations<!>SUPPLEMENTAL INFORMATION<!>AUTHOR CONTRIBUTIONS

<p>Although they dominate the consumer electronics market and are penetrating the electric vehicle market, Li-ion batteries (LIBs) are approaching the upper limit of energy densities that intercalation chemistries can provide. 1 State-of-the-art LIBs with the typical graphite and LiCoO 2 as anode and cathode can deliver a specific energy of $250 Wh/kg, which is an order of magnitude lower than gasoline can deliver. 2 To further enhance the energy density of batteries, more aggressive chemistries are required, one of which is a Li-metal anode. When coupled with a high Ni-content cathode such as LiNi 0.6 Mn 0.2 Co 0.2 O 2 (NMC622), a 500 Wh/kg battery becomes possible.</p><p>Li metal is considered the ultimate anode material because of its unique combination of the highest specific capacity of 3,860 mAh/g and the lowest electrochemical potential (À3.04 V versus standard hydrogen electrode) ever possible from any known materials. [2][3][4][5][6] However, its notoriety in both chemical and electrochemical stability, as demonstrated by limited Coulombic efficiency (CE) and dendrite formation during cycling, prevents its application in a battery environment. [7][8][9][10][11][12][13][14] Extensive work has been devoted to stabilizing Li-metal anodes through approaches including protective layers, [15][16][17][18] electrode designs at nanoscale, [19][20][21][22] electrolyte additives, 14,[23][24][25][26][27] and solid-state electrolytes 15,28 . Among these, ether-based electrolytes present the highest CE and the lowest overpotential, effectively suppressing dendrite growth owing to their low reactivity with Li metal. 2,4,12,14,17,22,[29][30][31][32][33][34][35] Especially, the highest cycling CE of 99.1% was recently realized in 1,2-dimethoxyethane (DME). 4 However, ether-based electrolytes are intrinsically unstable against oxidation on cathode surfaces, as characterized by their typical anodic limits of <4 V, 36 which is much lower</p><p>The Bigger Picture Interest in Li-metal batteries (LMBs) is reviving because higher energy densities can be enabled by the highest specific capacity and the lowest electrochemical potential of a Li-metal anode. However, challenges arising from the low Coulombic efficiency (CE) and dendritic nature of Li metal in carbonate electrolytes remain to be resolved. Here, by increasing LiFSI concentration in carbonate electrolytes (dimethyl carbonate [DMC], propylene carbonate, and ethylene carbonate/DMC) to 10 M, we achieved a high CE ($99.3%) of Li deposition and stripping, along with an anodic stability of >5.5 V. Pairing a Limetal anode in this electrolyte with LiNi 0.6 Mn 0.2 Co 0.2 O 2 (NMC622) at high loading (2.5 mAh/cm 2 ) created a NMC622jjLi cell, which showed a high capacity retention of 86% after 100 cycles at a high cutoff voltage of 4.6 V. LiF-rich and F-rich interphases formed on the Li-metal anode and cathode surfaces, respectively, are responsible for the successful suppression of Li dendrite growth as well as stabilization of the highvoltage cathode. than those of carbonate-based electrolytes. Thus, ether-based electrolytes can only be applied in low-voltage systems such as Li-S, Li-O 2 , and Li-LiFePO 4 . 18,22,32,34 For high energy density LMBs that require Li metal to be paired with a high-voltage, high-capacity cathode such as Ni-rich cathodes, a non-ether electrolyte that can simultaneously stabilize both Li-metal and cathode surfaces must be developed.</p><p>Organic carbonates are exclusively used as electrolyte solvents in almost all commercial LIBs today thanks to their intrinsically high oxidation stability (>4.3 V) and their unique capabilities of forming protective interphases on graphite anodes. However, these electrolytes cannot accommodate the much more stringent requirements presented when graphite and mild transition metal oxides (such as LiCoO 2 ) are replaced by the more aggressive Li-metal and Ni-rich NMC (nickel-manganese-cobalt) materials. 2,10,12,24 Here, we report that by simply increasing the Li bis(fluorosulfonyl)imide (LiFSI) concentration in carbonate electrolytes (propylene carbonate [PC], dimethyl carbonate [DMC], ethylene carbonate [EC]/DMC), a significantly high CE of $99.3% can be achieved with an extremely high cycling stability. The LiF-rich solid-electrolyte interphase (SEI) layer, which is formed mainly from the LiFSI salt reduction, effectively prevents dendrite growth and minimizes the sustained electrolyte decomposition. These highly concentrated electrolytes also stabilize the high-capacity Ni-rich NMC cathode at higher voltage (Figure 1). The simultaneous stabilization of both anode and cathode surfaces in these electrolytes leads to a high energy density NMCjjLi cell, which steadily delivers high energy density at a high cutoff voltage (4.6 V) for extended cycles.</p><!><p>CE truthfully monitors utilization and dendrite formation of metallic Li. A low CE indicates significant consumption of Li + and electrolyte, which will irreversibly result in poor cycling stability and the end of cell life. Li plating/stripping CE, defined as the ratio between the amounts of Li stripped and the amount plated on the Cu substrate, was examined in different electrolytes in 2,032 coin cells. Figures 2A-2D show the voltage profiles of the CujjLi cells at a current density of 0.2 mA/cm À2 in LiFSI-DMC electrolytes at varying salt concentrations. The charge-discharge voltage hysteresis and ionic conductivity of different electrolytes are summarized in Figure 2E, and the Li plating/stripping CEs in different electrolytes are compared in Figure 2F. Here, M stands for molar concentration, i.e., mole of salt dissolved in a liter of solvent. In diluted electrolyte (2 M LiFSI-DMC), the CE is only about 20%, indicating significant irreversible consumption of both Li + and electrolyte solvent as a result of the poor protection provided by the interphase, as revealed in the previous studies. 10 As the LiFSI concentration increases from 2 to 6 M, the CE quickly increased from 20% to 98.7%, eventually reaching 99.2% at 10 M, which is the highest CE ever reported for Li-metal anodes. Equally important is the overpotential between the plating and stripping processes, which becomes much lower in the concentrated carbonate electrolytes, despite their lower bulk ion conductivity than that of diluted electrolytes (Figure 2E), revealing that the interfacial resistances are dramatically reduced as a result of the high salt concentration. As a result of the improved CE and reduced interfacial resistances, the cycling stability of the Li anode in concentrated electrolytes improves dramatically. Although the CE in diluted electrolyte (2 M) never reached >40%, a relatively high CE of about 96% could by obtained in 4 M LiFSI-DMC electrolyte during the initial cycles. However, this CE lasted for only about ten cycles and then dropped to about 20%, which is similar to the diluted electrolyte (Figures 2B and 2F). In sharp comparison, 6 M and 10 M LiFSI-DMC electrolytes enabled up to 200 cycles without any deterioration detected. The Li plating/stripping behavior in Li/Li symmetrical cells was also evaluated in 6 M and 10 M LiFSI-DMC electrolytes (Figure S1). Both electrolytes showed good cycling stability. However, the overpotential in 6 M electrolyte increased at a faster pace than that in 10 M electrolyte. After 250 cycles, the overpotential in 6 M electrolyte was higher.</p><p>The highly reversible nature of Li plating/stripping in concentrated electrolytes is still preserved after DMC is replaced with other carbonate solvents, as shown in Figure 3 (LiFSI-EC/DMC system) and Figure S2 (LiFSI PC system). At 0.2 mA/cm À2 , an overpotential of about 10.2 mV (hysteresis, $20.5 mV) was observed for LiFSI-EC/DMC (Figures 3B and 3C), which increased to $62 mV and 100 mV at a current density of 2 and 5 mA/cm À2 , respectively. These values representing rate capability are comparable with concentrated DME ether electrolyte. 4 The Li plating/stripping CEs in the 1 M EC/DMC and the 10 M EC/DMC are compared in Figure 3D. A significantly higher CE of 97.5% is achieved in the initial cycles in the 10 M LiFSI-EC/DMC, which gradually ramps up to $99.3% after about 80 cycles. Again, this value remains the highest among all the reported Li-metal anode materials. In sharp contrast, the diluted electrolyte shows only a reversible CE of $84% for EC/DMC and $78% for PC (Figure S2).</p><p>We also assembled symmetric LijjLi cells to further confirm the cycling stability of Li-metal anode in an EC/DMC system. The long-term cycling stabilities of cointype LijjLi cells in 10 M and 1 M LiFSI-EC/DMC electrolytes are compared in Fig- ure 3E. The interfacial resistance of the LijjLi cell in concentrated electrolyte decreased in the first 60 hr, and then remained constant without any overpotential increase in the following 1,000 hr. However, the identical LijjLi cells in the diluted electrolyte cycled at the same current density demonstrated a significant impedance increase after only about 500 hr. After 700 hr, the overpotential of the cell was almost ten times higher than that using the concentrated electrolyte. The stripping/plating capacities (2 mAh/cm 2 ) in the LijjLi cell generally meet the requirements of commercial Li batteries imposed on the anodes in terms of areal capacity, current density, and cycle life. 37 These results further suggest that the better cyclability of Li-metal anodes in concentrated electrolytes could come from a more robust interphase that inhibits Li-metal dendrite growth and minimizes consumption of the electrolytes. Spectroscopic studies were conducted to understand the mechanism of high CE brought about by concentrated electrolytes. Raman spectra in Figures S3-S7 revealed that as the LiFSI salt concentration increases, the free carbonate molecules and free FSI À are dramatically decreased. To establish whether a relationship exists between the coordinated carbonate solvent with CE, 8.5 M LiTFSI-DMC, in which almost all DMC molecules were coordinated with Li + (Figure S8), was also studied as a comparison. Expectedly, the DMC structure in this electrolyte is quite similar to that in 10 M LiFSI concentrated electrolyte. However, a significantly lower Li plating/stripping CE arose from the LiTFSI concentrated electrolyte ($30%; Figure S9). Therefore, the different Li plating/stripping behaviors between 10 M LiFSI and 8.5 M LiTFSI electrolytes must come from the different interphase chemistry originating from the salt anion. We also evaluated the CE of Li plating and stripping in a saturated LiPF 6 EC/DMC electrolyte (<5 M) and highly concentrated LiFTFSI (Li[(FSO 2 )N(SO 2 CF 3 )]) EC/DMC electrolyte (8 M). Both electrolytes showed an improved CE for Li plating and stripping. However, because of the lower solubility of LiPF 6 in EC/DMC (Figure S10), the CE for Li plating and striping increased from 82% for 1 M LiPF 6 electrolyte to only $91% at the saturation of LiPF 6 (Figure S11), whereas the CE can reach >98.5% for 8 M LiFTFSI EC/DMC electrolyte (Figure S12), which is significantly higher than the Li plating/stripping CE (30%) in 8.5 M LiTFSI electrolytes. This difference arises from the much higher reactivity of LiFSI and LiFTFSI than LiTFSI, which accordingly results in much higher F content in the SEI on the Li-metal surface. 31 LiFSI concentrations significantly affected the morphology of the cycled Li metals. Scanning electron microscopy (SEM) images in Figures 4A-4D compare the Li-metal surfaces after being cycled at a current density of 0.5 mA/cm 2 and areal capacity of 1 mAh/cm 2 for 100 cycles in different electrolytes (1 M LiFSI and 10 M LiFSI-EC/ DMC). Significant amount of Li dendrites as well as dead Li formed on the surface of the Li-metal anode during cycling in the diluted electrolyte, which is similar to the morphology of Li metal cycled in the other carbonate electrolytes. 26,29 The formation of needle-like dendrites with diameter of 200-400 nm and length of several micrometers leads to an explosive increase in the specific surface area of the Li-metal anode, which accelerates the parasitic reactions with electrolyte and exhausts the Li sources, resulting in not only poor efficiencies of LMBs and extremely low volumespecific capacities of the Li-metal anodes 38 but also severe safety hazards. The Li foil (Figure 4A, inset) and the Cu electrode (Figure S13) after being cycled in 1 M LiFSI-EC/DMC electrolyte were found to be covered by a polymer-like brown film, indicating that the interphase formed in dilute carbonate electrolyte might mainly be contributed by the massive reduction of solvents. 37 This observation suggests that the poor protection by the interphase likely originates from its organic nature, which is ineffective in shielding bulk Li metal against sustained electron tunneling at certain ''hot-spots,'' leading to sustained electrolyte reduction and preferential Li growth, as demonstrated by a CE of <85% (Figure 3D). In contrast, the surfaces of the Li-metal and Cu electrode turn black after cycling in concentrated carbonate electrolyte, indicating that the composition and the morphology of the SEI layer in concentrated carbonate electrolyte differs from the SEI formed in diluted electrolyte. 4 The Li surface cycled in the concentrated carbonate electrolyte displays a round-shaped morphology with a dense and uniform structure (Figures 4C and 4D). Such morphology of Li deposition enabled by concentrated electrolytes should render three notable advantages to the LMBs: (1) The severe safety concern induced by dendritic and dead Li can be significantly relieved. Differing from the needle-like dendrites deposited in diluted electrolyte, which are a few hundred nanometers long and could easily penetrate the separators, the round-shaped structures reduce the possibility of penetration of the porous separators. (2) The surface area of the Li deposited in concentrated electrolyte is much smaller, hence minimized side reactions between the deposited Li and the electrolyte lead to a much higher Li deposition/stripping CE (as shown in Figures 2 and 3). (3) A much higher volumetric capacity would be available from the packing of Li metal upon its deposition from the concentrated carbonate electrolyte.</p><!><p>). The differences in the F1s, S2p, C1s, and O1s spectra between the two Li-metal anodes that were cycled in diluted and concentrated electrolytes are pronounced. One major difference lies in the significant variations in the elemental compositions in these two interphases. The one formed in the concentrated electrolyte is characterized by much higher F and S content but lower C and O content than those of the one formed in dilute electrolytes. The atomic ratio of F:C in the interphase is dramatically increased by more than ten times, from 0.42 in diluted electrolyte to over 5.05 in the concentrated electrolyte. More specifically, the accumulation of LiF species in the interphase is accompanied by the decline of carbon and oxygen species (C-C, C-H, C-O, C=O, etc.) on the Li-metal surface. Therefore, we can tentatively conclude that, because of the high population of FSI anions and their reactivity against reduction, the interphase formed by concentrated electrolyte tends to contain more LiF, which is contributed by the reduction of FSI rather than by solvent molecules. It is this chemical difference that is responsible for the distinct Li deposition/stripping behavior observed. LiF with high interface energy to Li can effectively prevent Li dendrite growth. 3 Molecular orbital energies of EC, DMC, and LiFSI were calculated by density functional theory (DFT) with the purpose of further understanding their respective reaction pathways. Figure 4G shows the energy values of the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) for those species. LiFSI has a lower LUMO energy (À1.70 eV) than those of EC (À0.92 eV) and DMC (À0.54 eV), indicating its higher tendency to react with Li metal than that of the solvent molecules under standard conditions. The preferential reduction of FSI À by Li metal is dramatically enhanced as the molar ratio between solvent and salt decreases from 9.5 in diluted electrolyte to 0.95 in concentrated electrolyte. In addition to thermodynamic considerations, the -SO 2 F group in FSI À is also kinetically more reactive than the carbonate solvents toward the Li-metal anode. 39 Splitting F À by cleavage of S-F bond leads to a thermodynamically stable LiF first, which precipitates on the Li-metal surface, followed by cleavage of the S-N bond. The broken fragment of F(SO 2 ) 2 N under the strong attraction of oxygen atoms to the Li surface leads to the formation of SO 2 , which promptly leaves the surface as a result of blocking by the LiF dominating layer formed earlier. This could be the main reason for the scarcity of sulfur-containing species in comparison with LiF (Figure 4F). Consequently, a LiF-rich interphase is formed in the highly concentrated LiFSI carbonate electrolyte, as demonstrated by the XPS results (Fig- ures 4E and 4F). LiF plays two critical roles in increasing the CE: (1) LiF itself is a good electric insulator and effectively blocks electron leakage through the interphase, 40 which was believed to be detrimental and one of the critical reasons causing sustained electrolyte decomposition and dendrite formation. 41,42 (2) LiF exhibits much higher interfacial energy to Li metal and meanwhile produces a reduction of as much as 0.13 eV in the activation energy barrier for Li diffusion at the electrolyte/Li-metal electrode interface. 43 Thus, the surface diffusivity should be increased by more than two orders of magnitude, which facilitates Li transport along the interface and promotes the formation of a uniform morphology of the deposited Li metal. This is consistent with the recent reports [44][45][46][47] that LiF can effectively suppress Li dendrite formation.</p><p>An ideal electrolyte for LMBs should not only provide a high Li plating/stripping CE but also be stable against oxidation on high-voltage cathode surfaces. Figure 5A demonstrates the anodic linear sweep of three different electrolytes (1 M LiFSI in EC/DMC, 10 M LiFSI in EC/DMC, and 4 M LiFSI-DME) from 3.0 V to 6.5 V at a scanning rate of 10 mV/s. At a high potential of 5.0 V, the anodic current density in 10 M LiFSI-EC/DMC electrolyte is only 1/100 that of 4 M LiFSI-DME or 1/7 that of 1 M LiFSI-EC/DMC electrolyte. As indicated by Dahn et al., 48 the oxidation of carbonate solvents (especially EC) at a high voltage is usually responsible for impedance growth and cell failure. The excellent oxidation stability of concentrated LiFSI-EC/ DMC electrolyte can be ascribed to the following two reasons: (1) the less stable organic component in possible cathode interphase is largely reduced because of the much lower presence of solvent in the concentrated electrolytes (Figure S15); and (2) a fluorine-rich interphase, which is mainly from anion oxidation, passivates the cathode surface and stabilizes the electrolyte solvents (Figure S16). According to XPS, the ratio of F:C (4.1) on such a stabilized cathode surface is significantly higher than the cathode surface cycled in 1 M LiPF 6 EC/DMC electrolyte (0.65). These F-containing species, including LiF, CF x , and S-F, form a dense cathode interphase, suppressing the parasitic reactions between the cathode and the electrolyte.</p><p>The charge-discharge and cycling behaviors of NMC622jjLi cells using different electrolytes were investigated (Figures 5B-5E and S17-S21). To utilize the high capacity of NMC, we adopted a much harsher charge-discharge protocol with a cutoff voltage of 4.6 V, which is much higher than most common charging protocols with a low cutoff voltage of 4.2 or 4.3 V using conventional electrolytes. Such a high cutoff voltage presents a rather rigorous test on the anodic stability of the electrolytes because it applies very severe electrochemical stress to induce side reactions. NMC622jjLi cells in 4 M LiFSI-DME cannot be charged to >4.2 V because of the poor anodic stability of ether solvents (Figure S17) although they possess a high Li-metal plating/stripping CE of $99% (Figure S18). In the diluted 1 M LiFSI-EC/DMC electrolyte, serious corrosion of the Al current collector by LiFSI (Figure S19) also make the first charge of NMC622 impossible (Figure S20). Because the charging of NMC622jjLi cells in 1 M LiFSI-EC/DMC and 4 M LiFSI-DME failed between 2.7 and 4.6 V, the cycling behavior of NMC622jjLi cells in highly concentrated 10 M LiFSI-EC/DMC electrolyte was compared with that of 1 M LiPF6 EC/DMC electrolyte (Figures 5B-5E).</p><p>Almost identical electrochemical performances can be obtained from the first cycle of NMC622jjLi cells in both diluted LiPF 6 electrolyte and the high concentration LiFSI carbonate electrolytes (Figure 5B). The slightly lower initial CE in 1 M LiPF 6 EC/DMC might be because of the oxidation of the carbonate solvents at the high voltage. Figures 5C and 5D show the charge-discharge voltage profiles at different cycles. Much higher voltage polarization with significant capacity decay was observed for the cell using 1 M LiPF 6 EC/DMC, suggesting that higher resistance was generated at the electrode interfaces as a result of the decomposition of the electrolyte solvent. Cycling performance and the corresponding CE of NMC622jjLi cells using these two different electrolytes are shown for the first 100 cycles (Figure 5E). The cell in 1 M LiPF 6 EC/DMC exhibited a CE of about 99% in the first 20 cycles. However, starting from the 20 th cycle, this CE sharply dropped to only about 95%, accompanied by a faster capacity decay rate. After 100 cycles, the cell in 1 M LiPF 6 EC/DMC retained only $52% of its original capacity. In contrast, the concentrated EC/DMC electrolyte enabled a high-energy-density NMC622jjLi cell with high and stable CE of >99.6% along with a capacity retention of $86% after 100 cycles, which represents a significant improvement for the aggressive chemistry of both Li-metal and NMC622 over traditional carbonate electrolytes, even with such a limited number of cycles. The enhancement in the electrochemical performance of NMC622jjLi cells in concentrated carbonate electrolytes is universal. Similar improvements in cycling performance were achieved for concentrated DMC electrolyte (Figure S21). After 150 cycles, the cell in concentrated DMC electrolyte retained a capacity of $80%. Yet, the cell in the dilute 1 M LiPF 6 DMC electrolyte dramatically dropped to 19% of the initial capacity.</p><!><p>Cathode NMC622 (LiNi 0.6 Mn 0.2 Co 0.2 O 2 ) electrode laminates ($13 mg active material cm À2 ) were supplied by SAFT America. The electrode laminates were punched into discs and further dried at 80 C under a vacuum overnight. All the solvents were purchased from Sigma-Aldrich, including EC, DMC, and PC. All the solvents were dried by molecular sieve (4 A ˚, Sigma-Aldrich) to make sure the water content was lower than 2 ppm, which was tested by a Karl-Fisher titrator (Metrohm 899 Coulometer). Lithium bis(fluorosulfonyl)imide (LiFSI, 99.9%) was purchased from American Elements. LiPF 6 (>99.99%) was purchased from Sigma-Aldrich.</p><!><p>The morphology and microstructure of the samples were investigated by SEM (Hitachi SU-70). XPS was conducted on a high-sensitivity Kratos AXIS 165 X-ray photoelectron spectrometer with Mg Ka radiation. All binding energy values were referenced to the C 1s peak of carbon at 284.6 eV. Before XPS characterization, the cycled electrodes were washed with the corresponding solvents several times to remove residual salts.</p><!><p>Electrolytes are prepared by adding LiFSI or LiPF 6 into various anhydrous solvents (DMC, PC, EC/DMC). The charge-discharge performances of the LMBs were examined by 2,032 coin-type cells. The same coin-type cells were used to investigate the cycling stability of Li plating/stripping in different electrolytes. The CE of Li plating and stripping was calculated from the ratio of the Li removed from the Cu substrate to that deposited in the same cycle. A three-electrode ''T cell'' was utilized to test the stability window of the different electrolytes with polished stainless steel as the working electrode and Li foils as the reference and counter electrodes with a Gamry 1000E electrochemical workstation (Gamry Instruments, USA). All cells were assembled in a glove box with water and oxygen content lower than 2 ppm and were tested at room temperature. The galvanostatic charge-discharge test was conducted on an Arbin battery test station (BT2000, Arbin Instruments, USA).</p><!><p>The Vienna ab initio Simulation Package (VASP) was used to perform DFT calculations, 49,50 and Perdew-Bruke-Ernzerhof (PBE) functional of the generalized-gradient approximation (GGA) was used for electron exchange and correlation. 51 Previous works have shown that GGA-PBE yields qualitatively the same trend for the ground state of higher acenes as the B3LYP function and a high-level wave function method. 52 The projector augmented wave method with an energy cutoff of 580 eV was used to describe the ion-electron interaction on a single k point. 53 The convergence condition for the energy was 10 À5 eV, and the structures were relaxed until the force on each atom was less than 0.001 eV/A ˚. For all solvents and the salt, a vacuum larger than 15 A ˚was used to simulate the molecules. Visualization of the LUMO and HOMO was done with Molekel software. 54</p><!><p>Supplemental Information includes 21 figures and can be found with this article online at https://doi.org/10.1016/j.chempr.2017.10.017.</p><!><p>Conceptualization, X.F. and C.W.; Methodology, X.F., L.C., X.J., and K.X.; Investigation, X.F., L.C., T.D., S.H., J.C., J.Z., and F.W.; Software, X.J. and J.J.; Writing -Original Draft, X.F.; Writing -Review & Editing, K.X. and C.W.; Funding Acquisition, C.W.; Supervision, C.W.</p>

Chem Cell

Native disorder mediates binding of dynein to NudE and Dynactin

We here report the molecular overlap of the linkage of three essential protein complexes that coordinate the formation of the mitotic spindle. These proteins are dynein, a large motor complex that moves machinery inside cells, and two of its regulators: a protein complex called dynactin, that is a dynein activator, and a protein called NudE whose depletion in mice produces a small brain and mental retardation. What is intriguing about the dynein/dynactin/NudE interplay is that dynactin and NudE bind to a common segment of dynein that is intrinsically disordered. Elucidating differences in their binding modes may explain how one regulator can be selected over the other even when both are present in the same cellular compartment. These results have far reaching impact not only in our understanding of processes essential for formation and orientation of the spindle but also offer a novel role for protein disorder in controlling cellular processes, and highlight the advantages of NMR spectroscopy in elucidating atomic level characterization of extremely complex dynamic cellular assemblies.

native_disorder_mediates_binding_of_dynein_to_nude_and_dynactin

Dynein structure and regulation<!>Dynein intermediate chain is an intrinsically disordered protein<!>Dynactin and NudE share common structural domains<!>Recognition sites identified by NMR and ITC<!>Structure of assembled IC<!>Disordered linkers for versatility in regulation

<p>Cytoplasmic dynein is a microtubule-based molecular motor that uses the energy derived from ATP hydrolysis to transport cellular cargo. Transport by dynein motors is essential in several aspects of cell behavior, including separation of chromosomes during mitosis, cell migration and transport of vesicles containing nutrients and other products [1]. Dynein is a massive 1.6 MDa protein complex composed of multiple subunits of different size. Figure 1 represents our current model for the configuration of its various subunits. There is no structure available for the entire complex, but new structural details of the dynein motor domain [2, 3], and of the light and intermediate chains [4-7] continue to emerge.</p><p>Commensurate with the numerous functions and activities of dynein within the cell, a multitude of adaptors and regulators have been identified [8]. The best characterized of the dynein regulatory proteins is dynactin; a multiprotein assembly that is essential for most, if not all cellular functions of the cytoplasmic dynein complex [9]. Dynactin is linked to dynein through its largest subunit, p150Glued [10]. Another dynein regulatory protein, NudE, first identified in A. nidulans as a protein required for even distribution of nuclei along the hyphae [11], is essential in processes including kinetochore and centrosome migration, organization of the Golgi complex, centrosome duplication and mitotic spindle positioning, and membrane transport [12-15].</p><p>NudE and p150Glued share a common binding motif on IC [16] which creates a paradox because dynactin, NudE and dynein co-localize in many cellular compartments [17], and raises the question as to how dynein regulation by either protein is coordinated. How does p150Glued binding to IC affect IC binding to NudE, and vice versa, and how does dynein select between different regulators?</p><!><p>The dynein intermediate chain (IC) is central to the structure of the dynein motor. It is composed of two domains. The extended N-terminal domain (N-IC) is indicated by grey solid and dotted lines (Figure 1). The C-terminal domain (C-IC), which interacts with the heavy chain, is predicted to form a relatively ordered and compact β-propeller structure indicated by the grey globular shapes in Figure 1. Two copies of IC are present in every dynein motor, which are bridged by the three dimeric light chains Tctex1, LC8, and LC7. In addition to the light chains, N-IC contains interaction motifs for several other proteins known to be integral to the function of dynein. These include p150Glued, NudE, huntingtin and the ZW10 subunit of the Rod RZZ complex [8].</p><p>With its many interactions, N-IC appears to be the key modulator of dynein assembly and attachment to cargoes. N-IC is representative of intrinsically disordered proteins (IDPs), an emerging class of proteins lacking well-defined structure. IDPs include proteins that are completely disordered and those comprised of a mixture of ordered and disordered residues. Disordered structures are defined as flexible ensembles of conformations that are, on average, aperiodic, extended and not well packed by other protein atoms. IDPs play diverse roles in the promotion of supramolecular assembly and regulation of function in various binding partners, and are themselves highly amenable to regulation through post-translational modification. IDPs are often located at the center of biological complexes where they may act as scaffolds presenting multiple binding domains and promoting spatial orientations that facilitate protein-protein interactions (reviewed in [18]). Often these IDPs when present in complexes fold upon binding [19], or retain their disordered structure in the complex, a phenomenon, referred to as fuzziness [20].</p><p>The assembly of the intrinsically disordered N-IC constitutes yet another class of disordered complexes. N-IC contains several disordered linear motifs that adopt unique structures when bound to dynein light chains. These induced structures complete the fold of the binding partners, while the linkers connecting the multiple linear motifs which are not involved in binding remain disordered [5, 6, 21, 22]. The linear motifs in dynein IC have the propensity to fold either as β-strands (recognitions motifs for Tctex1 and LC8) or α-helix (recognition motif for LC7), but only adopt this fold when bound to these partners. Figure 2 illustrates the different linear motifs in dynein IC in the apo and step-wise bound forms.</p><p>NMR spectroscopy and isothermal titration calorimetry as demonstrated below is a powerful combination for identifying recognition elements in disordered proteins and the energetics of multiple association steps.</p><!><p>The residues primarily involved in the interaction between dynein IC, dynactin p150Glued, and NudE include for all three proteins, segments whose secondary structure is predicted by standard sequence-based algorithms to be coiled-coil. NudE, has a predicted N-terminal coiled-coil domain and a largely unstructured C-terminal domain that associates with CENP-F, a nuclear matrix component required for kinetochore-microtubules interactions [23]. In solution, the N-terminal domain of NudE, nNudE, is a dimeric coiled-coil as predicted [24]. Solution studies of p150CC1 (residues 221-509) indicate a dimeric coiled-coil as predicted (data not shown). In contrast, NMR studies on apo IC1-143 which contains the binding sites for p150Glued, NudE, Tctex1 and LC8 show that while residues 3-36 are helical as predicted, there is no detectable population of stable coiled-coil, and the predominate conformations of unbound IC are disordered [21, 25]. In the apo form, IC1-143 is disordered but contains a short helical structure localized by NMR secondary chemical shifts to IC residues 1-40 [21].</p><!><p>A combination of NMR spectroscopy and titration calorimetry show that the binding site of p150Glued on IC corresponds to two segments: region 1 composed of residues 1-41, a sequence predicted to have coiled-coil secondary structure, and region 2, composed of residues 46-75, a predominantly unstructured segment with nascent helical propensity [21]. These binding regions were identified by peak disappearance in NMR spectra of 15N labeled IC upon titration with unlabeled p150Glued. The differentiation between region 1 and 2 is due to their disappearance at different ratios during the titration. No new peaks appear for the bound form, either due to the large 100 kDa complex or to exchange broadening associated with the dynamic nature of the complex, or to combination of both. The peaks that are retained in the spectrum belong to a segment that is completely disordered and thus has different relaxation behavior than the bound complex. Similar experiments with NudE show a different pattern of peak disappearance indicating that only region 1 is involved in binding.</p><p>Since the characterization of the bound complex is hampered by disappearance of the peaks at the binding site, an additional complementary technique is used to verify the binding boundaries. Smaller constructs that correspond to region 1 alone, and those that contain regions 1 and 2 were made and their binding to both p150 and NudE was characterized by ITC and compared to a larger domain of IC. With NudE, region 1 retains the full binding affinity, while with p150Glued, both regions 1 and 2 are required to match the binding affinity of NudE observed with region 1 alone [21, 24].</p><p>Features of this shared binding segment are: First, there are two noncontiguous IC recognition sequences for p150, only one for NudE. Second, these regions are helical in nature; region 1 is likely coiled-coil in the p150/IC and NudE/IC complexes as inferred from patterns of spectral exchange broadening and sequence-based structure prediction of the apo protein, while region 2 becomes helical with p150Glued but more disordered with NudE. Third, the intervening residues between IC regions interacting with dynactin remain disordered in the complex. Fourth, the affinity of one protein to IC is different due to pre-binding of the other. Spectra of IC when p150CC1 is titrated in a pre-formed nNudE/IC binary complex show that p150CC1 can displace nNudE and result in even more pronounced peak disappearance than with p150CC1 alone, suggesting that the binding affinity to p150Glued increases in the presence of NudE. In contrast, in the reciprocal experiment, NudE in excess does not appear to compete with p150CC1. The observation that the binding affinity to p150 increases in the presence of NudE even though a stable ternary complex is not formed, is quite puzzling, and so is the ability of p150Glued to out-compete NudE and not vice versa, especially when both bind with similar affinity.</p><!><p>Our interpretive model for IC in its assembled state with p150Glued or NudE and the light chains (Figure 3) is based upon a combination of structural data from X-ray crystallography (structure of Tctex1 and LC8 bound to a short segment of IC), dynamics and chemical shift mapping information from NMR spectroscopy (sites where p150 and NudE bind and the retained disorder in the linkers) as well as sequence-based prediction of structural propensity (coiled-coil structure of the p150/NudE binding region). Both helices of unbound IC (Figure 3, top) are included in the multi-region binding footprint of p150Glued, with the first comprising the entirety of region 1. The proposed coiled coil conformation for region 1 in the bound state derives from the complex exchange processes and sequence-based prediction of a coiled-coil in both IC and the p150Glued, or NudE; the coiled-coil assemblage depicted in the model could represent an IC/IC coiled-coil packed on p150Glued/p150Glued coiled-coil or NudE/NudE coiled-coil. The second, nascent helix in apo IC is contained within the p150Glued recognition motif in region 2, and shows less attenuation of peak intensity than those of region 1, presumably due to less complex exchange broadening processes, likely chemical exchange between the free and p150Glued–bound states of IC as well as structural fluctuation between nascent and fully-formed helix within IC. Thus the nascent helical structure depicted in the model for apo IC is proposed to persist and perhaps stabilize in the bound state, while residues ∼67-75 of region 2, which show less attenuation of peak intensity suggests retained disorder in this part of region 2 in bound p150Glued-IC. With NudE, there is no significant change in intensity in region 2 suggesting that this region remains flexible and does not interact with NudE.</p><p>Thus the bound IC structure differs depending on the partners with which it binds, and the difference is localized to what we term region 2, a stretch of a sequence that is highly disordered and susceptible to posttranslational modification and to alternative splicing.</p><!><p>Biological evidence suggests that NudE is required for localization of p150Glued at the nuclear envelope in prophase [26] implying that binding of NudE to IC enhances p150 binding. Our in vitro experiments support this observation; in the presence of both p150Glued and NudE, binding of p150Glued is tighter than when NudE is absent. This observation can be explained by evoking the ensemble properties common for intrinsically disordered proteins. Since both complexes associate with moderate IC-partner affinity, IC-NudE and IC-p150Glued complexes are rapidly interconverting ensembles of intrinsically disordered apo IC conformations and IC-partner complexes. Some population of the IC-NudE ensemble will have region 2 more exposed and more readily accessible to p150Glued binding. With region 2 bound to a protein – much larger than a residue-level modification – there may well be a shift in region 1 to conformations more favorable to p150Glued binding and/or less favorable to NudE binding. Then by mass action, in a mixture of NudE and p150Glued, an IC complex with the latter would be more highly populated.</p><p>Equally puzzling observation is the localization of both dynactin and NudE in the same cellular compartments. How is regulation of IC by dynactin versus NudE coordinated when both are present, and what determines which protein binds? Our in vitro results suggest that events that modify region 2 but do not significantly affect region 1 could interfere with p150Glued binding but have limited effect on NudE binding. Thus, residue-specific structural changes in and near region 2 likely either diminish or enhance dynactin binding, with limited effect on NudE binding. Disorder in region 2 and in the long linker following region 2 promotes local modifications like phosphorylation and splicing. The disorder in the 4-resiude linker separating region 1 and 2 minimizes effects on region 1 from residue-level modification in and near region 2 while its short length makes it likely that protein binding to region 2 affects the average structure and NudE affinity of region 1. The consequence of disorder in this small bi-segmental IC domain is that a modification localized to a short stretch in IC can modulate selection among multiple complex regulators of dynein function.</p>

PubMed Author Manuscript

3D Printed e-Tongue

Nowadays, one of the biggest issues addressed to electronic sensor fabrication is the build-up of efficient electrodes as an alternative way to the expensive, complex and multistage processes required by traditional techniques. Printed electronics arises as an interesting alternative to fulfill this task due to the simplicity and speed to stamp electrodes on various surfaces. Within this context, the Fused Deposition Modeling 3D printing is an emerging, cost-effective and alternative technology to fabricate complex structures that potentiates several fields with more creative ideas and new materials for a rapid prototyping of devices. We show here the fabrication of interdigitated electrodes using a standard home-made CoreXY 3D printer using transparent and graphene-based PLA filaments. Macro 3D printed electrodes were easily assembled within 6 min with outstanding reproducibility. The electrodes were also functionalized with different nanostructured thin films via dip-coating Layer-by-Layer technique to develop a 3D printed e-tongue setup. As a proof of concept, the printed e-tongue was applied to soil analysis. A control soil sample was enriched with several macro-nutrients to the plants (N, P, K, S, Mg, and Ca) and the discrimination was done by electrical impedance spectroscopy of water solution of the soil samples. The data was analyzed by Principal Component Analysis and the 3D printed sensor distinguished clearly all enriched samples despite the complexity of the soil chemical composition. The 3D printed e-tongue successfully used in soil analysis encourages further investments in developing new sensory tools for precision agriculture and other fields exploiting the simplicity and flexibility offered by the 3D printing techniques.

3d_printed_e-tongue

1. Introduction<!>2.1. 3D printer<!><!>2.1. 3D printer<!><!>2.1.1. Chemical treatment<!>2.1.2. LbL dip-coating<!>2.2. Electronic tongue<!>2.2.1. Soil samples<!>3.1. 3D printed IDEs<!><!>3.2. LbL deposition<!><!>3.2. LbL deposition<!><!>3.3. e-Tongue analysis of soil samples<!><!>3.3. e-Tongue analysis of soil samples<!><!>3.3. e-Tongue analysis of soil samples<!>4. Conclusions<!>Author contributions<!>Conflict of interest statement

<p>The continuous increase for food demand and limited productive crop areas have stimulated the development of new precision agriculture tools to avoid excessive and/or insufficient use of fertilizers and pesticides in soil management. Therefore, high-detailed data for soil characterization is fundamental for a precise soil management. However, current soil chemical analysis protocols are time-consuming and expensive procedures demanding alternative approaches for rapid and on-site soil characterization (Adamchuk and Viscarra Rossel, 2010).</p><p>There are basically two approaches for point-of-care soil characterization, one of them based on chemical extraction of specific soil macro-nutrients and then its detection by ion-selective electrodes (ISE) or ion selective field effect transistors (ISFET) (Artigas et al., 2001; Kim et al., 2007; Shaw et al., 2013). Despite the precise quantitative characterization, some parameters need to be tuned for each ion present on the soil sample, making it complicated for measurements of various nutrients or even the use of several instruments. The other approach is the direct measurement of the soil fertility parameters, and the most used techniques are optical spectroscopy (An et al., 2014; Vohland et al., 2014), capillarity electrophoresis (Smolka et al., 2017) and electronic tongues (Mimendia et al., 2014; Braunger et al., 2017). Optical spectroscopy provides several soil properties through rapid and simple measurements, but the lack of correlation between spectral bands and concentration of soil nutrients leads to models with high prediction errors. Electrophoresis is an interesting approach as it uses high electrical potential in order to separate ions based on their net charge. It provides accurate qualitative measurements of certain ions presented on samples, and also quantitative estimations of their concentrations. However, the need of high electrical potentials (~2000 V) may hamper some on-site applications. On the other hand, electronic tongue (e-tongue) sensors detect variations of the analyte dielectric constant, ensuring high sensitivity with no need of specific interactions (Riul et al., 2010). They have been widely used in quality control of foodstuff, beverages and pharmaceuticals, in addition to clinical and environmental analysis. Moreover, e-tongue devices provide rapid and continuous analysis of complex systems and fast experiments for either qualitative, semi-quantitative or quantitative analyses (Legin et al., 2003; Citterio and Suzuki, 2008; Shimizu et al., 2017).</p><p>One kind of e-tongue sensor uses interdigitated electrodes (IDEs) which are arrays of parallel plates capacitors in order to measure the analyte dielectric constant. The IDE geometry maximizes the capacitor effective area and then it increases its overall sensitivity (Olthuis et al., 1995; Igreja and Dias, 2011). The electrode fabrication usually involves expensive, complex and multistage micro-fabrication processes which still involves the use of toxic reagents. In that sense, there are great efforts to develop alternative techniques such as ink-jet printing, screen-printing and direct drawing processes exploiting conductive inks for electrode fabrication (Tomazelli Coltro et al., 2004; Coltro et al., 2010; Cummins and Desmulliez, 2012; Nakashima et al., 2012; Perinka et al., 2013; Chagas et al., 2015; Paula et al., 2018). However, these techniques still requires further steps to integrate the electrodes to assemble a functional device. The use of 3D printers in such task permits an easy integration of the electrodes to complex and intricate 3D structures in order to build up sensors in a straightforward manner (Gaal et al., 2017). Recent advances in thermoplastic materials used as filaments for Fused Deposition Modeling (FDM) 3D printing allowed an easy fabrication of 3D printed electrodes using a commercial conductive filament for electrochemical applications (Foster et al., 2017).</p><p>In this work we aim the development of planar 3D printed IDEs in order to assemble a proof of concept e-tongue. We have exploited this simple 3D-printed e-tongue sensor to discriminate soil samples enriched with important nutrients for crop production. Planar IDEs were printed in a home-made FDM 3D printer, being further functionalized with nanostrucutred Layer-by-Layer (LbL) films to be used as sensing units (Riul et al., 2003). Soil samples diluted in ultra-pure water were characterized via Principal Component Analysis (PCA) of the Electrical Impedance Spectroscopy (EIS) and a clear distinction among all samples was obtained.</p><!><p>A two nozzle home-made CoreXY FDM 3D printer was built based on the RepRap open hardware, displayed in Figure 1. It uses two commercial hot nozzles of 400 μm in diameter to extrude thermoplastics filaments of 1.75 mm in diameter. The molten filament is deposited over a hot bed to ensure a good adhesion of the first layer and to maintain a constant temperature gradient along the printed layers avoiding delamination. The hot bed is formed by a commercial heated plate coupled with a mirror, which provides a smooth and flat printing surface with building area of 200 mm × 200 mm. The printing heads are moved in the XY plane by two stepper motors following the H-frame type XY-positioning system (Itoh et al., 2004; Sollmann et al., 2010) and the hot bed is moved in the z-axis by another couple of stepper motors. The printer control is done by an open source Arduino microcontroller board Mega 2560, interfaced with a commercial RepRap Arduino Mega Pololu Shield (RAMPS).</p><!><p>Two noozle home-made CoreXY 3D printer used to produce the planar IDEs. Inset: 0.4 mm in diameter two nozzles setup.</p><!><p>The design of the interdigitated electrodes were done with Autodesk Inventor 2015 Student Edition and were further converted to STereoLithography (STL) format. The STL models were sliced using the free license software Slic3r optimized for two extruders. The slicing procedure converts the STL 3D models and transforms them into stacks of 2D printing planes, further interpreted by the printer hardware.</p><p>Planar IDEs, Figure 2a of several geometries were 3D-printed in order to verify the limits of our system and demonstrate the facility to tune the device geometry. For the e-tongue system planar IDEs were designed to have 3 pairs of fingers 9 mm long, 1 mm width and 1 mm spaced each other. The IDE base was comprised of 2 planes having 0.4 mm thickness that were printed with transparent Poly Lactic Acid (PLA) purchased from e3D. The conductive filament is a commercial PLA-based thermoplastic doped with graphene fibers, purchased from BlackMagic 3D. A profilometer Dektak 150 was used to estimate the root-mean-square (RMS) surface roughness of the printed conductive tracks.</p><!><p>(a) 3D printed IDE with 2 mm finger thick and 1.4 mm of finger separation. (b) 3D profilometry mapping of a printed IDE.</p><!><p>Before the dip-coating deposition, it was applied a chemical treatment on the surface of the printed IDEs in order to ensure the adhesion of the polyelectrolytes. The printed IDEs were placed in a solution of KMnO4-H2SO4 (50 mL), prepared from KMnO4 (194 mg) dissolved in H2SO4 (1 M). They were maintained in ultrasonic bath for 3 hours, and then washed with 1 L of ultra-pure water and HCl (1 M) (Martins et al., 2014). To remove the MnO2 adhered to the surface of the IDEs a further cleaning process was performed with a 1 M H2SO4 (25%) solution, and a 30% H2O2 (75%) solution. Ultra-pure water was provided from an Arium Comfort Sartorius system that was used also to prepare all polyelectrolyte solutions described below.</p><!><p>The LbL polymer film deposition was made in a home-made setup based on an Arduino board UNO and stepper motors. This setup allows a fully automated LbL film mounting with precise control of a large number of parameters for the LbL film deposition such as dipping velocities, time of immersion in each polyelectrolyte, wash and dry times (Hensel et al., 2018).</p><p>It was used three different LbL films in which the anionic layers used were copper phthalocyanine-3,4′,4′′,4‴-tetrasulfonic acid tetrasodium salt (CuTsPc), montmorillonite K (MMt-K), poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), and for all three LbL architectures poly(diallyldimethylammonium chloride) (PDDA) was used as the cationic layer. The aqueous CuTsPc solution was used at 0.5 mg/mL and pH 8, the MMt-K water solution was used at 1 mg/mL and pH 3, and the PEDOT:PSS solution was used at 0.2 mg/mL and pH 3. The cationic PDDA solution was prepared at 10 μL/mL and the pH was adjusted to be the same as the corresponding anionic polyelectrolyte forming the LbL film. The immersion time was 10 min for both anionic and cationic layers, and it was kept the same for all films deposited. 50 bilayers were deposited on each 3D-printed IDE and the LbL deposition was confirmed by the difference of the coated IDE impedance spectrum in air compared with that of the bare electrode also in air.</p><!><p>The e-tongue sensor was comprised of 4 sensing units, one bare IDE and three coated with nanostructured films described above. The sensor is based on the impedance measurement of the IDEs immersed in the liquid system, comparing the electrical response at a fixed frequency of different samples via Principal Component Analysis (PCA). Briefly, PCA is a multivariate statistical tool that reduces the dimensionality of the original data set facilitating correlation and visualization. This procedure is based on a linear transformation that maximizes the variance of the initial matrix and plot the new data on a new set of orthogonal axis called Principal Components without losing information (Rencher, 2012).</p><!><p>The soil samples were extracted from the same location and separated into seven pots with 1 L capacity. Each of them was added NH4NO3, NH4H2PO4, KCl, CaCl2(H2O)2, MgCl2(H2O)6, or (NH4)2SO4, in order to separately fertilize six soil samples with nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) or sulfur (S), respectively. A seventh sample was kept unfertilized as the control. All pots were maintained for 40 days in a greenhouse with daily irrigation to allow chemical reactions and full fertilization of the soils. To quantify the amount of macro-nutrients available to the plants, a portion of the samples was sent to a commercial laboratory for traditional chemical analysis.</p><p>The samples used for the 3D printed e-tongue analysis were diluted in 25 mL of ultra-pure water at 1 mg/mL. It was used a commercial Frequency Response Analyzer (FRA) Solartron 1260A with a Dielectric Interface 1269A to acquire the impedance spectra in ambient conditions. The data was analyzed at 1 kHz, as at the kHz frequency region the impedance of the system is known to be dominated by the film/electrode interface (Riul et al., 2003). The impedance spectrum was acquired for each soil sample and after the measurement the IDEs were thoroughly washed in ultra-pure water. A control EIS was then performed in ultra-pure water to verify cross-contamination of the electrodes.</p><!><p>An iterative process was used to optimize the printer parameters in order to print different planar IDEs. Figure 2a illustrates a particular IDE configuration that were printed within less than 10 min. It is worth mentioning that the geometry can be easily modified by simply changing the computational 3D model design, thus facilitating the prototyping process. Profilometry of a printed IDE finger showed a 6 μm RMS surface roughness on a square region on the top of the printed conductive track, Figure 2b.</p><p>Figure 3 illustrates the capacitance response in air among ten different 3D printed IDEs when compared with a gold IDE onto glass substrate with similar geometric parameters. As expected, the frequency response of the printed IDEs is analogous to the gold electrode. Moreover, besides the rapid prototyping offered by the 3D-printing technique it was observed an outstanding reproducibility of the geometric parameters of the printed devices as their capacitance spectra deviated in 1 pF range.</p><!><p>Capacitance spectra of a gold IDE and 10 different 3D printed electrodes.</p><!><p>In order to verify the layer-by-layer growth of the nanostructured films onto the polymeric substrate, the capacitance at 1 kHz was measured in air after each deposition step, Figure 4. Firstly, it was observed an initial non-linear trend in the measured capacitance between two different polyelectrolytes, attributed to the starting adsorption process of materials on the plastic electrodes in the LbL film build-up (Poghossian et al., 2006, 2013; Daikuzono et al., 2015). A linear trend growth was observed only after the twentieth five deposited layer, thus indicating a homogeneous adsorption process of materials on the electrode interface. The (PDDA/CuTsPc)50 film deposition was chosen to such analysis as it is easier to compare with recent ongoing studies on the capacitance change in the LbL dipping process made in our research group (Ferreira, 2016; Hensel et al., 2018).</p><!><p>Capacitance measurements in air at 1 kHz as function of the number of layers deposited for a (PDDA/CuTsPc)50 film grown onto a 3D printed IDE.</p><!><p>The final deposition process was confirmed by the difference between the impedance spectrum of the bare electrode and the coated IDE. Figure 5A illustrates the ratio of the real capacitance spectrum of a coated IDE to a bare IDE measured in ultra-pure water. Such graph indicates a change of the capacitive response of the coated IDE in comparison with the bare electrode in all the spectrum range. In particular, Figure 5B shows the comparison of the real capacitance values of the bare IDE and the coated electrodes at 1 kHz measured in ultra-pure water. As discussed previously, this frequency has a major contribution from the film/electrolyte interface (Riul et al., 2003), rendering easy the verification of the presence of thin films onto the printed IDEs.</p><!><p>(A) Ratio of the coated IDE capacitance spectrum in ultra-pure water to bare IDE response. The bar scale in the IDEs picture is 5 mm. (B) Comparison of the capacitance value at 1 kHz of the bare IDE and the coated electrode measured in ultra-pure water.</p><!><p>Figure 6 shows the frequency response of the real capacitance of a single measurement for each soil sample and each sensing unit. As expected at mid to low frequency region (104 Hz to 1 Hz) it can be observed a dispersion of the samples response, moreover, at this frequency range each sensing unit has a different shape of the spectra. It was also evaluated the relative capacitance spectra, Figure 7 which is the ratio between the real capacitance of the sample enriched with a nutrient (C) to the real capacitance of the control sample (C0). This analysis allows one to easily identify samples enriched with Mg and S, presenting good distinction from the control, while the phosphorus sample is grouped quite close to the control. This would be expected, since on tropical and poor soils, practically all the applied P must be retained by soil colloids (adsorbed and unavailable to the plants), which causes the similarity between P and control solutions.</p><!><p>Capacitance spectra of each macro-nutrient solution of all four sensing units.</p><p>Ratio of the capacitance spectrum of each macro-nutrient solution (C) to the spectrum of the control sample (C0) of all the four sensing units.</p><!><p>PCA analysis was applied to a set of three independent real capacitance measurements at 1 kHz from all seven samples, Figure 8A. A good correlation of the rough data and PCA decomposition was observed as the two first principal components add up to 99.36%. Moreover, a good distinction was achieved among the enriched aliquots even considering the high complexity involved in soil analysis. Nevertheless, an expected superposition between the phosphorus and the control samples was observed hindering their distinction as discussed above. One can overcome this superposition considering the third principal component, which accounts for 0.49% of the data variance creating a 3D extension of the PCA plot, Figure 8B. The data projection into the PC3 × PC1 plane shows clearly the separation of the control and phosphorus clusters. Finally, the 2D score plot is often used over the 3D extension because it is easier to see the data clustering, but in some cases it can hide important information and lead to false conclusions.</p><!><p>PCA score plot (A) 2D PC1 × PC2, and (B) 3D plot, evaluated at 1 kHz of the 3D printed e-tongue applied to soil analysis of seven distinct samples.</p><!><p>It is important to stress that this e-tongue system can contribute in the future for point-of-care systems applied in soil analysis and management. There is no need of complicated apparatus as impedance measurements can be taken at a fixed frequency simplifying the development of portable devices. Moreover, statistical analysis that does not require high computational demand can be easily integrated to create a portable tool for soil management. Despite the lack of quantitative information about the soil nutrients, the system can be used as a simplified apparatus to control deviations from the standard soil composition.</p><!><p>Using a home-made Core-XY FDM 3D printer and a commercial conductive filament, 3D printed IDEs have been successfully fabricated within 6 min with outstanding reproducibility. The electrodes were further functionalized with different nanostructured thin films via dip-coating LbL technique in order to develop a proof of concept 3D printed e-tongue. This system was then applied to soil analysis to discriminate soil aliquots enriched with different macro-nutrients (N, P, K, S, Mg, and Ca). The frequency response of the soil samples diluted in water were verified by electrical impedance spectroscopy, and then compared via PCA analysis. A good distinction of all samples was obtained despite the complexity of the soil chemical composition. Our results show that 3D printing technology potentiates the field of sensor fabrication with cost-effective and alternative materials for a rapid prototyping as well as greater flexibility in design, paving the way for more abundant developments.</p><!><p>GG: Fabrication of the printed electrodes, growth of the nanostructured LbL films for the e-tongue sensor and PCA analysis of the EIS of the soil samples. TdS: EIS measurements of the soil samples. VG: Project and setup of the Core-XY 3D printer. RH: Growth analysis of the nanostructured polymer films. LA: Preparation of the soil samples, discussions during the writing and revision of the manuscript. VR: Project of the Core-XY 3D printer, discussions during the writing and revision of the manuscript. AR: Principal investigator in this subject.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

Vinylimidazole coordination modes to Pt and Au metal centers

The coordination modes of 1-vinylimidazole to platinum and gold were studied. Complexes [PtCl 3 (Hvinylimidazole)]ÁH 2 O (1), [Au(vinylimidazole) 2 ] + [AuBr 2 ] À (2), [Hvinylimidazole] + [AuCl 4 ] À (3), and [Hvinylimidazole] + [AuBr 4 ] À (4) were prepared and structurally characterized. Compound 1 is the first structurally characterized transition metal complex containing a protonated vinylimidazole, which is coordinated through the vinyl group in the side-on position. In compound 2, the neutral ligands coordinate through the imidazole nitrogens to the reduced gold(I) center and the charge balancing counter anion [Au(I)Br 2 ] À has a short Au-Au contact with the cationic part. In 3 and 4, the acidic reaction conditions lead to the protonation of the imidazole nitrogen and an ion pair with tetrahalogenide gold(III)is obtained. The tendency to the different crystallized products is attributed to the combination of the metal and the halogen properties with the reaction conditions. Computational chemistry was used to explain the preference of the vinyl coordination type, as well as in the interpretation of the spectroscopic details and the nature of the intra-and intermolecular interactions present in the solid state.

vinylimidazole_coordination_modes_to_pt_and_au_metal_centers

Introduction<!>Reactions and structural studies<!>IR and FT-Raman spectroscopy<!>NMR spectroscopy<!>Stability of the coordination modes<!>Materials and methods<!>Crystal structure determination<!>Computational details<!>Conclusions<!>Conflicts of interest

<p>1-Vinylimidazole or N-vinylimidazole is a two nitrogen containing, five-membered, aromatic compound with the vinyl group at one of the nitrogen atoms. Imidazoles, in general, are important in biomolecules, [1][2][3][4] pharmaceutical applications [5][6][7][8][9][10] and in industrial utilization as corrosion inhibitors. 11 Vinylimidazole is particularly known for its homo and co-polymers. The monomer is easily polymerized through the vinyl group by exploiting UV-irradiation, heat or activator agents. Furthermore, vinylimidazole monomers can be quaternized to ionic liquids and polymerized through the side chains. 12,13 The active imidazole nitrogens in these polyionic hydrogels effectively bind metal atoms. Due to the mechanical stability, strength and recoverability of the materials, diverse applications in polymer anchored metal catalysts, 14 drug delivery, 15 and water purification 16,17 are produced.</p><p>In the context of transition metal complexes, the most probable coordination sites in 1-vinylimidazole are the imidazole nitrogen and the vinyl group. The substituent at the heteroatom blocks up the possibility for tautomerism typical of the original imidazole. The non-coordinated nitrogen atom is slightly basic and protonation of this site is possible. The high tendency to coordination to metal centers and formation of molecular complexes, as in the case of polymeric structures, is a source of diverse applications such as pharmacologically active species. 18,19 A survey of the CCDC database and literature reveals that exclusively nitrogen bound ligands are present in transition metal vinyl imidazole complexes. A quite large selection of complexes for lighter transition metals is published. However, going towards the heavier metals, the number of synthesized and characterized complexes is reduced. In group ten in the periodic table, Ni has several vinylimidazole derivatives. [20][21][22] For palladium, tetranuclear planar structures with one to four ligands have been reported, 23,24 while only the octahedral [PtCl 2 (vinylimidazole) 4 ]Cl 2 is crystallographically characterized for platinum. 25 Among the coinage metals, copper has several derivatives of this ligand. The most common coordination type is octahedral, but also tetrahedral complexes are known. Typically, these have two or four vinylimidazole ligands. [26][27][28][29][30][31][32][33][34][35][36][37] The catena compounds, where Cu centers with 1-vinylimidazole ligands and ReCl 6 -units are connected through chloro bridges, have shown magnetic properties. 38 Surprisingly, no compounds of Ag or Au have been reported. In group twelve, Zn 29,[38][39][40] and Cd 33,41 form MOF complexes with auxiliary linking ligands, and the vinylimidazoles coordinate monodentately.</p><p>Ruthenium has a unique cluster derivative [HRu 3 (CO) 10 (vinylimidazole)], where the deprotonated ligand acts as a bidentate bridging C,N-ligand. The imidazole carbon between the nitrogen atoms is utilized in coordination, but the vinyl group remains intact. 42 A similar type of ligand, 4-vinylpyridine typically coordinates through the nitrogen atom, as in the complexes of Cu, 43 Pt 44,45 and Zn. 46 The second isomer, 2-vinylpyridine, has been the subject of a relatively limited number of studies on the synthesis, structure and reactivity, which have involved metals Ru, [47][48][49][50][51][52] Os, 50,[53][54][55][56][57] Co, 58 Rh, 59,60 Ir, 61 Pd, 62 Pt 63,64 and Au. 65,66 2-vinylpyridine can form five-membered cyclometalated complexes with the high versatility of applications in the fields of biological, catalytic and luminescence properties. 64 In this work we concentrated on the reactions of vinylimidazole with late transition metals, Pt and Au, to clarify the relative reactivity of the imidazole group or the vinyl group towards the metal ions. The experimental crystal structures of the products were analyzed via X-ray diffraction, NMR, IR and Raman spectroscopy, and computational DFT methods.</p><!><p>The reaction of 1-vinylimidazole with K 2 PtCl 4 in water solution led to the formation of a white product, which by spectroscopic measurements and elemental analysis was interpreted as the square planar [PtCl 2 (vinylimidazole) 2 ] with nitrogen coordinated ligands. This is in good agreement with the earlier reported reactions in water, ethanol or acetone. 67 On the other hand, the reaction of 1-vinylimidazole with K 2 PtCl 4 in acidic medium generated a novel vinylimidazole derivative type [PtCl 3 (Hvinylimidazole)]ÁH 2 O (1), where the partially protonated ligand is bound to metal through the vinyl group in side-on geometry (Fig. 1a). The Pt(II) center has further three chloro ligands in the perpendicular plane against the CQC bond of 1.395(2) Å. The Pt-C(6) bond is 2.135(2) Å and Pt-C(7) is slightly shorter 2.109(2) Å, showing a strong side-on coordination type of the vinyl group. The sum of the van der Waals radii of Pt and C is 3.45 Å. The Pt-Cl bonds are 2.2972(5) and 2.2922(5) Å in the cis-position and 2.3056(5) Å in the trans-position to the vinylimidazole.</p><p>Reaction of vinylimidazole with AuBr 3 in organic aprotic THF solvent led to the formation of an ion pair [Au(vinylimidazole) 2 ] + [AuBr 2 ] À (2) (Fig. 2). In the cation, two neutral ligands are attached to the Au(I) center via imidazole nitrogens, the vinyl groups remaining unreacted. Anionic [Au(I)Br 2 ] À acts as the counter ion. Therefore, reduction of the metal with partial oxidation of the bromines has taken place during coordination.</p><p>The coordination at both gold atoms is almost ideal T-shape with Au-Au-N bond angles of 90.8(2)1 and 91.0(2)1. The Au-N distances are 2.018(5) and 2.017(5) Å. The CQC bond lengths in vinyl groups are considerably shorter, 1.306(10) and 1.300 (10) Å, than in 1. The Au-Au distance is 3.1200(5) Å, which is slightly shorter than the sum of the van der Waals radii of two gold atoms (3.32 Å). The cationic part is planar and the Br-Au-Br moiety lies perpendicular to this plane.</p><p>The ion pairs form a chain structure in the solid state via hydrogen bonds (Fig. 3). The bromine forms an intramolecular hydrogen bond of 3.0687(8) Å to H(2). The bromine atoms have also several short contacts of 2.8839(7)-3.0437(8) Å to hydrogens of neighboring ion pairs.</p><p>The nature of the hydrogen bonding network was further studied computationally by analyzing the charge density of an extended model by Quantum Theory of Atoms in Molecules (QTAIM). The model with four adjacent ion pairs was cut directly from the experimental crystal structure (see Fig. S1 in the ESI †) and analyzed using the DFT wavefunction. The results for the properties of the electron density are shown in Table 1.</p><p>The [Au(vinylimidazole) 2 ] + [AuBr 2 ] À ion pairs show a large number of relatively weak intermolecular hydrogen bonds to the adjacent ion pairs, explaining the packing of the ions into rows. The properties of the electron density, such as small r, the ratio between potential energy density and kinetic energy density |V|/G o 1, and interaction energy E INT o 10 kJ mol À1 , all point out to typical noncovalent intermolecular hydrogen bonds. In addition, the aurophilic Au(1)Á Á ÁAu(2) interactions at BCP 3 exhibit slightly larger strength with E INT of À26 kJ mol À1 . Notably, the aurophilic interactions to the neighboring cations have comparable strength (E INT = À13 kJ mol À1 at BCP 6), enhancing the self-assembly of the ion pairs. Furthermore, p-p interactions form between vinyl groups and the imidazole rings of the adjacent ion pairs, explaining the torsional behavior of the N-ligands as they stack together.</p><p>In the reaction of the ligand with either NaAuCl 4 or HAuCl 4 in water solution containing HCl, protonation of the ligand took place giving compound [Hvinylimidazole] + [AuCl 4 ] À (3) (Fig. 4) with a disordered structure. Thus, the protonated ligand does not coordinate to the metal like in the case of platinum. This type of ion pair was earlier known for instance for pyridine. 68 In the solid state, a network supported by hydrogen bonds H(3)-Cl(1) of 2.4256(5) Å and H(7B)-Cl(4) of 2.9059(5) and 2.9139(5) Å was obtained (Fig. 4).</p><p>The synthesis of [Hvinylimidazole] + [AuBr 4 ] À (4) from AuBr 3 in acidic HBr solution proceeded analogously to that of 3. The crystal structure and spectroscopic data showed a similar structure type to 3, but in this case with no disorder.</p><!><p>The appearance of the n(N-H) vibration at 3281, 3254 and 3256 cm À1 supports the protonation of the imidazole nitrogen in compounds 1, 3 and 4. The interpretation of the spectra was verified via computational simulation at the DFT level of theory on optimized molecular models. The experimental and simulated spectra are shown in Fig. S6-S8 (ESI †). Vinylic n(CQC) in the free ligand lies at 1630 cm À1 (computational value at 1626 cm À1 ). In 1 it is clearly shifted to a lower frequency of 1437 cm À1 (simulated 1441 cm À1 ), which is attributed to p-bonded CQC-ligands like ethylene and related ligands. 69 In a s-bonded ligand, the shift is typically to higher frequencies. In 2, 3 and 4, n(CQC) appears at 1640 cm À1 (sim. 1632), so the shift is not remarkable.</p><p>The Raman spectra were measured for complexes 1 and 2 in the solid state and for vinylimidazole in the liquid form, and also simulated at the DFT level of theory using single molecular models to facilitate interpretation. The computational values are given in Table 2, and an example of the interpretation of the most important signals for compound 1 is given in Fig. S4 and S5 (ESI †).</p><p>The full interpretation of the solid state Raman spectra was possible only for the platinum complex 1, which yielded the best quality experimental spectrum. Fig. S4 and S5 (ESI †) compare the experimental and simulated signals, which were further interpreted by animation of the vibrations. Even though the wavenumbers do not exactly match because all signals in the simulation do not scale similarly, the general appearance of the spectra is similar enough to allow interpretation (Table 2).</p><p>The coordination mode of 1 and 2 can most easily be seen in the stretching vibration of the CQC double bond of the vinyl group, which is strongly shifted to smaller wavenumbers in the platinum complex 1, but shows a similar value to that in the free vinylimidazoles for the gold complex 2. The same, but less clear trend is seen in the n(C-H) values of the CH 2 protons of the vinyl group, as well as in the scissor vibration of the CH 2 protons. Furthermore, clear Pt-C(vinyl) signals could be obtained in the spectrum of 1, verifying the side-on coordination of the metal.</p><p>The poor quality of the experimental Raman spectrum of compound 2, resulting from the degradation of the crystals during the measurement, did not allow all the signals to be resolved, most importantly the Au-N stretching frequency. However, indirect evidence on the coordination mode could be obtained by the absence of interaction with the vinyl group, since n(CQC) vinyl and n(C-H 2 ) vinyl were not shifted compared to the free 1-vinylimidazole. Additionally, there was no N-H stretching signal, which verified the coordination of the ligand in the non-protonated form.</p><p>It has been observed in the previous computational Raman studies of platinum complexes that the effect of temperature and isotopes of chlorine complicate the situation and some contradiction among the assignments exists. 70,71</p><!><p>The 1 H NMR spectra of all compounds were measured in d 6 -DMSO solvent and interpreted by comparison with the spectrum of the free ligand. The spectra are presented in Fig. S2 (ESI †).</p><p>Compound 1 shows two sets of sharp peaks with a very similar pattern to the free ligand shifted to slightly larger chemical shifts. One possibility to the two sets of signals is the existence of another isomer with a non-protonated and possibly N-coordinated ligand. This suggestion is supported by the observation that the imidazole protons (2, 4, and 5) in isomer b show the same chemical shifts as in the spectrum of 2, while the peaks of the crystallized product appear at the same location as in 3. The protonation of the vinylimidazole ligand was computationally found to change the charge distribution especially in the aromatic ring of the free ligand, which could explain the difference in the chemical shift values.</p><p>The spectrum of 2, as in the case of 1, has also a pattern of two signal sets shifted toward higher chemical shifts. In this case, the small difference in chemical shifts suggests quite similar structures for the isomers, indicating the presence of two compounds in solution, probably due to the rotation around the Au(1)-Au(2), Au(1)-N(3/3A), and N(1/1A)-C(6/6A) bonds. The major compound has reasonably sharp signals. However, the minor compound shows broad signals, which can be assumed to rise from hydrogen bonding of the protons to the near-by quadrupole bromines. Since the rotation in solution can be rather free around single bonds, in the minor isomer the protons can have short contact with bromines. The elemental analysis indicates the presence of one pure product. The NMR spectra of 3 and 4 are similar with one set of peaks describing one pure product without the possibility of fluxionality.</p><p>Overall, the most notable shift of the signals is observed at the imidazolic H(2) proton signal in those structures, where protonation of the ligand has taken place. This shift can be interpreted to have originated from the protonation of the imidazole nitrogen N3, which was found to modify considerably the calculated charge distribution of the whole imidazole ring and especially the charge of C(2) and H(2).</p><p>Both proton coupled and proton decoupled 13 C NMR spectra of the vinylimidazole ligand in the basic form as well as HCl treated vinylimidazole to simulate the protonation were measured (Fig. S3, ESI †), but only the proton decoupled spectra of the products (1-4) were obtained. Comparison of the experimental and the calculated [72][73][74] spectra is given in Table S1 (ESI †). The spectra show the remaining vinylimidazole assembly in all cases. In complexes 1, 3 and 4, containing the protonated ligands, the signal of C( 6) is slightly shifted to lower values as in the case of the protonated free ligand. Otherwise, the structure of the spectrum remains practically unchanged. The presence of different types of metals and paramagnetic species as well as the solvent effect has earlier shown to cause peculiarities in the spectrum appearance of 1-vinylimidazole and even the relative positions of the signals can change. 75,76</p><!><p>In order to find explanation for the preferred coordination mode in the Pt or Au vinylimidazoles, we performed computational optimization for models mimicking both N-coordination (A) of the deprotonated ligand and the side-on coordination (B) to the vinyl group of the protonated ligand. Since the HOMO-LUMO energy gap can give information on the relative stability of the models, the highest occupied and the lowest unoccupied orbitals along with the corresponding energies of the small molecular models are presented in Fig. 5.</p><p>There is a clear difference in the frontier molecular orbitals of the N-coordinated cations. With platinum, the strong involvement of the metal d orbitals in both the HOMO and LUMO stabilizes their energy, leading to a small energy gap. In contrast, the LUMO of the gold cation is expanded over the two ligands without contribution from the metal center, which destabilizes strongly the LUMO energy and leads to a large energy gap and hence to very stable coordination. The opposite is true in the CQC coordination mode B, where the LUMO of the Au complex concentrates over the MX orbitals, again stabilizing LUMO energy, whereas in the Pt complex the LUMO is expanded mostly on the ligand p orbitals. According to the frontier orbital energies, platinum would prefer coordination to the vinyl group and gold would prefer N-coordination of the 1-vinylimidazole, provided that the Au(III) center is reduced to Au(I). It should be noted that we also tested the side-on coordination with Au(I), and even though the stability of the product, De = 4.58 eV, was slightly larger than for Au(III), the preferred coordination mode would still be A. Experimentally, we tested also the reaction HAuCl 4 + vinylimidazole in THF solution. The crystals formed showed again the structure of 3. The 1 H NMR spectrum of the reaction mixture verified this, but two minor products were present, thus other coordination modes are possible for gold.</p><!><p>Commercially available reagents AuBr 3 (99%, Alfa Aesar), HAuCl 4 Á3H 2 O (Au 49.5%, Alfa Aesar), NaAuCl 4 Á2H 2 O (99%, Sigma Aldrich), K 2 PtCl 4 (99,9% Alfa Aesar) and 1-vinylimidazole (99%, Alfa Aesar) were used without purification. The organic solvents were dried using molecular sieves. Infrared spectra were measured from KBr pellets using a Shimadzu IRAffinity-1 Fourier transform infrared spectrophotometer in the range of 4000-400 cm À1 . The elemental analysis was performed on a varioMICRO V1.7. The 1 H NMR spectra were recorded on a Bruker Avance 400 MHz or Bruker AMX 400 MHz spectrometer. Raman spectra were recorded on a Renishaw inVia Raman Microscope with 514 nm excitation laser wavelength. Raman shifts ranging from 3200-100 cm À1 were collected.</p><!><p>The crystals of 1-4 were immersed in cryo-oil, mounted in a Nylon loop, and measured at a temperature of 150 K. The X-ray diffraction data were collected on a Bruker Kappa Apex II diffractometer using Mo Ka radiation (l = 0.71073 Å). The APEX2 77 program package was used for cell refinements and data reductions. The structures were solved by direct methods using the SHELXS-2018 78 program with the WinGX 79 graphical user interface. A numerical absorption correction (SADABS) 80 was applied to all data. Structural refinements were carried out using SHELXL-2018. 78 The crystallization solvent in 2 was heavily disordered and could not be resolved unambiguously. The contribution of the missing solvent to the calculated structure factors was taken into account by using a SQUEEZE routine of PLATON. 81 The missing solvent was not taken into account in the unit cell content.</p><p>The N-H hydrogen atoms in 1, 2 and 4 and O-H water hydrogen atoms in 1 were located from the difference Fourier map and constrained to ride on their parent atoms, with U iso = 1.2-1.5U eq (parent atom). All other hydrogen atoms were positioned geometrically and constrained to ride on their parent atoms, with C-H = 0.95 Å, N-H = 0.88 Å and U iso = 1.2U eq (parent atom). The crystallographic details are summarized in Table 3.</p><!><p>All calculations were performed by applying the Gaussian 09 software package. 82 The optimized geometry and simulated scaled infrared and Raman spectra of all the complexes were obtained by the PBE0 functional 83 with the 6-311++G(d,p) basis set for non-metal atoms and the Def2-TZVPPD basis set 84 for Pt and Au atoms.</p><p>To obtain the electronic properties of the solid state structure of 2, we performed topological charge density analysis with the QTAIM (Quantum Theory of Atoms in Molecules) 85 method, which allowed us to access the nature of the bonding via calculating different properties of the electron density at the bond critical points (BCPs). The analysis was done with the AIMALL program 86 13 C NMR (400 MHz, d 6 -DMSO, 298 K, d): 134.8 (C2), 128.9 (C4), 120.9 (C6), 118.6 (C5), 108.6 (C7) ppm.</p><p>[AuBr 4 ] À [Hvinylimidazole] + (4). AuBr 3 (33.6 mg, 0.077 mmol) was dissolved in 5 M HBr (3 ml) and one drop of vinylimidazole was added. After a few days, the red solid was filtered, washed with water and dried. Yield: 35 13 C NMR (400 MHz, d 6 -DMSO, 298 K, d): 134.9 (C2), 128.9 (C4), 120.9 (C6), 118.7 (C5), 108.7 (C7) ppm.</p><!><p>1-Vinylimidazole has different coordination modes towards platinum and gold. Reaction conditions were found to have a major effect on the obtained crystalline products, which was attributed to the protonation/deprotonation of the 1-vinylimidazole free nitrogen in acidic/basic medium. Under the acidic conditions, the first complex of this ligand with the vinyl group coordinated to platinum metal was structurally characterized. On the other hand, the protonated ligand did not directly coordinate to gold, but formed halide salts with [AuX 4 ] À as the counter anion. Under the basic conditions, the gold complex was observed as an ion pair with cation [AuL 2 ] + and anion [AuBr 2 ] À connected via aurophilic interactions.</p><p>According to the DFT calculations, the reduction of the Au(III) to Au(I) center leads to the formation of a very stable cationic N-coordinated complex with 1-vinylimidazole. For platinum, the calculations predict that the side-on coordination of the vinyl group is energetically more favorable than N-coordination, which explains the structure of complex 1. The stability of the molecular models was fully consistent with the experimentally obtained structures, which were verified via X-ray diffraction and IR, Raman, and NMR spectroscopy.</p><!><p>There are no conflicts to declare.</p>

Royal Society of Chemistry (RSC)

A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved Förster resonance energy transfer (TR-FRET)

Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR).Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03375-8.

a_15-min_non-competitive_homogeneous_assay_for_microcystin_and_nodularin_based_on_time-resolved_förs

Introduction<!>Common materials and reagents<!>Instruments<!>Plate readers<!><!>Anti-IC scFv-AP<!>Conjugations of anti-IC scFv-AP with acceptor fluorophore<!>BSA coating of microtiter wells<!>Homogeneous FRET assay and optimization of assay parameters<!>Performance of different AF680-labeled scFv-AP<!>Effect of incubation time on assay performance<!>Standard curves of different microcystin/nodularin variants<!>Heterogeneous IC-TRF assay<!>Detection of microcystin-LR from spiked water samples<!>Analysis of environmental water samples<!><!>Optimization of assay components and measurement parameters<!><!>Optimization of assay components and measurement parameters<!><!>Discussion<!>Conclusions<!>

<p>Toxic cyanobacterial blooms create local and global problems by contaminating the surface water resources with their potent toxins commonly known as cyanobacterial toxins or cyanotoxins. Microcystins and nodularins are the most commonly reported and troublesome cyanobacterial hepatotoxin having negative effect on animal and human health. Microcystins are also classified as possibly carcinogenic to humans [1]; chronic exposure to trace amounts of toxins has been connected to increased risk of hepatocellular carcinoma [2]. Altogether, microcystins and nodularins have been attributed as causative agents for various animal poisonings and identified as a threat for human health [3–5].</p><p>Though microcystins are found in freshwater bodies of all over the world and nodularins in predominantly less salty coastal brackish water (e.g., the Baltic Sea, coastal water of Southern Australia) [6], they share structure similarities. Both are monocyclic peptides; microcystins are composed of seven amino acids while nodularins are composed of five amino acids. An unusual β-amino acid adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid), which is essential for toxicity, is present in both microcystins and nodularins among with few other amino acid similarities. Structural variation occurs in both of them; however, it is much more prevalent in microcystins than in nodularins. Until now, over 250 microcystin congeners are reported in the literature along with about 10 congeners of nodularin [7, 8]. However, in the natural environment, many of these variants are present in minute quantities among the frequently reported microcystin congeners, such as microcystin-LR, -RR, -YR- LA, -WR, and dimethyl microcystin-LR, -RR depending on the geographical distribution [6, 9]. Microcystin-LR is the most commonly reported and widely distributed toxic microcystin variant. For nodularins, nodularin-R is the most dominant variant [10].</p><p>In 1998, WHO (World Health Organization) recommended a provisional guideline value of 1 μg/L of microcystin-LR in drinking water [11]. Recently, WHO revised the provisional guideline values of microcystin in drinking water and included recommended guideline values for recreational water. The guideline values for lifetime drinking water, short-term drinking water, and recreational water are 1 μg/L, 12 μg/L, and 24 μg/L of microcystin-LR equivalent [9]. WHO recommends that the public should be informed about cyanobacterial blooms in source waters when the water is used for recreation or for producing drinking water [9]. Simple and efficient methods for sensitive and quick screening within or below the WHO guideline level (1–24 μg/L of microcystin-LR equivalent for drinking water) are particularly in high demand.</p><p>Until now, there is no single analysis method sufficient alone for cyanotoxin monitoring. Existing analytical methods such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS) are time consuming, expensive, and require expertise. Some methods (mouse bioassay) are cumbersome and involve animal sacrifice, and many lack specificity and sensitivity (protein phosphatase inhibition assays, PPIAs) [12]. Immunoassays with sufficient sensitivity and specificity are a promising alternative method for microcystin/nodularin detection. Immunoassays are simple and easy to perform, and raw water can be directly applied to the assay without any sample processing. Thus, simple immunoassays are particularly promising tools for fast screening of large number of samples.</p><p>However, the available immunoassays (including commercial ones) for microcystin and/or nodularin are generally in the form of competitive format [13] as usual with immunoassays for low molecular weight targets (MW less than 2000 Da). Relying on one antibody recognition site, competitive assays are time consuming (2–3 h), requiring several incubation and washing steps, which must be strictly maintained. Furthermore, the generated signal is inversely proportional to the analyte concentration, which can complicate the interpretation of the result.</p><p>We have previously reported the development of sensitive non-competitive immunocomplex principle-based immunoassays for microcystin/nodularin [14, 15]. These assays utilize a unique antibody pair consisting of a monoclonal antibody binding to the adda group common in microcystin and nodularin, and an anti-immunocomplex (anti-IC) single-chain antibody fragment (scFv) recognizing the anti-adda monoclonal antibody (Mab) when bound to basically any microcystin or nodularin (at least those 11 tested so far) [14]. This unique generic anti-IC scFv was isolated from our synthetic antibody library [16, 17] by phage display. Both the previously developed assays [14, 15] are heterogeneous in nature, requiring an intermediate washing step to remove the unbound components before the signal development step.</p><p>Homogeneous immunoassays lacking the washing or separation steps are very appealing detection tools as they provide significant advantages regarding simplicity, rapidity, and less instrumentation requirement. As previously demonstrated by Pulli et al. [18] and Arola et al. [19], the fact that the two antibodies involved in the immunocomplex-based recognition of a low molecular weight analyte are inevitably brought into very close interaction provides an excellent basis for the utilization of the fluorescence/Förster resonance energy transfer (FRET) process for the signal generation in a homogeneous assay [20, 21]. In the FRET process, energy is transferred from a light-excited donor fluorophore through a dipole-dipole coupling interaction to an acceptor fluorophore, which then releases the energy as light at higher wavelength [20, 22, 23]. As the efficiency of the FRET process is highly dependent on the distance, being inversely proportional to the sixth power of it, the efficient energy transfer can typically only occur when the fluorophores are situated not more than 1–10 nm apart. By having the two antibodies needed for the immunocomplex formation labeled with the donor and acceptor fluorophore, respectively, FRET signal is likely obtained upon the recognition of the analyte by the antibodies. In time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) [24], a lanthanide ion–containing chelate is used as the donor compound. Due to the long fluorescence lifetime of such compounds, even >1000 μs [25], the FRET signal can be measured within an appropriate time window after the excitation, which helps to avoid interference due to the short lifetime auto-fluorescence or cross-talk between the fluorophores. This can result in significantly higher signal-to-background ratios and eventually improved assay sensitivity as compared to the standard FRET process.</p><p>Here, we describe a TR-FRET-based homogeneous non-competitive sandwich-type immunoassay for the detection of microcystin/nodularin using the aforementioned immunocomplex forming antibody pair: anti-adda Mab and anti-IC scFv (as fused to bacterial alkaline phosphatase). The assay enables us to have a sensitive and quantitative detection of microcystin/nodularin in a simple mix-and-measure approach, in a short time (<15 min). The assay is applicable for quantitative analysis of microcystin-LR with a sample detection limit of ~0.3 μg/L, satisfying the WHO guideline limit (1 μg/L), thus providing a powerful tool for rapid and sensitive screening for cyanobacterial cyclic peptide hepatotoxins.</p><!><p>Colorless assay buffer solution used in the TR-FRET assay, colored assay buffer used in assays other than the FRET assay, 96-well streptavidin-coated plates, wash concentrate for washing the 96-well plate, and enhancement solution to dissociate the lanthanide (europium) ion were from Kaivogen (Turku, Finland). MaxiSorp 96-well microtiter plates used in the FRET immunoassay were from Nunc A/S, Thermo Fisher Scientific (Roskilde, Denmark). Monoclonal antibody AD4G2 (adda specific) which binds microcystin or nodularin through the adda residue was purchased from Enzo Life Sciences, Inc. (USA). Bacterial anti-alkaline phosphatase polyclonal antibody (bAP Pab) purchased from LifeSpan Biosciences, Inc. (USA), was purified and labeled with europium (Eu-bAP Pab) to be used as a tracer in the immunocomplex (IC) assay based on time-resolved fluorescence (TRF) (referred here IC-TRF assay) according to an earlier report [14]. The near-infrared fluorescent label Alexa Fluor® 680 succinimidyl ester (AF680) were purchased from Molecular Probes, Invitrogen (Thermo Fisher Scientific Inc.). The immunoassays were carried out in room temperature (RT) of around 23 °C.</p><!><p>Delfia Plateshake for the shakings of the 96-well plates, plate washer, and enhancement solution dispenser were from Wallac, PerkinElmer Life and Analytical Sciences (Finland). Protein concentration was measured by a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific Inc.).</p><!><p>The luminescent signal of Eu(III) using time-resolved mode was measured by the multilabel counter Victor™ 1420 (PerkinElmer Life Sciences, Finland) by applying default factory settings (excitation 340 nm, emission 615 nm, delay 400 μs, gate time 400 μs). For TR-FRET measurement, the instrument was installed with a red-sensitive photomultiplier tube (R4632, Hamamatsu Photonics, Hamamatsu, Japan) and 730-nm bandpass emission filter of 10-nm bandwidth and 70% transmission maximum (Nabburg, Interferenzoptik Elektronik GmbH, Germany). TR-FRET was measured at 730 nm using a 340-nm excitation wavelength. The delay time of 75 μs and the measurement window of 50 μs were used according to the previous reports [26–28].</p><!><p>An intrinsically luminescent seven-dentate europium (7d-EuIII) chelate, MW: 674.46 g/mol [29] was used to label the anti-adda Mab to be used as a donor (Eu-anti-adda Mab) in the TR-FRET assay</p><!><p>A unique anti-IC scFv SA51D1 binder [14] was previously isolated from synthetic ScFvP antibody library [16, 17] by applying phage display technology using immunocomplex (anti-adda Mab bound to microcystin-LR) in the selection process. The selection process and screening of this binder have been described earlier [14]. The scFv as fusion with bacterial alkaline phosphatase (scFv-AP) was expressed in Escherichia coli strain RV308 in laboratory scale (5 L) fermentation at 26 °C. The scFv-AP was purified through ammonium sulfate precipitation, affinity chromatography (HisTrap Fast Flow Ni-NTA column, GE, USA), and size exclusion chromatography (Superdex 200 column, GE, USA) and eluted in TSA buffer, pH 7.5.</p><!><p>The anti-IC scFv-AP was labeled with the near-infrared fluorescent label Alexa Fluor 680 (AF680) to be used as an acceptor fluorophore in the FRET assay. The buffer of the purified scFv-AP was changed into PBS buffer pH 7.4 and then conjugated with AF680 using a reaction between the succinimidyl ester on the AF680 and the primary amino group on the scFv-AP. Aliquots of each 350 μg scFv-AP were mixed with either 5, 8, 10, or 15-fold (batch 1, 2, 3, and 4, respectively) molar excess of AF680 (dissolved in N,N-dimethylformamide from Sigma-Aldrich) in 50 mM carbonate buffer, pH 9.3 in 500-μL volume for 1 h at room temperature. The labeled products were purified by double gel filtration using NAP5 and NAP10 columns (GE Healthcare, UK) and eluted in TSA buffer, pH 7.5. According to the manufacturer's instruction, labeled protein concentration (M) was measured as [(A280 – A679 × 0.05) × dilution factor]/203,000, where the molar extinction coefficient of IgG is approximately 203,000 cm−1 M−1 and correction factor for absorption of the AF680 dye at 280 nm is 0.05. The labeling degrees [(A679 × dilution factor) / (184,000 × protein concentration (M)) where the approximate molar extinction coefficient of the AF680 dye at 679 nm is 184,000 cm−1 M−1] of the purified products were measured by absorbance together with appropriate wavelength and molar absorptivity of the AF680 (provided by the manufacturer). The absorption maximum for unconjugated AF680 dye (MW ~1150) is 679 nm and the emission maximum is 702 nm. For resulting AF680 conjugates, the theoretical absorption maximum is 684 nm and the emission maximum is 707 nm.</p><!><p>To prevent non-specific binding, low-fluorescence yellow 96-well MaxiSorp microtitration plates (Nunc, Roskilde, Denmark) were coated with BSA with saturation solution containing 0.1% BSA (Bioreba, Switzerland) in the presence of 0.1% (w/v) Germall II (ISP, Wayne, NJ) and 3% (w/v) trehalose (Sigma-Aldrich, St. Louis, MO) in 0.05 M Tris-HCl, pH 7.2. Briefly, 250 μL/well of saturation solution was added and incubated for 1 h at room temperature with slow shaking followed by aspiration of liquid. Plates were dried for 2 h and stored at +4 °C in a sealed bag until used in the FRET immunoassay.</p><!><p>The homogeneous assays were performed using 7d-EuIII chelate–labeled anti-adda Mab (Eu-anti-adda Mab) as a donor and fluorescent acceptor dye AF680 conjugated to anti-IC scFv-AP (AF680-scFv-AP) as an acceptor. In BSA-coated microtiter wells, toxin standard (0–100 μg/L of microcystin/nodularin) or sample was added followed by addition of reagent mixture (comprising Eu-anti-adda Mab and AF680-scFv-AP). Wells were then incubated (in room temperature with low shaking), and upon excitation at 340 nm, the sensitized emissions from AF680 generated by FRET were measured at 730 nm by a Victor instrument.</p><p>Combination of different amounts of Eu-anti-adda Mab (5–200 ng/well) and AF680-scFv-AP (10–200 ng/well) in a reagent mixture, effect of incubation time (2–60 min), and effect of reaction volume (60–100 μL) were tested on assay performance using microcystin-LR as standard. In addition, combination of different delay times (50–125 μs) and measurement windows (25–50 μs) were explored as measurement parameters.</p><p>Finally, in the optimized assay, 20 μL of sample/standard was mixed with 60 μL of reagent mixture (15 ng of Eu-anti-adda Mab and 120 ng of AF680-scFv-AP per well) and incubated for 15 min, and FRET measurement was carried out using 50 μs of measuring time with 75 μs of delay time.</p><p>The detection limit (the smallest detectable toxin concentration in the sample) was calculated from the standard curve based on the average response of blank + 3 times standard deviation of the blank. Concentrations of unknown samples were determined from the standard curve with the help of Origin software (OriginLab Corporation, Wellesley Hills, USA).</p><!><p>Four batches (batch 1, 2, 3, 4) of AF680-labeled scFv-AP (AF680-scFv-AP) were prepared using different excess (5x, 8x, 10x, 15x respectively) of AF680. All four batches of AF680-scFv-AP were compared for their performance in preliminary TR-FRET assay using microcystin-LR as standard. In BSA pre-coated plate, 80 μL of reagent mixture containing Eu-anti-adda Mab (0.25 μg/mL) and each batch of AF680-scFv-AP (2.5 μg/mL) were added in the presence of 20 μL of microcystin-LR. In the final 100-μL reaction volume, concentration range of microcystin-LR was 0–40 μg/L. TR-FRET measurement was performed after 5, 10, 15, 20, 25, and 30 min with the delay time of 75 μs and the measurement window of 50 μs.</p><!><p>The effect of incubation time on the performance of TR-FRET assay was observed using microcystin-LR as standard in duplicate wells. The assay was performed in 80-μL total reaction volume where 20 μL of microcystin-LR standard and 60 μL of reagent mixture were added. Microcystin-LR concentration in the final 80-μL reaction well ranged from 0, 0.05, to 250 μg/L. Signal was measured at different incubation time points: 2 min to 60 min.</p><!><p>A total of nine different purified microcystin variants and nodularin (microcystin-LR, 3-demethylmicrocystin-LR, microcystin-RR, 3-demethylmicrocystin-RR, microcystin-YR, microcystin-LY, microcystin-LF, microcystin-LW, nodularin-R) were (final concentration range in the 80-μL reaction well: 0–250 μg/L) analyzed to determine the specificity profile of the homogeneous TR-FRET assay. Toxins in the form of a lyophilized dried powder were obtained from Dr. Jussi Meriluoto's Lab (Åbo Akademi University, Turku, Finland) which were initially dissolved in 50% methanol (100–250 μM original stock). The successive working stocks and standard preparation were performed with reagent water. Toxin stocks were kept at −20 °C or 4 °C.</p><!><p>To compare the performance of the homogeneous assay to a similar heterogeneous assay, the environmental samples were also measured using the previously reported IC-TRF assay [14]. The assay concept was previously described [14] and was performed here with the following modification. In streptavidin-coated microtiter wells (Kaivogen, Turku, Finland), microcystin-LR standard (0–20 μg/L) and water samples were added as 20 μL/well in the presence of 60-μL reagent mixture (1 μg/mL of biotinylated anti-adda Mab, 1 μg/mL of scFv-AP, and 0.5 μg/mL of Eu-bAP Pab). The wells were incubated for 1 h at room temperature with slow shaking and washed four times. Then, 200 μL/well of enhancement solution (Kaivogen, Turku, Finland) was added, and after 5–10-min incubation at room temperature with shaking, TRF of Eu signal was measured with a Victor 1420 multilabel counter (Wallac, PerkinElmer Life and Analytical Sciences) using standard europium protocol where excitation wavelength was 340 nm and measurement wavelength was 615 nm.</p><!><p>Reagent water (Millipore) from the laboratory and two raw water samples from different Finnish lakes (Paalijärvi, Riihimäki, Finland 5.8.2009 and Tuusulanjärvi, Tuusula, Finland 24.6.2009) were selected for spiking experiment. The collected environmental samples were stored at −20 °C until use. Before spiking, all samples were tested for any possible presence of toxin by the heterogeneous IC-TRF assay [14]. Each sample was spiked with microcystin-LR (concentration range: 0, 0.2, 0.5, 1, 5, 10 μg/L). Toxin concentrations of the spiked and the corresponding unspiked water samples were measured by the TR-FRET assay using duplicate wells. The recovery was calculated as follows: %R = (spiked sample result by TR-FRET − unspiked sample result by IC-TRF assay)*100/known spike added concentration.</p><!><p>In total, 18 environmental samples collected during 2009 from Finland and Estonia [30] were analyzed by the developed TR-FRET-based homogeneous sandwich assay. For each sample, there were two parallel sets. One set comprising raw water as such (containing extracellular and any cell-bound toxin) was measured with the current TR-FRET assay as well as with the previously reported heterogeneous IC-TRF-based immunoassay [14]. Stored (−20 °C) raw samples were thawed-freeze-thawed to release the cell-bound toxin and used in the immunoassay without any concentration or further processing steps.</p><p>Another set of parallel water samples was previously filtered, toxins were extracted from the collected cells, and aliquots were made. Commercial ELISA and LC-MS were previously performed to determine the extracted intracellular microcystin/nodularin concentration and reported earlier [30]. From this same extracted sample set, one subset of aliquots was stored at −20 °C as dried form (the liquid was evaporated) until analysis by the current TR-FRET assay as well as by the heterogeneous IC-TRF immunoassay. Before analysis, the samples were reconstituted in reagent water accordingly based on known results [30].</p><!><p>The homogeneous non-competitive TR-FRET immunoassay concept for cyanobacterial cyclic peptide hepatotoxins microcystin (MC) and nodularin (Nod). Eu-chelate-labeled adda-specific monoclonal antibody and AF680-labeled anti-IC scFv-AP are added together with water sample. In the absence of toxin in water sample (a), the antibodies are free in the solution and FRET is not detected. In the presence of toxin (b), the anti-IC scFv binds specifically to the immunocomplex of anti-adda Mab and MC/Nod, bringing the labels in close proximity. Excitation of the Eu-chelate with UV light results in FRET between the labels, and sensitized emission of fluorescence signal can be detected at 730 nm in time-resolved mode</p><p>TR-FRET signal to blank ratio of the homogeneous assay using different batches of AF680-labeled scFv-AP in the presence of microcystin-LR in total 100-μL reaction well. TR-FRET measurement of the sensitized emission of AF680 at 730 nm was performed after 15-min incubation. Error bar indicates the standard deviation (SD) of the value</p><!><p>In order to optimize the reagent component in TR-FRET assay, varying amounts of Eu-anti-adda Mab (5–200 ng/well) and AF680-scFv-AP (10–200 ng/well) were evaluated in the assay in ~100-μL reaction well (see Supplementary Information (ESM), Fig. S1). Maximum average signals of 75,049 and 14,962 in the presence of 50 μg/L and 1 μg/L of microcystin-LR respectively were achieved using the highest tested Mab and scFv amounts (200 ng + 200 ng). However, in such case, the blank signal (10,607) is also increased reducing the corresponding signal to background ratios (S/B) (7.1 and 1.4 in the presence of 50 μg/L and 1 μg/L of microcystin-LR respectively). Decreasing amounts of Eu-Mab while maintaining the increased amount of AF680-scFv in a reaction well proportionately decreased the background signal. For example, with 20 ng of Eu-Mab + 200 ng of AF680-scFv, the S/B improved (15.2 and 4.8 in the presence of 50 μg/L and 1 μg/L of microcystin-LR respectively) more than twice. Several combinations of Eu-Mab + AF680-scFv provided S/B above 12 (at 50 μg/L of microcystin-LR) such as 10 ng Eu-Mab + 80 to 160 ng AF680-scFv or 20 ng Eu-Mab + 160 to 200 ng AF680-scFv.</p><!><p>Effect of total reaction volume in the microtiter well in the homogeneous assay using microcystin-LR as standard. Error bar indicates the standard deviation (SD) of the value</p><!><p>In the Victor fluorometer instrument, for TR-FRET measurement protocol, combination of different counting delay times (50–125 μs), measurement window time (25–50 μs), and flash energy level (EF; 130, 200, and default high 255) were explored to find out the suitable measurement parameters. Among these tested parameters [delay time/measurement window (EF)], measurement at 75/50 (EF high), 50/25 (EF high), and 75/25 (EF 200) seems to deliver best S/B.</p><p>Eventually, considering the signal level, S/B level, and acceptable cv%, in addition to minimal reagent consumption, in the subsequent experiments, 80-μL reaction volume was used. In such condition, 20 μL of sample/standard and 60 μL of reagent component comprising ~15 ng/well of Eu-anti-adda Mab and ~120 ng/well of AF680-scFv-AP were used. The measurement protocol included flash energy level of 255, counting delay time of 75 μs, and counting window time of 50 μs.</p><!><p>Effect of incubation time on the homogeneous assay performance using microcystin-LR (MC-LR) as standard. In BSA-coated microtiter wells, MC-LR (final concentration in 80-μL reaction well: 0.05 to 250 μg/L, plotted in logarithmic scale in X axis) was used to generate TR-FRET signal of the sensitized emission of AF680 at 730 nm (plotted in logarithmic scale in Y axis) at different incubation time points (2–60 min). Standard deviations of duplicate measurements are shown as error bar. Error bars are not visible when interfering with symbols</p><p>Standard curves of eight different microcystin variants and nodularin-R in the TR-FRET assay. Toxin concentrations in total 80-μL reaction well (0.05 to 250 μg/L) are plotted in logarithmic scale in X axis vs the corresponding TR-FRET signal (sensitized emission of AF680 at 730 nm) in logarithmic scale in Y axis. Standard deviations of duplicate measurements are shown as error bar. Error bars are not visible when interfering with symbols</p><p>Analysis of toxin-spiked water samples by TR-FRET assay</p><p>Coefficient of variations % (cv %) are of two replicate measurements. >dl (below detection limit); a, toxin concentration detected according to IC-TRF assay [14]</p><p>Microcystin/nodularin amount in the environmental water samples from Finland and Estonia</p><p>LC-MS results were adapted from Savela et al., 2014 [30]</p><!><p>For detection of microcystin or nodularin, compared to the highly expensive high-performance liquid chromatography (HPLC)– or liquid chromatography–mass spectrometry (LC-MS)–based methods, immunoassay-based methods offer advantages in terms of simplicity, cost-effectiveness, and wide accessibility. Furthermore, since raw water can be directly used, immunoassays are especially suitable for handling of large number of samples. Currently, several immunoassays for microcystins and nodularins are commercially available targeting the generic adda residue or specifically targeting the most common microcystin-LR [13, 31]. However, most of the available immunoassays are based on non-competitive methods that require several incubation and washing steps and hence consume several hours to complete the assays. Possibility of reduced assay time from hours to minutes while maintaining the sufficient sensitivity (for example below WHO guideline limit of 1 μg/L of microcystin for drinking water) translates into overall reduced cost as well as rapid decision-making possibilities in a critical situation.</p><p>Homogeneous immunoassays, avoiding washing/separation and usually also reagent addition steps, are highly appealing approaches for chemical analytics providing possibilities for the development of simple, rapid, and often cost-effective tools for the detection of specific analytes. In this study, we developed a homogeneous assay for the generic quantitative detection of microcystin and nodularin. This mix-and-measure-type assay combines the advantages of non-competitive immunocomplex-based recognition of the analyte with a sensitive TR-FRET measurement technology. Moreover, due to the unique recognition profile of the immunocomplex forming antibody pair used, the assay allows generic detection of microcystin/nodularin—a large group of related compounds.</p><p>The developed TR-FRET assays show maximal S/B ratio of between 10 and 15, depending on the toxin congeners used. These values are significantly higher than those in the two previously reported immunocomplex concept-based TR-FRET assays for morphine [18] and mycotoxin [19], where S/B values between 2 and 4 were observed. One of the factors helping to reach a good performance was the optimization of the labeling conditions for the acceptor-carrying antibody. Best performance was obtained using around 10-fold molar excess of the label. With lower excess, the labels apparently are less likely to hit the sites being at good distance for FRET in the immunocomplex, whereas with higher excess, the self-quenching between the closely located labels probably decreases the FRET efficiency. With the anti-adda Mab, relatively high Eu-labeling degree of 5.5 was obtained by using 1.6 mg/mL Mab and 100x molar excess of Eu-chelate. In theory, maximizing the number of Eu-chelates per antibody would be beneficial for FRET as the fluorescence of Eu-chelates is not prone to self-quenching. However, we decided not to try any further to increase the labeling degree due to the risk of affecting binding properties of the antibody. Indeed, another factor affecting the S/B ratio is the binding affinities of the antibodies involved in the immunocomplex formation. Good functionality of our assay is also reflected as the high sensitivity of detection, which, e.g., for microcystin-LR is ~0.3 μg/L, meeting the WHO guideline limit for microcystins in drinking water.</p><p>The assay facilitates rapid measurement; after mixing the reagents with the sample, the signal reaches saturation in 10–15-min incubation at RT. However, already after 2-min incubation, the signal for 1 μg/L of microcystin-LR can be reliably distinguished (Fig. 5). Compared to our previously reported heterogeneous immunoassay for microcystin/nodularin [14], the advantages of the present homogeneous assay include lower sample usage, together with reduced manual work and instrumentation requirements. The assay provides clear advantages for the screening of a large number of samples. However, such a simple and rapid homogeneous assay would, in theory, also very well lend itself for the analysis of individual samples in the field conditions. Concerning this, the main obstacle is the limited availability of portable instruments for the TRF-based measurements.</p><p>In comparison to our heterogeneous IC-TRF assay [14], there is more variation in relative signal levels obtained for different toxin congeners in the homogeneous assay. Especially, the signal for nodularin-R is clearly lower than that of microcystins. The results can potentially reflect somewhat lower affinities between the immunocomplex components in the case of nodularin-mediated interaction. Alternatively, the nodularin-mediated interaction might lead to the somewhat different orientation of the interacting antibodies compared to microcystin-based interaction, and the resulting changes in the relative positions for the FRET labels can affect the efficacy of the FRET. In any case, the homogeneous assay could recognize all the tested toxin variants (microcystin-LR, 3-demethylmicrocystin-LR, microcystin-RR, 3-demethylmicrocystin-RR, microcystin-YR, microcystin-LY, microcystin-LF, microcystin -LW, nodularin-R) and most importantly the most toxic and widely reported microcystin-LR variant.</p><p>For the practical assessment of the assay, microcystin-LR-spiked water (Table 1) and 18 true environmental water samples (Table 2) were analyzed and the results were compared to those obtained with reference methods (IC-TRF assay and LC-MS). The toxin concentration measured by the TR-FRET assay correlates well with other methods indicating the practical applicability of the assay for the assessment of toxin levels in both direct environmental water and the cell-extracted samples. Overall, water is a very favorable sample matrix for homogeneous FRET-based measurement as it does not, in contrast to, e.g., blood-based sample matrixes, by default contain intensively light-absorbing compounds which could interfere with the excitation, emission, or dipole-dipole coupling–based energy transfer processes in a FRET-based assay. However, should a water sample for some reason have an unusually strong color, a few different dilutions of the samples could, for safety's sake, be analyzed.</p><!><p>The presented generic and quantitative homogeneous assay is sensitive enough to be employed in screening of water samples for microcystins below WHO guideline value of drinking water (1 μg/L) and recreational water (24 μg/L). The performance of the assay was demonstrated by analyzing the toxin-spiked sample and real environmental water samples. Being simple and rapid, this mix-and-measure-type assay should be applicable for the analysis of large numbers of water samples for microcystin or nodularin levels. It should also be well-suited for automation and, hereby, useful for high-throughput screening applications.</p><!><p>(PDF 335 kb)</p><p>Published in the topical collection Analytical Applications of Biomimetic Recognition Elements with guest editors Maria C. Moreno-Bondi and Elena Benito-Peña.</p><p>Publisher's note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p>

PubMed Open Access

Isolation and Characterization of the Free Phenylphosphinidene Chalcogenides C6H5P=O and C6H5P=S, the Phosphorous Analogues of Nitrosobenzene and Thionitrosobenzene

AbstractThe structures and reactivities of organic phosphinidene chalcogenides have been mainly inferred from trapping or complexation experiments. Phosphinidene chalcogenide derivatives appear to be an elusive family of molecules that have been suggested as reactive intermediates in multiple organophosphorus reactions. The quest to isolate “free” phosphinidene chalcogenides remains a challenge in the field. Here, we present the synthesis, IR, and UV/Vis spectroscopic identification of hitherto elusive phenylphosphinidene oxide and phenylphosphinidene sulfide from the corresponding phosphonic diazide precursors. We isolated these higher congeners of nitroso‐ and thionitrosobenzene in argon matrices at 10 K. The spectral assignments are supported by B3LYP/6–311++G(3df,3pd) and MP2/cc‐pVTZ computations.

isolation_and_characterization_of_the_free_phenylphosphinidene_chalcogenides_c6h5p=o_and_c6h5p=s,_th

<!>Conflict of interest<!>

<p>A. Mardyukov, F. Keul, P. R. Schreiner, Angew. Chem. Int. Ed. 2020, 59, 12445.</p><p>In memory of Rolf Huisgen</p><p>While nitrosobenzenes are common, highly reactive reagents in organic chemistry,1, 2 their heavier isosteres based on phosphorus and sulfur remain unknown. Phosphinidene chalcogenides (R−P=E; E=O, S, Se) are reactive species that are isolobal with heteroatom‐substituted singlet carbenes (Scheme 1 B),3, 4, 5 but they have only rarely been observed directly.6, 7, 8 The closely related thionitrosobenzenes are also unknown in free form and are expected to be highly unstable.9, 10, 11 Despite considerable efforts, phosphinidene chalcogenides are only known as transient species in solution, and their existence can be inferred from product‐analysis studies and complexation experiments.3, 12, 13, 14, 15, 16 These transient species are very reactive toward many (organic) molecules and have thus been used as in‐situ reagents for phosphorus‐ and chalcogen‐ring formation.16, 17, 18, 19 Product analyses from trapping experiments revealed that R−P=E species undergo cycloaddition reactions along with the polarized phosphorus–chalcogen double bond either showing as [4+1] carbene‐like reactivity4, 20, 21, 22, 23 or [4+2] olefin‐like reactivity.15, 24 Very recently, Cummins and co‐workers reported an elegant approach to generate tert‐butylphosphinidene sulphide (tBuP=S) in solution under mild conditions using anthracene (C14H10) extrusion for the release of highly reactive tBuP=S in situ, which undergoes a Diels–Alder reaction with dienes.25 In 2017, Graham et al. reported the synthesis of four‐membered phosphorus–chalcogen (RPE)2 heterocycles (E=S, Se);12 subsequent reactions with N‐heterocyclic carbenes (NHCs) resulted in base‐stabilized phosphinidene sulfides. Schmidpeter and co‐workers26 prepared stable monomeric phosphorous monochalcogenides without bulky or intramolecularly coordinating substituents, which are stabilized through conjugation with a triphenylphosphoniumylidyl moiety (therefore named ylidylphosphorchalcogenides) and large contributions of zwitterionic resonance structures. Product studies suggested the transient generation of phosphinidene chalcogenides (thermally or photochemically) from various precursors,4, 15, 27 such as phospholene, phosphirane, or phosphanorbornadiene chalcogenides,28, 29, 30 and starting materials containing four‐membered rings with a P2E2 core (E=S, Se).3, 12</p><p>A) Photochemical generation of 1 and 2. B) Resonance structures of the phosphinidene chalcogenides. C) Triplet phosphinidene (5), phenyldioxophosphorane (6), and previously prepared heavier congeners of 6, namely 7 and 8.</p><p>The direct spectroscopic observation of phosphinidene chalcogenides, R−P=E, is extremely scarce. The stabilization of R−P=E species can be achieved through coordination to metal centers31, 32 or by using bulky substituents.33, 34 However, for E=O, only a few transition‐metal complexes containing an R−P=O moiety have been isolated.32, 35 Parent H−P=O has been identified as one of the emitting species in the chemiluminescence of white phosphorus and in the oxidation of phosphine.36 The spectroscopy and structure of H−P=O has been intensively studied.37, 38 The heavier congener H−P=S has been detected with neutralization–reionization mass spectrometry39 and rotational spectroscopy.40 Only in 2019, CH3−P=O was generated and efficiently trapped in argon matrices through photolysis or flash vacuum pyrolysis of methylphosphoryl diazide CH3P(O)(N3)2.41</p><p>As found for phosphinidene oxides and sulfides, dioxophosphoranes (phosphinidene dioxides, R−PO2) and thiooxophosphoranes (R−PS2) are unstable molecules thought to be generated in the thermolysis of suitable organophosphorus precursors.42, 43, 44 These transient species are highly electrophilic at the phosphorus atom and have thus found use as efficient phosphorylating agents. Recently, we reported the synthesis of previously elusive phenyldioxophosphorane (6, PhPO2), the phosphorus analogue of nitrobenzene, under matrix‐isolation conditions through the reaction of triplet phenylphosphinidene (5) with triplet molecular oxygen (3P‐O2).45 The heavier congeners of 7, namely (4‐methoxy)phenyl phosphine disulphide (6)46 and phenyl phosphine diselenide (8)47 (the monomeric forms of Lawesson's and Woollins' reagents, respectively), have been isolated and characterized spectroscopically as well (Scheme 1 C). Following our studies on the synthesis and reactivity of transient organophosphorus species including PhP,45 PhPO2,45 PhPCO,48 PhPS2,46 and PhPSe2,47 we report herein the first spectroscopic evidence of hitherto unknown "free" (that is, uncomplexed) phenylphosphinidene oxide Ph−P=O (1) and phenylphosphinidene sulfide Ph−P=S (2) by means of IR and UV/Vis spectroscopy (Scheme 1 A).</p><p>Phosphonic diazides represent potentially useful precursors for the generation of free phosphinidene chalcogenides that can be activated either thermally or photochemically, and we have chosen the UV photolysis of phenylphosphoryl diazide (3) in an argon matrix at 10 K with N2 as the only IR‐invisible byproduct. The IR spectrum of 3 is characterized by an intense group of signals centered at 2153 cm−1 (Figure 1). Irradiation (λ=254 nm) of matrices containing 3 results in very rapid and complete disappearance of its IR signals. From a number of experiments, we identify a set of IR bands (Figure 1 and Table S1, Supporting Information), the most prominent at 1185, 1098, 741, 689, and 462 cm−1, that show identical growth behavior upon photolysis. These signals are assigned to 1 based on comparison with B3LYP/6–311++G(3df,3pd) computations. The strong IR bands at 1185 and 1098 cm−1 are attributed to the P=O stretching and C−H deformation modes. The observed splitting of the vibrational bands is due to different trapping sites in the argon matrix. The measured P=O stretching frequency agrees well with a previously observed IR band at 1203 cm−1 for the stabilized neutral 2,4,6‐tri‐tert‐butylphenylphosphinidine oxide.33, 35 The bands at 741, 706, and 689 cm−1 are thus assigned to the C−H out‐of‐plane vibrational modes of the phenyl ring. Overall, the observed IR vibrational bands match the computed fundamentals of 1 (Table S1) very well.</p><p>a) IR spectrum of 3 computed at B3LYP/6–311++G(3df,3pd) (unscaled). b) IR difference spectra showing the photochemistry of 3 after irradiation at λ=254 nm in argon at 10 K. Downward bands assigned to 3 disappear while upward bands assigned to 1 appear after 15 min irradiation time. c) IR spectrum of 1 computed at B3LYP/6–311++G(3df,3pd) (unscaled).</p><p>Photochemical decomposition of 3 with 254 nm irradiation resulted in a slight yellow coloration of the matrix, which was colorless initially. The UV/Vis spectral analysis of the 254 nm photolysis of 3 shows that its strong absorptions at 192 and 217 nm gradually decrease while new absorptions grow. The spectrum obtained after complete decomposition of azide precursor 3 reveals strong absorptions at 192, 219, and 277 nm, a weak absorption at 337 nm, as well as several transitions with pronounced vibrational progressions extending from 430 nm up to 490 nm (Figure 2). Computational analysis using time‐dependent density functional theory (TD‐B3LYP/6–311++G(3df,3pd)) of the excitations of 1 exhibit a strong transition at 284 nm (f=0.222) and two weak transitions at 314 nm (f=0.011) and 474 nm (f=0.004) in good agreement with the experimentally observed UV/Vis spectrum (Figure 2). Likewise, in our earlier studies on reactive organophosphorus species,45, 46, 47, 48 the inspection of the molecular orbitals of 1 reveals that the weak absorption band in the visible region at 460 nm corresponds an n→π* transition, while the strong band at 277 nm is a π→π* transition.</p><p>Solid: UV/Vis spectrum of 3 isolated at 10 K in Ar. Dashed: UV/Vis spectrum of 1 at 10 K; the photochemistry of 3 after irradiation at λ=254 nm in Ar at 10 K. Inset: Computed TD‐B3LYP/6–311++G(3df,3pd) spectrum of 1.</p><p>Following the route for the synthesis of 1, we prepared 2 from the azide 4 by UV irradiation. Photolysis (λ=254 nm) of matrix‐isolated 4 in solid argon for 30 min at 10 K resulted in the complete disappearance of its IR bands and the appearance of a new set of IR bands. New strong bands appeared at 1185, 1084, 743, 706, 682, and 416 cm−1 upon UV irradiation (Figure 3 and Table S2), in good agreement with the computed values. For example, the band at 1185 and 1084 are assigned to the C−H deformation modes in 2. The bands at 743, 706, and 685 cm−1 are assigned to the C−H out‐of‐plane vibrational modes of the phenyl ring. The strong band at 682 cm−1 is assigned to the P=S stretching mode of 2; as expected, it absorbs at a lower frequency compared to 1 due to the considerably longer P=S bond (see below). The nature of the P=S bond in 1 can also be judged by comparison with the P=S stretching modes in related compounds. Several unstable thiophosphines (X−P=S; X=Br, F) have been characterized spectroscopically either in the gas phase or have been isolated in argon matrices.49 F−P=S shows an intense P=S stretching frequency at 720 cm−1,7 whereas Br−P=S absorbs at 712 cm−1.8 With the help of the computations, the other IR bands of medium intensity can also be attributed to 2 (Table S2).</p><p>a) IR spectrum of 4 computed at B3LYP/6–311++G(3df,3pd) (unscaled). b) IR difference spectra showing the photochemistry of 4 after irradiation at λ=254 nm in argon at 10 K. Downward bands assigned to 4 disappear while upward bands assigned to 2 appear after 15 min irradiation time. c) IR spectrum of 2 computed at B3LYP/6–311++G(3df,3pd) (unscaled).</p><p>The photochemistry of 4 was also investigated in the UV/Vis spectral region. Under reaction conditions similar to the IR experiments described above, irradiation of an argon matrix containing 4 results in a decrease in intensity of the bands at λ=194 and 220 nm, assigned to 4 and formation of new strong bands at λ max=192 and λ max=337 nm assigned to 2 (Figure 4 and Figure S2). The measured UV/Vis spectrum of 2 also displays a weak absorption in the 600–730 nm range with a pronounced vibrational fine structure. Similar to 1, all bands of 2 correlate well with the values of the electronic excitations at 191 and 218 nm (f=0.201 and 0.074), 331 nm (f=0.227), and 692 nm (f=0.0008) computed at TD‐B3LYP/6–311++G(3df,3pd).</p><p>Solid: UV/Vis spectrum of 4 isolated at 10 K in Ar. Dashed: UV/Vis spectrum of 2 at 10 K; the photochemistry of 4 after irradiation at λ=254 nm in Ar at 10 K. Inset: Computed TD‐B3LYP/6–311++G(3df,3pd) spectrum of 2.</p><p>Next, we compared the key geometrical and electronic parameters of the optimized structure of 1 with 2 (Figure 5 A). The P=O and P=S bond distances in 1 and 2 are 1.485 and 1.937 Å, respectively, at B3LYP/6–311++G(3df,3dp) (1.504 and 1.942 Å at MP2/cc‐pVTZ), thus showing double‐bond character. Wiberg bond indices of 1.74 and 1.54 were computed for the P=O and P=S bonds in 1 and 2, respectively, indicating significant double‐bond character. The P=O length in 1 is comparable to the corresponding values in previously reported compounds with R−P=O ligands as well as with an anionic molybdenum‐bound phosphinidene oxide complex with a P=O bond of 1.514 Å.32 The P=S bond distance in 2 is only slightly shorter than the P=S bond distance of 2.028 Å in the NHC adduct of aryl phosphine sulphide.12 The computed C−P bond distances in 1 and 2 are 1.819 and 1.820 Å, respectively, at B3LYP/6–311++G(3df,3dp) (1.824 and 1.822 Å at MP2/cc‐pVTZ) with bond‐dissociation energies (BDE) of 73.4 and 68.5 kcal mol−1 (including zero‐point vibrational‐energy corrections, ZPVEs). No significant geometric differences were observed in the phenyl rings. This indicates there is negligible electronic delocalization of the P=E moiety with the phenyl ring (see below). According to our computations, 1 and 2 are planar C s‐symmetric structures with a 1A′ electronic ground state.</p><p>A) Selected bond lengths [Å] and angles [°] of 1 and 2 at B3LYP/6–311++G(3df,3pd). The values in parentheses were computed at MP2/cc‐pVTZ; B), C) Molecular orbitals of 1 (B) and 2 (C) at B3LYP/6–311++G(3df,3pd).</p><p>The NPA atomic charges of 1 and 2 are +0.33 and +0.07 e at phosphorus, while the oxygen and sulfur atoms have considerable negative charges of −0.34 and −0.33 e, respectively. Sulfur forms weaker and longer bonds than oxygen, owing to the larger sulfur atom with larger and more diffuse orbitals, resulting in poorer orbital overlap with the phosphorus orbitals as compared to the oxygen atom. Indeed, the HOMOs (highest occupied molecular orbitals) in 1 and 2 are in‐plane orbitals combining the σ(P−C) orbital, the P lone pair, and an in‐plane p‐orbital of the chalcogen. Carbene‐like features are evident from the finding that the HOMO entails the P lone pair and the LUMO the empty pz orbital on phosphorus; the isolobal analogy to a singlet carbene holds.4 The HOMO−1 of 1 is mainly localized over the phenyl ring, while in 2, the HOMO−1 displays pronounced π‐bonding between the phosphorus and sulfur atoms (Figure 5 B,C). When sulfur is present instead of oxygen, there is a higher destabilization of the HOMO energy, leading to a smaller HOMO–LUMO gap; this indicates that 2 is more basic than 1. Thus, the estimated ΔE=E HOMO−E LUMO value for 2 (0.10 eV) is smaller than that of 1 (0.14 eV). The resulting smaller HOMO–LUMO gap correlates well with the UV/Vis absorption maxima of 2, which is red‐shifted for 2 (λ max≈660 nm) compared to 1 (λ max≈460 nm).</p><p>In summary, we present the generation and isolation of phenylphosphinide oxide and phenylphosphinidene sulfide using a combination of photolysis, matrix‐isolation IR, and UV/Vis spectroscopic methods as well as quantum‐chemical computations. The bis‐azide precursors are easy to prepare and readily undergo thermal or photochemical decomposition to give 1 and 2. The facile generation of 1 and 2 opens the door for further experimental studies on their (photo)reactivity.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Gender differences between hypocretin/orexin knockout and wild type mice: age, body weight, body composition, metabolic markers, leptin and insulin resistance

Female hypocretin knockout (Hcrt KO) mice have increased body weight despite decreased food intake compared to wild type (WT) mice. In order to understand the nature of the increased body weight, we carried out a detailed study of Hcrt KO and WT, male, and female mice. Female KO mice showed consistently higher body weight than WT mice, from 4 to 20 months (20\xe2\x80\x9360%). Fat, muscle, and free fluid levels were all significantly higher in adult (7\xe2\x80\x939 months) as well as old (18\xe2\x80\x9320 months) female KO mice compared to age-matched WT mice. Old male KO mice showed significantly higher fat content (150%) compared to age-matched WT mice, but no significant change in body weight. Respiratory quotient (\xe2\x88\x9219%) and metabolic rates (\xe2\x88\x9214%) were significantly lower in KO mice compared to WT mice, regardless of gender or age. Female KO mice had significantly higher serum leptin levels (191%) than WT mice at 18\xe2\x80\x9320 months, but no difference between male mice were observed. Conversely, insulin resistance was significantly higher in both male (73%) and female (93%) KO mice compared to age- and sex-matched WT mice. We conclude that absence of the Hcrt peptide has gender-specific effects. In contrast, Hcrt-ataxin mice and human narcoleptics, with loss of the whole Hcrt cell, show weight gain in both sexes.

gender_differences_between_hypocretin/orexin_knockout_and_wild_type_mice:_age,_body_weight,_body_com

<!>Ethical approval<!>Animals<!>Body weight<!>Body composition<!>Metabolic studies<!>Blood and serum analysis<!>Statistical analysis<!>Body weight<!>Body composition<!>Metabolic parameters<!>Serum leptin levels<!>Insulin resistance and insulin release<!>Weight of adipose tissue<!>Discussion

<p>The hypothalamic neuropeptide, hypocretin (Hcrt), also known as orexin, has been implicated in energy metabolism, body weight control, sleep–wake regulation, reward-motivated behaviors and addiction (Hara et al. 2001; Mochizuki et al. 2004; Zhang et al. 2007; Tsuneki et al. 2008; McGregor et al. 2011; Mahler et al. 2012). Several studies have shown that loss of Hcrt neurons causes narcolepsy with cataplexy in humans (Peyron et al. 2000; Thannickal et al. 2000). Similar symptoms are observed in hypocretin knockout (KO) and Hcrt-ataxin mice (Chemelli et al. 1999; Hara et al. 2001).</p><p>In humans, increased body mass index (BMI) has been noted in both men and women narcoleptics compared to the general population. Poli et al. (2009) reported that patients with narcolepsy with cataplexy (all HLA DQBI*0602 positive, with CSF levels of orexin A < 110 pg/mL) had a higher BMI and BMI-independent metabolic alterations, including increased waist circumference, high density lipo-protein-cholesterol levels, and glucose/insulin ratio (insulin resistance index), compared to patients with idiopathic hypersomnia (with CSF levels of orexin A > 300 pg/mL). This suggests that loss of Hcrt neurons may lead to altered metabolic control, resulting in obesity, insulin resistance, and increased risk of type 2 diabetes. Despite increased BMI, normal or decreased leptin levels were seen in both men and women narcoleptics, compared to the general population. Donjacour et al. (2013) reported no changes in total ghrelin or leptin levels in narcoleptic patients on or off sodium oxybate, compared to the general population. On the other hand, Kok et al. (2003) reported that male narcoleptics had a large reduction in leptin levels compared to control humans, suggesting that lowered leptin levels may predispose narcoleptic humans to weight gain. Beitinger et al. (2012) reported that narcoleptic, nonobese patients, who have low levels of Hcrt, showed a tendency to decreased insulin sensitivity. Honda et al. (1986) showed that noninsulin-dependent diabetes mellitus was significantly increased in narcoleptics, compared to the general Japanese population, irrespective of their obesity index.</p><p>In animals, several authors have reported increased body weight in female but not male Hcrt KO mice compared to wild type (WT) littermate controls (Fujiki et al. 2006; Tsuneki et al. 2008). This is despite the fact that both sexes showed decreased food intake (Tsuneki et al. 2008) and decreased locomotor activity (Kayaba et al. 2003; Mochizuki et al. 2006; Tsuneki et al. 2008). Tsuneki et al. (2008) further reported that impaired glucose tolerance, decreased insulin sensitivity, and increased serum insulin levels were observed in both male and female Hcrt KO mice, compared to gender-matched WT mice. However, increased serum leptin levels were only observed in female mice. Fujiki et al. (2006) reported that obesity was more prominent in female, than male Hcrt KO and Hcrt-ataxin transgenic (TG) mice compared to WT controls, and was associated with higher serum leptin levels, suggesting a gender-specific alteration of leptin-hypocretin signaling. Hara et al. (2001) reported that male Hcrt-ataxin TG mice exhibited increased body weight, decreased food intake and decreased locomotor activity compared to WT mice. Zhang et al. (2007) similarly reported that male Hcrt-ataxin TG mice exhibited decreased feeding and drinking behavior, as well as decreased energy expenditure and decreased locomotor activity compared to their WT littermates. This may account for the increased body weight observed in these male Hcrt-ataxin TG mice.</p><p>Female Hcrt KO mice have increased body weight despite decreased food and water intake compared to female WT mice. In order to understand the nature of the increased body weight, we carried out a detailed study of Hcrt KO and WT, male and female mice. The mice were followed from 4 to 20 months, a longer period than has been used in prior studies. We also analyzed, for the first time, changes in the amount of fat, muscle, free fluid content and white adipose tissue (WAT), and brown adipose tissue (BAT), as well as respiratory quotient (RQ), and metabolic rate (MR). The mice were then sacrificed and blood glucose, serum insulin, and leptin levels were measured.</p><!><p>All procedures were approved by the Institutional Animal Care and Use Committee of the University of California at Los Angeles (UCLA) and the Veterans Administration Greater Los Angeles Health Care System (VAGLAHS). Every measure was taken to minimize pain or discomfort in the animals.</p><!><p>Hcrt KO mice, with a mixed C57BL/6J-129/SvEv background, and their WT littermates were obtained from our breeding colony at the Division of Laboratory Animal Medicine at UCLA. All mice used in this study were derived from heterozygote parents. Between 4 and 5 littermates or age-matched mice, of each genotype and gender, were used (four male WT, five male KO, five female WT and five female KO). The animals were housed under standard light (12 h light/dark cycle, lights on at 7:00 hours) and temperature conditions (24°C ± 1°C), and were given free access to normal laboratory diet (PicoLab Rodent Diet 20 irradiated 5053 from Lab Diet/PMI Nutrition International, St Louis, MO, USA).</p><!><p>The mice were weighed every month from 9 to 18 months. Between 18 and 20 months, the mice were transferred to the core Mouse Metabolic Syndrome Phenotype Facility at UCLA, for body composition and metabolic studies. Since female Hcrt KO mice started showing significant increases in body weight at 9 months (the first time point in the initial phase of our study), we monitored another group of female mice (five KO and five WT) starting at 3 months, and subjected them to body composition and metabolic analyses at 7–9 months.</p><!><p>The amount of total fat, muscle, and free fluid content of each mouse, was determined by NMR measurement, using the minispec mq series from Bruker (Bruker Optics Inc. Billerica, MA, USA). Each mouse was placed in the chamber for approximately 1–2 min. One reading was taken for each mouse, during the light phase.</p><!><p>Indirect calorimetry was recorded using the Comprehensive Laboratory Animal Monitoring System (Columbus Instruments, Columbus, OH, USA). The mice were housed individually in calorimeter chambers. After 3 days of acclimatization, one reading was taken per mouse, during the light phase. Metabolic parameters were calculated as described below (Funato et al. 2009): Respiratory Quotient (RQ)=VCO2 produced /VO2 consumed Metabolic Rate (MR)=VO2 consumed (mL/min)/ Body weight (kg)</p><!><p>After completing the studies on body composition and metabolic changes, the mice now aged about 20 months, were fasted overnight, and then sacrificed by carbon dioxide inhalation. Blood was collected by cardiac puncture, into red capiject tubes with a serum separator, left at 24°C for 20 min, and then centrifuged for 20 min at 142 g. Blood glucose levels were assayed with a glucometer (ReliOn Ultima), and serum leptin and insulin levels with the Mouse Leptin (Cat # 90030) and the Ultra Sensitive Mouse Insulin (Cat # 90080) ELISA kits respectively (Crystal Chem Inc., Downers Grove, IL, USA), according to manufacturer's instructions (Funato et al. 2009). The homeostasis model assessment of insulin resistance (HOMA-IR) and beta-cell activity (HOMA-%β) was calculated using fasting glucose and insulin concentrations, with the following formula (Matthews et al. 1985): HOMA-IR = glucose (mg/dL)× insulin(μU/mL)/405 HOMA- %β=(360× insulin )/( glucose-63 )</p><!><p>Three-way repeated measures anova with post hoc Tukey test, was used to determine the interaction between gender, genotype, and age, for body weight analyses, among old (18–20 months) male and female mice, while two-way anova with post hoc Newman–Keuls, was used to determine genotype and gender-specific differences, in the analyses of body composition, metabolic parameters and serum components. Unpaired Student's t-test was used to determine the differences in body weight, body composition, and metabolic parameters between adult (7–9 months) KO and WT female mice. The significance level was set at p < 0.05.</p><!><p>A significant effect of genotype (F = 13.78, p = 0.003), age (F = 31.35, p < 0.0001), interaction between gender and genotype (F = 15.07, p = 0.002), gender and age (F = 5.64, p < 0.0001), and genotype and age (F = 2.93, p = 0.004) on body weight, was observed among old male and female mice. Post hoc Tukey comparisons indicated that female KO mice had significantly higher body weight (ranging from 50 to 60%, p < 0.01) than female WT mice, starting as early as 9 months and persisting until 18 months (Fig. 1a). Male KO mice did not show any significant changes in body weight compared to male WT mice, throughout this period. On the other hand, female WT mice had significantly lower body weight compared to male WT mice between 9 and 17 months (ranging from 37 to 14%, p < 0.01), while female KO mice had significantly higher body weight compared to male KO mice between 16 and 18 months (averaging around 18%, p < 0.01). Since female KO mice started showing significant increases in body weight at 9 months (the first time point in the initial phase of our study), we monitored another group of female mice (five KO and five WT) starting at 3 months. We found that the difference in body weight between female KO and WT mice actually started to become significant as early as 4 months (20%, p < 0.05, Fig. 1b), and continued to increase with age.</p><!><p>Two-way anova showed significant effects of gender (F = 17.68, p = 0.0008) and genotype (F = 19.76, p = 0.0005) in the fat, muscle, and free fluid content. Post hoc Newman–Keuls revealed that old female KO mice had significantly more fat, muscle, and free fluid (90%, 27%, both p < 0.01, and 86%, p < 0.05, respectively) levels compared to female WT mice (Fig. 2a and c). In contrast, the only significant difference between old male mice was the fat content, with male KO mice having significantly more fat (150%, p < 0.01, Fig. 2a) than male WT mice. Muscle and free fluid levels were not significantly different between old male KO and WT mice. Furthermore, female KO mice had significantly more fat (82%, p < 0.01) and free fluid (60%, p < 0.05) content than male KO mice, while female WT mice had significantly more fat (141%, p < 0.05), but less muscle (−25%, p < 0.01) mass than male WT mice. The unpaired Student's t-test indicated that adult (7–9 months) female KO mice also had significantly higher body fat, muscle, and free fluid (272%, 19%, both p < 0.01, and 42%, p < 0.05, respectively) levels than adult female WT mice (Fig. 2d–f).</p><!><p>After completing the analysis for body composition, metabolic parameters, including RQ and MR were measured. Two-way anova indicated a significant effect of genotype, on both RQ (F = 4.97, p < 0.05) and MR (F = 4.85, p < 0.05), with KO mice showing lower RQ (−19%) and lower MR (−14%) than WT mice, irrespective of gender (Fig. 3a and b). Adult female KO mice also had significantly lower (−23%, p = 0.003) MR compared to age-matched female WT mice, but RQ was not significantly different (Fig. 3c and d).</p><!><p>The mice were between 18 and 20 months old when they were sacrificed. Two-way anova indicated a significant effect of genotype (F = 9.88, p = 0.007), and the interaction between gender and genotype (F = 5.87, p = 0.03), on serum leptin levels. Post hoc Newman–Keuls comparisons showed that female KO mice had the highest serum leptin levels, compared to all the other groups (female KO vs. female WT= 191%, p < 0.01and female KO vs. male KO = 111%, p < 0.01, Fig. 4a). No significant differences in serum leptin levels between male KO and male WT mice or between male and female WT mice were observed. The mice at 20 months were of drastically different body weight, with female KO mice still being the heaviest (Fig. 4b). After adjusting for body weight, female KO mice still showed the highest serum leptin/body weight values compared to all the other groups (female KO vs. female WT = 93%, p < 0.05 and female KO vs. male KO = 95%, p < 0.05, Fig. 4c). Serum leptin levels paralleled the amount of body fat and body weight, with female KO mice having the highest levels of all three parameters.</p><!><p>The HOMA-IR was calculated using fasting blood glucose and serum insulin concentrations (Fig. 5a). Two-way anova revealed a significant effect of genotype on insulin resistance (F = 23.35, p = 0.0002), with both male and female Hcrt KO mice showing higher insulin resistance compared to sex-matched WT mice (male KO vs. male WT = 73%, p < 0.05 and female KO vs. female WT = 93%, p < 0.01). Although not significant, male KO mice showed a trend toward higher insulin release from beta cells (HOMA-%β) compared to male WT mice (90%, p = 0.09, Fig. 5b).</p><!><p>Since both adult and old female Hcrt KO mice showed significantly larger body weight, compared to age-matched female WT mice, we also analyzed the weight of BAT and WAT (abdominal) in these mice (Fig. 6). Two way anova revealed a significant effect of genotype (F = 21.67, p = 0.0003) on BAT, as well as age (F = 18.13, p = 0.0006) and genotype (F = 47.21, p < 0.0001) on WAT. Both old (20 months) and adult (10 months) female KO mice had significantly more BAT (69% and 84%, p < 0.05, respectively, Fig. 6a) than age-matched WT females. Similarly, both old and adult female KO mice had more WAT (290% and 452%, p < 0.01, respectively, Fig. 6b) compared to age-matched female WT mice. Furthermore, old KO females exhibited more WAT compared to adult KO females (61%, p < 0.01), but no significant difference was noted between old and adult WT female mice.</p><!><p>In the present study, we observed that female Hcrt KO mice were 20% heavier than female WT mice starting as early as 4 months, with the difference increasing with age until 20 months (50–60%), when the animals were sacrificed. This is a longer time interval than previously reported by other investigators. No significant difference in body weight between male KO and male WT mice was noted. Tsuneki et al. (2008) similarly reported that female KO mice became 20% heavier than female WT mice at 9 months (36 weeks), however, no difference in body weight between male KO and male WT mice was observed. Fujiki et al. (2006) also reported that KO mice became heavier than WT mice at just 3.5 months (100 days). This difference was significant only in female mice, although male mice started showing a slight increase in body weight at 18 months.</p><p>Besides changes in body weight, we show for the first time, that female Hcrt KO mice, as young as 7–9 months and as old as 18–20 months exhibited significant increases in the major components of the body (fat, muscle and free fluid), compared to age-matched female WT controls. This would account for the larger body weight of female KO mice compared to female WT mice. The difference in the sum total of fat, muscle, and free fluid content (17.14 g) between old female Hcrt KO and WT mice parallels the body weight difference (15.16 g) between them, and is more than double the difference in sum total (8.01 g) and body weight (6.83 g) between old male Hcrt KO and WT mice. The difference in the sum total of the major components of the body (14.83 g) between adult female Hcrt KO and WT mice also closely mimics the difference in their body weight (14.40 g), and closely resembles the changes seen in old female mice. The reason for the greater difference in the sum total of body fat, muscle, and free fluid content between female and male mice is not clear. The increase in body fat in old male Hcrt KO mice, compared to old male WT mice, did not result in increased body weight of these mice. This could be due to the fact that other factors (such as the weight of various internal organs and bone density) not measured in this study, may have counterbalanced the change in body fat. Table 1 shows the changes in the major components of the body, after normalizing for the different body weight. Hcrt KO mice, regardless of age or gender, showed increased body fat percentage and decreased muscle percentage, compared to wild type mice. Free fluid percentage, however, was significantly higher only in old female KO mice compared to old female WT mice.</p><p>In this study, we report for the first time, that both adult and old female Hcrt KO mice had significantly larger deposits of brown and white adipose tissues, compared to age-matched female WT mice. The mechanism involved in the increased fat content in Hcrt KO mice compared to WT mice remains to be determined. We further showed that KO mice had lower MR compared to WT mice, irrespective of age or gender. The decrease in MR results in increased body fat, seen in all Hcrt KO mice. Sellayah et al. (2011) recently showed that after 6 weeks on a high fat diet, male WT mice gained 15% of their initial body weight, and energy expenditure was increased by 13.5%, compared to male WT mice fed a normal diet. In contrast, male OX (orexin, hypocretin)-null mice on a high fat diet gained 45% body weight, with no change in energy expenditure, compared to male OX-null mice fed a normal diet. Conversely, Funato et al. (2009) reported that both male and female mice over-expressing orexin (CAG/Orexin transgenic mice) did not show any increase in body weight, produced by a high fat diet, as seen in sex-matched WT mice. These CAG/Orexin mice demonstrate ectopic peptide production in several brain regions, all of which have been implicated in various homeostatic, circadian, learned and/or hedonistic aspects of food intake, taste preference or energy homeostasis (Saper et al. 2002). Funato et al. (2009) also showed that male mice over-expressing orexin had higher energy expenditure with effective mass correction, compared to male WT mice, fed a high-fat diet. Taken together, our findings, as well as those of Funato et al. (2009) and Sellayah et al. (2011), indicate that orexin over expression prevents body weight changes, while lack of orexin increases body weight, due to reduction in energy expenditure/MR.</p><p>Sellayah et al. (2011) also reported that 6-week-old OX-null male mice showed lower food intake and physical activity, compared to WT male mice, regardless of whether they were on a normal or high-fat diet. Tsuneki et al. (2008) similarly reported that food intake in 1-year-old Hcrt KO mice, fed normal chow, decreased by 27% in male, and 22% in female mice compared to sex-matched WT mice. These authors also showed that locomotor activity decreased by more than 50% in both male and female KO mice, compared to sex-matched WT mice, fed normal chow. We previously reported that male Hcrt KO mice were less motivated to work for a food reward, in the light phase, compared to male WT littermates (McGregor et al. 2011). No performance differences were observed when they were required to bar press to avoid foot shock, in either the light or the dark phase, or when working for a food reward, in the dark phase.</p><p>Sex differences and body weight are known to significantly affect serum leptin levels (Frederich et al. 1995; Niskanen et al. 1997). In the current study, we showed that 20-month-old female Hcrt KO mice had significantly higher body weight, body fat, muscle, and free fluid levels, as well as serum leptin levels, compared to age-matched female WT mice. Tsuneki et al. (2008) similarly reported that 9-month-(but not 2 month) old female Hcrt KO mice had larger body weight and higher serum leptin levels, compared to female WT mice of the same age, when maintained on a normal diet. No differences in serum leptin levels or body weight, were seen between male KO and WT mice at any age.</p><p>We further observed that both male and female Hcrt KO mice showed higher insulin resistance (HOMA-IR) compared to sex-matched WT mice, at 20 months. This is consistent with the findings of Tsuneki et al. (2008), who reported that insulin sensitivity is decreased (higher insulin resistance), in both male and female KO mice, compared to sex-matched WT mice, at 9 months. This suggests that insulin resistance begins in adult Hcrt KO mice and persists until old age (9–20 months). It is interesting to note that male Hcrt KO mice did not show any significant difference in body weight compared to male WT mice, however, they did show significant increases in body fat content, insulin resistance, and also insulin release. This suggests that body fat, rather than body weight, is a more useful indicator of the increased risk of type-two diabetes.</p><p>Other investigators have also reported differences in hypocretin neurotransmission between male and female wild type animals. Higher levels of Hcrt receptors (both type 1 and type 2) in male rats compared to female rats have been reported (Jöhren et al. 2001). Both receptors are located in brown adipose tissue, however, Hcrt-induced adipogenesis and adipose tissue developmental differentiation are dependent on receptor 1 signaling, as indicated by studies in the Hcrt ligand and Hcrt receptor KO mice (Sellayah and Sikder 2012). Funabashi et al. (2009) reported that female, but not male, rats (3 months old) showed increased activation of Hcrt neurons, and increased feeding during rebound, after 48 h fasting. Pirnik et al. (2008) reported a positive correlation between body weight, fat gain, and increased activation of Hcrt neurons in female, but not male mice. Jöhren et al. (2002) reported significantly higher prepro-orexin mRNA levels in the hypothalamus of female rats compared to male rats, although Brownell and Conti (2010) showed that male mice had more Hcrt neurons than female mice (2251 Hcrt neurons in males vs. 1805 in females at 100 days). These authors also showed that the number of Hcrt neurons decreased with age, by the same percentage, in male and female mice (by 800 days, male mice lost 15%, while female mice lost 16% of their Hcrt neurons). They went on to suggest that the decline in the number of Hcrt neurons may help to explain the physiological changes in sleep and energy homeostasis regulation, during aging. We speculate that this decrease in the number of Hcrt neurons may also account for the increased body weight and body fat percentage, observed in aged mice (Sellayah and Sikder 2014).</p><p>Unlike our animal studies, human studies indicate that both male and female narcoleptics have increased BMI, compared to patients with idiopathic hypersomnia (Poli et al. 2009), psychiatric controls, including depression, alcohol dependence, schizophrenia or substance abuse patients (Dahmen et al. 2009), patients with neurological disorders (Schuld et al. 2000), as well as to healthy controls (Nishino et al. 2001). A major difference between human narcoleptics and Hcrt KO mice (animal model of narcolepsy) is that, in humans the Hcrt cell is lost in its entity (on average 90% of the Hcrt cell population is lost), as is the case in the Hcrt-ataxin mouse (another animal model of narcolepsy). However, in the Hcrt KO mouse, there is no expression of the Hcrt peptide, due to a mutation of the prepro-orexin gene, but the cells remain intact. Hence, any changes observed in the Hcrt KO mouse compared to the WT mouse, would arise solely from the loss of the Hcrt peptide, and any compensation due to this loss. On the other hand, any changes observed in human narcoleptics compared to control humans, would be due to the loss of any, or all, associated neurotransmitters, including Hcrt, dynorphin, and neuronal activity-regulated pentraxin (NARP) (Blouin et al. 2005; Crocker et al. 2005), resulting from the loss of the Hcrt neurons.</p><p>Schuld et al. (2000) reported no differences in BMI between medicated (tricyclic antidepressants and/or psycho-stimulants) and drug naïve narcoleptic patients, suggesting that the higher BMI in narcoleptic patients is a consequence of the disease-related behavior, including reduced locomotor activity and increased amounts of sleep, which may result in lower energy expenditure, rather than the drug treatment. Chabas et al. (2007) observed that narcoleptic patients had lower energy expenditure than controls, however, the two groups were not BMI matched. Fronczek et al. (2008) found no difference in basal MR between male narcoleptic patients and age and BMI-matched controls. Dahmen et al. (2001) studied a mixed group of narcoleptics (2 males and 11 females) and controls (8 males and 22 females), and showed that the basal MR and energy expenditure, of nonobese (BMI < 30) narcoleptics were significantly lower than BMI-matched controls. Lammers et al. (1996) reported decreased daily food intake in narcoleptic humans compared to controls, while Middelkoop et al. (1995) showed that the total intensity of physical activity did not differ between the two groups.</p><p>We previously reported that the weight, throughout development (from birth to 12 months), did not differ between genetically mutated narcoleptic Doberman Pinchers and breed-matched controls (John et al. 2004). Whereas narcoleptic mice and humans show a loss of the Hcrt peptide and/or neuron, and thus lower Hcrt levels, narcoleptic canines have normal levels of Hcrt, but a mutation in the Hcrt receptor 2 (John et al. 2004). This suggests that Hcrt signaling through receptor 1, rather than receptor 2, plays a role in obesity. Furthermore, the different neurological changes, as well as species variability, can account for differences in weight regulation between narcoleptic humans, mice, and dogs.</p><p>We conclude that absence of the Hcrt peptide has gender-specific effects. Only female Hcrt KO mice show increased body weight, muscle and free fluid content, as well as higher serum leptin levels, compared to female WT mice. On the other hand, both male and female KO mice show greater body fat and higher insulin resistance, compared to sex-matched WT mice. In contrast, prior work has shown that Hcrt-ataxin mice and human narcoleptics show weight gain in both sexes. These subjects lose Hcrt cells, not just the Hcrt peptide, indicating that Hcrt peptide loss produces gender-specific changes.</p>

PubMed Author Manuscript

A catalytically active [Mn]-hydrogenase incorporating a non-native metal cofactor

Nature carefully selects specific metal ions for incorporation into the enzymes that catalyze the chemical reactions necessary for life. Hydrogenases, enzymes that activate molecular H2, exclusively utilize Ni and Fe in [NiFe]-, [FeFe]-, and [Fe]-hydrogeanses. However, other transition metals are known to activate or catalyze the production of hydrogen in synthetic systems. Here, we report the development of a biomimetic model complex of [Fe]-hydrogenase that incorporates a Mn, as opposed to a Fe, metal center. This Mn complex is able to heterolytically cleave H2 as well as catalyze hydrogenation reactions. Incorporation of the model into an apoenzyme of [Fe]-hydrogenase results in a [Mn]-hydrogenase with enhanced occupancy-normalized activity over an analogous semi-synthetic [Fe]-hydrogenase. These findings represent the first instance of a non-native metal hydrogenase showing catalytic functionality and demonstrate that hydrogenases based on a manganese active site are viable.

a_catalytically_active_[mn]-hydrogenase_incorporating_a_non-native_metal_cofactor

Introduction<!>Synthesis, characterization, and catalytic activity of Mn models<!>Reconstitution and activity of [Mn]-hydrogenase<!>Discussion<!>Synthesis of complex 3<!>Synthesis of complex 4 and 4(18-crown-6)<!>Computational methods<!>Preparation of apoenzymes<!>Reconstitution of [Mn]-hydrogenase<!>Enzyme activity assay of the [Mn]-hydrogenase holoenzyme<!>Infrared spectroscopy of enzyme

<p>Nature judiciously chooses metal ions to catalyze biochemical reactions. Understanding these choices is not only important for fundamental knowledge of biocatalysis, but might also provide guidelines for designing synthetic catalysts. Structure-activity studies where native metal ions are replaced by non-native analogues represent a direct approach for unraveling the choices made by nature. However, abiological replacement of native metals that maintain the activity of the native enzyme activity have rarely been reported.1–3</p><p>When it comes to the activation of dihydrogen (H2), nature chooses only Fe and Ni, as in [NiFe]-, [FeFe]-, and [Fe]-hydrogeanses.4 It is not yet clear whether this choice is due solely to the higher bioavailability of Fe and Ni relative to other metals, or due to their unique ability to activate H2 in biological systems. Many other transition metals are known to activate or evolve hydrogen in synthetic systems. A [RuRu] analogue of [FeFe]-hydrogenase was the only semi-synthetic hydrogenase that contained a non-native metal, but it was inactive.5 [Fe]-hydrogenase is an attractive platform to address the questions concerning the choices of metals in biological H2 activation.6 This enzyme catalyzes the hydrogenation of methenyl-tetrahydromethanopterin (methenyl-H4MPT+) to form methylene-H4MPT, a reaction involved in microbial methanogenesis.7 Unlike [NiFe]- and [FeFe]-hydrogenases, [Fe]-hydrogenase has only one Fe center per active site. In its resting state, the low-spin FeII center is coordinated by one H2O and two cis-CO molecules, a cysteine-thiolate, as well as an acyl carbon and a pyridinyl nitrogen from a guanylylpyridinol moiety (Fig. 1a).8,9 During catalysis, water likely dissociates, thereby opening a binding site for H2. Considering that Mn(I) and Fe(II) are isoelectronic, and further inspired by the recent development of Mn-catalyzed hydrogenation,10 we decided to prepare a Mn(I) model of the active site of [Fe]-hydrogenase. Through reconstitution of the apoenzyme of [Fe]-hydrogenase with this Mn(I) model, we have obtained the first [Mn]-hydrogenase. This artificial hydrogenase is active for the native [Fe]-hydrogenase reactions, indicating Mn as a viable metal for biological H2 activation in the enzyme.</p><!><p>The reaction of 2-hydroxy-6-methylpyridine (1) with two equivalents of n-BuLi gave a dilithiated intermediate (2), which was not isolated but treated directly with Mn(CO)5Br followed by acidic workup to give the targeted Mn(I) model 3 (Fig. 1b). The infrared (IR) spectrum of 3 in tetrahydrofuran (THF) exhibits four CO peaks at 1948, 1967, 1983 and 2068 cm-1 (Supplementary Fig. 13), indicative of a Mn tetra(carbonyl) species. The peak at 1651 cm-1 was attributed to the acyl group. The NMR spectra of 3 (in deuterated THF) is characteristic of a diamagnetic species (Supplementary Fig. 8), indicating a low-spin configuration of Mn(I). The 2-OH proton gave a broad 1H peak at 11.26 ppm. In the 13C NMR spectrum [in deuterated acetonitrile (CD3CN)] (Supplementary Fig. 9), three types of CO carbon were observed at 213.6, 215.9, and 220.3 ppm, respectively. The acyl carbon gave a peak at 280.8 ppm. CO/13CO exchange was observed by 13C NMR when 3 was treated with an excess of 13CO in deuterated THF or CD3CN, suggesting that one or more CO ligands readily dissociate from the Mn center (Supplementary Fig. 1-2). The solid-state molecular structure of 3 was determined by X-ray crystallography (Fig. 1c). The Mn ion possesses an octahedral coordination geometry. Three CO ligands are co-planar, while the fourth CO ligand occupies a position orthogonal to the other three. The acylmethylpyridinol ligand coordinates in a bidentate fashion via the acyl C and pyridonal N.</p><p>A H2/D2 exchange assay was employed to test the activity of 3 towards H2 activation.11 The reaction was conducted in a high-pressure NMR tube, where D2 (8 bar) and H2 (12 bar, total pressure of mixed gas 20 bar) were added to a solution of 3 in THF-d8. The reaction was monitored by NMR spectroscopy. Complex 3 alone did not mediate H/D exchange after two days at 25 °C. However, in the presence of a base (e.g., 1-methylpyrrolidine (MP) or triethylamine (Et3N)), the formation of HD was observed within 3 h at 25 °C (Supplementary Fig. 3), indicating H2 activation. A D2/H+ exchange experiment was then conducted, in which 20 bar of D2 was introduced to a CD3CN-d3 solution of 3 containing 3.0 equivalent of MP at 25 °C. H2 and HD were detected after 1 h (Supplementary Fig. 5). This result indicates that H2 splitting is a heterolytic process, which is the same as in the enzymatic reaction.</p><p>We previously reported that deprotonation of the 2-OH group was a key step in enabling H2 activation by a semi-synthetic [Fe]-hydrogenase.12 After deprotonation, the anionic 2-O− group served as an internal base to assist in the heterolytic splitting of H2 at the Fe center.12,13 The need for a base for H2 splitting by 3 suggested that a similar mechanism was operating in the activation of H2 by 3. To verify this hypothesis, we sought to intentionally deprotonate the 2-OH group in 3. A clean reaction was observed upon treatment of 3 with potassium hydride (KH) in THF at room temperature (Fig. 1d). In the 1H NMR spectrum of the in-situ formed complex 4, all the peaks of 3 except the 2-OH peak were still observed, with a significant upfield shift (Supplementary Fig. 10). 1H NMR spectra similar to that of 4 were obtained when 3 was treated with an excess of MP or Et3N, suggesting the formation of a similar deprotonated species. To facilitate isolation and crystallization, 4 was treated with a K+-selective crown ether, 18-crown-6 to give complex 4(18-crown-6) (Fig.1d). 4 and 4(18-crown-6) exhibit similar NMR spectra (Supplementary Fig. 10-11). The IR spectrum of 4(18-crown-6) in THF exhibits four ν(CO) peaks at 1901, 1944, 1972, 2052 cm-1, and a ν(acyl C=O) peak at 1616 cm-1 (Supplementary Fig. 14). These peaks are shifted to lower values compared to the corresponding peaks in 3, indicating a more electron-rich Mn center upon deprotonation of the 2-OH group. The X-ray structure of 4(18-crown-6) confirmed the deprotonation of the 2-OH group (Fig. 1e). The 2-O− group has a strong interaction (2.6097(14) Å) with the K cation, which is further coordinated by the 6 O atoms of 18-crown-6. Both 4 and 4(18-crown-6) were able to activate H2 without an external base, as observed in the H2/D2 exchange assay (Supplementary Fig. 4). This result supports that deprotonation of 2-OH group is a key step in H2 activation by 3 in the presence of a base.</p><p>Complexes 3 and 4 are hydrogenation catalysts. To optimize the reaction conditions, hydrogenation of benzaldehyde (5a) was employed as a test reaction with complex 3 as the catalyst. After screening various reaction parameters, the best conditions were: 1 mol% complex 3 with 20 mol% MP as base under 50 bar H2 at 80 °C in THF (Fig. 1f). Under these conditions, the yield of hydrogenation was quantitative. Although H2 activation was observed at 25 °C, a higher temperature (e.g., 80 °C) was necessary for catalytic hydrogenation, suggesting that hydride transfer was more difficult than H2 activation. Under similar conditions using 4 (5 mol%) rather than 3 as the catalyst and without the base, hydrogenation was also achieved in a 96% yield. Similar reaction conditions could be applied for the hydrogenation of other substrates including: ketone (5b), primary imine (5c), and secondary imine (5d; Fig. 1g), moderate yields were obtained (Supplementary Fig. 6). The hydrogenation of an olefin substrate was less efficient (5e; Fig. 1g). The reaction profiles of hydrogenation reactions were monitored by 19F NMR spectroscopy using 4-CF3 benzaldehyde (5f) as the substrate (Supplementary Fig. 7). No induction period was observed, suggesting that 3 and 4 were the true pre-catalysts or catalysts. The initial rates of reactions catalyzed by 3 and 4 were similar, but the reaction catalyzed by 4 was slower, probably due to some decomposition of 4. The catalytic activity of 3 for hydrogenation is modest compared to some state-of-the-art Mn and Fe catalysts (Supplementary Table 2). The ligand environment of 3 is less electron donating than those of highly active Mn complexes, leading to a less hydridic Mn-H intermediate, which might be the origin of the subdued activity.</p><p>Density functional theory (DFT) computations (PBE0-dDsC/TZ2P//M06/def2-SVP theoretical level, see Computational Methods in Supplementary Information) suggest the following catalytic cycle for the hydrogenation of 5a catalyzed by 3 (Fig. 2a and Supplementary Fig. 21). Deprotonation of the 2-OH group in 3 by the base MP followed by substitution of one CO ligand (trans to the acyl ligand) by H2 gives intermediate 7b. Heterolytic cleavage of the coordinated H2 in 7b leads to a hydride complex 7c, which hydrogenates 5a to give the alcohol-bound complex 7e. Replacing the alcohol product in 7e by H2 yields the product and regenerates 7b. The substitution of CO in 7a by H2 as well as the transition state associated with hydride transfer represent the highest points on the potential energy surface (Fig. 2b), with similar values of about 26 kcal/mol. Deprotonation and H2 splitting are facile processes. Given the accuracy of the DFT method, the computed reaction energy profile qualitatively agrees with the experimental results.</p><!><p>The activity of 3 in H2 splitting and hydrogenation catalysis contrasts the inactivity of its Fe(II) analogue in H2 activation.12 This reactivity difference is consistent with recent observations that Mn(I) complexes are often more active or stable than Fe(II) complexes in hydrogenation reactions.10 However, it was unclear whether Mn would be competent for biological H2 activation. The study of semi-synthetic [RuRu]-hydrogenase showed that even though H2 activation on synthetic Ru complexes was facile, in a hydrogenase environment this metal was inactive.5 To test possible Mn-catalyzed hydrogenase activity, complex 3 was employed to reconstitute a [Mn]-hydrogenase. The protocol previously used for the reconstitution of semi-synthetic [Fe]-hydrogenases was also applicable here.12 3 was first dissolved in a solution containing 99% methanol and 1% of acetic acid. The solution containing two equivalents of 3 was mixed with a solution of [Fe]-hydrogenase apoenzyme from Methanocaldococcus jannaschii heterologously produced in Escherichia coli in the presence of 2-mM GMP (GMP = guanosine monophosphate). While 3 is soluble in pure methanol, active enzyme was not reconstituted using a methanol solution of 3 in the absence of acetic acid. The role of acetic acid is unclear as the IR spectra of complex 3 in pure methanol and in mixtures of methanol and acetic acid are essentially the same (Supplementary Fig. 15-16), suggesting that an acetate-bound form of 3 was not the major species in the reconstitution medium as was the case for the reconstitution of semisynthetic [Fe]-hydrogenase.12</p><p>The activity of this [Mn]-hydrogenase was measured photometrically for both the forward (reduction of methenyl-H4MPT+ with H2) and the reverse (H2 production from methylene-H4MPT) reactions. The absorbance of methenyl- H4MPT+ at 336 nm was used as a spectroscopic probe of the reaction rate. To our delight, the [Mn]-hydrogenase was active for the native reactions of [Fe]-hydrogenase (Fig. 3a-d). The activity largely remained identical for multiple samples; the averaged apparent specific activity was 1.5 ± 0.1 U/mg and 0.09 ± 0.01 U/mg for the forward and reverse reaction, respectively. As observed in the reconstitution of semisynthetic [Fe]-hydrogenase,12 the absence of GMP in the reconstitution solution resulted in a 2-fold decrease of the activity of the reconstituted enzyme (Table 1) although the IR spectrum was not changed (Supplementary Fig. 17). Interestingly, the [Mn]-hydrogenase is biased towards the forward reaction, which is around 20 times faster than the reverse reaction. The native [Fe]-hydrogenase and semi-synthetic [Fe]-hydrogenase have only slightly higher rates for the forward reaction (Table 1).12,14 The forward reaction was conducted at pH 7.5 while the reverse reaction was conducted at pH 6.0. The bias of the [Mn]-hydrogenase toward the forward reaction might be due to the higher pKa of the 2-OH group compared to [Fe]-hydrogenase and semi-synthetic[Fe]-hydrogenase,12 which hinders its deprotonation at pH 6.0, thereby decreasing the base function and the rate of the reverse reaction. Control experiments showed that complex 3 or the apoenzyme of [Fe]-hydrogenase alone did not afford the activity.</p><p>In [Fe]-hydrogenase, the Fe center is bound to Cys176 thiolate. The reconstituted Cys176Ala variant of [Mn]-hydrogenase has a specific activity of 0.12±0.19 U/mg for the forward reaction, which is 8% of the native variant of the reconstituted [Mn]-hydrogenase. This result contrasts the findings on semi-synthetic [Fe]-hydrogenase, where reconstituted Cys176Ala variants were inactive (Table 2).12 This suggests that binding of the Mn complex to Cys176-S is not absolutely needed for [Mn]-hydrogenase to be active. In the native [Fe]-hydrogenase, the iron complex component of the FeGP cofactor is connected to the protein via hydrogen bonds involving the backbone NH of Cys176, the hydroxyl group of Thr13, the carboxy group of Asp251, and water molecules (Supplementary Fig. 18). These interactions might lead to specific binding of the Mn complex even in the absence of Cys176-Fe bonding, albeit with an unoptimized orientation.</p><p>Different batches of samples of [Mn]-hydrogenase have nearly identical activity, however, they exhibit different IR spectra in the region of ν(CO) vibrational bands. For example, the spectral shape and positions of two samples are similar and resemble those of complex 3 (Supplementary Fig. 19a-b) but their intensities are different. A third sample has a spectrum similar to that of native [Fe]-hydrogenase (Supplementary Fig. 19). This result suggests that various amount of Mn complexes are unspecifically bound to the protein in different samples in addition to a similar amount of specifically bound complex, which gives rises to the hydrogenase activity. Assuming that the IR spectrum of Figure S19c originates from specifically bound Mn mimic because only two CO bands at 1985 and 1965 cm-1 were detected, we estimated the occupancy of the active site of [Mn]-hydrogenase as 20%, by comparing its CO peak area with that of the reconstituted native [Fe]-hydrogenase (Supplementary Fig. 19). Considering this occupancy, the actual specific activity is about 7.5 U/mg (forward reaction) and 0.45 U/mg (reverse reaction). The corresponding turnover frequencies (TOFs) are 5 s−1 and 0.3 s−1 for the forward and reverse reactions, respectively. These TOFs are 3 orders of magnitude higher than those of complex 3 alone in hydrogenation reactions (Supplementary Table 3), underscoring the important role of the protein environment for catalysis. They are also significantly higher than those of state-of-the-art synthetic Mn and Fe catalysts (Supplementary Table 3).</p><p>To probe whether the unspecifically bound Mn complex contributed to the activity of [Mn]-hydrogenase, reconstitution was conducted using a mutated apoenzyme of [Fe]-hydrogenase, in which the binding residues for the FeGP cofactor were triply mutated (T13V, C176A and D251A). Although unspecific binding of complex 3 to the triple mutant apoenzyme was confirmed by IR spectroscopy (Supplementary Fig. 20), this variant of [Mn]-hydrogenase had no enzymatic activity (Table 2). Moreover, F420-dependent methylene-H4MPT dehydrogenase (Mtd), which does not bind the FeGP cofactor but binds the H4MPT substrates, was also used for reconstitution. Unspecific binding of complex 3 by Mtd was again observed (Supplementary Fig. 20), but this sample also showed no enzymatic activity (Table 2). Taken together, these data indicate that the unspecific bound Mn complex is catalytically inactive.</p><!><p>The active sites of both the semi-synthetic [Mn]- and [Fe]-hydrogenase lack the guanosine monophosphate (GMP) moiety and two methyl groups in the pyridinol group of the native cofactor (Fig. 1a), which should be the principle reason for their specific activity being only a few percent of native [Fe]-hydrogenase.12 Direct comparison of the semi-synthetic [Mn]- and [Fe]-hydrogenases, however, reveals the relative catalytic competency of the two different metals. The occupancy-normalized specific activity of [Mn]-hydrogenase is 25% higher than in the analogous [Fe]-hydrogenase for the forward reaction (3 U/mg at 50% active site occupancy),12 which is the physiological reaction during methanogenesis. Thus, Mn appears to be more active than Fe for enzymatic H2 activation.</p><p>In summary, a Mn(I) model of the active site of [Fe]-hydrogenase that is capable of splitting H2 and catalyzing hydrogenation reactions has been prepared. Reconstitution of the apoenzyme of [Fe]-hydrogenase with this Mn(I) model leads to a [Mn]-hydrogenase, which is active for the [Fe]-hydrogenase reactions. These findings represent the first demonstration of catalytic functionality of a non-native metal in a hydrogenase enzyme. The molar activity of this semi-synthetic [Mn]-hydrogenase is higher than its Fe analogue, raising an intriguing question – why does nature choose Fe over Mn?</p><!><p>2-Hydroxy-6-Methylpyridine 1 (1.3 g, 12 mmol) was dissolved in 60 mL of dry THF in a Schlenk flask. To this solution, n-BuLi (2.5 M in hexane, 9.6 mL, 24 mmol) was added dropwise at 0 °C and the solution was further stirred for 30 min at 0 °C. In another schlenk flask a THF (80 mL) solution of Mn(CO)5Br (3.3 g, 12 mmol) was cooled to -78 °C. The solution of deprotonated 1 was then added dropwise to the Mn(CO)5Br solution at -78 °C. The resulting mixture was allowed to slowly warm to room temperature and further heated to 50 °C. After stirring at 50 °C overnight, the mixture was cooled to room temperature and 1.5 mL of 37% HCl aqueous solution was then slowly added. THF solvent was removed 30 min later. The residue was further purified by silica gel chromatography in glovebox (1.3 g, 36%) using ethyl acetate/hexane as eluent. Single crystal suitable for X-ray test was obtained via layer diffusion of pentane to a THF solution of complex 3 at -22 °C.</p><!><p>To a solution of complex 3 (1.0 g, 3.3 mmol) in THF (5 mL) was added KH (132 mg, 3.3 mmol) slowly under stirring at room temperature. When there was no more H2 gas formed, a THF solution of 18-crown-6 (958 mg, 3.6 mmol) was added at once to the mixture. The resulting mixture was further stirred at room temperature for 2 h before it was filtered through a Teflon membrane to remove all the solid impurity. A layer of Et2O was then added on top of the THF solution and the mixture was stored at -22 °C. Crystal of 4(18-crown-6) was obtained in 75% yield (1.5 g).</p><!><p>The geometries of all species were optimized at the M06/def2-SVP theoretical level using the "ultrafine" integration grid and the SMD implicit solvent model for tetrahydrofuran as implemented in Gaussian09. Refined energy estimates were obtained using single point computations on the optimized M06 geometries at the PBE0-dDsC/TZ2P level as implemented in ADF. Reported free energies are derived from the PBE0-dDsC electronic energies, M06 enthalpy and vibrational only entropy contributions, and solvation corrections using the COSMO-RS model.</p><!><p>The [Fe]-hydrogenase-encoding gene (hmd) from M. jannaschii8 and the gene encoding F420-dependent methylene-H4MPT dehydrogenase (mtd) gene from Archaeoglobus fulgidus15. The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus were cloned into the expression vector pET24b. Genes of the hmd mutants (C176A and T13V-C176A-D251A) were synthesized using the template of the wild type hmd gene by GenScript. The E. coli strain BL21(DE3) containing the expression vector for hmd or mtd was cultivated and the gene expression was induced. The over-produced protein was purified as described previously8.</p><!><p>Complex 3 was dissolved in solution containing 99% methanol and 1% acetic acid. The reconstitution of [Mn]-hydrogenase was performed in the anaerobic tent (95% N2/5% H2) at 8 °C. The 2 ml reconstituted system contains 0.5 mM complex 3, 0.25 mM apoenzyme, 2 mM guanosine monophosphate (GMP) and 100 mM sodium acetate buffer pH 5.6. The mixture was incubated on ice for 1 h. Then the mixture was washed by 10 mM MOPS/KOH pH 7.0 containing 2 mM DTT through a 30 kDa cut-off ultrafilter with at least totally1000 folds dilution to remove the unbound complex 3. Finally, the reconstituted [Mn]-hydrogenase holoenzyme was concentrated to ~50 mg/ml for enzyme activity assay. The reconstituted enzyme was quickly frozen in liquid nitrogen and store at –75 °C. The reconstitution of semisynthetic [Mn]-hydrogenase using the mutated apoenzyme and Mtd, and semisynthetic [Fe]-hydrogenases were performed using the same method in the presence of 2 mM GMP as described above. The native [Fe]-hydrogenase with the FeGP cofactor was prepared as described previously.8</p><!><p>The reduction of methenyl-tetrahydromethanopterin (methenyl-H4MPT+) to methylene-H4MPT (the forward reaction) and the dehydrogenation of methylene-H4MPT to methenyl-H4MPT+ (the reverse reaction) was measured. For the forward reaction, 20 µM (final concentration) methenyl-H4MPT+ was added to a 0.7 ml solution containing 120 mM potassium phosphate buffer pH 7.5 containing 1 mM EDTA under 100% H2 gas phase at 40 °C. The reaction was started by injecting 10 µl reconstituted [Fe]-hydrogenase sample. Reduction of methenyl-H4MPT+ was detected by measuring the decrease of the absorbance at 336 nm. For the reverse reaction, 20 µM (final concentration) methylene-H4MPT was added to a 0.7 ml solution containing 120 mM potassium phosphate buffer pH 6.0 containing 1 mM EDTA under a 100% N2 gas phase at 40 °C. The reaction was started by injecting 10 µl reconstituted [Mn]-hydrogenase sample. Dehydrogenation of methylene-H4MPT was detected by measuring the increase in the absorbance at 336 nm. The activities were calculated using the extinction coefficient of methenyl-H4MPT+ (ε336 nm = 21.6 mM−1cm−1).14 One unit (U) activity is the amount of enzyme catalyzing the decrease of 1 μmol/min methenyl-H4MPT+ (the forward reaction) or increase of methenyl-H4MPT+ (the reverse reaction). For the kinetic measurements shown in Fig.3, the absorbance was measured with an Ultrospec 1100pro spectrophotometer (GE Healthcare); the spectra were recorded on a Specode S600 diode-array spectrophotometer (Jena Analytik). The 20 µM methenyl-H4MPT+ and 20 µM methylene-H4MPT (final concentration) was used as the substrate for the forward and reverse reaction, respectively, 0.007 mg/ml reconstituted enzyme (final concentrations) was added to the 0.7 ml reaction mixture; the light path of the cuvette was 1 cm. The spectra were recorded every 10 s. As a control, complex 3 (14 uM, final concentration) or the apoenzyme (0.014 mg/ml) was added to the assay instead of the reconstituted enzyme.</p><!><p>The samples for IR spectroscopy were prepared in amber-colored 1.5-ml Eppendorf tubes. The sample solutions contained 150 mg/ml (4-mM) semisynthetic [Mn]-hydrogenase in 10-mM MOPS/NaOH pH 7.0. The sample solutions were prepared in the anaerobic tent with the gas phase 95%N2/5%H2 and then frozen in liquid nitrogen. The frozen samples in tubes were stored in a Dewar filled with liquid nitrogen until the measurements were taken.</p><p>All IR spectra were obtained with an FTIR spectrometer (Bruker, Vertex 70V) in an attenuated total reflection (ATR) optical configuration with a Si prism of 45° incident angle and 2 active reflections (Smith Detection, DuraSamplIR IITM). Spectra were obtained with a resolution of 4 cm-1. Five micro-litres of the sample solutions were dropped onto the effective area of a Si prism (3 mm diameter) and concentrated by slowly evaporating the solvent under mild flow of argon gas. The hydration of the sample was estimated by the relative intensities of the water band (OH stretching mode) at approximately 3500 cm-1 against the amide II band of the protein at approximately 1550 cm-1. Spectra were measured successively during the concentration process. We selected a spectrum of a mildly hydrated sample that provided enough intensity to analyze the cofactor bands. A baseline correction was made on the selected spectrum to eliminate contributions of the broad background from the water overtone band at approximately 2000 cm-1. Typically, 512 spectra were averaged to obtain a sufficient signal-to-noise ratio. Measurements were performed in the dark by covering the spectrometer with blackout fabric to avoid light-induced decomposition of the sample. Intensities of the observed bands from various samples were at arbitrary concentrations. For quantitative comparison of the obtained spectra, intensities of the CO bands were normalized by the peak intensities of the amide II band of each spectrum.</p>

PubMed Author Manuscript

Escherichia coli FtnA Acts as an Iron Buffer for Re-assembly of Iron-Sulfur Clusters in Response to Hydrogen Peroxide Stress

Iron-sulfur clusters are one of the most ubiquitous redox centers in biology. Ironically, iron-sulfur clusters are highly sensitive to reactive oxygen species. Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron-sulfur clusters, the iron released from the disrupted iron-sulfur clusters will further promote the production of deleterious hydroxyl free radicals via the Fenton reaction. Here, we report that ferritin A (FtnA), a major iron-storage protein in Escherichia coli, is able to scavenge the iron released from the disrupted iron-sulfur clusters and alleviates the production of hydroxyl free radicals. Furthermore, we find that the iron stored in ferritin A can be retrieved by an iron chaperon IscA for the re-assembly of the iron-sulfur cluster in a proposed scaffold IscU in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential. The results suggest that E. coli ferritin A may act as an iron buffer to sequester the iron released from the disrupted iron-sulfur clusters under oxidative stress conditions and to facilitate the re-assembly of the disrupted iron-sulfur clusters under normal physiological conditions.

escherichia_coli_ftna_acts_as_an_iron_buffer_for_re-assembly_of_iron-sulfur_clusters_in_response_to_

Introduction<!>Protein Preparation<!>Measurements of hydroxyl free radicals<!>Iron binding analyses<!>Iron-sulfur cluster assembly in IscU<!>Hydrogen peroxide disrupts the IscU [2Fe-2S] cluster and promotes the production of hydroxyl free radicals<!>Ferritin A (FtnA) alleviates the production of hydroxyl free radicals by scavenging the iron released from the IscU [2Fe-2S] cluster<!>The iron-bound FtnA is not an efficient iron donor for the iron-sulfur cluster assembly in IscU<!>IscA can retrieve iron from the iron-bound FtnA<!>Apo-IscA mediates the iron-sulfur cluster assembly in IscU when the iron-bound FtnA is used as the iron source in vitro<!>Discussion<!>Disruption of the IscU [2Fe-2S] cluster by H2O2<!>The IscU [2Fe-2S] cluster promotes the production of hydroxyl free radicals in the presence of H2O2<!>FtnA alleviates the iron-mediated production of hydroxyl free radicals<!>FtnA binds the iron released from the IscU [2Fe-2S] cluster in the presence of H2O2<!>The iron-bound FtnA is not an efficient iron donor for the iron-sulfur cluster assembly in IscU<!>Apo-IscA can retrieve the iron from the iron-bound FtnA<!>Apo-IscA promotes the FtnA-mediated iron-sulfur cluster assembly in IscU<!>A proposed model for the iron binding of IscA and FtnA under oxidative stress and normal physiological conditions

<p>Iron-sulfur clusters represent one of the major iron-containing cofactors in living organisms. Throughout evolution, iron-sulfur clusters have become integral parts of diverse physiological processes ranging from respiratory electron transfer, sugar metabolism, nitrogen fixation, photosynthesis, amino acids biosynthesis, heme and biotin biosynthesis, intracellular iron homeostasis, RNA modification, DNA synthesis and repair, and the regulation of gene expression (Beinert et al. 1997, Kiley and Beinert 2003, Johnson et al. 2005, Rouault and Tong 2005, Fontecave 2006, Lill and Muhlenhoff 2006). It is now clear that the biogenesis of iron-sulfur clusters is not a spontaneous process. Increasing evidence has suggested that the iron-sulfur cluster assembly requires multiple proteins which are highly conserved from bacteria to humans (Johnson et al. 2005, Rouault and Tong 2005, Lill and Muhlenhoff 2006). In Escherichia coli, at least two gene clusters iscSUA-hscBA-fdx (Zheng et al. 1998) and sufABCDSE (Takahashi and Tokumoto 2002, Outten et al. 2004) have been identified as critical for the biogenesis or repair of iron-sulfur clusters. Whereas deletion of iscSUA-hscBA-fdx severely decreases the iron-sulfur cluster assembly activity in E. coli, deletion of both iscSUA-hscBA-fdx and sufABCDSE results in an inviable phenotype (Takahashi and Tokumoto 2002). Among the proteins encoded by the gene clusters, IscS is a cysteine desulfurase containing a pyridoxal-5-phosphate (Flint 1996, Cupp-Vickery et al. 2003). IscS catalyzes desulfurization of L-cysteine and transfers sulfane sulfur for the iron-sulfur cluster assembly in a proposed scaffold IscU (Agar et al. 2000, Smith et al. 2001, Urbina et al. 2001, Kato et al. 2002, Smith et al. 2005) which eventually transfers the assembled clusters to target proteins (Wu et al. 2002, Unciuleac et al. 2007). Two heat shock cognate proteins, HscB and HscA, specifically interact with IscU (Silberg et al. 2004, Tapley and Vickery 2004) and stimulate the transfer of the assembled clusters from IscU to apo-ferredoxin in an ATP-dependent reaction (Chandramouli and Johnson 2006). SufS and IscS are paralogs with 30% sequence identity. The catalytic activity of SufS is relatively low, but can be stimulated by SufE and SufBCD complex (Loiseau et al. 2003, Outten et al. 2003). Recent studies further indicated that SufS transfers sulfane sulfur to SufB via SufE (Layer et al. 2007). IscA and SufA are also paralogs with 46% sequence identity. It was reported that both IscA and SufA can host transient iron-sulfur clusters, thus being proposed as alternative scaffold proteins (Krebs et al. 2001, Ollagnier-de-Choudens et al. 2001, Wollenberg et al. 2003, Sendra et al. 2007). However, unlike IscU (Agar et al. 2000), IscA and SufA show a strong iron binding affinity with an iron association constant of 2.0×1019M−1 in the presence of the thioredoxin reductase system (Ding and Clark 2004, Ding et al. 2005, Yang et al. 2006, Lu et al. 2008). Furthermore, the iron-bound IscA and SufA can efficiently provide iron for the iron-sulfur cluster assembly in IscU (Ding et al. 2004, Ding et al. 2005, Yang et al. 2006, Lu et al. 2008). These results suggested that the primary function of IscA/SufA may be to recruit intracellular iron and deliver iron for the biogenesis of iron-sulfur clusters. Based on these studies, we proposed that IscA/SufA (an iron donor) and IscS/SufS (a sulfur donor) may work in concert to coordinately deliver the iron and sulfur for the iron-sulfur cluster assembly in scaffold proteins IscU or SufBCD complex (Yang et al. 2006, Lu et al. 2008).</p><p>Ironically, iron-sulfur clusters are highly sensitive to reactive free radicals (Djaman et al. 2004, Jang and Imlay 2007). Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron-sulfur clusters (Kiley and Beinert 2003), the iron released from the disrupted iron-sulfur clusters will further promote the production of deleterious hydroxyl free radicals via Fenton reaction (Keyer and Imlay 1996). The mechanisms underlying the sequestration of the iron released from the disrupted iron-sulfur clusters and the subsequent repair of the iron-sulfur clusters have not been fully understood. Here, we report that ferritin A (FtnA), a major iron storage protein in E. coli (Hudson et al. 1993, Abdul-Tehrani et al. 1999), is able to scavenge the iron released from the disrupted iron-sulfur clusters and alleviates the iron-mediated production of hydroxyl free radicals in the presence of hydrogen peroxide. Furthermore, we find that the iron stored in FtnA can be retrieved by IscA for the iron-sulfur cluster assembly in IscU in the presence of the thioredoxin reductase system. The physiological roles of ferritins in cellular response to oxidative stresses and in repair of the disrupted iron-sulfur clusters will be discussed.</p><!><p>The DNA fragment encoding ferritin A (FtnA) was amplified from E. coli genomic DNA. Two primers, FTN-1, 5' GAGCACTACCATGGTGAAACCAGAAA-3' and FTN-2, 5'-GAGCATTAGTTAAGCTTGTCGAGGGT-3' were used for the PCR amplification. The PCR product was digested with two restriction enzymes HindIII and NcoI, and ligated into an expression vector pET28b+. The cloned DNA fragment was confirmed by direct sequencing using T7 primers. Recombinant FtnA was prepared following the procedures as previously described (Yang et al. 2002). The gel filtration analyses showed that recombinant FtnA was a homopolymer of 24 subunits with a molecular weight of ~ 440 kDa as reported previously by others (Hudson et al. 1993). The E. coli thioredoxin 1 (Veine et al. 1998) and thioredoxin reductase (Mulrooney 1997) were prepared as described in (Ding et al. 2005). The purity of purified proteins was greater than 95% as judged by the SDS electrophoresis analysis followed by staining with the Coomassie blue.</p><!><p>Hydroxyl free radicals were measured following the procedure described by Halliwell et al. (Halliwell et al. 1987). Briefly, hydroxyl free radicals generated in solution degrade 2-deoxyribose and form a malondialdehyde-like compound which reacts with thiobarbituric acid to generate a chromogen. In the experiments, the IscU [2Fe-2S] cluster or freshly prepared Fe(NH4)2(SO4)2 was incubated with potassium phosphate buffer K2PO4 (10 mM) (pH 7.4), NaCl (60 mM), 2-deoxyribose (4 mM) at 37°C for 10 min before hydrogen peroxide (0.5 mM) was added to initiate the Fenton reaction. The reactions were continued at 37°C for additional 25 min. A developing solution containing 1% thiobarbituric acid and 2.8% trichloroacetic acid (400 µl) was then mixed with the above incubation solutions (600 µL), and boiled for 15 min. The mixtures were centrifuged at 14,000 rpm for 15 min. The amounts of the chromogen in the supernatants were measured from the emission at a wavelength of 553 nm using an excitation wavelength of 532 nm in a Perkin-Elmer LS-3 Fluorometer.</p><!><p>The iron binding in proteins was analyzed after the proteins were incubated with freshly prepared Fe(NH4)2(SO4)2 in the presence of the thioredoxin reductase system, followed by re-purification of the protein using a Mono-Q column (0.98 ml) as described previously (Ding et al. 2005). The re-purification procedure did not affect the iron binding in frataxin A, as over 95% of the total iron content remained in the protein after passing through the Mono-Q column. The total iron content in the eluted samples was determined using the Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) (Department of Geology and Geophysics/LSU) or an iron indicator ferrozine (Yang et al. 2006). The iron-ferrozine complex was measured at 564 nm using an extinction coefficient of 27.9 mM−1cm−1 (Cowart et al. 1993). Both iron analysis methods produced similar results.</p><!><p>For the iron-sulfur cluster assembly reactions, IscU (50 µM) was pre-incubated with cysteine desulfurase IscS (1 µM) and Fe(NH4)2(SO4)2 (400 µM) in the presence of the thioredoxin reductase system (thioredoxin-1 (5 µM), thioredoxin reductase (0.5 µM) and NADPH (500 µM)) at 37°C for 5 min. If indicated, the iron-bound IscA or FtnA was used instead of Fe(NH4)2(SO4)2 as the iron donor. The iron-sulfur cluster assembly was initiated by adding L-cysteine (1 mM) and monitored in the Beckman DU640 spectrometer. After incubation, the IscU [2Fe-2S] cluster was re-purified using a Mono-Q column as described previously (Yang et al. 2006).</p><!><p>IscU is a well characterized scaffold protein for the biogenesis of iron-sulfur clusters (Agar et al. 2000, Unciuleac et al. 2007). Purified E. coli IscU [2Fe-2S] cluster had a distinctive absorption peak at 456 nm (Agar et al. 2000, Yang et al. 2006). The [2Fe-2S] cluster in IscU was relatively stable in the presence of the thioredoxin reductase system. However, when hydrogen peroxide was added to the incubation solution, the absorption peak at 456 nm of the IscU [2Fe-2S] cluster was quickly eliminated (Figure 1). The total iron and sulfur content analyses of the re-purified IscU confirmed that the IscU [2Fe-2S] cluster was converted to apo-IscU by hydrogen peroxide.</p><p>The iron released from the disrupted iron-sulfur clusters could potentially promote the production of hydroxyl free radicals via the Fenton reaction (Keyer and Imlay 1996). To test this idea, we utilized the 2-deoxyribose method (Halliwell et al. 1987) to measure the production of hydroxyl free radicals in solutions as described in the Materials and Methods. Figure 2 shows that the production of hydroxyl free radicals was almost linearly proportional to the concentration of the IscU [2Fe-2S] cluster in the incubation solution. The amount of hydroxyl free radicals generated by the IscU [2Fe-2S] cluster was also close to that when an equivalent amount of ferrous iron were used in the incubation solutions, suggesting that both iron released from the IscU [2Fe-2S] cluster by hydrogen peroxide contributed to the production of hydroxyl free radicals. Taken together, these results showed that hydrogen peroxide disrupts the IscU [2Fe-2S] cluster and that the iron released from the disrupted iron-sulfur clusters promotes the production of hydroxyl free radicals in the presence of the thioredoxin reductase system.</p><!><p>To avoid the excessive production of deleterious hydroxyl free radicals, the iron released from the disrupted iron-sulfur clusters must be efficiently sequestered. Although IscA is an iron binding protein and provides iron for the biogenesis of iron-sulfur clusters under physiologically relevant conditions (Ding and Clark 2004, Ding et al. 2005, Yang et al. 2006), IscA fails to bind any iron in the presence of hydrogen peroxide (Ding et al. 2007). Indeed, we found that addition of apo-IscA had no effect on the IscU [2Fe-2S] cluster-mediated production of hydroxyl free radicals in the presence of hydrogen peroxide (Figure 3). Thus, other cellular proteins likely exist to scavenge the iron released from the disrupted iron-sulfur clusters under oxidative stress conditions.</p><p>Increasing evidence indicated that ferritins, a large family of iron-storage proteins found in bacteria and mammalian cells (Andrews et al. 2003, Carrondo 2003, Liu and Theil 2005, Sargent et al. 2005, Galatro and Puntarulo 2007), may have an important role in cellular defense against oxidative stresses (Abdul-Tehrani et al. 1999, Epsztejn et al. 1999, Cozzi et al. 2000, Bou-Abdallah et al. 2005, Zhao et al. 2006, Velayudhan et al. 2007). In E. coli, there are at least four ferritin-like proteins: ferritin A (FtnA), ferritin B (FtnB), bacterioferritin (Bft), and Dps (Andrews et al. 2003). To examine whether ferritins could scavenge the iron released from the disrupted iron-sulfur clusters under oxidative stress conditions, we have used ferritin A, a major iron storage protein in E. coli cells (Hudson et al. 1993, Abdul-Tehrani et al. 1999), as an example. Figure 3 shows that when apo-FtnA was incubated with the IscU [2Fe-2S] cluster and hydrogen peroxide in the presence of the thioredoxin reductase system, the production of hydroxyl free radicals was dramatically decreased.</p><p>To further test whether the iron released from the IscU [2Fe-2S] cluster was sequestered by apo-FtnA, both FtnA and IscU were re-purified after incubation with hydrogen peroxide using an anion exchange Mono-Q column. Figure 4A shows that the re-purified FtnA had a significant increase of the absorption peak at around 320 nm, indicative of iron binding in the protein. The total iron content analyses of the re-purified FtnA showed that over 90% of the iron in the IscU [2Fe-2S] cluster was transferred to apo-FtnA after incubation with hydrogen peroxide (Figure 4B). These results demonstrate that apo-FtnA is able to scavenge the iron released from the disrupted iron-sulfur clusters and alleviates the production of hydroxyl free radicals in the presence of hydrogen peroxide.</p><!><p>As a major iron-storage protein in E. coli, it is possible that FtnA may act as an iron donor for the biogenesis of iron-sulfur clusters. To test this idea, the iron-bound FtnA (the ratio of iron to the FtnA monomer was about 4) was incubated with IscU, cysteine desulfurase IscS and L-cysteine in the presence of the thioredoxin reductase system at 37°C anaerobically. Figure 5A shows that little or no iron-sulfur clusters were assembled in IscU after 30 min incubation. Increase of the iron-bound FtnA concentration by 5 fold in the incubation solution did not significantly increase the iron-sulfur cluster assembly in IscU (data not shown). In contrast, when the iron-bound IscA was used as the iron donor, an absorption peak at 456 nm of the IscU [2Fe-2S] cluster quickly appeared as we reported previously (Yang et al. 2006). Thus, unlike the iron-bound IscA, the iron-bound FtnA is not an efficient iron donor for the iron-sulfur cluster assembly in IscU.</p><!><p>In the presence of the thioredoxin reductase system IscA acts as a strong iron binding protein with an iron association constant of 2.0×1019M−1 (Ding and Clark 2004, Ding et al. 2005). This led us to speculate that IscA may be able to compete with FtnA for iron binding under normal physiological conditions.</p><p>In the experiments, apo-IscA was incubated with the iron-bound FtnA (the ratio of iron to the FtnA monomer was about 4) in the presence of the thioredoxin reductase system at 37°C for 1 hour to establish the iron binding equilibrium. IscA and FtnA were then re-purified using a Mono-Q column. The total iron content analyses showed that a significant amount of iron was transferred from the iron-bound FtnA to apo-IscA after the incubation (Figure 6B). Under the same experimental conditions, IscU failed to retrieve any iron from the iron-bound FtnA in the presence of the thioredoxin reductase system (data not shown), suggesting that IscA, but not IscU, can acquire the iron from the iron-bound FtnA under physiologically relevant conditions.</p><p>The non-denaturing polyacrylamide electrophoresis and the gel filtration chromatography studies indicated that IscA and the iron-bound FtnA did not form any stable protein complexes (data not shown), indicating that the iron-bound FtnA and apo-IscA did not form a stable protein complex to facilitate the iron transfer. Because the iron mobilization from ferritins requires reduction of ferric iron to ferrous iron (Liu and Theil 2005), and IscA scavenges "free" ferrous iron only in the presence of the thioredoxin reductase system (Ding et al. 2005), we propose that the iron transfer from the iron-bound FtnA to apo-IscA is through the binding competition for ferrous iron in the presence of the thioredoxin reductase system.</p><!><p>Since the iron-bound IscA can provide iron for the iron-sulfur cluster assembly in IscU (Figure 5), and apo-IscA can retrieve iron from the iron-bound FtnA (Figure 6), we reasoned that apo-IscA may promote the iron-sulfur cluster assembly in IscU when the iron-bound FtnA was used as the iron source. Indeed, addition of apo-IscA significantly increased the iron-sulfur cluster assembly in IscU under the experimental conditions (Figure 7). Re-purification of IscU from the incubation solution further revealed that the iron-sulfur clusters were assembled in IscU, but not in IscA (data not shown). Thus, the iron stored in FtnA can be mobilized by IscA for the iron-sulfur cluster assembly in IscU in the presence of the thioredoxin reductase system.</p><!><p>Ferritins are a large group of iron-storage proteins found in diverse organisms (Andrews et al. 2003, Carrondo 2003, Liu and Theil 2005, Sargent et al. 2005, Galatro and Puntarulo 2007). In mammals, ferritins consist of 24 subunits of two types, H and L (Liu and Theil 2005). Under aerobic conditions, ferrous irons are rapidly oxidized to the mineral ferrihydrite by the built-in di-iron ferroxidase in ferritin H subunits (Sargent et al. 2005). Up to 4500 iron atoms can be stored inside an approximately 8 nm nanocage of the ferritin polymer (Andrews et al. 2003, Carrondo 2003, Liu and Theil 2005, Sargent et al. 2005). Over-expression of human H subunit ferritin in cultured HeLa cells and eryhtroid cells renders the cells resistive to hydrogen peroxide (Epsztejn et al. 1999, Cozzi et al. 2000). In vitro electron paramagnetic resonance (EPR) spin-trapping study also indicates that human H subunit ferritin can detoxify the iron-mediated production of hydroxyl free radicals (Zhao et al. 2006). In mitochondria (Galatro and Puntarulo 2007) and microbes (Andrews et al. 2003, Carrondo 2003), ferritins exist as a homopolymer composed of 24 identical "H-type" subunits. In bacteria, genetic studies suggest that ferritins also have an important role in the cellular defense against oxidative stresses (Abdul-Tehrani et al. 1999, Velayudhan et al. 2007). Here, we report that purified ferritin A (FtnA), a major iron-storage protein in E. coli (Hudson et al. 1993, Abdul-Tehrani et al. 1999), is able to scavenge the iron released from the disrupted iron-sulfur clusters and alleviates the iron-mediated production of hydroxyl free radicals in the presence of hydrogen peroxide in vitro (Figure 3 and Figure 4). The results provide biochemical evidence showing that ferritins can effectively prevent the production of hydroxyl free radicals due to the disruption of iron-sulfur clusters under hydrogen peroxide stress.</p><p>While the mechanism by which iron enters ferritins has been well characterized (Liu and Theil 2005), much less is known on how the iron is released from ferritins. At least two models have been proposed for the iron release from ferritins. In the first model, the iron-bound ferritins are degraded in lysosomes, resulting in iron release to cytoplasm (Radisky and Kaplan 1998, Kidane et al. 2006). However, the questions as how ferritins enter lysosomes and how iron is released to cytoplasm still remain to be addressed. Also, there are no lysosomes for possible degradation of ferritins in bacteria. In the second model, the ferritin pores formed in the interface of the subunits may reversibly unfold and fold to allow the iron release from ferritins (Liu et al. 2003, De Domenico et al. 2006). Recent studies further revealed that some specific peptides are able to promote the iron release from ferritins (Liu et al. 2007), supporting the notion that the iron release from ferritins may be regulated by other proteins. Here we show that in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential (Aslund and Beckwith 1999), the iron bound in FtnA can be retrieved by an iron binding chaperon IscA without any degradation of FtnA (Figure 6). Since IscA only binds ferrous iron (Ding et al. 2005), we propose that ferric iron stored in FtnA is first reduced to ferrous iron by the thioredoxin reductase system, making the iron accessible for IscA to bind. Nevertheless, we were unable to mobilize all iron from FtnA for the iron binding in IscA under the experimental conditions. It is likely that additional protein partners are required to completely release iron from ferritins (Liu et al. 2007).</p><p>The finding that IscA can retrieve the iron from the iron-bound FtnA provides new evidence for the notion that IscA is a physiological iron donor for the assembly or repair of iron-sulfur clusters (Ding et al. 2005, Yang et al. 2006, Lu et al. 2008). We show here that IscA and FtnA have very different iron binding properties. Under normal physiological conditions (e.g. in the presence of the thioredoxin reductase system), IscA is a strong iron binding protein that can retrieve iron from the iron-storage protein FtnA (Figure 6). However, under oxidative stress conditions (e.g. in the presence of hydrogen peroxide), IscA fails to bind any iron (Ding et al. 2007) (Figure 3), whereas FtnA becomes a potent iron binding protein to scavenge the iron released from the disrupted iron-sulfur clusters. The iron binding in FtnA effectively prevents the production of hydroxyl free radicals (Figure 3), likely because the build-in ferroxidase of FtnA converts ferrous iron to mineral ferric iron and alleviates the Fenton reaction (Bou-Abdallah et al. 2005). When normal physiological conditions are re-established, IscA retrieves the iron from FtnA for the re-assembly of the disrupted iron-sulfur clusters in proteins. Figure 8 summarizes the interplay of IscA and FtnA for the iron binding under normal physiological and oxidative stress conditions. The dynamic iron binding equilibrium between IscA and ferritins is expected to prevent the iron-mediated production of hydroxyl free radicals under oxidative stress conditions and yet ensure the iron supply for the biogenesis and/or repair of iron-sulfur clusters under normal physiological conditions</p><!><p>Purified IscU [2Fe-2S] cluster (50 µM) was incubated with different concentrations of H2O2 in the presence of the thioredoxin reductase system at 37°C. A), the UV-Vis absorption spectra of the IscU [2Fe-2S] cluster before (spectrum a) and after (spectrum b) incubation with 2 mM H2O2 for 20 min. B), the disruption kinetics of the IscU [2Fe-2S] cluster by H2O2. Disruption of the IscU [2Fe-2S] cluster by H2O2 was monitored at an absorption peak of 456 nm. The H2O2 concentrations in the incubation solutions were 0 mM (open circles), 0.5 mM (closed squares), 1 mM (closed diamonds), and 2 mM (closed circles), respectively.</p><!><p>Purified IscU [2Fe-2S] cluster (0 to 10 µM) was mixed with 2-deoxyribose (4 mM), potassium phosphate buffer (10 mM) (pH 7.4) and NaCl (60 mM) at 37°C for 10 min before H2O2 (0.5 mM) was added to initiate Fenton reaction. The amounts of hydroxyl free radicals were measured as described in the Materials and Methods, and plotted as a function of the IscU [2Fe-2S] cluster concentration. The Y-axis at 100% represents the amount of hydroxyl free radicals produced when 20 µM freshly prepared Fe(NH4)2(SO4)2 was used in the incubation solution. Closed circles: the IscU [2Fe-2S] cluster; open circles: apo-IscU.</p><!><p>Different concentrations of apo-IscA and apo-FtnA were mixed with the IscU [2Fe-2S] cluster (5 µM), 2-deoxyribose (4 mM), potassium phosphate (10 mM) (pH 7.4), and NaCl (60 mM) at 37°C for 10 min before H2O2 (0.5 mM) was added to initiate the Fenton reaction. The relative amounts of hydroxyl free radicals were measured as described in the Materials and Methods.</p><!><p>Purified IscU [2Fe-2S] cluster (50 µM) was incubated with apo-FtnA (100 mM) and H2O2 (2 mM) in the presence of the thioredoxin reductase system at 37°C for 20 min. FtnA and IscU were re-purified from the incubation solution using a gel filtration column (Superdex-200, Amersham Bioscience). A), the UV-Vis absorption spectra of the re-purified FtnA before and after incubation with the IscU [2Fe-2S] cluster and H2O2. B), the total iron contents in FtnA and IscU before and after incubation with H2O2.</p><!><p>A), purified apo-IscU (50 µM) was pre-incubated with IscS (1 µM) and the iron-bound FtnA (containing 50 mM iron in 12.5 µM monomeric FtnA) in the presence of the thioredoxin reductase system at 37°C for 5 min before L-cysteine (1 mM) was added to initiate the iron-sulfur cluster assembly under anaerobic conditions. The UV-Vis absorption spectra were taken at 0 min (thin line) and 30 min (thick line) after addition of L-cysteine. B), as in A) except the iron-bound FtnA was replaced with the iron-bound IscA (containing 50 µM iron in 25 µM monomeric IscA) in the pre-incubation solution. The absorption peak at 456 nm indicates the assembly of the IscU [2Fe-2S] cluster.</p><!><p>The iron-bound FtnA (containing 200 µM iron in 50 µM monomeric FtnA) was incubated with apo-IscA (50 µM) in the presence of the thioredoxin reductase system at 37°C for 1 hour, followed by re-purification of IscA and FtnA using a Mono-Q column. A), the elution profile of IscA and FtnA from the Mono-Q column. The SDS polyacrylamide gel electrophoresis showed that IscA was eluted in fraction 7 and 8, and FtnA in fraction 11 and 12. B), total iron contents in the eluted IscA and FtnA fractions after apo-IscA was incubated with the iron-bound FtnA in the presence of the thioredoxin reductase system (closed squares) or the thioredoxin reductase system missing thioredoxin reductase (closed circles).</p><!><p>A) the iron-bound FtnA (containing 200 µM iron in 50 µM monomeric FtnA) was pre-incubated with apo-IscU (50 µM) and IscS (1 µM) in the presence of the thioredoxin reductase system at 37°C for 30 min. The UV-Vis spectra were taken 0 min (thin line) and 30 min (thick line) after L-cysteine (1 mM) was added under anaerobic conditions. B), as in A) except apo-IscA (50 µM) was included in the pre-incubation solution. The absorption peak at 456 nm indicates the formation of the IscU [2Fe-2S] cluster.</p><!><p>Under oxidative stress conditions, the IscU [2Fe-2S] cluster is disrupted, and the iron released from the disrupted iron-sulfur clusters is scavenged by FtnA. Under normal physical conditions, IscA retrieves iron from the iron-bound FtnA and transfers the iron for the re-assembly of iron-sulfur clusters in IscU.</p>

PubMed Author Manuscript

Acetylation of A\xce\xb240 Alters Aggregation in the Presence and Absence of Lipid Membranes

A hallmark of Alzheimer\xe2\x80\x99s disease (AD) is the formation of senile plaques comprised of the \xce\xb2-amyloid (A\xce\xb2) peptide. A\xce\xb2 fibrillization is a complex nucleation-dependent process involving a variety of metastable intermediate aggregates and features the formation of inter- and intramolecular salt bridges involving lysine residues, K16 and K28. Cationic lysine residues also mediate protein\xe2\x80\x93lipid interactions via association with anionic lipid headgroups. As several toxic mechanisms attributed to A\xce\xb2 involve membrane interactions, the impact of acetylation on A\xce\xb240 aggregation in the presence and absence of membranes was determined. Using chemical acetylation, varying mixtures of acetylated and nonacetylated A\xce\xb240 were produced. With increasing acetylation, fibril and oligomer formation decreased, eventually completely arresting fibrillization. In the presence of total brain lipid extract (TBLE) vesicles, acetylation reduced the interaction of A\xce\xb240 with membranes; however, fibrils still formed at near complete levels of acetylation. Additionally, the combination of TBLE and acetylated A\xce\xb2 promoted annular aggregates. Finally, toxicity associated with A\xce\xb240 was reduced with increasing acetylation in a cell culture assay. These results suggest that in the absence of membranes that the cationic character of lysine plays a major role in fibril formation. However, acetylation promotes unique aggregation pathways in the presence of lipid membranes.

acetylation_of_a\xce\xb240_alters_aggregation_in_the_presence_and_absence_of_lipid_membranes

INTRODUCTION<!>Lysine-16 Is Preferentially Acetylated over Lysine-28.<!>Acetylation of A\xce\xb240 Extends the Lag Phase and Extent of Aggregation.<!>Acetylation of A\xce\xb240 Inhibits Fibril Formation and Oligomerization.<!>Exposure to TBLE Membranes Promotes Fibrillization of Acetylated A\xce\xb240.<!>TBLE Promotes Unique Aggregate Morphologies of Acetylated A\xce\xb240.<!>Acetylation Reduces the Affinity of A\xce\xb240 for TBLE Membranes.<!>A\xce\xb240-Induced Cytotoxicity Is Reduced by Acetylation in a Dose Dependent Manner.<!>DISCUSSION<!>Peptide Preparation.<!>Mass Spectrometry.<!>Thioflavin-T Fluorescence Assay (ThT).<!>Lipid Vesicle Preparation.<!>Ex Situ Tapping Mode Atomic Force Microscopy (TMAFM).<!>Preparation of TBLE/Polydiacetylene Vesicles (PDA).<!>Caspase 3/7 Toxicity Assay.

<p>Two prominent pathological features associated with Alzheimers disease (AD), a fatal neurodegenerative disorder, are cerebrovascular, diffuse, and neuritic plaques composed predominantly of the amyloidogenic peptide amyloid-β (Aβ) and neurofibrillary tangles comprised of the protein tau. These proteinaceous deposits of tau and Aβ are predominantly comprised of β-sheet-rich fibrous aggregates termed amyloid fibrils. Aβ aggregation can be a multifaceted and complex process that involves a variety of metastable, intermediate species that may or may not be directly on-pathway to fibril formation.1 The aggregate species that can be formed include a variety of soluble Aβ oligomers (AβOs), insoluble protofibrils, and annular aggregates.1–3 Beyond the heterogeneity of smaller intermediate aggregate structures, Aβ fibrils can form a variety of distinct structures and morphologies, often termed polymorphs, as a result of preparatory and/or environmental conditions during the aggregation process.4–6</p><p>Specific domains or amino acids within the Aβ peptide appear to play a prominent role in dictating the rate of aggregation and the resulting aggregate morphologies.7 For example, specific regions within the Aβ peptide have been implicated as being particularly amyloidogenic.7–10 Using site-directed spin labeling electron paramagnetic resonance, two β-strand forming domains in Aβ were identified (residues 11–21 and 29–39, respectively) that were separated by a β-turn.11 NMR studies further support the two β-strand regions separated by a β-turn motif in a number of fibril structures.12–14 Various fragments of Aβ display different propensities to aggregate.7 The first β-strand forming stretch of Aβ also contains a hydrophobic core (residues 16–21) with enhanced amyloidogenic properties.15 Furthermore, a number of point mutations associated with familial forms of AD clearly alter aggregation rates and aggregate morphologies.16</p><p>Lysine is of particular interest as it contains a positive charge at the end of an aliphatic chain. There are two lysine residues in Aβ: lysine-16 (K16) and lysine-28 (K28). Electrostatic interactions facilitated by these lysine residues in Aβ play an essential role in Aβ folding, nucleation, oligomerization, fibril formation, and toxicity.17,18 Specifically, a salt bridge forms between D23 and K28 that stabilizes a β-turn in a variety of fibril structures.18–20 K16 is the first amino acid of the previously mentioned hydrophobic core. Additionally, K16 can be involved in intermolecular salt bridge in some fibril structures of Aβ;18,21 however, K16 is solvent-exposed in other fibril structures.22 Beyond fibril formation, lysine residues are involved in oligomer formation, as substituting alanine for each lysine altered Aβ assembly and highlighted a role of K16 in Aβ toxicity.23</p><p>In addition to playing a role in Aβ aggregation, lysine can play a prominent role in the interaction of Aβ with lipid membranes. In general, lysine facilitates a variety of peptide-membrane interactions, as it contains the ability to electrostatically interact with charged lipid headgroups while the alkyl chain allows for hydrophobic interactions within the lipid tails.24 Interaction with cellular membranes, comprised predominately of lipids, can profoundly influence Aβ aggregation,25 promote unique aggregation pathways,26–28 nucleate aggregation,29–34 and/or stabilize specific Aβ aggregate species.26,32,35,36 It is even suggested that the binding of Aβ to the plasma membrane is a key step in Aβ toxicity.37,38 A variety of model lipid membranes alter Aβ conformation and exert enormous influence on the aggregation state, but the exact impact of membranes on Aβ aggregation is specific to lipid composition.39 In addition to influencing Aβ aggregation,40,41 membranes can be directly targeted by Aβ, resulting in membrane damage and dysfunction through altered mechanical properties42 and/or the formation of discrete pores.32,35,43 While lipid composition and biophysical properties of membranes play a role in Aβ/lipid interactions, physicochemical properties of the residues within Aβ also influence surface interactions.16,44 For example, disease-related point mutations within Aβ alter the aggregate species observed on model lipid membranes, demonstrating the importance of hydrophilicity and electrostatic properties of specific amino acids within Aβ.16</p><p>Here, our aim was to understand how acetylating lysine alters Aβ40 aggregation in the presence and absence of lipid membranes. Aβ40 was acetylated by exposure to sulfo-N-hydroxysulfosuccinimide acetate (NHSA) producing acetylated K16 (AcK16) and K28 (AcK28). The lipid system used was total brain lipid extract (TBLE), which contain a physiologically relevant mix of lipid components. Aβ does not appear to be extensively acetylated in vivo. Rather, this study was motivated by the dual role lysine plays in both Aβ aggregation and membrane binding. In addition, this study demonstrates that simple, targeted chemical changes in Aβ profoundly impact aggregation, membrane interaction, and ultimately toxicity. In the absence of lipid vesicles, acetylation of Aβ40 reduced both oligomerization and fibrillization, with extensive acetylation resulting in complete inhibition of fibril formation. However, the presence of TBLE lipid vesicles appeared to invoke a distinct aggregation mechanism, resulting in fibril formation even when Aβ40 was extensively acetylated. Furthermore, acetylation reduced Aβ-related toxicity in cell culture.</p><!><p>To determine the extent at which K16 and K28 were accessible for acetylation and determine experimental conditions under which the impact of acetylation on aggregation could be studied, Aβ40 (20 μM) was exposed to NHSA, an established agent for acetylating primary amines.45,46 Aβ40 was treated with varying concentrations of NHSA ranging from 0.02 mM to 2.5 mM (resulting in a range of peptide to NHSA molar ratios of 1:1 to 1:125). As there are two lysine residues in Aβ40 that can be acetylated (K16 and K28), Aβ40 was digested with pepsin to generate peptide fragments containing only one of the lysine residues so that the extent of acetylation of each discrete lysine could be determined by MS.</p><p>The percent of acetylated K16 (AcK6) and K28 (AcK28) was determined by comparing the abundance of the peptic fragments of different charge states containing just one lysine that was acetylated or not (Figure 1). When no NHSA was added to Aβ40, no acetylation of lysine was observed. At lower concentrations of NHSA, ranging from 0.02 mM to 0.16 mM, K16 is preferentially acetylated over K28 with K16 acetylation being ~2–3× more abundant than K28 (Figure 1B). With the 0.16 mM treatment of NHSA, ~47% of K16 was acetylated, but only ~17% of K28 had been modified. Effectively, these lower concentration treatments of NHSA resulted in conditions at which K16 was the predominately acetylated lysine; however, most of the available lysine residues (both K16 and K28) were not acetylated. At NHSA concentrations of 0.50 mM, ~78% of K16 was acetylated, while ~48% of K28 was acetylated, resulting in a condition in which the vast majority of Aβ was acetylated with AcK16 being the most abundant form (~1.6×). Further increasing the concentration of the acetylating agent to 1.0 mM and 1.5 mM produced an increase in the acetylation of K28 to about ~70–78% and reduced the ratio of AcK16:AcK28 to about 1:1. While AcK16 and AcK28 were observable with all treatments of NHSA, the NHSA concentrations of 0.02 mM to 0.50 mM represent a regime in which K16 is predominantly more acetylated than K28; whereas, for concentrations of 1.0 mM and above of NHSA both K16 and K28 are acetylated to similar and significant extents. Further supporting the notion that K16 was more accessible for acetylation compared to K28 was the reproducibility of the NHSA reaction from experiment to experiment (Figure S1). While the abundance of AcK16 and AcK28 always increased with larger NHSA concentration and was relatively reproducible, the extent of K28 acetylation varied to a greater extent.</p><!><p>Having established the extent of acetylation of K16 and K28 exposed to varying amounts of NHSA, we next determined the impact of AcK16 and AcK28 on the aggregation of Aβ40. ThT assays were performed with 20 μM Aβ40 that had been exposed to increasing concentrations of NHSA (Figure 2). In general, increasing acetylation of Aβ40 inhibited aggregation. That is, as the concentration of NHSA increased, the lag phase was extended and the total amount of β-sheet content decreased. The addition of 0.16 mM and 0.32 mM NHSA did not significantly change the lag phase associated with Aβ40 compared to control (Aβ40 that was not exposed to NHSA). Aβ40 in the absence of an NHSA treatment, with 0.16 mM, or 0.32 mM treatment of NHSA exhibited similar lag phases of 6.8 ± 0.16 h, 6.4 ± 0.22 h, and 7.0 ± 0.17 h, respectively (Figure 2B), and the relative slope of the growth phase, Rslope (h−1), was also not significantly changed (0.40 ± 0.058 h−1, 0.33 ± 0.037 h−1, and 0.27 ± 0.046 h−1 respectively, Figure 2C). Treatment with 0.16 mM, 0.32 mM, and 0.50 mM NHSA reduced the total extent of fibrillization as determined by the relative fluorescent signal of the steady-state plateau region (RFss) of the ThT curve (reduced by 23%, 45%, and 53% respectively, Figure 2D). Increasing the concentrations of NHSA to 0.50 mM resulted in a statistically significant extension of the lag phase, 7.7 ± 0.13 h. In addition, the slope of the growth phase at 0.50 mM NHSA significantly deviates from the control, 0.15 ± 0.040 h−1 (Figure 2C), and the total extent of fibril formation (measured as RFss) was reduced by 58%. Finally, treatment with 1.0 mM NHSA and 1.5 mM NHSA completely suppressed fibril formation over the 20 h ThT assay, although there is a hint of a potential fibril nucleation event after ~18 h. As such, accurate lag phase and growth phases could not be determined; however, the relative fluorescence at the end of the experiment is less than 20% of the control for both conditions.</p><!><p>While the ThT assays demonstrate that acetylation of lysine via exposure to NHSA reduces fibrillization of Aβ40, these assays do not provide information concerning the potential of acetylation to promote polymorphic fibril structure or AβO formation. In light of previous observations that substituting lysine in Aβ with alanine results in altered fibril morphology,23 we incubated Aβ40 under the exact same conditions as used in the ThT assays and sampled these solutions at multiple time points for AFM analysis. Beyond looking at fibril morphology, this has the added benefit of allowing for the assessment of AβO formation. To analyze the population and dimensions of Aβ fibrils and AβOs, criteria based on aggregate morphologies were used for automated image analysis.47 AβOs were defined in AFM images as being larger than 1 nm in height, having an aspect ratio of between 1.0 and 2.5 (indicating a nonfibrillar morphology), and occupying a surface area less than 9,500 nm2. The surface area criteria were based on our observation that the smallest fibrils occupied a surface area of ~10,000 nm2 and that oligomers occupied an area typically 5 times smaller than this. Fibrils were identified as aggregates that were at least 1 nm in height with an aspect ratio greater than 2.5 and occupying a surface area of at least 10,000 nm2. Upon solubilization, an initial aliquot of Aβ40 was sampled for all conditions (effectively time point 0 h), and no aggregates were observed.</p><p>Based on AFM images and consistent with the ThT assays, increasing acetylation of Aβ40 impaired aggregation (Figure 3). AβO growth was quantified by counting the number of discrete oligomers per unit area observed on mica (Figure 4A); fibril growth was quantified by the percent of the mica surface covered by fibrils (Figure 4B). This approximation was used to quantify the extent of fibril formation due to fibril bundling and heterogeneity in length, making it difficult to manually, or with an algorithm, determine the exact numbers of fibrils. When nonacetylated Aβ was incubated, a large population of oligomers was observed within 3 h with a peak population appearing between 5 and 7 h, as measured by the number of AβOs per unit area (Figure 4A). Fibrils appeared after 7 h of incubation, and the fibril density on the mica surface steadily increased, as measured by the % area of the surface occupied by fibrils (Figure 4B). The appearance of fibrils coincided with a steady decline in the AβO population.</p><p>When treated with NHSA concentrations of 0.16 mM, 0.32 mM, and 0.5 mM, AβOs also appeared after 3 h of incubation; however, the AβO population was significantly reduced in an acetylation-dependent manner (Figure 3 and Figure 4A). With all three of these NHSA treatments, fibrils were still observed at later time points. For both 0.16 mM and 0.32 mM concentrations of NHSA, fibrils could be observed after 7 h of incubation, but fibrils were not observed by AFM for Aβ40 treated with 0.5 mM NHSA until after 9 h of incubation (Figure 3 and Figure 4B). The density of fibrils observed on mica was also decreased as a function of acetylation (Figure 4B). Consistent with the ThT assays, treatment with 1.0 mM and 1.5 mM NHSA resulted in no observable fibrils over the 11 h incubation time. In addition, the number of AβOs observed at any given time point was significantly reduced (~20 times), suggesting that extensive acetylation arrests Aβ40 aggregation at the oligomeric phase. It should be noted that the detection limit of AβOs associated with this AFM analysis is likely on the order of octamers. That is, smaller AβOs may still have formed but would be difficult to observe with this assay. Hexameric and smaller AβOs have been observed for Aβ40 in which the lysine residues have been substituted for with alanines.23 With that being said, size analysis demonstrates that reaching a condition in which Aβ40, is significantly acetylated results in smaller and fewer AβOs. Specifically, AβOs formed without NHSA treatment or with NHSA treatments of 0.32 mM or less resulted in oligomers with a broad height distribution with a mode of ~1.5–3.5 nm (Figure 4C). NHSA treatments of 0.5 mM or greater resulted in oligomers that had a mode height of ~1–1.5 nm (Figure 4C). As the size of AβOs observed under a specific condition did not change as a function of time, and so few oligomers were observed at extensive NHSA treatments, the height histograms presented were compiled over all time points.</p><p>To determine if acetylation promoted unique fibril polymorphs, morphological features of fibrils grown under the different treatments of NHSA were determined by AFM image analysis (Figure 5). Height profiles were taken perpendicular to the axis of each fibril for Aβ40 fibrils formed at NHSA concentrations of 0.0 mM, 0.16 mM, 0.32 mM, and 0.50 mM (Figure 5A). The 1.0 mM and 1.5 mM NHSA condition were not analyzed as fibrils were not observed. Profiles were taken in a manner to avoid fibril crossings in order to represent the height scale of single fibril species better. The fibril height profiles of Aβ40 at 0.0 mM, 0.16 mM, 0.32 mM, and 0.50 mM NHSA concentrations all had a mode fibril height of ~2.5 nm (Figure 5A), suggesting that the fibrils formed under these conditions were not polymorphic. As fibril height can vary along the contour of the fibrils, the average height along fibril contour of every individual fibril was determined. As fibril heights did not appear to change as a function of time, data time points of 7 h and beyond were compiled for each variable to produce histograms of the average fibril contour height (Figure 5B). The median fibril contour height for NHSA concentrations of 0.0 mM, 0.16 mM, 0.32 mM, and 0.50 mM was 2.4, 2.2, 3.1, and 2.5 nm, respectively, with an interquartile range of 1.0, 0.8, 0.4, and 0.6 nm, respectively (Figure 5B), confirming the absence of any polymorphic fibril structures for Aβ40 under these acetylating concentrations.</p><!><p>As the presence of lipid membranes has been implicated as a modifier of Aβ aggregation39,48 and lysine plays a prominent role in peptide/lipid interactions,49–51 we next wanted to determine how the presence of lipid membranes modifies the aggregation of Aβ40 under the various acetylating conditions. ThT assays were performed in the presence of large unilamellar vesicles (LUV) of TBLE. TBLE was chosen as a model system due to its physiologically relevant mixture of membrane lipids and its extensive use in studying Aβ membrane interactions.7,16,52,53 In short, Aβ40 (20 μM) was incubated in the presence of TBLE LUVs at a lipid to peptide ratio of 30:1 under the same experimental and instrumental conditions as the assays performed in the absence of lipids.</p><p>There are examples in the literature of the presence of lipids both accelerating54–56 or slowing39,57 Aβ aggregation. While the lipid composition of the membrane plays a role,39,58,59 the preparatory history of Aβ may also influence the Aβ/lipid interaction.53 With the Aβ preparation protocol used here, TBLE membranes slowed aggregation compared to aggregation in the absence of lipids (compare the ThT assay curves of Aβ40 without NHSA treatment in Figure 2 and Figure 6).60 For example, the presence of TBLE extended the lag phase of Aβ40 aggregation ~5 h. While acetylation associated with NHSA treatment of 1.0 mM or greater completely prevented fibrillization in the absence of TBLE vesicles, Aβ40 formed fibrils under all acetylating conditions based on the ThT assay curves (Figure 6), and this was later confirmed by AFM images of these solutions at the end of the ThT assay (Figure 7). In the presence of TBLE, the lag phase decreased with increasing acetylation, with the following lag-phase times exhibited: Aβ40, 9.8 ± 0.67 h; 0.16 mM NHSA, 7.8 ± 0.40 h; 0.32 mM NHSA, 7.2 ± 0.31 h; 0.50 mM NHSA, 6.8 ± 0.34 h; 1.0 mM NHSA, 7.1 ± 0.36 h; and 1.5 mM NHSA, 6.8 ± 0.99 h (Figure 6B). This trend suggests that Aβ acetylation promotes the formation of the critical nucleus associated with fibril initiation. This is in stark contrast to the impact of acetylation in the absence of TBLE, in which the lag phase was extended. In addition to the shortened lag phase in the presence of TBLE vesicles, acetylating conditions of 0.5 mM, 1.0 mM, and 1.5 mM NHSA all exhibited a statistically significant decrease in the slope of the growth phase in the presence of TBLE compared to control (Aβ40, 0.43 ± 0.042 h−1; 0.5 mM NHSA, 0.26 ± 0.026 h−1; 1.0 mM NHSA, 0.17 ± 0.027 h−1; 1.5 mM NHSA, 0.18 ± 0.031 h−1). Concurrent with this shortened lag phase, the overall β-sheet load, as measured by relative fluorescence at steady state, systematically decreased with increasing acetylated lysine content. The reduction in β-sheet formation started with ~20–35% reduction at 0.16 mM NHSA and 0.32 mM NHSA concentrations to ~60% reduction for 1.0 mM NHSA and 1.5 mM NHSA (Figure 6D).</p><!><p>While not resulting in unique fibril morphologies in the absence of lipids, point mutations in Aβ associated with familial forms of AD aggregate into unique polymorphic structures in the presence of TBLE bilayers.16 This suggests that acetylation could promote polymorphic fibrils in the presence of TBLE, despite not producing polymorphs in the absence of lipids. To determine if acetylated forms of Aβ40 produced polymorphic fibrils, Aβ40 (20 μM) was incubated in the presence of TBLE LUVs at a lipid to peptide ratio of 30:1. After 20 h samples were deposited on a mica substrate, and AFM image analysis was performed for all conditions. Height profiles of fibrils demonstrated a fibril height of 2.5–3.0 nm (Figure 7A), like that observed in the absence of TBLE (Figure 5A). However, there are several regions along the contour of the fibrils formed in the presence of TBLE that appeared thicker. That is, the morphology along the contour of the fibril deviated to a greater extent. As a result, the height along fibril contours had a much broader distribution when compared to fibrils in the absence of lipids. Median heights of fibrils in the presence of TBLE were 3.6 nm (0.0 mM NHSA), 3.3 nm (0.16 mM NHSA), 3.3 nm (0.32 mM NHSA), 3.9 nm (0.50 mM NHSA), 2.8 nm (1.0 mM NHSA), and 3.3 nm (1.5 mM NHSA), and the distribution of heights defined by the interquartile range were 2.6 nm (0.0 mM NHSA), 2.0 nm (0.16 mM NHSA), 1.9 nm (0.32 mM NHSA), 2.0 nm (0.50 mM NHSA), 2.1 nm (1.0 mM NHSA), and 2.1 nm (1.5 mM NHSA) (Figure 7B). An example of this broadening can be observed for Aβ40 and 0.50 mM NHSA in Figure 8B. Collectively, it appears that TBLE does not promote a fibril polymorph in acetylated Aβ40; however, the increased variation in morphology along the fibril contour may indicate that lipids are being incorporated into or decorating the observed fibrils. This does not appear to be dependent on acetylation. Rather, it appears to be a consequence of fibril formation in the presence of TBLE vesicles.</p><p>While the morphology of fibrils formed in the presence of TBLE did not appear to be altered by acetylation, increased acetylation promoted the formation of annular aggregates in the presence of TBLE (Figure 7A and Figure 8). For conditions of 1.0 and 1.5 mM NHSA, a large population of annular aggregates formed (2.6 ± 0.4 to 6.4 ± 0.6 per 25 μm2, respectively) that were not readily observed in the absence of lipid or with lower levels of acetylation. These annular aggregates were considerably thicker than fibrils with heights ranging from ~5–10 nm (Figure 8B). The inner diameter of these annular aggregates ranged from ~60–200 nm with a median size of ~114 nm (Figure S9B). Many annular aggregates appeared to be strongly associated with fibril aggregates as they were colocalized on the mica surface, with some fibrils appearing to protrude directly from annular aggregates (Figure S9A).</p><!><p>As the presence of TBLE vesicles clearly influenced the aggregation of acetylated Aβ40, a polydiacetylene (PDA) lipid-binding assay was performed to measure the total interaction between Aβ40 and TBLE membranes (Figure 9). Polydiacetylene is a lipid moiety that is easily incorporated into vesicles and photo-cross-linked, resulting in hybrid vesicles with enhanced fluorescence in response to mechanical stress associated with peptide binding and aggregation. By monitoring the fluorescence of these vesicles as a function of time, the total interaction of Aβ with the TBLE/PDA vesicles can be monitored. Control experiments were performed with the various doses of NHSA, and a fluorescent response was not invoked in these control experiments. Upon exposure of TBLE/PDA vesicles to Aβ40, the fluorescent signal steadily increases as the peptide binds and aggregates on the vesicles. As the extent of Aβ40 acetylation was increased, the total interaction between the peptide and vesicles was gradually reduced. As binding to and aggregation on the vesicle can both induce PDA response, it is important to interpret this assay with the results of the ThT assay in mind. As the shortest lag phase for any condition was ~7 h as determined by the ThT assay, the PDA response for the first 7 h is dominated by the initial binding of Aβ to the membrane. It appears that acetylation reduces the overall affinity of Aβ40 for TBLE vesicles. As the steady-state phase of fibril formation is reached for all conditions by 11 h of incubation (as determined by the ThT assay), the total fibril load has its largest contribution to the total PDA signal. In this regard, the magnitude of the PDA decreases with increasing acetylation in a similar fashion in comparison with the ThT assay. Lastly, at ~17 h the PDA/TBLE fluorescence signal associated with exposure to β that had not been treated with NHSA or treated with 0.16 mM NHSA began to decrease instead of maintaining a plateaued maximum response. This drop-in signal eventually reaches that of the maximum response of other experimental conditions. Extensive fibrillization on lipid vesicles can lead to PDA vesicle instability that results in flocculation, and these two conditions were associated with the most substantial fibril load. As a result, this likely reflects a loss in signal to the instability of the PDA/TBLE vesicles upon prolonged exposure to late-stage aggregates that alter the colloidal suspension.</p><!><p>To determine the impact of acetylation on the Aβ-induced cytotoxicity, mouse neuroblastoma X Rat neuron hybrid, ND7/23, cells were exposed to 5 μM Aβ40 that had been treated with varying doses of NHSA (0.0, 0.16, 0.32, 0.50, 1.00, and 1.50 mM). Cell death was determined with a Caspase 3/7 toxicity assay 24 h after exposure to the different Aβ preparations (Figure 10). Unmodified Aβ40 (0 mM treatment of NHSA) resulted in 21.7 ± 0.8% toxicity. The percent toxicity decreased in a dose dependent manner with increasing acetylation. The smallest treatment with NHSA (0.16 mM), a condition for which a third of available lysine residues were acetylated, significantly reduced Aβ toxicity (15.5 ± 1.0%) compared to control. Under conditions when approximately two-thirds of available lysine residues are acetylated (0.5 mM NHSA treatment), Aβ-induced toxicity dropped below 10%, and when 85–90% of the available lysine residues were acetylated (1.5 mM NHSA treatment), the percent toxicity was only 4.3 ± 1.4%.</p><!><p>Understanding how individual amino acids of Aβ affect aggregation and peptide-membrane interactions is significant as it provides the possibility for focused, therapeutic targeting and may provide more effective treatment strategies. In this work, we explored the role of lysine residues in the aggregation of Aβ40 in the presence and absence of membranes through removal of the positive charge of lysine side chains via acetylation. In summary (Figure 11), we demonstrate that 1) acetylation reduces the aggregation propensity of Aβ40, eventually abolishing fibril formation; 2) acetylation impedes the formation of AβOs; 3) under our experimental conditions, TBLE membranes slow Aβ40 aggregation; however, acetylation of Aβ40 promotes aggregation in the presence of TBLE compared to control; 4) acetylation of Aβ40 reduces the total Aβ/membrane interaction; 5) the combination of acetylation and the presence of TBLE can promote the formation of annular aggregates of Aβ40. This work suggests that the Aβ aggregation process (in both the presence and absence of lipid bilayers) is highly dependent on the charge state of lysine residues and that the Aβ aggregation mechanism is unique on TBLE bilayers.</p><p>Through increasing the concentration of NHSA, both K16 and K28 could be acetylated, but K16 was more readily acetylated. The preferential acetylation of K16 is consistent with structural studies of monomeric Aβ demonstrating that K16 is solvent-exposed while K28 is partially protected.22,61 The experimental conditions created two regimes of Aβ acetylation: one in which AcK16 predominated (0.16 mM and 0.32 mM NHSA) and the other in which the abundance of AcK16 to AcK28 was similar (1.0 mM and 1.5 mM NHSA). As the complete absence of fibrils was observed only when K28 was appreciably acetylated, it is tempting to associate K28 acetylation with this observation. However, as substitution of either K16 or K28 with alanine impacted Aβ aggregation,23 it is likely that acetylation of both lysine residues plays a role in the observed inhibition of aggregation. There are plausible mechanisms associated with acetylation of either lysine. K16 is associated with an amyloidogenic core sequence in Aβ that plays a crucial role in fibril formation,9,10 and as a result, altering this region is likely to impact aggregation. There is a precedent for such a notion as the Flemish mutation in Aβ, which changes an alanine to glycine and is at the end of this amyloidogenic core sequence, significantly reduces the fate of Aβ fibrillization.62–64 Indeed, low concentration treatments of NHSA, when K28 is not appreciably acetylated, exhibited significant (although not absolute) reduction in fibril and oligomer formation. A potential mechanism for AcK16 inhibition of aggregation is provided by computational studies of Aβ42[K16A] demonstrating that the substitution of lysine results in a loss of intrapeptide contacts between the hydrophobic core and C-terminus of Aβ, therefore, reducing the propensity of β-sheet formation required for aggregation.65 With regard to K28, acetylation would disrupt its ability to participate in a salt bridge with D23 or E22. This salt bridge is associated with the turn region of a β-hairpin observed in an Aβ fibril structure.17 The removal of K28's positive charge through acetylation would, therefore, destabilize Aβ fibrils. Support for this mechanism can be seen in the success of a variety of Aβ fibril inhibitors directly targeting this salt bridge.66–70 Furthermore, artificially creating the salt bridge between D23 and K28 via the addition of a lactam linker increases the rate of fibril formation by removing the entropic barrier imposed by the establishment of the salt bridge and effectively removing the lag phase.71 Changes in the net charge of Aβ40 from −3 to −4 or −5 may also play a role in impeding fibril formation as increases in the solvation energy would reduce the strength of the hydrophobic collapse of Aβ that is associated with the initial stages of aggregation.72,73</p><p>Beyond inhibiting fibril formation, acetylation of Aβ reduces oligomerization. This finding appears to be in contradiction to the oligomerization of Aβ40[K16A] and Aβ40[K28A] which displayed similar oligomerization distributions to wild-type Aβ40.23 However, the detection limitations of the AFM analysis used here would not allow for the detection of the small oligomers (hexamers or smaller) observed by Sinha et al. With regard to the oligomers observable by AFM, these tend to be classified as high molecular weight (HMW) oligomers, and a number of toxic HMW AβOs have been reported.74–77 The smaller, low molecular weight (LMW) AβOs tend to be less toxic or even benign compared with their HMW counter-parts.74,76–78 The analysis suggests that even if acetylation does not alter the formation of LMW AβOs, the more toxically relevant HMW oligomers are reduced by acetylation. With regards to potential mechanisms for this inhibition of AβO formation, molecular dynamics suggests that Aβ oligomerization is governed through electrostatic interactions40 and that reducing electrostatic attraction promotes smaller ordered oligomers.41 Such a scenario is consistent with our observation that high levels of acetylation resulted in fewer and smaller AβOs, as acetylation would reduce the electrostatic attraction between Aβ peptides.</p><p>The impact of acetylation on Aβ aggregation and toxicity suggests that targeting K16 and K28 can be an effective way to manipulate aggregation for potential therapeutic purposes. Indeed, there are several indications that such a strategy may be effective. A molecular tweezer, CRL01, that selectively binds to lysine side chains inhibits the formation of AβOs and fibrils.79 Aspirin, which can act as an acetylating agent,80–82 can reduce the risk of AD83 and protects HEK239 cells against cytotoxicity associated with exposure to Aβ fibrils.84 More broadly, aspirin reduces protein aggregation in several amyloid-based, neurodegenerative diseases,85,86 and this protection was directly linked to its ability to acetylate proteins in C. elegan models of Huntington's disease and AD.85,87 However, aspirin's ability to acetylate proteins is not lysine-specific.88</p><p>Lipid membrane environments have been shown to influence the aggregation of a number of amyloid-forming proteins29,58,89–94 and can even induce unique aggregation pathways.26,91 The impact of membranes on Aβ aggregation is sensitive to several biochemical and biophysical factors.37 For example, membrane thickness regulates Aβ aggregation, as POPC and DOPC catalyzed fibrillization at a wide range of lipid to peptide ratios but DLPC stabilized cytotoxic oligomers at stoichiometric or higher lipid to peptides ratios.95 Interestingly, Aβ adopts a partially folded α-helical structure spanning from K16-E22 when binding zwitterionic lipids.96 The inclusion of a charged lysine (K16) in this α-helix forming stretch associated with lipid binding implies potential importance of the positive charge associated with this residue in interacting with membranes and that removing the positive charge, e.g., by acetylation, should profoundly impact Aβ's interaction on membranes. In a similar fashion, mutations, Arctic (E22G) and Italian (E22K) of the negatively charged glutamic acid, at the end of this sequence promote morphologically distinct fibrils on supported TBLE bilayers.16 Ganglioside clusters within lipid membranes facilitate the Aβ/membrane interaction and promote fibrils formation;97 however, these fibrils differ than those formed in aqueous solution in morphology, secondary structure, and cytotoxic properties.39 Importantly, ganglioside clusters appear to trap K28.98 Beyond a role in fibril formation, specific lipid interactions may stabilize AβO intermediates, as has been demonstrated with polymethacrylate copolymer encased lipid-nanodiscs.99,100</p><p>A unique, lipid-induced aggregation pathway would be consistent with our results with regard to Aβ aggregation in the presence and absence of TBLE vesicles. While acetylation extended the lag-phase Aβ aggregation in the absence of TBLE vesicles, the opposite was observed in the presence of such vesicles, and Aβ was able to form fibrils even after extensive acetylation associated with high concentration treatments of NHSA. Despite the shortened lag phase associated with acetylation of Aβ in the presence of TBLE vesicles, the total fibril load was reduced, and polymorphic annular aggregates were observed. Collectively, this suggests that, in the presence of lipids, acetylation enhances the formation of the critical nucleus associated with initiating fibrillization but promotes secondary aggregation pathways. The enhanced nucleation could be related to enhanced diffusion along the membrane surface. That is, once bound to the lipid membrane, the removal of the cationic nature of lysine via acetylation could reduce the interaction with anionic head-groups, which may allow for freer diffusion of Aβ peptides along the membrane surface and increase the ability of peptides colliding and coalescing into a critical nucleus. A plausible scenario for the observed decrease in the slope of the growth phase, associated with the reduction in fibril elongation and β-sheet load, is that the inability to form a salt bridge between K28 and other residues increases the dissociation rate of monomers from the growing fibril ends relative to the association rate, resulting in an overall slower elongation.</p><p>As mentioned earlier, the preparatory history of Aβ influences Aβ aggregation in the presence and absence of lipids interaction.53,101–103 The impact of preparatory history on aggregation suggests that environmental differences strongly impact aggregation, and lipids can be considered an environmental factor. Under our experimental conditions, extensive acetylation (>85% of available lysine residues acetylated) completely arrested Aβ40 fibrillization in the absence of lipid; however, fibrils still formed with extensive acetylation when incubated with TBLE. This demonstrates that complete inhibition of fibril formation due to acetylation may not be universal across preparation protocols and environmental conditions. With that being said, both K16 and K28 participate in intermolecular and intramolecular electrostatic interactions that stabilize a variety of fibril structures,17,18 and it is likely that acetylation would universally retard fibrillization, albeit to varying extents. While K28 typically forms salt bridges with D23,18–20 acetylating K28 may have a more pronounced impact of aggregation compared to K16, which appears to be solvent exposed in fibrils under some experimental conditions.22 While the formation of the salt bridge between K18 and D23 may facilitate and stabilize fibrils, it does not appear to be required. For example, the Iowa mutation (D23N) in Aβ, which removes a negative charge involved in salt bridge formation with K28, slows aggregation but does not completely suppress fibril formation.44 However, the Iowa mutation does promote a distinct polymorphic fibril structure when aggregating on lipid membranes.16</p><p>While Aβ does not appear to be extensively acetylated in vivo, several other amyloid-forming proteins, including tau, α-synuclein (α-syn), and huntingtin (htt), are acetylated. Acetylation of tau is elevated in human tauopathy brains with acetylation of various lysine residues impacting different aspects of tau biology, i.e., turnover, aggregation, and interactions with microtubules.104–107 Importantly, acetylation modifies tau-associated toxicity,105,106,108 and tau acetylation alters susceptibility to Aβ-associated toxicity.107 Enzymes that regulate tau acetylation can be targeted to reduce Tau toxicity.105,109,110 N-Terminal acetylation occurs in α-syn in vivo,111–113 which enhances its propensity to form α-helical structures when binding lipids and reduces aggregation.114,115 Acetylation of lysine residues within the first 17 amino acids of htt have been identified by mass spectroscopy,116 and acetylation of some of these residues reduces htt's ability to bind lipid membranes and reduces toxicity in cell cultures.117 However, there are conflicting reports with regards to the impact of acetylation of htt on aggregation, as chemical acetylation reduced aggregation in htt-exon1 fusion protein117 but synthetic htt peptides with selected acetylated lysine residues demonstrated a minimal impact on aggregation.118 The discrepancy may lie in the extent of acetylation, as the chemical acetylation was nonselective compared to the precise acetylation of specific residues in the synthetic peptide. Collectively, it appears that the cationic nature of lysine residues in general plays a role in amyloid formation that can be manipulated by acetylation.</p><p>Aspirin-induced lysine acetylation impedes the aggregation of SOD1, leading to the hypothesis that targeting the net charge of native or misfolded proteins may represent a viable therapeutic target.119 As Aβ is not extensively acetylated in vivo, targeting enzymes to promote its acetylation or deacetylation is likely not a viable therapeutic strategy. However, targeting the net charge of Aβ, as suggested by the SOD1 study, to influence its propensity to aggregate and/or bind lipid membranes may be an effective strategy, provided a method to selectively target lysine residues within Aβ can be developed. In particular, the reduced affinity of acetylated Aβ for lipid membranes could underlie the observed reduced toxicity as a function of acetylation, as binding to and damaging the plasma membrane plays a role in A.β toxicity120,121.</p><!><p>Synthetic Aβ40 (Invitrogen) was equilibrated to room temperature for 30 min. Aβ40 was dissolved in 10% NH4OH(w/v) to acquire an alkaline stock solution of Aβ40 (0.5 mg/mL). The stock solution was unperturbed for 10 min followed by 5 min of bath sonication. The Aβ40 stock solution was aliquoted and snap-frozen. Aliquots were lyophilized overnight and stored at −80 °C until the day of experimentation. For experiments, individual aliquots of Aβ40 were equilibrated to room temperature. An alkaline solution, 60 mM NaOH(aq), was used to solubilize Aβ40 (target concentration of 1.5 mg/mL), creating what is referred to as the Aβ40·NaOH solution. The Aβ40·NaOH solution was incubated at room temperature for 10 min, after which it was gently pipet mixed. The actual concentration of the Aβ40·NaOH solution was measured (NanoDrop, ThermoScientific) via absorbance at 280 nm with an extinction coefficient of 1440 M−1 cm−1. The pH of the Aβ40·NaOH solution was controlled to be ≥11.23. For all experiments, the Aβ40·NaOH solution was diluted into a 20 mM HEPES 150 mM NaCl buffer, pH = 7.23.</p><!><p>For the MS experiments, the Aβ40·NaOH solution was divided into 10 μg aliquots. These aliquots were diluted in the HEPES buffer and treated with NHSA at increasing molar ratios of NHSA:Aβ40. Samples were allowed to incubate at room temperature for 1 h before the NHSA reaction was quenched with 1 M Trizma (Sigma-Aldrich' St. Louis, MO). All samples were snapfrozen and lyophilized overnight. The following day, samples were dissolved in 1 M acetic acid containing 0.5 mg/mL of pepsin (10:1 pepsin:Aβ40). Samples were left overnight protected from light and incubated at room temperature. On the third day samples were analyzed via liquid chromatography (LC)-MS (Thermofisher Scientific Q Exactive Mass Spectrometer, an LC-MS/MS with orbitrap). To remove residual salts, chromatography was performed utilizing a C18 column (ZORBAX RRHD Eclipse XDB 80 Å C18, 2.1 × 100 mm, 1.8 μm; Agilent, Santa Clara, CA) and a mobile phase of 0.1% formic/water (v/v) (A) and 0.1% formic acid/acetonitrile (v/v) (B) with a gradient of 5%B for 0–2 min, 40%B for 5 min, 50%B for 6 min, 95%B for 7–8 min, and 5% B for 8.5–10 min with a flow rate of 300 μL/min. A full MS scan was performed for the mass-to-charge range (m/z) of 200–2000 with a resolution of 70,000 and a resolution of 17,500 for the Top 5 tandem MS (MS/MS) method that was used. Mass spectral analysis was performed using BioPharma Finder to determine the percent abundance of AcK16 and AcK28. This was carried out by comparing lysine-specific fragments of K16 (YEVHHQKLVF) and K28 (FAEDVGSNKGAIIGL) of Aβ40 to the same peptide fragments containing a 43.05 Da mass shift, for the +1, +2, and +3 charge states. LC-MS/MS analyses for several notable reagent:analyte molar ratios were performed in duplicate (see the Supporting Information) to demonstrate the reproducibility in % K acetylation assignments.</p><!><p>Monitoring fibril formation was achieved with thioflavin T (ThT; Sigma-Aldrich' St. Louis, MO) assays. Assays were carried out in a Costar 96-well black clear flat bottom plate. The Aβ40·NaOH solution was added to 20 mM HEPES 150 mM NaCl solutions containing 3 mol equiv of ThT to Aβ40 and varied molar equivalents of NHSA to Aβ40 at a pH of 7.23. The resulting solutions contained 20 μM Aβ40 and the addition of 0.16, 0.32, 0.50, 1.00, and 1.50 mM NHSA Additionally, a 3 mm borosilicate bead was introduced to facilitate homogeneous mixing across wells.122 The assay plate was sealed with plate tape and incubated at 37 °C for 24 h on a SpectraMax M2Multi-Mode plate reader (Molecular Devices, Sunnyvale, CA). Fluorescence intensities were measured in 5 min intervals at excitation and emission wavelengths of 440 and 484 nm, respectively, with shaking in between reads. Assays performed in the presence of TBLE were performed in the same manner with the addition of TBLE vesicles prepared as described below.</p><p>Data analysis was performed utilizing GraphPad Prism 6 to average replicates and perform baseline corrections and error analysis reported as the standard error mean (SEM). Additionally, through the use of a Boltzmann sigmodal function (eq 1) kinetic parameters such as the nucleation time, commonly referred to here as lag phase (tlag), was obtained by fitting the individual traces of ThT curves using the following equation:123,124 (1)Y=(yi+mix)+yf+mfx1+e−(x−xaτ)</p><p>Utilizing GraphPad Prism 6 the initial lag phase and plateau regions of the curves are defined by linear fits where the initial fluorescence and slope are defined by yi and mi and the final fluorescence and slope are defined as yf and mf, respectively. The time to reach the half-maximum of the curve is defined by xo, and the elongation time constant of the growth phase is defined as τ. As a nonideal sigmoidal function, no constraints were applied. A least-squares fitting operation was then performed for a maximum iteration limit of 1000. The lag-phase time was calculated from the kinetic parameters, tlag = xo − 2τ. The slope of the growth phase is associated with the growth of fibrils. The slope was calculated by normalizing the ThT traces to the mean maximum response of the control group (no NHSA added) within each independent experiment for comparison across experiments. To obtain the relative slope of the growth phase, Rslope (h−1) the linear fits of the growth phase were performed by defining the boundaries of the fit to only include the linear region between the lag phase and the plateau region as visually determined for each ThT curve. The maximum signal of fluorescence at steady state was calculated averaging the signal over the plateau region of the sigmoidal curve. Initial starting points were visually determined to be near the inflection point where the sigmoidal curve enters the plateau region. The fits were performed from these starting points to the end of the data set. Once the boundaries were defined, the average fluorescence signal within this plateau region was determined. For comparison between trials, these were converted to the relative fluorescence at steady state (RFss) by dividing each value by the control experiment (no NHSA for that experiment). All kinetic parameters were then averaged from triplicates of independent experiments to determine the mean ± SEM. Significance was reported as greater than 95% confidence calculated using a standard Student's t test to determine variations in parameters across varied NHSA concentrations.</p><!><p>A 5 mg amount of TBLE (Avanti Polar Lipids, Alabaster, AL) was hydrated with a 20 mM HEPES 150 mM NaCl buffer, pH = 7.23, and allowed to incubate for 30 min at 60 °C and 1400 rpm in a Thermomixer. Lipid samples were vigorously pipetted and scraped to remove all traces of TBLE from the walls of the centrifuge tube; after which, the samples were put through 7 freeze–thaw cycles and 30 min of bath sonication at 37 °C. Lipid stocks were again heated to 60 °C before being extruded through a 100 nm polycarbonate membrane filter. Resulting vesicle solutions were kept at 37 °C before being diluted into a HEPES solution at a final concentration of 0.432 mg/mL within a 96-well plate for a protein to lipid ratio of 30:1.</p><!><p>Incubations of Aβ40 (20 μM) were performed in a similar manner to previous ThT assays with the same concentrations and ratios of Aβ40 to NHSA. Solutions were incubated at 37 °C and shaken and read at the same intervals as previous ThT assays; however, at the time points of 0, 1, 3, 5, 7, 8, 9, 11, and 24 h the 96-well plate was removed from the plate reader, and two 2 μL samples were taken from each solution. The experiment was done in this manner in order to track the progress of Aβ aggregate formation. Once samples were taken, they were allowed to incubate for 1 min before being washed with 100 μL of 18 ultrapure water. Samples were dried with compressed air immediately after the wash. After sampling, the plate was returned and incubated until the next time interval. Samples were imaged via atomic force microscopy (AFM) under tapping mode on a Nanoscope V Multimode (Veeco, Santa Barbara, CA) with a closed-loop vertical engage J-scanner. Cantilevers used for images were a diving-board-shaped silicon oxide cantilever with a resonance frequency of ~300 kHz and a spring constant of 40 N/m (Budget Sensors).</p><p>AFM images were processed utilizing Matlab equipped with the image processing toolbox (MathWorks, Natick, MA) as described previously.47 To correct for background curvature, height images were flattened using a second polynomial flattening algorithm. To aid in automatically identifying individual aggregates, AFM images were converted into binary maps after establishing a height threshold of 1.0 nm. Individual aggregates were determined from the binary maps using a recognition algorithm. Physical features of individual aggregates (e.g., height, volume, diameter, shape factor, length, aspect ratio, length, and area covered) were measured. Average heights of fibrils were determined by measuring the fibrils along their contour length while excluding any cross over regions of overlapping fibrils. The range of fibril contour length, or the spread of fibril heights, was calculated as the interquartile range which involves taking the median of the lower and upper of a given data set. Populations of oligomers and fibrils, along with the percent area covered by fibrils, were plotted using GraphPad Prism 6 to average across replicates and perform error analysis reported as a standard error mean (SEM).</p><!><p>Separately, 8.4 mg of 10,12-tricosadiynoic acid (PDA) (GFS Chemicals, Columbus, OH) and 5.0 mg of TBLE were dissolved in a 1:1 chloroform:ethanol solution. The two were combined in a glass vial at a 60:40 mass ratio of PDA to TBLE, and solvents were removed under vacuum to produce a TBLE/PDA film. This film was hydrated with 8 mL of 20 mM HEPES 150 mM NaCl buffer (pH = 7.23) at 75 °C. The solution was probe sonicated at 180 W continuously for 10 min. The resulting solution was stored protected from light in a 4 °C fridge overnight. The following day the TBLE/PDA solution was allowed to reach ambient temperature before transferring it to a 100 mL beaker. The solution was stirred at 300 rpm for 10 min under cover of a black box adapted with a TLC lamp that irradiated the solution at 254 nm for the full length of time. PDA assays were performed in a Costar 96 black clear flat bottom well plate with the addition of 3 mm borosilicate glass beads. Peptide-lipid interactions were observed fluorescently at room temperature with excitation and emission wavelengths of 485 and 560 nm utilizing a SpectraMax M2Multi-Mode plate reader (Molecular Devices, Sunnyvale, CA). Data analysis was performed by averaging triplicates and performing baseline corrections with GraphPad Prism 6.</p><!><p>For cell based assays, synthetic Aβ40 was initially prepared as previously described above. Disaggregated Aβ40 was added to a 20 mM HEPES 150 mM NaCl buffer, pH = 7.23 along with NHSA. Acetylated forms of Aβ40 were obtained by incubating 20 μM Aβ40 in a 20 mM HEPES 150 mM NaCl buffer, pH = 7.23, along with the simultaneous addition of NHSA at 0.0, 0.16, 0.32, 0.50, 1.00, or 1.50 mM NHSA. The subsequent solutions were kept at room temperature for 1 h without agitation. The NHSA reaction was quenched with a lysine solution for a final concentration of 2.7 mM. Dialysis was performed against H2O at 4 °C in 2 h intervals for a total of 4 h (Slide-A-Lyzer MINI Dialysis Devices, MWCO 3.5K, Thermo fisher Scientific). Samples of Aβ40 were alkalified with 10% NH4OH (w/v) and lyophilized to produce monomeric acetylated forms of Aβ40.</p><p>Mouse neuroblastoma X Rat neuron hybrid, ND7/23 (Sigma-Aldrich, St. Louis, MO), was grown in a Dulbecco's Modified Eagle Medium augmented with 4.5 g/L glucose, 110 mg/L sodium pyruvate, 1% penicillin-streptomycin (stabilized with 10,000 units penicillin and 10 mg streptomycin/mL), 2 g/L sodium bicarbonate, 584 mg/L L-glutamine (Sigma-Aldrich, St. Louis, MO), 5 mM HEPES (Fisher BioReagents, Waltham, MA), and 10% HyClone fetal bovine serum (Thermo Scientific, Waltham, MA) at 37 °C and 5% CO2. Cells were seeded at 5 × 103 cells/well and grown for 24 h. Aβ40 previously acetylated with NHSA treatments of 0.0, 0.16, 0.32, 0.50, 1.00, or 1.50 mM was added to cells for a final concentration of 5 μM Aβ40. After 24 h of incubation the Caspase 3 reagent (Anaspec, Sensolyte AMC Caspase 3/7 assay kit) was added and allowed to shake for 45 min while protected from light at room temperature. Fluorescence intensities were measured at 45 min, 1, 3, and 4 h at Ex/Em = 354 nm/442 nm on a Synergy H1 (BioTek, Winooski, VT) plate reader. Data analysis was performed by subtracting the fluorescence readings of untreated cells from Aβ40 exposed cells and then dividing by the difference of the positive control, the proteasome inhibitor MG132, and untreated cells to produce a measure of relative Caspase 3/7 activity. Relative Caspase 3/7 activities were compared using a two-way ANOVA, followed by a Dunnett's multiple comparisons test.</p>

PubMed Author Manuscript

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.

preservation_of_immunorecognition_by_transferring_cells_from_10%_neutral_buffered_formalin_to_70%_et

<!>Material and methods<!>Results<!>Discussion

<p>Ten percent neutral buffered formalin (NBF) is used almost universally for diagnostic pathology because of its ability to preserve consistently the morphological details of cells and tissues (Grizzle et al. 2008, Grizzle 2009).</p><p>The reaction of 10% NBF with proteins in cells and tissues is thought to occur by three reactions. In the first reaction, formaldehyde reacts rapidly primarily with the amino and thiol groups of amino acids to form hydroxymethyl derivatives (Pearse 1968). Subsequently, in the case of primary amino groups, some hydroxymethyl groups may undergo a condensation reaction to form an imine, also called a Schiff base (Metz et al. 2004). This second reaction occurs more slowly. These two initial reactions may influence immunostaining based on the current approach to fixation, which typically limits fixation to less than 24 h for a thin (e.g., 3 mm) piece of tissue. As the time of exposure to 10% NBF increases, a third reaction occurs in which the imine reacts with other side chains including glutamine, asparagine, tryptophan, histidine, arginine, cysteine and tyrosine in a cross-linking fashion (Metz et al. 2004). These molecular changes may mask some antigen epitopes, which would reduce immunostaining (Arnold et al. 1996). The relatively slow penetration of formaldehyde (< 1 mm/h) affects the fixation reactions in tissues. In addition, processing tissues fixed in 10% NBF to paraffin also affects immunorecognition (Grizzle et al. 2008, Otali et al. 2009).</p><p>Fixation by 10% NBF for longer than 48 h has been studied extensively, but less is known concerning fixation in 10% NBF for shorter periods, e.g., < 12 h. Also, the effects of various concentrations of ethanol following initial fixation in 10% NBF, has not been studied adequately (Otali et al. 2009). It has been reported anecdotally and generally accepted that transferring thin tissues and cells that have been fixed in 10% NBF for more than 24 h to 70% ethanol prevents further loss of immunorecognition of some epitopes. There are few studies in the literature, however, to support this belief and to our knowledge, there is no study of the optimal time for transferring tissues from 10% NBF to 70% ethanol (Leung et al. 2011).</p><p>Ethanol is a dehydrating agent, but it also may act on the hydroxymethyl adducts that have not been cross-linked to catalyze the formation of reactive imines by removing the hydrogen atom from the nitrogen of the original amine end group and the hydroxyl group from the 10% NBF adduct (Dapson 2007).</p><p>Immunorecognition by monoclonal and polyclonal antibodies likely depends on many factors including how both the epitopes/antigens and the antibody identifying the epitopes react; this is indicated by the differential effects of fixation in 10% NBF and/or other cross-linking fixatives that may modify the epitope (Arnold et al. 1996). Other less commonly considered effects of cross-linking fixatives that may affect immunorecognition include steric hindrance of epitope-antibody reactions resulting from cross-linking and local chemical effects caused by reactive hydroxymethyl groups and/or imines.</p><p>Previously, we reported that fixation interacts with tissue processing to decrease immunorecognition (Otali et al. 2009). In that study, cells grown on microscope slides were processed to paraffin as a model to study the interaction of fixation by 10% NBF with cumulative processing steps to paraffin. In the study reported here, cells were grown on coverslips, because this approach frequently is used in research and constitutes a simplified model for enhancing our understanding of the potential short term effects of 10% NBF fixation and transfer to 70% ethanol.</p><p>We grew cells on coverslips specifically to evaluate whether transfer of cells from 10% NBF to 70% ethanol decreases the loss of immunorecognition caused by fixation in 10% NBF. Our study also was designed to determine the optimal time for transfer of cells from 10% NBF to 70% ethanol and how various times in ethanol affect immunorecognition. The results suggest that there is a clear benefit to transferring cells, and potentially tissues, from 10% NBF to 70% ethanol prior to immunohistochemical analysis.</p><!><p>The effects of fixation of cells by 10% NBF for 5 min, 12, 15, 18, 36, 108 and 180 h were compared with fixation for 12 h in 10% NBF followed by transferring the cells to 70% ethanol for 3, 6, 24, 96 and 168 h so that the total time in the two solutions would be equivalent (Table 1). Fixation for 5 min was considered minimal, because "no fixation" usually resulted in detachment of cells from the coverslips during immunostaining.</p><p>Two cell lines were used: DU145 (prostate cancer) and SKOV3 (ovarian cancer) obtained from American Type Culture Collection (ATCC). The cell lines were maintained in RPMI 1640 and DMEM, respectively, with 10% fetal calf serum plus supplements, MEM vitamin solution (Gibco, Grand Island, NY) 1-glutamine (Gibco), antibiotic-antimycotic solution of penicillin, streptomycin and amphotericin B (Gibco) in an incubator with 5% CO2 at 37° C.</p><p>DU145 or SKOV3 cell lines were trypsinized, re-suspended in their respective media and plated uniformly on 22 × 22 mm sterile coverslips in six-well plates at a cell concentration of 150,000/ml for DU145 and 175,000/ml for SKOV3. Plating was carried out over several days in a decreasing time schedule. When confluence reached about 70%, usually after two days, the cells on the coverslips were rinsed quickly twice in PBS, pH 7.4, and either fixed in 10% NBF alone (Richard Allan Scientific, Kalamazoo, MI) for 5 min, 12, 15, 18, 36, 108 or 180 h or fixed in 10% NBF for 12 h, then placed in 70% ethanol (AAPER Alcohol and Chemical Co. Shelbyville, KY) for 3, 6, 24, 96 or 168 h (Table 1). Fixation was carried out at room temperature and all fixation times were synchronized to enable immunostaining of all experimental variations to be performed at the same time.</p><p>When the designated end point of fixation had been reached, the fixed cells on the coverslips were rinsed in Tris buffer, pH 7.6, for 10 min and permeabilized. For the permeabilization step, the cells on the coverslips were dehydrated through graded concentrations of ethanol, i.e., 70, 95%, and absolute ethanol for 2 min at each concentration, treated with acetone (Fisher Scientific, Fairlawn, NJ) for 15 sec, then rehydrated through graded concentrations of ethanol, i.e., absolute, 95, and 70%, before washing in Tris buffer for 2 min (Rodriguez-Burford et al. 2002). Endogenous peroxidase was quenched by exposure to 3% aqueous H2O2, for 5 min and rinsing with Tris buffer. To reduce nonspecific staining, 3% goat serum was added to the cells on the coverslips for 1 h at room temperature. The cells on the coverslips were stained with a monoclonal antibody to the proliferative nuclear marker, PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:18,000, a monoclonal antibody to the proliferative biomarker, Ki67, clone MIB-1 (Bio-genex, San Ramon, CA) diluted 1:60, monoclonal antibodies to anti-keratins AE1/AE3 (Boehinger Mannheim Corp., Indianapolis, IN) 5 μg/ml, diluted 1: 40 and a monoclonal antibody to EGFr (Zymed, San Francisco, CA) 3 μg/ml, diluted 1:5. Dilutions of antibodies were in phosphate buffer EDTA (PBE), pH 7.6. These relatively dilute concentrations of antibodies were chosen so that staining was relatively weak to make assessment of effects on immunorecognition easier. For each antibody and cell line, a control was included in which the primary antibody was replaced with 3% goat serum. Next, the cells on the coverslips were rinsed with Tris buffer, pH 7.6, and incubated with multispecies biotinylated goat anti-mouse/rabbit secondary antibody for 10 min (Signet, Dedham, MA) and HRP-conjugated streptavidin for 5 min (Signet). Color was developed with diaminobenzidine (DAB) for 7 min (Bio-genex, San Ramon, CA) to produce an insoluble chromogen. The cells on the coverslips were rinsed with deionized water, counterstained with Mayer's hematoxylin (Sigma-Aldrich, St. Louis, MO) for 1 min 15 sec, blued in tap water, dehydrated though graded concentrations of ethanols: 70, 95%, and absolute ethanol, before clearing in thee changes in xylene (Fisher Scientific, Fairlawn, NJ). The cells on the coverslips were mounted on microscope slides using Permount (Fisher Scientific). Immunostaining for each experiment was repeated independently three times.</p><p>Blinded evaluations were performed by a board certified diagnostic pathologist (W.E.G.). The immunostaining for a specific cell line for all three experiments under each condition was evaluated during the same session. Two parameters were evaluated: percentage of cells stained and immunostaining score as described in (Grizzle et al. 1998, Otali et al. 2009). Briefly, the intensity of nuclear staining was determined in 1.0 increments from 0 for no staining to 4 for strongest staining. The proportion of cells stained at each intensity level was estimated and multiplied by the staining intensity. The total immunostaining score is the sum of the products of the proportion of cells stained at each staining intensity multiplied by the staining intensity, e.g., 40% of cells staining at intensity of 2 would yield 0.4 × 2 = 0.8 and 60% of cells staining at 3 would yield 0.6 × 3 = 1.8. Adding the components of the immunostaining score for this example would be 2.6. Similarly, 100% of cells with no staining would give a total immunostaining score of 1×0 = 0, and 100% of cells staining at maximum intensity would give an immunostaining score of 1 × 4 = 4, thus immunostaining scores range from 0 to 4. Five random fields were evaluated for each variable for each experiment and the values obtained from the three replicate independent experiments were used to calculate means and standard deviations. Where appropriate, intracellular localization was evaluated separately, e.g., EGFr cellular membrane staining was evaluated separately from cytoplasmic staining for EGFr. Because of the pattern of staining of Ki67-MIB-1 at low concentrations, only staining of mitotic cells was evaluated.</p><p>Because our hypothesis was based on prior studies that indicated that the percentage of cells stained and the immunostaining score would decrease with longer fixation in 10% NBF, right-tailed pair-wise t-tests were performed to assess the differences in immunostaining between experimental time periods. We considered the difference statistically significant if p ≤ 0.01. As expected, we observed a general trend toward a decreasing immunostaining score with increasing duration of fixation in 10% NBF. In addition, preliminary data indicated that transfer of specimens into 70% ethanol would minimize the decreased immunorecognition caused by fixation in 10% NBF; thus, right-tailed tests also were performed for these data, which also followed the trends observed in the preliminary studies.</p><!><p>Immunostaining scores were more sensitive than the percentage of cells stained. For both DU145 and SKOV3 cells, as the duration of fixation in 10% NBF increased, immunostaining scores decreased so that by 180 h there was no observable PCNA staining (Fig. 1A, B).</p><p>After fixation by 10% NBF for 5 min, the PCNA immunostaining scores differed from each subsequent experimental time period for both DU145 and SKOV3 cells (Table 2). A statistically significant decrease (p < 0.01) was observed after 18 h in DU145 cells. In SKOV3 cells, a statistically significant decrease (p < 0.01) occurred after 36 h.</p><p>Immunostaining scores for cells that were fixed for 12 h in 10% NBF followed by 70% ethanol for various periods showed no statistically significant decreases in immunostaining throughout the 180 h of the study for either DU145 or SKOV3 cells (Table 3).</p><p>With regard to the percentage of cells stained, there was no statistically significant decrease in staining in either DU145 or SKOV3 cells until after more than 36 h fixation in 10% NBF (p < 0.01; Table 4). There were no cells that were stained for PCNA in either cell line after fixation for 180 h. Figure 1C (IV) shows no DU145 cells stained for PCNA after fixation in 10% NBF for 180 h.</p><p>There were no statistically significant differences in percentages of DU145 and SKOV3 cell staining for all periods of fixation in 10% NBF for 12 h followed by transfer to 70% ethanol (Table 5).</p><p>A statistically significant decrease (p < 0.01) in immunostaining scores for all periods was observed for cytokeratins AE1/AE3 in DU145 after fixation for 5 min in 10% NBF compared to other fixation times as shown in Table 6. For SKOV3 cells, a statistically significant decrease (p <0.01) in immunostaining scores was observed after ≥ 12 h fixation in 10% NBF (Table 6). Figure 2A, B demonstrate that in both DU145 and SKOV3 cells, the extent of the decrease in immunostaining scores for cytokeratins AE1/AE3 could be important for interpreting staining after fixation for approximately 36 h in 10% NBF. Figure 2C (IV) shows DU145 cells with significantly reduced staining for cytokeratins AE1/AE3 after fixation for 180 h in 10% NBF.</p><p>After fixation for 12 h in 10% NBF and transfer to 70% ethanol, there were no statistically significant changes in immunostaining scores for DU145 or SKOV3 between 12 h and any subsequent times (Table 3).</p><p>Compared to fixation for 5 min in 10% NBF, there was a statistically significant decline (p < 0.01) in the number of DU145 cells stained for cytokeratins AE1/AE3 after fixation for > 18 h in 10% NBF (Table 4); the number of stained SKOV3 cells decreased after 36 h (p < 0.01) (Table 4).</p><p>There were no significant differences in the percent of DU145 and SKOV3 cells stained for cytokeratins AE1/AE3 after fixation in 10% NBF for 12 h followed by transfer to 70% ethanol (Table 5).</p><p>Comparing cytoplasmic immunostaining scores for EGFr in DU145 cells after fixation for 5 min in 10% NBF with other periods of fixation, statistically significant decreases (p < 0.01) were observed at 12 h and subsequent times (Table 7). Although there were statistically significant changes in cytoplasmic immunostaining scores for EGFr in SKOV3 (Table 7), the relative differences were too small to be of practical importance (Fig. 3B).</p><p>Cytoplasmic immunostaining scores for EGFr after fixation for 12 h in 10% NBF compared to fixation for 12 h in 10% NBF followed by experimental periods in 70% ethanol showed no statistically significant differences for either DU145 or SKOV3 cells (Table 3).</p><p>Comparing the percentages of DU145 cells stained for cytoplasmic EGFr after fixation in 10% NBF for 5 min with other periods, a statistically significant difference was observed only at 180 h exposure to 10% NBF (p<0.01) (Table 4).</p><p>The percentage of cells stained for cytoplasmic EGFr in SKOV3 cells after fixation for 5 min in 10% NBF was not statistically different compared to any of the other experimental periods (Table 4).</p><p>The percentage of cells with cytoplasmic staining for EGFr after fixation for 12 h in 10% NBF compared to fixation for 12 h in 10% NBF followed by experimental time periods in 70% ethanol showed no statistically significant differences for either DU145 or SKOV3 (Table 5).</p><p>Membrane immunostaining scores for EGFr staining in DU145 cells after fixation for 5 min in 10% NBF compared to the subsequent experimental time periods showed statistically significant differences (p < 0.01) at 18 h and after ≥ 36 h fixation (Table 8). No statistically significant differences in SKOV3 cells were observed at any of the time intervals. (Table 8) compared to 10% NBF fixation for 5 min.</p><p>Membrane immunostaining scores for EGFr for DU145 or SKOV3 cells after fixation for 12 h in 10% NBF followed by transfer to 70% ethanol showed no statistically significant differences at any of the experimental time periods (Fig. 4A, B; Table 3). Figure 4C shows DU145 cells at selected times after fixation by 10% NBF and fixation for 12 h in 10% NBF followed by transfer to 70% ethanol.</p><p>The percentage of both DU145 and SKOV3 cells stained were significantly decreased in DU145 cells after 18 h and in SKOV3 cells after 108 h; however, these statistical changes were too small to be of experimental importance (Table 4). Transfer of both DU145 and SKOV3 to 70% ethanol preserved immunorecognition (Table 5).</p><p>After fixation in 10% NBF for 5 min, staining of mitoses in DU145 cells with the MIB-1 antibody to Ki67 showed a statistically significant decrease (p < 0.01) after 108 or 180 h (Table 8); however, in SKOV3 cells, staining at all subsequent experimental time points were variable (Table 8). Figure 5A, B shows that a significant decrease in MIB-1 staining occurs in DU145 and SKOV3 cells after 108 h and 36 h, respectively, compared to fixation for 12 h in 10% NBF followed by transfer to 70% ethanol for the same amount of time. No statistically significant differences were observed for either DU145 or SKOV3 cells fixed in 10% NBF for 12 h followed by transfer to 70% ethanol for the experimental time periods, (Table 3).</p><!><p>Cells and tissues fixed initially in 10% NBF sometimes may be transferred to 70% ethanol to reduce the loss of immunorecognition that may occur with longer fixation times. Our study demonstrates that this approach may improve immunodetection of some antigens, although the benefits vary with the antigen-antibody pair, and the cell lines and tissues investigated.</p><p>We used cell lines grown on coverslips as a model to evaluate the effects of fixation by 10% NBF compared to 10% NBF followed by transfer to 70% ethanol to study the effects of fixation on immunohistochemistry of solid tissue.</p><p>Both the proportion of cells stained and immunostaining scores of the cells studied were used to assess the effects of fixation (Grizzle et al. 1998). As expected, the immunostaining score was more sensitive for identifying the effects of fixation on immunorecognition than the percentage of cells stained (Grizzle et al. 1998, Poczatek et al. 1999).</p><p>These results agree with previous reports that immunorecognition of specific antibodies of some specific antigens decreases after fixation in 10% NBF and that this decrease is related directly to the duration of fixation (Otali et al. 2009). Frequently, a statistically significant decrease in immunorecognition after fixation for only 12 h in 10% NBF was observed; however, the decrease usually was not large enough to be important for interpretation of staining until after fixation in 10% NBF for 18 h. The extent and time varied with the cell line and antigen-antibody pair. For EGFr, there was less variation in pattern and intensity of immunostaining compared to other antibody-antigen combinations after fixation in 10% NBF or fixation for 12 h in 10% NBF followed by transfer to 70% ethanol.</p><p>For all other antigen-antibody pairs studied, transfer from 10% NBF to 70% ethanol after 12 h resulted in improved immunorecognition. The results show the optimal time for transfer to 70% ethanol is between 18 and 36 h of fixation in 10% NBF. This varied slightly with the antigen-antibody pair and the cell line. Our study supports the general approach of transferring cells grown on coverslips, and by analogy tissues, from 10% NBF to 70% ethanol to preserve immunorecognition.</p><p>Previously, using a "cell model" of fixation and tissue processing, it was demonstrated that exposure of cells fixed initially by 10% NBF followed by 70% ethanol preserved immunorecognition (Otali et al. 2009). Transfer to 70% ethanol may be useful for immunohistochemistry, because ethanol may facilitate the penetration of antibodies into cells and tissues (Farmilo and Stead 2001). Documented reports on the role that ethanol plays in fixation of cells and tissues and the extent of the effects of ethanol on immunohistochemistry are conflicting. Clearly, 70% ethanol acts as a dehydrating agent for cells and tissues. Some reports indicate that the effects of ethanol on fixation depend on the type of tissue. Ethanol, by extracting lipids, may variably affect tissue antigenicity of specific antigens. For example, in mice, ethanol treatment markedly reduced the detection of Gb3 in normal kidney tissue, but only minimally in neurons. This variation was attributed to differences in the lipid composition of the tissues (Kolling et al. 2008).</p><p>Improved immunohistochemical staining of mammary cancers following fixation in ethanol or alcoholic formalin has been reported for keratins and p53 compared to fixation with 10% NBF (Arnold et al. 1996). A study that compared the expression of p185erbB-2 in ethanol fixed cell blocks and fine needle aspirates of formalin fixed breast tissue in paraffin blocks reported variable results, although no empirical evidence was presented (Williams et al. 2009).</p><p>Fowler et al. (2008) compared the structural properties of RNase A after fixation in 10% formalin, 10% NBF plus ethanol dehydration, or 100% ethanol without prior fixation in 10% NBF. These investigators reported that fixation in 10% NBF for one week did not alter significantly the secondary structure of RNase A. They reported that unfixed RNase A incubated in 100% ethanol for the same period recovered its native structure after ethanol was removed and the RNase A was reconstituted in phosphate buffer. When formaldehyde fixed RNase A was incubated in ethanol for 1 week, then re-hydrated in phosphate buffer, there was a significant decrease in near UV light spectral band intensity, which indicated that the changes were not reversible. Both native and formalin fixed RNase A have been reported to undergo structural transition from the α and β conformation to nearly all β conformation as ethanol concentration was increased from 80 to 100%. Fowler et al. (2008) suggested that exposure to ethanol after formalin fixation causes protein aggregation, which likely stabilizes methylene-bridge crosslinks, hydrogen bonds and van der Waals interactions; this interpretation does not support the preservation of immunorecognition that was observed in these studies.</p><p>The permeabilization step during immunostaining required the cells on the coverslips be taken through a series of increasing concentrations of ethanol to acetone, then reversed. This method was used because it gave better immunostaining than other alternatives, e.g., using only acetone or using Triton X-100 and subsequently washing in Tris buffer. This approach of permeabilizing cells (Rodriguez-Burford et al. 2002) is unlikely to have biased experimental observations or interpretations.</p><p>Immunorecognition of PCNA, cytokeratins AE1/AE3, and Ki67-MIB-1 decreased significantly after fixation in 10% NBF > 12 h; however, changes in immunorecognition were not large enough to make a practical difference until after exposure to 10% NBF for 18 h. The immunorecognition of EGFr was affected less by fixation in 10% NBF. By contrast, fixation for 12 h with 10% NBF followed by transfer to 70% ethanol between 3 and 168 h prevented significant loss of immunostaining for any of the antigens studied.</p><p>Although the results of our study apply most directly to immunostaining of cells, the approach also could be a preliminary model to test whether the immunorecognition by tissues fixed in 10% NBF might benefit from transfer to 70% ethanol prior to processing to paraffin. This study is underway.</p><p>To evaluate immunohistochemical staining, the staining score, which considers both the proportion of cells stained and their intensity of staining, is more sensitive than using only the percentage of cells stained for identifying subtle changes in immunostaining as described previously (Poczatek et al. 1999).</p>

PubMed Author Manuscript

5-Carboxylcytosine and Cytosine Protonation Distinctly Alter the Stability and Dehybridization Dynamics of the DNA Duplex

Applications associated with nucleobase protonation events are grounded in their fundamental impact on DNA thermodynamics, structure, and hybridization dynamics. Of the canonical nucleobases, N3 protonation of cytosine (C) is the most widely utilized in both biology and nanotechnology. Naturally occurring C derivatives that shift the N3 pKa introduce an additional level of tunability. The epigenetic nucleobase 5-carboxylcytosine (caC) presents a particularly interesting example since this derivative forms Watson-Crick base pairs of similar stability and displays pH-dependent behavior over the same range as the canonical nucleobase. However, the titratable group in caC corresponds to the exocyclic carboxyl group rather than N3, and the implications of these divergent protonation events toward DNA hybridization thermodynamics, kinetics, and base pairing dynamics remain poorly understood. Here, we study the pH-dependence of these physical properties using model oligonucleotides containing C and caC with FTIR and temperature-jump IR spectroscopy. We demonstrate that N3 protonation of C completely disrupts duplex stability, leading to large shifts in the duplex/single-strand equilibrium, a reduction in the cooperativity of melting, and an acceleration in the rate of duplex dissociation. In contrast, while increasing 5-carboxyl protonation in caC-containing duplexes induces an increase in base pair fluctuations, the DNA duplex can tolerate substantial protonation without significant perturbation to the duplex/single-strand equilibrium. However, 5-carboxyl protonation has a large impact on hybridization kinetics by reducing the transition state free energy. Our thermodynamic and kinetic analysis provides new insight on the impact of two divergent protonation mechanisms in naturally occurring nucleobases on the biophysical properties of DNA.

5-carboxylcytosine_and_cytosine_protonation_distinctly_alter_the_stability_and_dehybridization_dynam

Introduction<!>pH-Dependent FTIR Melting Experiments on caC- and C-Containing Oligonucleotides<!>pH-Dependent Relaxation Kinetics Monitored with Temperature-Jump Spectroscopy<!>Self-Consistently Modeling pH-Dependent DNA Hybridization Thermodynamics and Kinetics<!>Impact of N3 and 5-Carboxyl Protonation on the Thermodynamic Stability of DNA<!>N3 and 5-Carboxyl Protonation Distinctly alter DNA Hybridization Kinetics<!>Proposed Mechanistic Impact of N3 and 5-Carboxyl Protonation on DNA Hybridization<!>Protonated caC Sites Cooperatively Impact Duplex-to-Single-Strand Thermodynamics and Kinetics<!>Conclusions<!>Synthesis and Purification of 5\xe2\x80\xb2-TA(caC)G(caC)G(caC)GTA-3\xe2\x80\xb2<!>Oligonucleotide Sample Preparation<!>Equilibrium FTIR and 2D IR Measurements<!>Transient T-jump IR Spectroscopy

<p>The physical and biological properties of DNA are highly sensitive to environmental factors such as temperature,1–4 counterions,5 and pH.6–9 In particular, pH effects can greatly impact nucleic acids through direct protonation or de-protonation of specific nucleobase sites, leading to changes in secondary structure6, 10–11 and duplex dissociation.7, 12–13 Proton concentrations across different intracellular compartments are highly regulated and vary substantially from pH 8.0 in mitochondria to pH 7.2 in the nucleus to pH 4.7 in lysosomes.14 Different local environments regulate DNA properties and can potentially lead to damage. Even before the determination of DNA's double helical structure,15 it had been shown that polymeric DNA dissociates under alkaline and acidic conditions.8 Since then, many details related to the identities of protonated species,6–7, 16–18 conformational changes,7, 16 and their thermodynamic impact on duplex DNA (dsDNA) have been investigated.7, 13, 19 It has been demonstrated that the thermal melting temperature of DNA is greatly reduced under acidic conditions and largely stems from perturbations to G:C base pairing.6, 17–18, 20 As a result, the degree of protonation tolerated in dsDNA and its thermodynamic impact is highly-dependent on the level of G:C content.</p><p>In general, the pH-dependent properties of nucleic acids observed in biology are driven by protonation at the N3 position (Fig. 1) of cytosine (C) due to its relatively high and tunable pKa compared to other protonatable bases.10 In addition to initiating mismatches and G:C Hoogsteen base pairs,21 N3 protonation drives the formation of C quadruplex structures known as intercalated motifs (i-motifs).10 Recently, i-motif structures have been observed in vivo, particularly in human promoter regions, suggesting that they may play a significant role in gene regulation.22</p><p>While N3 protonation of C is correlated to most pH-induced function of canonical nucleic acids, numerous modified nucleobases exist in vivo that have unique pH-dependent properties. For example, 5-bromination lowers the pKa of uracil from ~10 to ~8,24 allowing for significant N3 deprotonation under physiological conditions that may facilitate damage to nucleic acids.25 More recently, cytosine derivatives involved in the active DNA demethylation pathway, where 5-methylcytosine (mC) is sequentially oxidized to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and finally 5-carboxylcytosine (caC),26–27 have been discovered in mammals. In this context fC and caC are selectively excised by thymine DNA glycosylase (TDG) to give an abasic site which is repaired with C by a base excision repair (BER) pathway to complete the demethylation cycle.27 Neither mC nor hmC are associated with significant shifts in N3 pKa (pKa,N3). In contrast, fC exhibits a reduced pKa,N3 of 2.4 but shows no pH-dependence in its excision activity, while caC has two titratable groups with pKa values of 2.1–2.4 (pKa,N3) and 4.2–4.8 (C5-COO−, pKa,COO−),23, 28–29 and shows accelerated excision under mildly acidic conditions.28, 30 The pH-dependent behavior of the caC nucleobase stands out among the epigenetic cytosine derivatives since it has a pKa in a similar range to C, but the titratable group associated with this protonation is the exocyclic carboxyl group rather than N3. While addition of a proton at N3 occupies a hydrogen bond acceptor site that blocks the formation of a Watson-Crick base pair (Fig. 1), protonation of the exocyclic carboxyl group situated in the major groove would appear less perturbative, but could nevertheless lead to unique pH-dependent thermodynamic or kinetic effects in modified dsDNA.</p><p>In nucleic acid nanotechnology, the sensitivity of DNA to C protonation has motivated the design of numerous pH-driven nanodevices.31–32 In particular, the formation of i-motif and DNA triple helices in C- and CG-rich sequences, respectively, require C protonation and offer tunability of nucleic acid secondary structure.10, 32 These secondary structures are highly sensitive to sequence composition and environmental factors, which can shift pKa,N3 by 0.5–1 units.10 Such shifts allow for the construction of uniquely pH-sensitive DNA tools for a variety of applications. For example, pH-driven conformational switches have been used to enhance the location specificity of drug-delivery agents,33 increase control over DNA strand displacement equilibria,34 and develop sensors to map the pH of biological environments.35 While C N3 protonation is the primary mechanism behind these pH-devices, more recent work has demonstrated enhanced tunability through the incorporation of modified nucleobases.36–38 In particular, substitution of C to caC has been shown to destabilize i-motif formation in many sequences with high sensitivity to the position of modification as well as destabilize DNA triplex formation under physiological conditions.38–39 Destabilization of each type of structure is most likely due to the reduction in pKa,N3 upon 5-carboxylation. However, the dynamic properties, such as folding rate and mechanism, of pH-driven devices are also essential to their function. As has been shown for 5-bromocytidine,36 caC may be able to tune specific aspects of i-motif folding or DNA triplex association dynamics important for nanotechnology.</p><p>The pH-dependent properties of canonical and modified DNA utilized in biology and nanotechnology are determined by the impact of nucleobase protonation on local and global base pairing properties. Therefore, a detailed understanding of the pH-dependence of hybridization thermodynamics, kinetics, and structural dynamics is warranted. The incorporation of caC may lead to unique pH-dependences in these properties compared to canonical DNA that are nevertheless tunable over the same pH range due to the fact that pKa,COO− is similar to pKa,N3 in C, the 5-position is oriented in the major groove, and protonation of the exocyclic carboxyl group may not directly disturb caC:G base pairing. So far, the biophysical impact of caC has primarily been studied at neutral pH, showing only minor perturbations to double-stranded DNA.39–42 However, the protonated carboxyl group is much more electron-withdrawing43 and caC has a reduced pKa,N3, which has been proposed to weaken G:caC base pairing.23 Additionally, simulations have suggested that 5-carboxyl protonation may tune local solvation and increase local base pair fluctuations.42</p><p>Despite interest in the pH-dependent properties of C and caC, a detailed understanding of nucleobase protonation on duplex dissociation, particularly the kinetics and base pairing dynamics involved in the process, remains incomplete. Here, we characterize the role of 5-carboxyl and N3 protonation on DNA duplex thermodynamics and dehybridization dynamics of a ten-nucleotide sequence using FTIR and temperature-jump infrared (IR) spectroscopy. The mid-IR spectrum of nucleic acids is sensitive to nucleobase-specific changes in base pairing and protonation,23, 44–48 making IR spectroscopy particularly useful for the study of pH-dependent hybridization of DNA. We demonstrate that N3 protonation of C drastically shifts the duplex/single-strand equilibrium and reduces the barrier to dissociation without significantly impacting the hybridization transition state energy. In contrast, caC-modified dsDNA can handle essentially complete protonation of the 5-carboxyl sites without significantly shifting the duplex-to-single-strand transition. Instead, 5-carboxyl protonation leads to highly sloped melting curve baselines that are interpreted as a reduction in the internal base pairing within duplexes. Protonation of caC still leads to a large reduction in the dissociation barrier, but, in contrast to N3 protonation of C, does so through lowering the transition state free energy rather than destabilizing the duplex state. Our results demonstrate that caC and C impart distinct pH-dependent properties onto dsDNA, providing insight into the biological function of these protonation events as well as their potential utility in nucleic acid nanotechnology.</p><!><p>As an initial assessment of the pH dependence of hybridization, Fig. 2 shows FTIR temperature series of the sequence 5′-TAXGXGXGTA-3′, when X = caC (Fig. 2a,b), and when X = C (Fig. 2d,e), prepared at pH* 6.8 and 3.7 and ranging from 3 to 97 °C in ca. 4.5 °C steps. Samples are prepared in deuterated buffer to eliminate the background from the H2O bend vibration.49 D2O has been shown to have a negligible influence on DNA thermodynamics relative to H2O.50 The solution pD is 0.44 greater than the measured pH*,51 so the deuteron concentration at pH* 6.8 corresponds to standard physiological values. The in-plane ring vibrations, carbonyl stretches, and −ND2 bends of the nucleobases absorb in the 1500–1750 cm−1 frequency range and each base contributes a unique vibrational fingerprint to the oligonucleotide spectrum.44, 46–47</p><p>At pH* 6.8 and 3 °C, both sequences exist overwhelmingly as duplexed DNA. The corresponding spectra of these XG-rich duplexes (Fig. 2a,d) appear similar to one another, although the carboxyl group of caC mixes strongly with the in-plane base vibrations and results in an additional absorption near 1570 cm−1.23 As the temperature increases, the spectrum in this frequency range is reshaped significantly and several prominent changes are indicative of dehybridization. Below 1600 cm−1, the spectrum is dominated by G ring mode absorptions whose intensity increases as X:G base pairing is disrupted. A similar increase corresponding to A ring mode absorption is observed near 1625 cm−1. At higher frequency, the spectrum is congested with multiple overlapping peaks, with the growth in intensity near 1665 cm−1 corresponding to T, X, and G carbonyl absorptions. The increase due to G at this frequency is accompanied by a drop in absorption near 1685 cm−1 since this G carbonyl mode is shifted 20 cm−1 when engaged in a Watson-Crick base pair. The highest frequency T carbonyl at 1690 cm−1 can also contribute to this intensity reduction upon the loss of T:A base pairing.</p><p>Reducing the pH* of the oligonucleotide solution significantly alters the FTIR spectrum, particularly at low temperature, as evident through comparison of the pH* 6.8 spectra in Fig. 2a,d against the pH* 3.7 spectra in Fig. 2b,e. FTIR spectra measured at intermediate pH* points at both low and high temperature are shown in Fig. S1. When X = caC, a loss in absorbance is observed near 1575 and 1625 cm−1 as well as a shift of the 1650 cm−1 feature to higher frequency and gain at 1700 cm−1. These spectral changes are consistent with the pH*-dependent FTIR spectra of 2′-deoxy-5′-carboxylcytidine (dcaC) (Fig. S1c & S2). Therefore, we can assign the pH*-dependent changes of X = caC to protonation of the caC nucleobase. Adenine (AMP N1 pKa: 3.9–4.0) and guanine (GMP N7 pKa: 3.0–3.3) can also be protonated under acidic conditions,52–53 but comparison of the spectra in Fig. 2b,e against the pH-dependent FTIR spectra of these nucleosides suggests there is little of these protonation events over the studied pH* range (Fig. S4). The FTIR spectra of X = caC additionally reveal a reduction in amplitude and shift of the 1685 cm−1 guanine mode to lower frequency as caC is protonated. The loss of this feature at neutral pH reports on DNA duplex dissociation, and is always accompanied by an increase in absorbance of the G peaks centered at 1575 and 1665 cm−1, as discussed above. For X = caC, the absorption at 1575 cm−1 instead decreases in amplitude due to shifts in the caC ring vibration upon protonation, suggesting that the loss of the 1685 cm−1 feature at low temperature does not result from duplex dissociation. The frequency and amplitude of the 1685 cm−1 peak is known to be sensitive to DNA conformation as well as the local base pairing and stacking environment,44–45, 47, 54–55 and therefore numerous factors (Fig. S6 & S7) could be responsible for its modulation as discussed in the SI.</p><p>From pH* 6.8 to 3.5, X = C exhibits a shift in the 1650 cm−1 peak to higher frequency and growth of a band near 1700 cm−1 at low temperature (Fig. 2e). Both of these spectral changes are consistent with pH*-dependent FTIR spectra of 2′-deoxycytidine (dC), suggesting N3 of C is protonated when X = C over this pH* range, in agreement with the reported pKa,N3 of 4.2–4.5.17, 23, 29 The significant loss and gain at 1685 and 1660 cm−1, respectively, as well as the intensity gain at 1575 cm−1 also suggest changes to guanine either through protonation or a reduction in base pairing. The pKa of guanine's N7 position (3–3.3) is much lower than pKa,N3 of C,52 and signatures of guanine protonation (Fig. S4) are not present in either the low or high-temperature oligomer spectra. Instead, the intensity changes in the guanine features must be attributed to the loss of G:C base pairing due to N3 protonation of C.</p><p>To further characterize the pH*-dependent spectral changes from N3 protonation, we performed an FTIR titration of the X = C sequence from pH* 6.8 to 1.8 at 3 °C (Fig. 3a). Over this pH* range, DNA dissociates to single strands due to protonation of C, A, G, and the phosphate backbone. The spectral changes as the pH* of the solution decreases in Fig. 3a are dominated by features that indicate the loss of G:C base pairing coincident with N3 protonation of C, as assigned above. Evidence of N1 and N7 protonation of A and G, respectively, is only observed at the lowest pH* points sampled (Fig. S4 & S5). Therefore, the 2nd SVD component of the FTIR spectra across this pH* range should report on the N3 C protonation-driven duplex-to-single-strand transition and can be used to estimate the fraction of intact duplexes at 3 °C as a function of pH*. When fit to a Henderson-Hasselbalch equation, the 2nd SVD component duplex-to-single-strand and N3 protonation trend show an inflection point of 3.9 ± 0.1 that corresponds to the acid denaturation melting point (pHm) of the duplex/single-strand equilibrium at 3 °C. In analogy to the melting temperature (Tm) in thermal denaturation experiments, pHm corresponds to the pH where half of the possible duplex species are intact. The value of pHm for X = C is consistent with those previously measured for polymeric DNA.7</p><p>Our pH*-dependent FTIR measurements of X = C and X = caC may be used to estimate the number of protonated C and caC sites, respectively, per duplex (χ) as a function of pH*. For X = caC, an apparent pKa,COO− of 5.1 is determined from SVD across pH* at 3 °C (Fig. 3b). The apparent pKa,COO− shows no measurable change from 3 to 97 °C (Fig. S3), suggesting that pKa,COO− of the X = caC duplex and single-strand are similar. Therefore, we assume that the 5-carboxyl protonation equilibrium for X = caC is identical in the duplex and single-strand and does not strongly depend on temperature. The small change in pKa,COO− with temperature is consistent with previous temperature-dependent measurements of nucleic acid protonation.56 Additionally, the X = caC sequence is assumed to be highly duplexed at low temperature across the pH* range studied here based on the spectroscopic signatures consistent with extensive caC:G base pairing observed at pH* 3.7 at 3 °C discussed above. The resulting degree of X = caC duplex protonation as a function of pH* is shown in Fig. 3c. The X = caC duplex is almost completely deprotonated at pH* 6.8 whereas an average of 5+ sites per duplex are protonated below pH* 4.4. Further details regarding our estimation of the degree of protonation are presented in the SI.</p><p>As discussed above, the pH*-induced duplex-to-single-strand transition in the X = C sequence appears to be driven by N3 protonation of C. The convolution of these two processes indicates that N3 protonation of C occurs over the same pH*-range as duplex denaturation, but it is not clear how protonation is distributed among duplex and single-strand species. Assuming the effective pKa,N3 and pHm at 3 °C are equivalent for X = C, the fraction of intact duplex species (θext) and degree of N3 protonation in the duplex and single-strand can be related as a function of pH* (See SI for details). As shown in Fig. 3c, X = C is essentially only protonated in the single-strand while the duplex cannot tolerate N3 protonation at even one site. This result may also be interpreted as a significant reduction of pKa,N3 in the duplex relative to the single-strand,57 leading to a sharp decline in duplex fraction at pH* values where single-strand protonation becomes favorable.</p><p>Having identified the spectroscopic signatures of dehybridization and nucleobase protonation, we can assess the pH* dependent melting behavior of the two model sequences. To take into account the global changes to the spectrum upon melting, a SVD is performed on the FTIR temperature series at each pH* to determine a melting curve.45, 58 The melting curves measured from pH* 6.8 to 3.5 are shown in Fig. 2c and 2f for the X = caC and C sequences, respectively. While the two sequences have nearly identical melting profiles at pH* 6.8, they exhibit starkly different trends with decreasing pH*. The X = caC melting profile shows only minor shifts in the inflection point of the sigmoidal melting transition with descending pH*, but the low-temperature baseline slope steepens significantly under acidic conditions. In contrast, the melting transition of X = C shifts drastically to lower temperature and broadens as pH* is reduced. Baseline slopes are often observed in thermal melting curves and are attributed to factors such as evaporation, temperature-dependent changes in sample path length, or DNA base solvation.59–60 However, a comparison of spectral changes along the low-temperature baseline when X = caC (Fig. S8) reveals gains in intensity at 1575 and 1660 cm−1, indicating the loss of G:C base pairing, at pH* 3.7 that are not observed under neutral conditions. This observation as well as the abnormally large changes in baseline slope with decreasing pH* when X = caC suggest that the low-temperature baseline is related to changes in base pairing and base stacking within the duplex state. Based on the pH* and temperature dependence of the mid-IR spectra, the pH* dependence of the melting curves, and the assignment of the extent of protonation in the duplex state, we conclude that there is distinct pH-dependent melting behavior due to divergent protonation events in C- and caC-containing duplexes. Whereas the X = C sequence protonates at N3 C over this pH* range and appears unable to tolerate protonation in the duplex state, the X = caC sequence protonates at the exocyclic 5-carboxyl group, resulting in a duplex much more robust to reductions in pH.</p><!><p>In addition to equilibrium melting experiments, we employed transient temperature jump (T-jump) measurements to assess the kinetic and dynamic impact of nucleobase protonation in the X = C and caC oligonucleotides. The difference heterodyned dispersed vibrational echo (t-HDVE) spectrum, which can be interpreted like a pump-probe difference spectrum,61 is used to track changes to the DNA ensemble at delays following the T-jump. Illustrative time traces tracked at the most intense difference feature of 1670 cm−1, which contains contributions from G:C and A:T base pairing, are shown for the X = C and caC sequences at pH* 6.8 and 3.7 in Fig. 4. For both sequences and at each pH*, three distinct timescales are observed: (1) a small amplitude rise within ~200 ns (λns), (2) a larger rise near ~100 μs (λμs), and (3) decay of the difference signal due to thermal relaxation and re-hybridization in ~ 2 ms. As shown in the SI using temperature-jump two-dimensional IR spectroscopy (t-2DIR, Fig. S9), λns is primarily assigned to fraying of the A:T termini while λμs corresponds to the duplex-to-single-strand transition. Previous T-jump measurements of short DNA oligomers containing A:T termini have revealed a 10–100 ns AT response that was assigned to fraying of the termini.45, 62 Spectral changes associated with λμs are similar to an equilibrium thermal difference spectrum (Fig. S9) between the initial (Ti) and final (Tf) temperatures, indicating the response corresponds to the duplex-to-single-strand transition.</p><p>For each sequence and pH* condition, T-jump experiments were performed with a fixed jump magnitude (ΔT ≈15 °C) and varying Ti along the duplex-to-single-strand transition region (Fig. 4a,d). Under neutral conditions, X = C and X = caC show similar temperature-dependent relaxation kinetics. In each case, λμs increases exponentially with temperature, as observed in previous T-jump experiments of DNA dissociation.1–2, 58, 62–65 Additionally, the magnitude of the λ μs response varies with the expected change in equilibrium base pairing (Fig. 4a,d) between Ti and Tf. In contrast to λμs, the temperature-dependence of λns is negligible within our experimental resolution. The magnitude of the λns response remains unchanged across the low Ti sampled, but sharply reduces at high temperature. Since λns primarily corresponds to fraying of the A:T termini as well as other changes in base pairing, the amplitude of its response is expected to decrease as Ti approaches a condition where the remaining duplexes in solution are largely frayed at equilibrium. More details relating the signal change of λns to duplex thermodynamics are discussed in the SI.</p><p>As the solution pH* is reduced, both X = C and X = caC exhibit drastically different relaxation kinetics compared to those at neutral conditions. The λμs response becomes increasingly non-exponential (Fig. S14) and faster. Additionally, the variation of λμs and its associated signal amplitude with temperature are greatly reduced, signifying a reduction in the barrier to dissociation and cooperativity of the duplex-to-single-strand transition, respectively. For X = caC, the signal change of the duplex/single-strand response decreases at low pH*, consistent with the apparent change in the melting curve shown in Fig. 4d and adds further support that the highly sloped low-temperature baseline observed upon 5-carbxoyl protonation arises from changes in internal duplex base pairing. Overall, our results demonstrate N3 and 5-carboxyl protonation have distinct and significant impacts on DNA duplex stability and dehybridization kinetics.</p><!><p>To quantify the impact of protonation on the hybridization of our model sequences and to provide a consistent framework with which to discuss all of the experimental results, we propose a model that self-consistently describes the thermodynamics and kinetics of duplex formation. The melting of short oligonucleotides is typically assumed to occur in an all-or-nothing fashion, where DNA strands are fully base paired or separated.60, 66 However, the apparent loss of A:T and G:C base pairing at low temperature and asymmetric melting curves exhibited by X = caC suggests a degree of pH-dependent pre-melting within the DNA duplex. To account for both the sigmoidal duplex-to-single-strand transition and the loss of base pairing at lower temperatures that gives rise to a sloping baseline, we interpret the melting curve as a total base pairing fraction, θ(T) that can be separated into external (θext) and internal (θint) contributions (see SI for additional details):67–68 (1)θ(T)=θint(T)θext(T) Here, θext refers to the fraction of intact duplexes containing at least one base pair and is related to the duplex/single-strand (D ⇋ 2S) equilibrium constant, Kd, that can be described by an external enthalpy (ΔH°ext) and entropy (ΔS°ext). The average fraction of intact base pairs within the duplex is given by θint. For polymeric DNA, reductions in θint are the dominant factor in duplex dissociation and typically exhibit a more gradual and less cooperative dependence on temperature relative to θext.67 The low temperature changes in base pairing that manifest as asymmetry in the melting curve also appear to accumulate non-cooperatively with temperature, consistent with the expected profile of θint. For the purpose of modelling θint, we derive an expression for the average fraction of intact base pairs with respect to the equilibrium constant for forming or breaking a single base pair (see SI for details) that is described by an internal enthalpy (ΔH°int) and entropy (ΔS°int).However, it is an oversimplification to assign low-temperature loss of base pairing to the discrete loss of base pair contacts for such a short duplex. Instead, it is more likely that the steepening duplex baseline of X = caC upon 5-carboxyl protonation corresponds to loosening of caC:G base pairing along continuous structural coordinates or an increase in base pair structural fluctuations as suggested previously.42</p><p>The model can be extended to incorporate the T-jump results by assuming a two-state equilibrium of self-complementary oligomers, where the observed relaxation rate λμs is related to the association (ka) and dissociation (kd) rates:69 (2)λμs=kd+4[S]Tfka The single-strand concentration ([S]) at the final temperature (Tf) is obtained from the melting curve. In practice, we use a maximum entropy inverse-Laplace transform approach (MEM-iLT)70 to extract λns and λμs, the observed rates of each process (Fig. S15). To determine both the activation enthalpy (ΔHǂ) and entropy (ΔSǂ) of hybridization we fit ka and kd to a Kramers model in the high friction limit:71 (3)kd=Cdoλnsη(T)exp(ΔSd‡R)exp(−ΔHd‡RT) (4)ka=Caoλnsη(T)exp(ΔSa‡R)exp(−ΔHa‡RT) where λns is the observed rate of the 10–100 ns response measured in the t-HDVE kinetic traces, and η(T) is the temperature-dependent viscosity of D2O.72 Here, λns is taken as an estimate for the rate of diffusive hybridization in analogy to similar treatments in protein folding studies.73–75 This stems from our assignment of λns as largely corresponding to terminal A:T fraying dynamics, which have been shown to be diffusive in nature.62 The temperature-dependence of λns is negligible within the error of our measurement over the surveyed temperature range, therefore the mean over the lowest three Ti is used in the Kramers analysis. An additional unit parameter is included in the dissociation (C°d) and association (C°a) pre-exponential factor fixed at 1 Pa∙s and 1 Pa∙s∙M−1, respectively, which amounts to assuming that the pre-exponential factor is equivalent for association and dissociation, effectively placing any additional contributions into ΔSǂ.</p><p>For a two-state process on a 1D free energy surface, the standard free energy difference between the duplex and single-strand states (ΔG°ext) is equal to the difference between the dissociation (ΔGǂd) and association (ΔGǂa) activation free energies. Therefore, the duplex/single-strand equilibrium constant can be expressed in terms of enthalpic and entropic barriers: (5)Kd(T)=exp((ΔSd‡−ΔSa‡)R)exp((ΔHa‡−ΔHd‡)RT) where the numerators in the arguments of the exponentials are equal to ΔS°ext and ΔH°ext, respectively. Using eqs. 1–5, the thermodynamic and kinetic data can be globally fit to self-consistently describe the pH*-dependent hybridization thermodynamics and kinetics of X = C and X = caC. In total, the thermodynamic and kinetic data is described by six parameters: ΔHǂd, ΔHǂa, ΔSǂd, ΔSǂa, ΔHint, ΔSint. An additional parameter (A) that scales the melting 2nd SVD components by a value between 0 and 1 at the lowest temperature measured is also needed (See SI). In an all-or-nothing melting model, thermal melting curves are normalized to 1 along the duplex baseline, reflecting that all duplexes are intact at the lowest temperature. However, sequences with substantial changes in internal base pairing prior to duplex dissociation may not be fully intact at the lowest temperature measured, and the normalization amplitude (A) used in fitting the thermal melting curves is proposed to report on the degree of internal base pairing at the lowest temperature sampled (3 °C).</p><p>Fig. 5 shows fits using eqs. 1–5 to the 2nd SVD component melting curves and λμs across temperature for X = caC and X = C. T-jump experiments were performed at select pH* points among the measured equilibrium melting experiments. Therefore, melting 2nd SVD components acquired at pH* 6.0, 5.5, and 3.5 were fit without λμs to eq. 1 using ΔH°ext, ΔS°ext, ΔH°int, and ΔS°int. As shown in Fig. S16 & S17, consistent thermodynamic values are determined when fitting the 2nd SVD components with or without kinetic data. The profiles of θint and θext determined from the fits for X = caC are shown in Fig. 5c. Upon a reduction in pH*, the profile of θext exhibits a small shift toward lower temperature and broadens slightly while θint becomes more sharply decreasing along temperature. The value of A for X = caC is assumed to account for deviations in θint from 1 at 3 °C and was found to be insensitive to pH* within the accuracy of our measurement and model (Fig. S16). Therefore, the average value of 1/A across pH* (0.825) was applied to each 2nd SVD component and the average fit error across pH* is used to estimate the error in A.</p><p>A comparison of the FTIR temperature series from pH* 6.8 to 3.5 for X = C (Fig. 2f) shows substantial broadening and shifting of the melting transition as pH* is reduced, indicating a loss of melting cooperativity and overall destabilization of the duplex, respectively. This provides further evidence that the low pH* samples do not begin from a duplex fraction of unity at 3 °C. The thermal 2nd SVD components measured for the X = C sequence can be modeled using eq. 1, where both θint and θext contribute to the melting profile. However, our FTIR titration analysis suggests that the X = C sequence cannot tolerate N3 protonation in the duplex state, and it is a reasonable approximation that θint is independent of pH*. Therefore, we fit the pH* 6.8 2nd SVD component to eq. 1, determining θint, θext, and a 3 °C normalization offset of 0.825. Under neutral conditions, the slope of θint is expected to arise from fraying of the A:T termini (Fig. S8). The normalized 2nd SVD components at all pH* points were scaled at 3 °C (Fig. 5d) by the value of θext determined from the FTIR titration of X = C (Fig. 3a) and the form of θint at pH* 6.8 (dashed line in Fig. 5f) was assumed constant across all values of pH*. As shown in Fig. 5, both the thermal 2nd SVD components and temperature trend in λμs are well fit within this treatment.</p><p>Fig. 6 presents the trends in thermodynamic and kinetic parameters determined from the fits to thermal 2nd SVD components and λμs across pH* for X = caC and X = C. Over the studied pH* range, X = caC exhibits reductions in ΔH°ext and ΔS°ext of ca. 40 kJ/mol and 150 J/molK, respectively. X = C shows substantially greater reductions in ΔH°ext and ΔS°ext of ca. 140 kJ/mol and 400 kJ/mol, further demonstrating the large disruption of duplex formation upon N3 protonation shown here and by others.7, 12–13 5-carboxyl protonation also leads to a reduction in θint as well as increase in ΔH°int and ΔS°int. The dissociation barriers ΔHǂd and ΔSǂd both decrease upon 5-carboxyl and N3 protonation while opposite trends are observed in ΔHǂa and ΔSǂa between the two types of protonation. At pH* 6.8, each sequence exhibits negative association barriers as observed for DNA hybridization in many previous works.1, 62–64, 76 However, ΔHǂa and ΔSǂa each become more negative upon 5-carboxyl protonation and less negative upon N3 protonation. Overall, the activation parameters resemble those in activation energy (E) and pre-exponential factor (A) determined from an Arrhenius analysis of the kinetic data (Fig. S19–S21). Further discussion of the hybridization thermodynamic and kinetic parameter trends with pH* follows.</p><p>Examining the trends in thermodynamic and kinetic parameters in Fig. 6 across pH* reveals distinct behavior due to 5-carboxyl and N3 protonation. For X = caC, each parameter remains almost unchanged between pH* 6.8 and 5.0 and then changes sharply across lower pH* values with titration-like behavior. In contrast, X = C exhibits large, but more gradual changes across the pH* window studied. The trends for X = caC can each be well fit by the Hill equation77 with consistent apparent pKa values of ca. 4.5 and variable Hill coefficients (n). For fitting of binding curves, n > 1 indicates positive cooperativity between binding sites that leads to sharpening of the binding transition. The trends in external thermodynamics as well as enthalpic and entropic barriers reveal sharp transitions that fit to values of n > 1.</p><!><p>Similar to previous studies,6–7, 12–13 the X = C duplex is greatly destabilized as a result of N3 protonation, and our analysis suggests that this specific sequence essentially cannot tolerate any protonation in the duplex state. However, the degree of duplex destabilization from N3 protonation and maximum number of protonated C bases is expected to vary with sequence composition. Previous work with polymeric DNA and short RNA sequences has shown that duplex stability is dependent on the GC content in the sequence.7, 12–13, 18 UV CD measurements suggested that up to 50% of C bases in polymeric DNA could be protonated prior to signs of complete duplex dissociation at low temperature.6 Instead, C protonation leads to disruption of many GC base pairs prior to full dissociation of the duplex.18 It is clear that N3 protonation generally destabilizes the duplex state, but the degree and nature of the destabilization will depend on the context of the GC base pairs. Therefore, while X = C completely dissociates upon a N3 protonation event, other sequences may be able to tolerate some degree of protonation depending on the number and location of GC base pairs present. Regardless, it is well-established that the thermodynamics of nucleic acid hybridization in both polymers and oligonucleotides is additive and can be decomposed into the nearest-neighbor contributions of discrete dinucleotide steps.78–79 It is thus reasonable to expect the present results to apply generally for these base pairs and their local sequence context whether in short duplexes or longer polymeric DNA.</p><p>In contrast to the X = C sequence, protonation of the exocyclic carboxyl group of caC is much less perturbative to the duplex/single-strand equilibrium, shifting the melting inflection point down by ~6 °C in going from pH* 6.8 to 3.5. As a result, the X = caC duplex can tolerate near complete protonation of caC sites (Fig. 3), and this leads to measurable changes in internal base pairing in the duplex. As caC is protonated, clear signatures of the loss of G:C base pairing appear along the low-temperature baseline of X = caC (Fig. S8). For short oligonucleotides like those studied here, it is unlikely that the low-temperature G:C changes correspond to complete loss of discrete base pair contacts. Instead, these signatures suggest loosening of hydrogen bonding or base stacking along continuous structural coordinates. Recent MD simulations of caC-containing duplexes demonstrated that 5-carboxyl protonation increases the degree of structural fluctuations within the modified X:G base pair.42 Motions along these structural coordinates alter the intramolecular base pair distance and orientation, which are expected to alter guanine and C vibrational modes. Therefore such structural fluctuations may account for the observed melting curve baselines.47 The physical origin for this base pair loosening may stem from weakened caC:G base pairing due to the electron-withdrawing nature of the protonated carboxylic acid group, consistent with similar base pair loosening observed for DNA containing 5-formylcytosine.68</p><p>Assignment of the two pKa values observed for caC (2.1–2.4 and 4.2–4.8) has been debated in recent reports.23, 80–81 Using FTIR titrations and simulated IR spectra of 2'-deoxy-5-carboxylcytosine, we previously concluded that the pKa values at 4.2–4.8 and 2.1–2.4 correspond to protonation of the 5-carboxyl and N3 positions, respectively.23 The pH-dependent behavior of X = C and X = caC observed in the current study further support these assignments. Overall, protonation of X = C is shown disrupt the DNA duplex to a much greater degree than protonation of X = caC from pH* 6.8 – 3.5. If X = caC was protonated at the N3 position in the studied pH* range, similar pH-dependent behavior to X = C would be expected. Therefore, we confidently assign the pKa value at 4.2–4.8 in the caC nucleobase and within duplex DNA to protonation of the 5-carboxyl position.</p><!><p>To date, the impact of nucleobase protonation on dissociation and hybridization has remained elusive from a dynamical perspective, despite the importance of these processes in biology and nanotechnology applications. Not only do our results add insight to how N3 protonation impacts the stability of short oligonucleotides as well as the tunability of base pairing stabilities through caC protonation, but we have also characterized the impact on the kinetics of hybridization.</p><p>Just as N3 protonation greatly destabilizes the DNA duplex, it also increases the rate of duplex dissociation. For example, kd increases by nearly a factor of 500 at Ti 55 °C between pH* 6.8 and 3.7 (Fig. S19). 5-carboxyl protonation leads to a far more modest increase in the dissociation rate at Ti 55 °C by a factor of 7–10 (Fig. S20) between pH* 6.8 and 3.7. Global fitting of the kinetics and thermodynamics indicate that both N3 and 5-carboxyl protonated oligonucleotides are still well described by a two-state duplex/single-strand model. Over the pH* range studied, each type of protonation results in a similar reduction of 80–100 kJ/mol in ΔHǂd and 200–250 J/mol∙K in ΔSǂd. The trend upon N3 protonation suggests that ΔHǂd and ΔSǂd would continue to decrease at pH* <3.5. However, due to significant protonation of adenine and eventually guanine at pH* <3.5, we have restricted our study to the pH* window between 6.8 and 3.7. In contrast to the dissociation barriers, both ΔHǂa and ΔSǂa become less negative for X = C and more negative for X = caC as pH* decreases.</p><p>For both dissociation and association, the enthalpy and entropy are highly pH*-dependent, and therefore simplistic free energy diagrams may be informative for understanding the impact from each type of protonation. Fig. 7 presents free energy plots along a 1D hybridization coordinate for X = C and X = caC at 37 °C. These three-level surfaces are composed of the duplex/single-strand free energy change (left), hybridization transition state (TS, center), and single-strand (right). The single-strand free energy is assumed to be independent of pH* and is set as the reference state. At 37 °C, X = C and X = caC exhibit reductions in ΔGǂd of 25 and 20 kJ/mol, respectively, in going from pH* 6.8 to 3.7. ΔGǂa is essentially independent of N3 protonation at this temperature. Across the studied pH* range, linear relationships are observed between ΔGǂd/ΔGǂa and ΔG°ext for each sequence (Fig 7c). For X = C, a slope of 1.04 is observed when plotting ΔGǂd against ΔG°ext and a slope of 0.06 is observed when plotting ΔGǂa against ΔG°ext, indicating that reduction in ΔGǂd at low pH* is due to destabilization of the duplex relative to the single-strand rather than a change in the transition state energy. In contrast, X = caC exhibits a slope of 1.77 for ΔGǂd vs. ΔG°ext and 0.77 for ΔGǂa vs. ΔG°ext, indicating that both destabilization of the duplex relative to the single-strand and a reduction in transition state free energy occur upon 5-carboxyl protonation.</p><!><p>One can gain further insight into the mechanistic implications of the thermodynamic and kinetic results if we assume that the hybridization transition state involves the formation of some small subset of native contacts followed by rapid downhill formation of the remaining base pairs, in analogy to the classic nucleation-zipper mechanism of helix-to-coil transitions in biomolecules.2–3 At neutral pH*, both sequences exhibit negative values of ΔHǂa (Fig. 6b). This result has been observed routinely in DNA hybridization,1–2, 4, 58, 62–64, 76 and is assumed to account for base pair formation in the transition state. A large entropic penalty due to loss of translational and configuration freedom accompanies this initial base pair formation, causing the rate of association to decrease as temperature increases.</p><p>Interestingly, ΔHǂa approaches 0 kJ/mol and ΔSǂa becomes less negative with increasing N3 protonation of the X = C oligonucleotide. Multiple possibilities could account for the reduction in magnitude of ΔHǂa. The X = C single-strand is expected to become highly protonated as pH* decreases, as evidenced by the pH-driven denaturation observed in Fig. 3. For GC-rich sequences such as the one studied here, hybridization is assumed to initiate at C:G base pairs, but protonation at N3 interferes with the ability to form C:G contacts. Therefore, the number of contacts in the transition state may remain unchanged but offer less enthalpic stabilization due to the competition between base pair hydrogen bonding and protonation at N3. In addition, the reduction in magnitude of ΔSǂa may result from changes in solvation and sodium cation environment around the protonated single-strand as noted previously.82 As more N3 sites are protonated, the negative charge density of the single-strand is reduced, and the number of bound sodium cations and excluded chloride and phosphate anions decrease. Analysis of the FTIR titration (Fig. 3) on X = C suggests that N3 protonation can only occur in the single-strand. As a result, the charge density and counterion environment around the protonated single-strand and deprotonated duplex are quite different, and hybridization of the former must involve the loss of N3 protons, gain of associated sodium cations, and exclusion of additional anions. These changes likely contribute to the thermodynamic and kinetic effects observed for N3 protonation in the X = C sequence.</p><p>The decrease in the magnitude of ΔHǂa and ΔSǂa at low pH* could also reflect a reduction in the number of base pairs formed in the hybridization transition state. ΔHǂa ranges from −35 to 0 kJ/mol over the pH* range studied here. The average enthalpic benefit associated with forming a base pair in the CG region of X = C is estimated to be −21 kJ/mol based on the unified nearest-neighbor parameters for DNA hybridization.78 Therefore, the change in ΔHǂa suggests that the number of base pair contacts in the transition state shifts from ~2 to 0 upon protonation. For the X = C sequence, the reduction of ΔHǂa to 0 kJ/mol would suggest that no base pairs are formed in the transition-state at low pH*. Even in this case, ka is 100 times slower (~107 M−1s−1) than the estimated diffusion-limited association rate constant determined from fluorescence correlation spectroscopy (FCS) measurements of short oligonucleotides,76 indicating that an entropic barrier to duplex formation dictates DNA hybridization kinetics even without the formation of base pair contacts in the transition state.</p><p>While protonation of the X = caC sequence also increases the rate of duplex association, ΔHǂa and ΔSǂa are both observed to increase in magnitude as pH* is lowered. Again, this could be interpreted as changes to the energetics of initial contact formation or reflect a change in the structure of the hybridization transition state. 5-carboxyl protonation has the potential to impact intra- and inter-strand electrostatics, caC:G base pairing, base stacking, and major groove solvation. Our pH*-dependent FTIR results indicate that caC sites within X = caC are essentially completely deprotonated at pH* 6.8, adding substantial negative charge to the duplex and single-strand states. Protonation of caC should reduce electrostatic repulsion between the exocyclic carboxyl group and the phosphate backbone. However, the absence of negative charge may alter the degree of hydration in the major groove. Our model for internal base pairing suggests that 5-carboxyl protonation increases the enthalpic stabilization of base pair formation (Fig. 6c) even though contacts become loosened overall. Such an increase in ΔH°int may explain the increase in magnitude of ΔHǂa as pH* decreases, but not the increase in entropic penalty. The change in ΔSǂa is more complex as 5-carboxyl protonation can alter factors such as base hydration and single-strand flexibility.83 Both the increase in magnitude of ΔHǂa and ΔSǂa and decrease in ΔHǂd and ΔSǂd could also be explained by an increase in the number of base pair contacts associated with the hybridization transition state, which could in turn result from the increased structural fluctuation of caC:G base pairs upon 5-carboxyl protonation.42</p><p>Our results demonstrate that N3 and 5-carboxyl protonation significantly perturb DNA hybridization and dissociation kinetics, but in a nucleobase-specific manner that can be rationalized in terms of duplex destabilization and changes to the transition state for a two-state process. However, it is important to note that while the nature of the transition state may change, so too may the distribution of transition states. As shown in Fig. 4 & S14, both sequences exhibit non-exponential relaxation kinetics at low pH* that may stem from inhomogeneity among the duplex ensemble or the transition state. This spread of rates may be modulated by base pairing fluctuations, solvent and ion interactions, as well as distributions of protonated species. A more detailed discussion regarding the origin of the non-exponential kinetics is presented in the SI.</p><!><p>5-carboxyl protonation reduces the overall magnitude of the thermodynamic parameters for duplex dissociation (ΔH°ext, ΔS°ext, ΔG°ext) in an apparently cooperative manner. As shown in Fig. 6, trends in ΔH°ext, ΔS°ext, and ΔG°ext for X = caC follow a Hill profile with Hill coefficient >1, signifying positive cooperativity. Typically, Hill equations are applied to identify cooperativity in binding problems,84 but here the protonation of X = caC is noncooperative (n = 1, Fig. 3). Instead, 5-carboxyl protonation seems to cooperatively shift the thermodynamics of the duplex-to-single-strand transition. Our analysis indicates that nearly all caC sites in the duplex state become protonated from pH* 6.8 to 3.5, and therefore it is possible that multiple protonated caC:G base pairs interact. It seems most likely that such cooperativity would arise from adjacent protonated base pairs, where increased structural fluctuations of each may amplify the weakening of hydrogen bonding and base stacking interactions. Similar effects were predicted for C methylation in which simulations of adjacent methylated CpG dinucleotides influenced base pair fluctuations in a more than additive manner.85</p><p>Similar to the shifts in duplex/single-strand equilibrium, 5-carboxyl protonation appears to alter DNA hybridization kinetics in a cooperative manner as both dissociation and association barriers show sharp transitions as a function of pH* and exhibit a Hill coefficient greater than 1 (Fig. 6). Additionally, the pH* trends in dissociation barrier show a sharper transition (larger Hill coefficient) than observed for the external thermodynamic terms. As shown in the 1D free energy surfaces (Fig. 7b), the pH*-dependence of ΔGǂd at 37 °C has contributions from duplex destabilization and a reduction in free energy of the transition state as pH* is lowered. The former corresponds to ΔG°ext while the latter is only captured by ΔGǂd and ΔGǂa. Therefore, the more cooperative manner in which ΔGǂd varies as a function of pH* relative to ΔG°ext suggests that protonation of multiple 5-carboxyl sites leads to a cooperative change in the free energy of the hybridization transition state in addition to the duplex state. A comparison of ΔHǂd with ΔH°ext and ΔSǂd with ΔS°ext reveal the same trends in apparent cooperativity with 5-carboxyl protonation.</p><p>N3 protonation leads to a larger reduction in DNA duplex stability and dissociation barrier than 5-carboxyl protonation, but it does so almost linearly as a function of pH* (Fig. 6a) over the range studied. This more gradual reduction reflects a non- or anti-cooperative impact of N3 protonation on the duplex-to-single-strand transition and is consistent with our FTIR titration results that estimate the X = C duplex cannot tolerate N3 protonation, making cooperative interactions between multiple protonated sites in the duplex very unlikely.</p><!><p>We have investigated the impact of cytosine and 5-carboxylcytosine protonation on the thermodynamics and kinetics of DNA dissociation and hybridization using FTIR and optically-induced temperature-jump IR spectroscopy. Our results demonstrate that between pH* 6.8 and 3.5, DNA containing C and caC are predominantly protonated at the N3 and 5-carboxyl positions, respectively. Each protonation is shown to have a distinct impact on the thermodynamics and kinetics of dissociation and association between model DNA oligonucleotides. Protonation at N3 completely disrupts the ability of the DNA to duplex, shifts the melting inflection point by >20 °C, and reduces the cooperativity of the duplex-to-single-strand transition. These thermodynamic properties are accompanied by a speedup in the rate of duplex dissociation and increasingly stretched exponential kinetics at low pH*, with the reduction in dissociation barrier primarily achieved through destabilization of the duplex state. 5-carboxyl protonation leads to highly sloped melting baselines that reflect an accumulation of disrupted base pairing contacts in the duplex state, but perturbations to the duplex/single-strand equilibrium are comparatively minor. Regardless of the identity of X, the dissociation free energy barrier is reduced to a similar degree over the pH* range studied. However, N3 protonation does reduces the barrier through destabilization of the DNA duplex while 5-carboxyl protonation reduces the dehybridization transition state energy. Unlike with N3 protonation, X = caC duplexes can be highly protonated, and protonation of multiple sites is observed to alter duplex/single-strand thermodynamics and kinetics in a cooperative manner.</p><p>Protonation of N3 in cytosine plays critical roles in DNA damage, triplex association, and i-motif formation and is utilized to create pH-driven nanodevices. Additionally, the potential importance of the protonation equilibrium of 5-carboxylcytosine in cytosine demethylation has recently come to light. Each of these functions and applications rely on protonation-driven perturbations to double helical DNA, and this work demonstrates the unique ability of N3 and 5-carboxyl protonation to alter the stability, hybridization kinetics, and base pairing dynamics of nucleic acids. The loosening of caC:G base pairing and reduction in dissociation barrier initiated by caC protonation may assist in its selective recognition within the cytosine demethylation pathway.27–28, 30, 86 Such pH-dependent base pair loosening as well as the overall reduction of pKa,N3 upon substitution of caC for C may also prove useful in tuning the physical properties of DNA triplex and i-motif formation. Recent work has shown that caC can effectively fine tune the thermodynamic stability of both structures,38–39 and the results and analysis in the present work provide physical insight into the observed thermodynamic response of the DNA triplex and i-motif to incorporation of caC as well as the prediction of pH-dependent dynamical behavior upon protonation of the 5-carboxyl group.</p><!><p>Unmodified and 5caC phosphoramidites were purchased from Glen Research and DNA oligomers were synthesized at 1 μmol scale in several batches. After oligomer synthesis, the beads were treated with 0.1 M K2CO3 in 1:1 MeOH/water at 42 °C for 16 hours, and then acetic acid was added to neutralize the pH to 7.0. Oligomers were subsequently purified with dialysis in ultrapure water at 4 °C for 48 hours and lyophilized to a powder form.</p><!><p>The sequence 5′-TACGCGCGTA-3′ was purchased from Integrated DNA Technologies (IDT) at desalt grade purity. Samples were purified further with 3 kD cutoff centrifugal filters (Amicon). For IR spectroscopic measurements, all labile protons were exchanged in deuterium oxide (D2O, Cambridge Isotopes, 99.9%). Samples were prepared at a concentration of 1 mM in deuterated 20 mM sodium phosphate buffer at pH* 6.8 with 16 mM NaCl. Units of pH* indicate the measured pH of a deuterated solution using a standard glass electrode pH meter. The pKa values reported here were first converted to represent the value that would be determined in a non-deuterated solution.51 Solutions were prepared at reported pH* values through the addition of concentrated DCl. To minimize HOD content, samples were lyophilized after addition of DCl and re-dissolved in the appropriate volume of pure D2O.</p><!><p>FTIR spectra were measured with a Bruker Tensor FTIR spectrometer at 1 cm−1 resolution. Samples were placed between two 1 mm thick CaF2 windows separated by a 50 μm Teflon spacer enclosed within a home-built brass jacket. The jacket temperature is controlled with a recirculating chiller (Ministat 125, Huber). The sample temperature for a given chiller bath temperature was determined using a thermocouple attached to the center of the CaF2 window. The FTIR titration of X = C was performed using a home-built flow cell with a 50 μm path length and 1 mm CaF2 windows. Samples prepared at each pH* condition were flowed into the sample cell using a syringe pump (Harvard Apparatus).</p><p>Two-dimensional (2D IR) measurements were performed on a previously described setup with a BOXCAR geometry.87–88 2D IR spectra were collected with ZZZZ polarization and at a fixed waiting time (τ2) of 150 fs. The coherence time (τ1) was scanned from −60 to 2500 fs and −60 to 2000 fs for rephasing and non-rephasing surfaces, respectively, with a 4 fs step size.</p><!><p>The details of our temperature-jump (T-jump) spectrometer have previously been described in detail.87–88 In brief, the output of a frequency-doubled Nd:YAG (YG 980, Quantel) was sent through an optical parametric oscillator (OPO) to generate 2 μm pulses (5 ns, 20 mJ, 20 Hz) used to pump the O-D stretch overtone of D2O. Time-dependent changes to the DNA structure are monitored between 5 ns and 50 ms with nonlinear infrared spectroscopy. Transient heterodyne detected vibrational echo (t-HDVE) infrared spectra were acquired in ZZZZ polarization and the local oscillator was stepped in 5 fs intervals between −10 and 10 fs relative to maximum interference with the DVE signal. t-HDVE spectra were processed with Fourier Transform Spectral Interferometry,61, 89 and the recovered dispersed pump-probe (t-DPP) is used throughout the study. Transient 2D IR (t-2DIR) measurements were acquired with undersampling along τ1 at a fixed τ2 of 150 fs. A τ1 step size of 16 fs was used. Rephasing and non-rephasing FIDs were scanned from −60 to 1750 fs and −60 to 1250 fs, respectively. Initial temperatures (Ti) were set using a recirculating chiller connected to a brass sample jacket as for FTIR measurements. The temperature-jump magnitude (ΔT) was determined through monitoring the change in mid-IR D2O transmission and was set by adjusting the voltages applied to the Nd:YAG flashlamps or by using a polarizer to attenuate the 2 μm output. In this study, ΔT was set to ~15 °C for each measurement.</p>

PubMed Author Manuscript

Functional Analysis of Two Lebocin-Related Proteins from Manduca sexta

Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1\xe2\x80\x934 that are located close to the C-termini. In addition, we found that synthetic Leb-B22\xe2\x80\x9348 peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.

functional_analysis_of_two_lebocin-related_proteins_from_manduca_sexta

1. Introduction<!>2.1 Insects, pathogenic bacteria, and fungi<!>2.2 Sequence analysis<!>2.3 Genome walking and RACE<!>2.4 Tissue distribution and microbial induction of lebocin mRNAs and time-course induction of lebocin proteins<!>2.5 Recombinant protein expression and in vitro cleavage assay<!>2.6 Peptide synthesis<!>2.7 Antimicrobial activity assay<!>2.8 Agglutination of GFP-E. coli by synthetic peptides<!>3.1 Cloning and sequence analysis of M. sexta lebocin-related genes<!>3.2 Tissue distribution and transcriptional induction of Leb-B and Leb-C genes<!>3.3 Induced expression of lebocin proteins in hemolymph<!>3.4 Expression and purification of recombinant fusion proteins and in vitro cleavage assay<!>3.5 Antimicrobial activities of synthetic peptides<!>3.6 Agglutination of GFP-E. coli by Leb-B22\xe2\x80\x9348 peptide<!>4. Discussion

<p>Antimicrobial peptides (AMPs) with antibacterial or antifungal activities are produced in invertebrates and vertebrates as a defense mechanism (Bulet et al., 2004). Most AMPs are cationic or amphipathic peptides that can destroy bacterial membrane or inhibit intracellular functions of bacteria (Brogden, 2005; Yeaman and Yount, 2003). AMPs can be classified into α-helix containing peptides, cysteine-containing cyclic peptides and specific residue-rich peptides (Bulet et al., 2004; Lemaitre and Hoffmann, 2007). Insect AMPs are produced by the fat body (an equivalent organ of human liver), hemocytes and epithelial cells (Lemaitre and Hoffmann, 2007). AMP expression is mainly regulated by the Rel family of transcription factors, Dorsal, Dif and Relish, in Drosophila melanogaster via activation of the Toll and IMD (immune deficiency) pathways (Hetru and Hoffmann, 2009; Lemaitre and Hoffmann, 2007). Bacterial peptidoglycan (PG), lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are major bacteria-associated molecular patterns recognized by insects and activate expression of AMPs (Lemaitre and Hoffmann, 2007; Rao and Yu, 2010).</p><p>In the tobacco hornworm, Manduca sexta, six classes of AMPs have been identified: attacin (Kanost et al., 1990), cecropin (Dickinson et al., 1988), a lebocin-related precursor protein (Rayaprolu et al., 2010), moricin (Dai et al., 2008), gloverin (Zhu et al., 2003), and defensin (Genbank accession number: HQ400765). Lebocin was originally identified in Bombyx mori (Chowdhury et al., 1995; Furukawa et al., 1997; Hara and Yamakawa, 1995b), and it belongs to a family of lepidoptera-specific proline-rich AMPs (Otvos, 2002). Lebocin precursor proteins have also been identified in Samia cynthia (Bao et al., 2005), Trichoplusia ni (Liu et al., 2000), Pseudoplusia includens (Lavine et al., 2005), and M. sexta (Rayaprolu et al., 2010). Lebocin precursor proteins are secreted proteins that can be processed to produce active lebocin peptides and active B. mori lebocins are 32-residue peptides located closely to the C-termini of precursor proteins (Chowdhury et al., 1995; Furukawa et al., 1997). Glycosylation of the Thr15 in the 32-residue active B. mori lebocins is critical for antibacterial activity (Hara and Yamakawa, 1995b). B. mori lebocins 1–3 show lower antibacterial activities than cecropin B1, and synthetic lebocin peptides show even lower or no activities compared with natural products (Hara and Yamakawa, 1995b). B. mori lebocin-3 can increase permeability of E. coli-type liposomes and reduce minimal inhibitory concentration of cecropin D at low ionic-strength conditions (Hara and Yamakawa, 1995a).</p><p>Lebocin precursor proteins from other lepidopteran insects align well only with the N-terminal segments of B. mori lebocin precursor proteins, but other parts of lebocin sequences are quite different (Bao et al., 2005). It is unclear how active lebocin peptides are generated from precursors, and which peptides are active against microorganisms. Here we report an integrated study of molecular cloning, protein expression and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) in M. sexta. We determined the cleavage sites in M. sexta lebocin precursors by protein sequencing of the fragments recovered from recombinant proteins treated with induced plasma. We also verified activity of synthetic peptides against bacteria and fungi, and found that active M. sexta Leb-B/C peptides were located at the N-termini, which are different from B. mori lebocins that are located closely to the C-termini. In addition, we showed that synthetic peptide Leb-B22–48, the active lebocin peptide of Leb-B, not only had higher antibacterial activity but also caused agglutination of E. coli cells, a property that has not been reported before.</p><!><p>M. sexta eggs were purchased from Carolina Biological Supply (Burlington, NC, USA), and larvae were reared on artificial diet at 25°C (Dunn and Drake, 1983). The fifth instar larvae (Day 2) were used for bacteria injection, plasma preparation, hemocytes and fat body collection. Dry yeast (Saccharomyces cerevisiae) and Gram-positive Micrococcus luteus were purchased from Sigma-Aldrich (MO, USA). Escherichia coli strain XL1-blue was purchased from Stratagene (CA, USA). Salmonella typhimurium was kindly provided by Professor Michael O'Connor, Staphylococcus aureus and Bacillus cereus were provided by Professor Brian Geisbrecht, S. cerevisiae (BY4741) and Cryptococcus neoformans (alpha) were provided by Professor Alexander Idnurm, School of Biological Sciences at University of Missouri-Kansas City. GFP-E. coli K12 was a gift from Professor Brenda Beerntsen at University of Missouri-Columbia.</p><!><p>Sequences of lebocin-related precursor proteins were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/). Multiple sequence alignment was performed using Mega5 with a gap opening penalty of 10 and gap extension penalty of 0.2 (Tamura et al., 2011). Signal peptide sequences were predicted with SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al., 2004).</p><!><p>Genomic DNA was prepared from hemocytes of M. sexta larvae with PureLink Genomic DNA Mini Kit (Invitrogen). Genomic DNA was digested with Dra I, EcoR V, Pvu II and Stu I, respectively, and purified by phenol-chloroform extraction and ethanol precipitation. Purified fragments were ligated with a synthetic adaptor GenomeWalkerAP with T4 DNA ligase (New England BioLabs). The primary PCR was performed with AP1 and MsLebocinGSP1 primers (Table 1) using a two-step PCR: 7 cycles of 94°C for 25 sec, 72°C for 3 min, 32 cycles of 94°C for 25 sec, 55°C for 30 sec, and 72°C for 3 min. Nested PCR was performed with AP2 and MsLebocinGSP2 primers (Table 1) with 1μL of 1:100 diluted primary PCR product as the template. To determine cDNA sequences, 5′ and 3′ RACE were performed using smarter race kit (Clontech). PCR products were cloned into pGEM-T vector (Promega) and sequenced at DNA Core Facility, University of Missouri-Columbia.</p><!><p>Naïve or E. coli XL-1 blue immunized fifth instar larvae (24h after injection) were dissected, hemocytes, fat body, midgut, epidermis and malpighian tubules were collected and washed 3 times in anti-coagulant (AC) saline (4 mM NaCl, 40 mM KCl, 8 mM EDTA, 9.5 mM citric acid-monohydrate, 27 mM sodium citrate, 5% sucrose, 0.1% polyvinylpyrollidone, 1.7 mM PIPES). Total RNA was prepared with TRI reagent (Sigma-Aldrich). Reverse transcription was performed with Oligo-dT primer and ImProm-II reverse transcriptase (Promega) following the manufacturer's instructions. For real-time PCR analysis, fifth instar larvae were injected with saline, heat-killed E. coli strain XL1-blue (108 cells/larva), dry M. luteus (120 μg/larva) or yeast (S. cerevisiae) (107 cells/larva). Twenty-four hours after injection, hemocytes and fat body were collected separately. Total RNA and cDNA were prepared as described above. Real-time PCR was performed with SYBR Premix (Takara) on a 7500 system (Applied Biosystems) with msLebocin1-RT-N and msLebocin1-RT-C (for Leb-B), msLebocin2-RT-N and msLebocin2-RT-C (for Leb-C), rpS3-N and rpS3-C (for ribosomal protein subunit 3 (rpS3), an internal control) primers (Table 1). Data from three replicates of each sample was analyzed with SDS software (ABI) using a comparative method (2−ΔΔCt) and these experiments were repeated with 3 different biological samples.</p><p>To determine time-course induction of lebocin proteins by Western blot analysis, the fifth instar larvae were injected with E. coli, M. luteus or yeast (S. cerevisiae) as described above (4 larvae in each group). Hemolymph (~50 μL) was collected from each larva every two hours after injection. Hemolymph samples were centrifuged at 3000g for 10 minutes and cell-free plasma samples were collected. Plasma samples (0.125 μL from each larva, 0.5 μL in total from four larvae) were analyzed by 10% Tris-Tricine gels. Western blot was performed using rabbit anti-Leb-B antiserum (see below) (1:2000 dilution) (Cocalico Biologicals, Inc) and Goat anti-Rabbit IgG, HRP-conjugate (1:10000 dilution) (Millipore). Images were developed using Amersham ECL Plus Western Blotting Detection Reagents on a typhoon phosphorimager (GE Healthcare).</p><!><p>Full-length (Leb-B22–143 and Leb-C21–143) or various fragments (Leb-B22–48, Leb-C21–48, Leb-B49–92, Leb-C49–92 and Leb-B93–143) (Table 2) of Leb-B and Leb-C were expressed as soluble Thioredoxin (TRX) fusion proteins at 16°C after IPTG induction in Rosetta (DE3) cells using pET32a vector (Novagen). Bacterial pellets were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and sonicated for 10 min on ice. Cell lysate was centrifuged for 20 min at 18,000g. The clear supernatant was used for protein purification using Ni-NTA agarose. Leb-B (residues 22–143) was also expressed using the H6pQE60 vector (Lee et al., 1994) in E. coli XL1-blue cells as inclusion bodies, purified under denaturing conditions with Ni-NTA agarose (Qiagen), and used as an antigen to produce rabbit polyclonal antiserum against Leb-B at Cocalico Biologicals, Inc (Pennsylvania, USA).</p><p>To determine cleavage of recombinant fusion proteins by proteinases in M. sexta larval plasma, induced plasma samples were collected from the fifth instar larvae at 12 hours after injection of E. coli XL-1 blue (107 cells/larva). Recombinant fusion proteins (1 μg each protein) were treated with 0.5 μL induced plasma in a total of 10μL in 10 mM Tris buffer at room temperature overnight. The cleavage products were separated on 15% SDS-PAGE and analyzed by Western blotting using mouse anti-His-Tag monoclonal antibody (Sigma-Aldrich). To determine the cleavage sites, TRX-Leb-B fusion protein (1 μg) was treated with induced plasma (0.5 μL) in a total of 10 μL at 16°C overnight. The cleavage products were separated on 10% Tris-Tricine gels and visualized by Coomassie Blue staining, or proteins were transferred to nitrocellulose membranes and analyzed by Western blotting using polyclonal rabbit anti-Leb-B antiserum or monoclonal mouse anti-His-Tag antibody (Sigma-Aldrich). For protein sequencing, the cleavage products were separated on Tris-Tricine gels and transferred to PVDF membrane (0.22 μm), the membrane was stained with 0.05% Coomassie brilliant blue R-250 prepared in water, and the small peptides were cut out and sent for protein sequencing at Biomolecular Resource Facility at the University of Texas Medical Branch.</p><!><p>Six peptides (10 mg each) based on the sequences of Leb-B and Leb-C (Leb-B22–48, Leb-C21–48, Leb-B49–92, Leb-B93–108, Leb-B109–143, and Leb-B117–143) (Table 2) were synthesized by Shanghai Apeptide Co., LTD (Shanghai, China) and purified by HPLC to > 95%.</p><!><p>Antimicrobial activity of synthetic peptides was tested against four pathogenic bacterial strains: S. aureus, B. cereus, S. typhimurium and S. marcescens, and two fungal strains: S. cerevisiae (BY4741) and C. neoformans. A zone inhibition assay was performed for initial tests as described previously (Rao and Yu, 2010). A broth micro-dilution assay was used to generate growth curves (Rayaprolu et al., 2010). Briefly, overnight bacterial cultures were subcultured in tryptic soy broth (#091010717, MP Biochemicals) or fungal cultures were subcultured in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) and grown to mid-log phase. The bacterial and fungal cultures were centrifuged at 1,000g for 10 minutes at 4°C and washed once with 10 mM Tris, 0.1 mM EDTA. The bacterial cells were diluted to 5×105 cfu/ml in 5% tryptic soy broth and fungal cells were diluted to 1×105 cfu/ml in YPD medium, and the diluted bacterial cultures (190 μL) or fungal cultures (95 μL) were mixed with synthetic peptide (4 mg/mL, to a final concentration of 200 μg/mL) in 96-well plates. B. cereus and S. marcescens were cultured at 30°C with 220 rpm shaking, S. aureus and S. typhimurium were cultured at 37°C with 220 rpm shaking, and S. cerevisiae and C. neoformans cells were cultured at 30°C with 220 rpm shaking. OD600 was measured with Powerwave XS plate reader every hour (BioTek, VT, US). Bacterial growth curves were generated using the Graphpad Prism version 4.0 for Windows (GraphPad Software, La Jolla California USA, http://www.graphpad.com/).</p><!><p>GFP-E. coli overnight culture (1 μL) was incubated with each synthetic peptide (5 μL, 4 mg/mL in H2O) or BSA (5 μL, 4 mg/mL in H2O) in a total of 10 μL in 10mM Tris buffer and incubated at room temperature for 30 minutes. Samples of bacterial cells were then applied to microscope slides and observed using an Olympus BX50 fluorescence microscope.</p><!><p>In a project to clone regulatory sequence of M. sexta lebocin gene by genome walking, we obtained two genomic fragments encoding two proteins that are homologous to lebocin-like proteins from other lepidopteran insects. To confirm the cDNA sequences, we performed 5′ and 3′ RACE to clone and sequence cDNA clones. We named the two clones as lebocin-related protein B (Leb-B, Genbank accession #: GU563900) and lebocin-related protein C (Leb-C, Genbank accession #: GU563901). Leb-B and Leb-C cDNAs were 90% identical, while protein sequences were 78% identical, but both proteins showed very low similarity (~20% identity) to a previously reported M. sexta lebocin-related protein (called Leb-A in this study) (Rayaprolu et al., 2010). Protein sequence alignment showed that the 143-residue Leb-B and Leb-C are similar to lebocin-related proteins from Samia cynthia (57 and 53% identity), Antheraea pernyi (56 and 51% to ApLeb-1, 40 and 42% to ApLeb-2), Pieris rapae (41 and 40%), Trichoplusia ni (30%), Pseudoplusia includens (33 and 32%), and to B. mori lebocins 1 to 4 (34–39%) (Fig. 1A). The 32-residue B. mori active lebocin peptides (Fig. 1A, boxed region) are not similar to the corresponding regions in other lepidopteran lebocin-related proteins. We also noticed presence of the RXXR motifs in all lebocin-related proteins (Fig. 1A). Thus M. sexta Leb-B/C may be precursor proteins that can be processed in the hemolymph by certain convertases (Devi, 1991; Veenstra, 2000).</p><!><p>Antimicrobial peptide (AMP) genes are generally present in insects at very low basal levels. Upon detection of pathogens, AMP genes are transcriptionally up-regulated due to activation of the Rel-family of transcription factors (Ganesan et al., 2011). To study induction of M. sexta Leb-B and Leb-C genes, total RNA was prepared from different tissues of naïve and microorganism-induced larvae for cDNA synthesis. Two different pairs of primers were used to differentially amplify Leb-B and Leb-C genes (Fig. 2A). RT-PCR results showed that Leb-B and Leb-C mRNAs were not detected in the five tissues of naïve larvae, and Leb-B transcript was greatly induced in hemocytes, fat body, epidermis and malpighian tubule but weakly induced in midgut, while Leb-C transcript was greatly induced in all five tissues (Fig. 2B). Real-time PCR showed that both Leb-B and Leb-C mRNAs were induced in fat body and hemocytes after larvae were injected with E. coli, M. luteus and S. cerevisiae (Fig. 2C and D). In fat body, Leb-B transcript was induced to a higher level than Leb-C, but in hemocytes, Leb-C was induced to a higher level than Leb-B (Fig. 2C and D).</p><!><p>To confirm induced expression of M. sexta Leb-B and Leb-C proteins in hemolymph, we used a time-course Western blot to study accumulation of Leb-B and Leb-C proteins in the hemolymph after larvae were injected with microorganisms. Rabbit polyclonal anti-Leb-B antiserum was generated using recombinant Leb-B precursor as an antigen for the Western blot analysis. In the E. coli, M. luteus and yeast (S. cerevisiae) induced plasma samples, lebocin proteins (in between 14.4 and 24.9 kDa protein markers, arrows) became visible as early as 6h post-injection (Fig. 3A and 3B) and no later than 8 h post-injection (Fig. 3C). The protein concentration in hemolymph continued to increase until 12h post-injection. We also observed accumulation of small peptide(s) in between 3.4 and 7.8 kDa protein markers (Fig. 3, arrowheads). These results suggest that active M. sexta lebocins may be generated from precursor proteins upon immune stimulation.</p><!><p>To study cleavage of lebocin precursor proteins, we expressed full-length and various fragments of Leb-B and Leb-C as fusion proteins to Thioredoxin (TRX)-tag at the N-termini (Table 2) (Fig. 4). The amino acid sequences of the fragments were selected based on the RXXR motifs as potential cleavage sites. All seven recombinant fusion proteins were purified to homogeneity as analyzed by SDS-PAGE (Fig. 4A), and they were all recognized by monoclonal anti-His-tag antibody (Fig. 4B). Five (lanes 1, 2, 5–7), but Leb-B22–48 and Leb-C21–48 fusion proteins (lanes 3 and 4), were also recognized by rabbit polyclonal antibody to Leb-B (Fig. 4C).</p><p>To determine cleavage of fusion proteins by proteinases in the hemolymph, purified fusion proteins and the control TRX-tag were treated with induced plasma from bacteria-injected M. sexta larvae. Western blot analysis showed that all seven fusion proteins but the control TRX-tag were cleaved by induced larval plasma, as cleavage products were detected after incubation of fusion proteins with induced plasma (Fig. 5, arrowheads), indicating that there are cleavage sites in Leb-B and Leb-C as well as in the five fragments (Leb-B22–48, Leb-C21–48, Leb-B49–92, Leb-C49–92 and Leb-B93–143) (Table 2). Leb-B22–143, Leb-C21–143, Leb-B22–48, Leb-C21–48 and Leb-B93–143 were cleaved more completely than Leb-B49–92 and Leb-C49–92. There may be more than one cleavage sites in Leb-B/C because naïve plasma sample could also cleave fusion proteins incompletely and generated more than one cleavage products (data not shown).</p><p>To further determine the cleavage sites, we recovered cleavage products from Leb-B fusion protein with Ni-NTA beads and separated them on Tris-Tricine gel (Fig. 6). Multiple cleavage products were confirmed by Western blot using anti-His-Tag and anti-Leb-B antibodies (Fig. 6A and B, lane 3). We observed three smaller cleavage products (Fig. 6C, Bands a, b and c in lane 3), suggesting that there may be at least three cleavage sites in Leb-B. Bands a, b and c were excised separately and sent for sequencing, and the first five amino acids were determined as EAGDK, SVNEP and SFDSR for Bands a, b and c, respectively (Fig. 6D), which perfectly matched the amino acid sequences right after the three RXXR motifs in Leb-B (Figs. 1A and 6E). The three bands were C-terminal fragments of Leb-B after cleavage at the three RXXR sites, and the calculated molecular masses of Bands a, b and c are 12.1, 7.2 and 5.3 kDa, respectively, which also matched the apparent masses of the three fragments in Tris-Tricine gel (Fig. 6C, arrows). In the induced plasma sample alone, anti-Leb-B antibody also recognized a protein and a peptide (Fig. 6B, lane 2, asterisks), which may represent Leb-B precursor protein and processed lebocin peptide(s), respectively.</p><!><p>Since we determined the cleavage sites in Leb-B, six peptides (Leb-B22–48, Leb-C21–48, Leb-B49–92, Leb-B93–108, Leb-B109–143, and Leb-B117–143) (Table 2) were synthesized to verify activity of M. sexta lebocin peptides. We initially measured antibacterial activities of synthetic peptides using zone inhibition assay (Rao and Yu, 2010), but did not see inhibition zones (data not shown), maybe because these synthetic peptides have low activity as B. mori active lebocins (Hara and Yamakawa, 1995b). We then performed the micro-dilution assay (Rayaprolu et al., 2010) using four pathogenic bacterial strains and two fungal strains. We found that Leb-B22–48 and Leb-C21–48 had high activity against Gram-positive S. aureus, B. cereus, and Gram-negative S. typhimurium (Fig. 7A–C), Leb-C21–48 also had high activity against Gram-negative S. marcescens (Fig. 7D) and the fungus C. neoformans (Fig. 7F). Leb-B49–92 and Leb-B93–108 had high activity against S. aureus (Fig. 7A), Leb-B109–143 had low activity against S. typhimurium (Fig. 7C), Leb-B22–48 and Leb-B109–143 had low activity against S. marcescens (Fig. 7D), and Leb-B49–92, Leb-B93–108, Leb-B109–143 and Leb-B117–143 had low activity against B. cereus (Fig. 7B). These results suggest that Leb-B22–48 and Leb-C21–48, which were located at the N-termini of Leb-B and Leb-C, may be the major active M. sexta lebocin peptides.</p><!><p>When testing antimicrobial activities of synthetic peptides, we also incubated an E. coli strain that expresses GFP with synthetic peptides and observed bacterial cells under fluorescence microscope. Surprisingly, we found that synthetic Leb-B22–48 peptide could agglutinate E. coli cells (Fig. 8b and h); while other five synthetic peptides, including Leb-C21–48 that had high antibacterial activity as Leb-B22–48, didn't have this agglutination activity (Fig. 8c–g). These E. coli cells formed large clumps in the presence of Leb-B22–48, but lysis of bacterial cells was not observed (Fig. 8h).</p><!><p>Induced production of antimicrobial peptides (AMPs) is an important humoral response in insects (Lemaitre and Hoffmann, 2007). Fat body and hemocytes are major tissues to produce AMPs and secrete them into hemolymph. Epidermal cells also can synthesize AMPs and initiate localized immune responses (Lemaitre and Hoffmann, 2007). AMP gene expression is mainly regulated by the Rel-family of transcription factors, which bind to cis-regulatory elements in the promoters of AMP genes and initiate transcription (Ganesan et al., 2011). We found that M. sexta Leb-B and Leb-C mRNAs were up-regulated in several tissues that may directly contact pathogens (Fig. 2B). Gram-negative E. coli, Gram-positive M. luteus and yeast (S. cerevisiae) all were able to stimulate expression of Leb-B and Leb-C in hemocytes and fat body, and Leb-B was induced to a higher level in fat body than Leb-C, while Leb-C was induced to a higher level in hemocytes than Leb-B (Fig. 2C and D). Induced expression of Leb-B and Leb-C proteins and accumulation of processed peptide(s) in the hemolymph were also confirmed by Western blotting analysis (Fig. 3).</p><p>Most AMPs are synthesized as precursor proteins or pro-proteins, and they require proteolytic processing to produce active AMPs. Proteinases in insect hemolymph are important for cleavage of signaling molecules and activation of the prophenoloxidase system and immune signaling pathways (Gál et al., 2007; Jiang and Kanost, 2000; Jiang et al., 1999; Kim et al., 2008; Volz et al., 2005). Analysis of lebocin protein sequences showed that lebocin precursor proteins contain several RXXR motifs (Fig. 1A), which can be recognized by processing enzymes (Devi, 1991; Veenstra., 2000), suggesting that active lebocins are generated from precursor proteins by processing enzymes in vivo. Two RXXR motifs are highly conserved in all lebocin precursors, while M. sexta Leb-B/C and B. mori lebocins 1–4 precursors contain two more RXXR motifs (Fig. 1A). In order to study processing of M. sexta Leb-B/C in vitro, recombinant Leb-B and Leb-C precursors and different fragments of Leb-B and Leb-C containing RXXR motif(s) were expressed as fusion proteins to a thioredoxin (TRX)-tag. These fusion proteins were soluble and purified under native conditions, but they did not have antimicrobial activity (data not shown). All seven recombinant fusion proteins (Leb-B and Leb-C (4 RXXR motifs), Leb-B22–48, Leb-C21–48, Leb-B49–92, Leb-C49–92 (each contains one RXXR motif), and Leb-B93–143 (two RXXR motifs)), but not the TRX-tag alone, could be processed by induced plasma from M. sexta larvae (Fig. 5), indicating that these RXXR motifs indeed were recognized by processing enzymes in the hemolymph. Using Leb-B fusion protein and protein sequencing of cleavage products, three cleavage sites were determined (Fig. 6). Leb-B precursor was cleaved mainly at the first three RXXR motifs, RTVR45–48, RYAR89–92, and RFVR105–108 (Figs. 1A and 6E). Naïve plasma could also process the fusion proteins, but the cleavage was incomplete compared to the induced plasma (data not shown), suggesting that some proteinases also require activation.</p><p>Leb-B and Leb-C are both 143-residue long with a 21-residue signal peptide for Leb-B and 20-residue signal peptide for Leb-C. Leb-B and Leb-C shared 78% identity, but they showed low similarity (~20% identify) to a previously reported lebocin-related protein (Leb-A) in M. sexta (Rayaprolu et al., 2010). Leb-A contains several repeats of a 27-residue fragment, and it also contains multiple RXXR motifs. To test antimicrobial activity of processed peptides from M. sexta Leb-B and Leb-C, several peptides based on the cleavage of recombinant Leb-B by induced plasma were synthesized (Table 2). We initially used a zone inhibition assay to test activity of synthetic peptides, but didn't observe formation of inhibition zones (data not shown), suggesting that antibacterial activity of these synthetic peptides may be low. This may be because synthetic peptides lack necessary glycosylations that are important for antibacterial activities as in the case of B. mori lebocins (Hara and Yamakawa, 1995b). High salt concentration (171 mM NaCl) in the normal LB agar plate may also inhibit the activity of synthetic peptides (Hara and Yamakawa, 1995b). Therefore, we used a modified micro-dilution assay (Rayaprolu et al., 2010) to test antimicrobial activities of these peptides in 5% tryptic soy broth (4.2 mM NaCl) or in YPD medium (1% yeast extract, 2% peptone, 2% dextrose).</p><p>Leb-B22–48 and Leb-C21–48 (N-terminal fragments) showed very low similarity, but both fragments contain 5 proline residues, and they may be proline-rich active peptides. Antimicrobial activity assay showed that both synthetic Leb-B22–48 and Leb-C21–48 peptides had higher activity against Gram-positive S. aureus and B. cereus, as well as Gram-negative S. typhimurium and S. marcescens than other four peptides (Fig. 7). Leb-B49–92 and Leb-C49–92 are almost identical and differ only in 4 residues, while Leb-B93–143 and Leb-C93–143 are completely identical. Synthetic Leb-B49–92, Leb-B93–108, Leb-B109–143, and Leb-B117–143 peptides all showed some activity against B. cereus (Fig. 7B), Leb-B49–92 and Leb-B93–108 had high activity against S. aureus (Fig. 7A), whereas Leb-B109–143 had low activity against S. typhimurium and S. marcescens (Fig. 7C and D). These results suggest that lebocin precursors may be processed by proteinases to produce different peptides, and these processed peptides may function together to exert maximal activity against different microorganisms. Among the six peptides, only Leb-C21–48 had activity against the fungus C. neoformans (Fig. 7F), and Leb-C21–48 also had higher activity against Gram-negative S. marcescens than Leb-B22–48 (Fig. 7D), suggesting that Leb-C21–48 may have higher activity and broader spectrum against microorganisms than Leb-B22–48. We also observed that Leb-B precursor could be expressed and purified from E. coli, but re-folded Leb-B precursor did not have activity against bacteria (data not shown). However, Leb-C precursor could not be expressed in E. coli since bacterial culture stopped growing and became clear after addition of IPTG (data not shown), suggesting that some products of Leb-C may be toxic to E. coli.</p><p>Sequence alignment showed that the N-terminal fragments of B. mori lebocins 1–4, P. rapae, P. includens, and T. ni lebocin precursors share high similarity, the N-terminal fragments of M. sexta Leb-B, S. cynthia and A. pernyi lebocin-1 precursors share high similarity, while the N-terminal fragments of M. sexta Leb-C and A. pernyi lebocin-2 have high similarity, which are more similar to those of B. mori lebocins 1–4 than to M. sexta Leb-B, S. cynthia and A. pernyi lebocin-1 precursors (Fig. 1B). All these N-terminal fragments are proline-rich (4–6 proline residues). The active peptides of B. mori lebocins 1–4 did not align with the corresponding regions in other lebocin precursors (Fig. 1A). Since the N-terminal fragments (Leb-B22–48 and Leb-C21–48) of M. sexta Leb-B and Leb-C were active peptides (Fig. 7), we think that the N-terminal proline-rich fragments of lebocins are likely active peptides after processing by proteinases in lepidopteran insects. We also found an interesting result that Leb-B22–48 not only had higher antibacterial activity than other peptides, but also caused agglutination of E. coli cells (Fig. 8b and h), a property that has not been reported for lebocins so far. Leb-C21–48 though had high antibacterial activity as Leb-B22–48 (Fig. 7), couldn't agglutinate E. coli cells (Fig. 8g). But lysis of the agglutinated E. coli cells by Leb-B22–48 was not observed (Fig. 8h). These results suggest that even though lebocin peptides may have lower direct antibacterial activity compared to other AMPs, agglutinating activity of some lebocin peptides can help other AMPs to kill bacterial cells more effectively, as it has been reported that B. mori lebocins can decrease minimum inhibitory concentration of cecropin D (Hara and Yamakawa, 1995a). Future work is to investigate how lebocin peptides act together with other AMPs to kill different bacteria.</p>

PubMed Author Manuscript

Effect of polyelectrolyte complex formation on the antibacterial activity of copolymer of alkylated 4-vinylpyridine

Polymers bearing quaternized 4-vinylpyridine (QVP) groups are known for their antibacterial activities and these polymers can form polyelectrolyte complexes (PEC) with polyanions through electrostatic interactions. PEC formation can be used to adjust the antibacterial activity of polymers of QVP, deliver active molecules, or design antibacterial supramolecular structures. However, the antibacterial activity of PECs of QVP polymers has not been investigated. In this study, a copolymer of QVP was mixed with polyacrylic acid in various molar ratios of components to form PECs. Hydrodynamic diameters and zeta potentials of formed PECs were determined by dynamic and electrophoretic light scattering spectroscopy techniques. The zeta potentials of PECs changed between –24 and +16 mV with variation in the ratio of components. Antibacterial assays against E. coli revealed a relation of PEC formation with antibacterial activity since MIC values changed between 125–1000 μg/mL according to the ratio of components.

effect_of_polyelectrolyte_complex_formation_on_the_antibacterial_activity_of_copolymer_of_alkylated_

1. Introduction<!>2.1. Materials<!>2.2. Equipment<!>2.3. Synthesis of poly(QVP-co-OEGMA) copolymer<!>2.4. Preparation of complexes of poly(QVP-co-OEGMA) and PAA<!>2.5. Antibacterial tests<!>3. Results and discussion<!><!>3. Results and discussion<!><!>3. Results and discussion<!><!>3. Results and discussion<!><!>3. Results and discussion<!><!>3. Results and discussion<!><!>3. Results and discussion<!><!>3. Results and discussion<!><!>Conclusion

<p>Cationic polymers attract considerable interest in the field of biomedical applications as antibacterial agents due to their inherent bacteria-killing activity and easy synthesis and modification [1,2]. Polymers bearing quaternary ammonium groups are the most prominent polycations used for antibacterial purposes [3]. These polymers interact cooperatively with negatively charged groups of the bacterial membrane and kill bacteria by disrupting its membrane. Hydrophobic groups are also added to the polymer structure to increase the activity of polycations by interacting with hydrophobic alkyl chains of lipids [4]. However, hydrophobic groups in the structure of polycations may decrease the solubility of the polymer in aqueous solutions. Therefore, adding hydrophilic monomers or groups such as PEG, HEMA, or HPMA into the structure of the polycation becomes a crucial design parameter for higher antibacterial activity [5,6].</p><p>Copolymers of quaternized 4-vinylpyridine (QVP) with oligoethylene glycol methyl ether methacrylate (OEGMA) were investigated in depth by Youngblood and his colleagues with the purpose of developing biocompatible and effective antibacterial agents [5,7]. In the structure of QVP and OEGMA copolymer, while QVP and OEGMA monomers are the quaternized ammonium groups and hydrophilic groups, respectively, hexyl groups attached to QVP groups are the hydrophobic moieties. By changing the quantities of monomers and chain length of OEGMA, the polymer's antibacterial activity and biocompatibility can be adjusted [8]. These polymers can also be used to develop more complex novel materials with the driving force of intermolecular interactions between the cationic structure of the polymer and an oppositely charged molecule such as pharmaceutical peptides/proteins, genetic materials, or natural/synthetic polyanions [9,10].</p><p>Electrostatic interactions between polycations and polyanions cause the formation of polyelectrolyte (or polyion) complexes (PEC). These complexes can be produced from synthetic polyelectrolytes and can also be found in nature, such as DNA-histone complexes. PECs have been investigated intensely for a long time in drug delivery applications [11], gene therapy studies [12], vaccine adjuvants [13], etc. Beyond these applications, PECs have been used as antibacterial materials, such as coatings [14] or hydrogels [15]. However, examples regarding the antibacterial activity of PECs are limited and a detailed correlation between the antibacterial activities of PECs with their properties needs to be studied in detail.</p><p>PECs are formed by cooperative electrostatic interactions between polycations and polyanions and the structure of the formed PECs depends on numerous parameters, such as the ratio of (+) and (–) charged groups, concentration, ionic strength of the solution, charge density of polyelectrolytes, chain length of polyelectrolytes, ionization degree of the charged groups, location of the charged group in the polymer structure, hydrophobic or hydrophilic groups in the polymer backbone, etc. [16]. In addition, PECs can reversibly dissociate to their polymeric components under certain conditions [17]. Thus, changes in the parameters of PEC formation or environmental conditions can affect size, shape, zeta potential, and consequently the biological activity of the PEC [18,19]. It is clear that the correlation of the composition and formation parameters of PEC with its antibacterial activity can induce the development of novel antibacterial materials with variable characteristics, which can also be sensitive to environmental stimuli.</p><p>In this study, an antibacterial cationic copolymer was used to form PEC with an anionic polymer and the effect of the complexation on antibacterial activity of the polycation was investigated. For this purpose, an antibacterial copolymer of QVP and OEGMA was synthesized and this copolymer was mixed with polyacrylic acid (PAA) to form PECs in varying mole ratios of polyelectrolytes. Physicochemical properties and antibacterial activity of the complexes were examined to reveal a relationship between the structure of the PEC and its biological activity.</p><!><p>OEGMA500 (Mn = 500 g/mol), 4-vinylpyridine (4VP), and PAA (Mw = 30 kg/mol) were purchased from Aldrich. 4,4-azobis(4-cyanovaleric acid) (ACVA) and 1-bromohexane were obtained from Sigma-Aldrich. Diethyl ether and methanol were obtained from Merck. NaCl, NaH2PO4.5H2O, and Na2HPO4.12H2O were obtained from Riedel-de Haen. Ultra-pure water was obtained from a Millipore MilliQ Gradient system. All other chemicals used were analytical grade.</p><!><p>1H-NMR spectra were acquired from Bruker Avance III 500 MHz and deuterated DMSO was used as the solvent.</p><p>GPC (Viscotek TDA302) with refractive index (RI) and right-angle light scattering (LS) detectors was used to determine the molecular weight and molecular weight distribution of poly(QVP-co-OEGMA). Eprogen CatSEC300 was the GPC column and the flow rate was 0.4 mL/min. A solution of 0.1 M acetic acid with 0.1 M NaCl was used as the mobile phase. Polyethylene oxide (Mn = 5 kg/mol, Mw/Mn = 1.05) was used for the calibration of detectors. All samples were filtered by 0.45 μm syringe filters before GPC analysis.</p><p>The degree of quaternization of the copolymer was determined by FTIR spectrum acquired by Shimadzu IRPrestige-21 FTIR spectrometry with Pike MIRacle ATR device.</p><p>4VP groups of the copolymer were quaternized with microwave heating by using a Milestone Microsynth microwave oven. The reaction temperature of 80 °C was maintained by the preset maximum microwave energy of 200 W.</p><p>Hydrodynamic diameters (Dh) and zeta potentials of polymers and PECs were acquired by dynamic (DLS) and electrophoretic (ELS) light scattering spectrometry (Zetasizer Nano ZS, Malvern). All DLS and ELS measurements were acquired at 25 °C. General purpose (NNLS) analysis model was chosen from the data acquisition software of equipment for fitting the correlation function in DLS measurements. To determine their zeta potentials in ELS measurements, 1/10 diluted solutions of complexes were used.</p><!><p>A random copolymer of poly(4VP-co-OEGMA500) was obtained by polymerization of 4VP and OEGMA500 together. 4VP and OEGMA500 were dissolved in DMF with initial concentrations of 1.5 M and 0.17 M, respectively. The solution was purged with N2 gas for at least 30 min and the reaction vessel was tightly sealed. The mixture was heated to 70 °C and polymerization was initiated with the addition of ACVA. The concentration of ACVA was 5.6 mM. After 24 h, the polymer was precipitated with cold diethyl ether. The precipitate was dissolved and precipitated twice to remove the remaining monomer and DMF. The final product was dried overnight in vacuum at 50 °C and then characterized by GPC and 1H-NMR spectroscopy.</p><p>4VP groups of the copolymer were quaternized by the method used in our previous study [20]. Briefly, the obtained copolymer was dissolved in 2-propanol with a 10% concentration. 1-bromohexane was used as the quaternization agent and added to the solution with the mole number 5 times more than the 4VP groups in the dissolved copolymer. The reaction vessel, closed with a septum, was kept in the microwave oven for the reaction for 3 h. The reaction temperature was 80 °C, maintained by a maximum microwave energy of 200 W. The polymer was precipitated with cold diethyl ether and dried. The quaternized copolymer was characterized by FTIR spectrometry.</p><!><p>Stock solutions of poly(QVP-co-OEGMA) and PAA were prepared separately in 0.02 M phosphate buffer at pH 7.0. Stock solutions were filtered with a 0.2 μm syringe filter. The stock solutions of polymers were mixed in various [NR++4]/[COO-] ratios (0.1, 0.5, 1, 2, 3, 5, 10, 20, and 50). [NR+4]/[COO-] is the molar ratio of positively charged amine groups of poly(QVP-co-OEGMA) to negatively charged carboxylic acid groups of PAA. Eq. (1) shows the molar ratio calculation where Cpoly(QV P−co−OEGMA) and CPAA are concentrations of polymers and Mpoly(QV P−co−OEGMA) and MPAA are molecular weights of polymers. In Eq. (1), while NNR4+ is the number of quaternized pyridine rings in one poly(QVP-co-OEGMA) chain, NCOO- is the number of ionized carboxylic acid groups in one PAA chain. pKa of PAA is 4.5 and all carboxylic acid groups in PAA chains are fully ionized at pH 7.0 [21]. In all PEC solutions, the concentration of poly(QVP-co-OEGMA) was 2 mg/mL. [NR+4]/[COO-] ratios were adjusted by changing the PAA concentration.</p><!><p>The broth microdilution method [22] was used as the quantitative method to evaluate the antibacterial activity of polymers and PECs against E. coli (ATCC number: 25922). Polymer and PEC solutions were serially diluted in liquid growth medium using a 96-well plate. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of samples were determined after incubation at 37 °C for 24 h. The bacterial culture medium (106 cfu/mL) was added into each well and the final volume of the wells was adjusted to 200 μL. The negative control wells contained broth and bacteria only. While only broth was used for the blank control wells, a mixture of vancomycin antibiotic solutions (4 mg/L) and bacteria was used in positive control wells. MIC values were determined both spectrophotometrically (OD600 nm) and by a standard plate counting method.</p><!><p>The study investigates the effect of PEC formation on the antibacterial activity of poly(QVP-co-OEGMA). For this purpose, a random copolymer of 4VP and OEGMA500 was synthesized using free radical polymerization. The obtained copolymer was characterized by GPC and 1 H-NMR spectrometry. Figure 1 gives the GPC chromatograms of poly(QVP-co-OEGMA) acquired from RI and LS. As seen in Figure 1, the synthesized polymer has a unimodal distribution with PDI of 1.3 and Mw of the copolymer is 114 kg/mol.</p><!><p>GPC chromatograms of poly(4VP-co-OEGMA) acquired from RI (black line) and LS (blue line) detectors.</p><!><p>In Figure 2, the 1H-NMR spectrum of the synthesized copolymer is given, in which protons of pyridine rings are clearly observed at 8.26 and 6.59 ppm. The peaks at 4.13 and 3.50 ppm belong to the CH2 protons on the side chain of OEGMA. Peak areas of these peaks allow determining the monomer quantities in the copolymer chain. Although 90 mol% of monomers were 4VP in the feed of polymerization, 97 mol% of the monomers in the final copolymer chain are 4VP and the remaining 3 mol% of monomers are OEGMA. This result complies with previous studies about the reactivities of these monomers [23,24], in which 4VP is more reactive than OEGMA and therefore less OEGMA monomers were included in the structure of the copolymer chain than the polymerization feed.</p><!><p>1H-NMR spectrum of poly(4VP-co-OEGMA) in DMSO.</p><!><p>To obtain a positively charged copolymer, amine groups of 4VP rings were quaternized with 1-bromohexane in the next step. Figure 3 gives the FTIR spectrum of the copolymer after the quaternization reaction. The FTIR spectrum allows to easily determine the quaternization and degree of quaternization of 4VP rings [20,25]. In Figure 3, the band at 1725 cm-1 belongs to the C=O group of the OEGMA monomer. Actually, C=O gives a strong and sharp band, but the molar ratio of OEGMA is very low in the copolymer chain according to the NMR spectrum. Therefore, a weak C=O band is observed in the copolymer's FTIR spectrum. The band at 1100 cm-1 belongs to C–O–C ether groups in the side chain of OEGMA. Since there are 10 times more ether groups than C=O groups in the OEGMA monomer, the ether band gives a stronger band. The pyridine ring is characterized by the band at 1600 cm-1 , however, this band is observed as a very small shoulder next to the band at 1640 cm-1 , which belongs to the quaternized pyridine ring. As seen from the spectrum, the band of the quaternized pyridine ring at 1640 cm-1 gives a strong and sharp peak. The ratio of absorbances of the peaks at 1600 and 1640 cm-1 directly gives the degree of quaternization in the copolymer chain as 91% [20,25]. The band at 1468 cm-1 also shows the quaternized pyridine rings of the copolymer. The band of nonquaternized pyridine rings at 1415 cm-1 is not observed in the spectrum due to the high degree of quaternization.</p><!><p>FTIR spectrum of quaternized poly(QVP-co-OEGMA).</p><!><p>The structure of poly(QVP-co-OEGMA) synthesized in this study depends on the polymer produced by Youngblood and his colleagues [5,7,8]. They showed that even 1% of hydrophilic monomers, such as OEGMA, in the structure of quaternized PVP dramatically increases the antibacterial activity of the polymer due to increased solubility and less interaction with proteins. Consequently, the polymer can interact with the membranes of bacteria. Alkyl groups attached to vinylpyridine rings are not only used for quaternization of the polymer, but also for increased interaction with hydrophobic lipid chains of the membrane to be disrupted. It has been shown that longer alkyl chains attached to the vinylpyridine group increase antibacterial activity [26]. However, solubility becomes a major problem when long alkyl chains are attached to the polymer after quaternization. Alkyl groups of up to 6 carbons used in quaternization of vinylpyridine are appropriate alkylating agents since their chain length is long enough to interact with lipids and short enough not to cause insolubility.</p><p>While antibacterial activity, biocompatibility, and effect of monomer composition or type of hydrophilic monomer on the antibacterial activity of the synthesized quaternized copolymer were studied extensively in previous studies [5,7,27], the effect of complexation with an oppositely charged molecule was not studied for this copolymer. Complexation with an oppositely charged molecule may allow a design of novel antibacterial materials delivering a different type of agents for more effective antibacterial applications or other drugs as dual effect systems. Also, the interaction of the copolymer with oppositely charged molecules under physiological conditions or using the copolymer for coating metallic or ceramic materials with electrostatic forces can affect the antibacterial activity of the copolymer. Therefore, in this study, we investigated the effect of the complexation of quaternized poly(4VP-co-OEGMA) with an oppositely charged polymer of PAA on the antibacterial activity of quaternized poly(4VP-co-OEGMA). For this purpose, we simply mixed the solutions of poly(QVP-co-OEGMA) with PAA in different molar ratios of [NR+4]/[COO−]. All solutions were clear, except for the slightly turbid solution of [NR+4]/[COO−] = 1.</p><p>Poly(QVP-co-OEGMA), PAA, and the complexes prepared in different molar ratios of these polymers were analyzed with dynamic and electrophoretic light scattering spectroscopy techniques to determine their hydrodynamic diameters and zeta potentials. In all samples, the concentration of poly(QVP-co-OEGMA) was fixed to 2 mg/mL and the concentration of PAA was changed to obtain the desired [NR+4]/[COO−] ratio.</p><p>Size and zeta potential are important physicochemical parameters that directly affect the biological activity of macromolecular systems. Figure 4 gives the hydrodynamic diameters and polydispersity index (PDI) values of the polymers and their complexes. While the diameters of poly(QVP-co-OEGMA) and PAA are 14.2 and 2.1 nm (Figure 4A), respectively, the PDI values of polymers are 0.47 and 0.34 for poly(QVP-co-OEGMA) and PAA (Figure 4B), respectively. These 2 polymers were mixed in varying [NR+4]/[COO−] ratios. In [NR+4]/[COO−] ratios of 0.1 and 0.5, hydrodynamic diameters close to 25 nm are larger than both poly(QVPco-OEGMA) and PAA, which reveals PEC formation with the help of intermolecular forces between oppositely charged polymers. In addition, PDI values of complexes prepared at [NR+4]/[COO−] = 0.1 and 0.5 ratios are lower than 0.3, which reveals a monodisperse particle formation [28] by mixing polydisperse polyelectrolytes. Poly(QVP-co-OEGMA) and PAA form PEC particles by binding together with electrostatic attractions.</p><!><p>Hydrodynamic diameters (by volume) (A) and PDI values (B) of poly(QVP-co-OEGMA), PAA, and complexes of these polymers prepared in varying [NR+4]/[COO-] ratios.</p><!><p>Increasing the [NR+4]/[COO−] ratio to 1 caused the formation of larger PEC particles and turbidity in the solution, which can be a result of the neutralization of most of the charged groups with an oppositely charged group. This can lead to the formation of more hydrophobic structures and aggregation of PEC complexes together to form larger particles. In Figure 4A, while the diameter of the complex is given as 66 nm, there are also particles with a diameter of 516 nm. Relative abundances of these particles are 50.1% (66 nm) and 45.6% (516 nm), and the remaining particles are much larger aggregates (>5 μm). The PDI value of the complex at [NR+4]/[COO−] = 1 ratio is above 0.6, which exhibits the formation of random heterogeneous aggregates. The turbidity in the solution, large diameters of particles, and high PDI value observed at the ratio of [NR+4]/[COO−] = 1 reveal that a complex coacervation occurs at this ratio of polyelectrolytes. It is known that complex coacervation depends on the extended interactions between soluble complexes and coacervation tends to be maximized when the ratio of opposite charges in polyelectrolytes is close to 1 [29].</p><p>When the ratio was increased to [NR+4]/[COO−] ratios greater than 1, smaller PEC particles were formed with hydrodynamic diameters between 15–20 nm and PDI values between 0.2 and 0.39. All formed PEC particles, except the one with [NR+4]/[COO−] = 1, can have free charged chains not interacting with the oppositely charged groups and these free charged groups on PEC particles can repel other PEC particles and prevent the formation of larger aggregates. However, it is important to indicate that diameters and PDI values of PECs exhibit opposite trends above the ratio of [NR+4]/[COO−] = 1. Both the diameter and PDI values of complexes approach the values of free poly(QVP-co-OEGMA) as the ratio of components is increased above [NR+4]/[COO−] = 1. It can be said that the PEC exhibits characteristics of free poly(QVP-co-OEGMA) at very high [NR+4]/[COO−] ratios due to the fact that most parts of the polycation chain are free and not interacting with PAA.</p><p>Figure 5 shows the zeta potentials of individual polymers and the PECs formed by mixing oppositely charged polymers in different [NR+4]/[COO−] ratios. As expected, while the zeta potential of PAA is –4 mV, poly(QVP-co-OEGMA) has a zeta potential of +11 mV. The difference in absolute values (4 and 11) of the zeta potentials of PAA and poly(QVP-co-OEGMA) is the result of different molecular weights of the polymers and the number of charged groups on the related polymer chains. PECs with [NR+4]/[COO−] ratios lower than 1 have negative zeta potentials because of the free carboxylic acid groups in noninteracting PAA chains due to an insufficient number of positively charged QVP groups.</p><!><p>Zeta potentials of poly(QVP-co-OEGMA), PAA, and complexes of these polymers prepared in varying [NR+4]/[COO-] ratios.</p><!><p>The PEC of [NR+4]/[COO−] = 1 has a zeta potential of –14.7 mV, which reveals that free PAA chains are available on the formed PEC particles. However, in this [NR+4]/[COO−] ratio, the numbers of oppositely charged groups are almost equal. Much bigger particles in solution refer to much more PAA and poly(QVP-co- OEGMA) that exist in a single PEC particle, and as a result, there can be a larger number of smaller free PAA fragments on these particles. In addition, these small PAA fragments on PEC particles do not have enough charge density to repel and form smaller PEC particles (Figure 6).</p><!><p>Schematic representation of formed PECs at different N/P ratios.</p><!><p>When the [NR+4]/[COO−] ratio was increased above 1, positively charged particles were obtained and the zeta potential value of PECs with [NR+4]/[COO−] ratios of 2 to 50 were between +10.5 and +15.8 mV. These values show the free poly(QVP-co-OEGMA) chains on the formed PEC particles that do not interact with PAA chains and repel other PEC particles to prevent the formation of larger aggregates.</p><p>Above and below [NR+4]/[COO−] = 1, oppositely charged groups of each polymer attract each other and form PEC particles. However, since the number of oppositely charged groups is not equal, there are noninteracting free charged groups in the PEC structure that repel other PEC particles.</p><p>At [NR+4]/[COO−] = 1, since there are almost equal numbers of oppositely charged groups, there is a stronger attraction between PAA and poly(QVP-co-OEGMA). In this ratio, formed PEC particles will be less charged and this may cause the particles to coalesce to form larger PEC particles, as observed in the size distribution. When less charged particles merge together, the charge of the final particle will be related to the sum of all these small numbers of charges. Here, it is significant to mention that environmental conditions, especially the ionic strength and pH, can directly affect the final PEC structure and zeta potential.</p><p>By changing the concentration of PAA and keeping the concentration of poly(QVP-co-OEGMA) constant, a set of PEC particles were obtained in a close range of hydrodynamic diameters (except [NR+4]/[COO−] = 1) but with different charges. This allowed us to observe the effect of PEC particle's charge, size, and quantity of PAA on the antibacterial activity of poly(QVP-co-OEGMA). Table 1 gives MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of PAA, poly(QVP-co-OEGMA), and the PECs of these polymers against E. coli obtained from broth microdilution method. In Table 1, given MIC or MBC values are calculated from the concentration of poly(QVP-co-OEGMA).</p><!><p>MIC and MBC values of PAA, poly(QVP-co-OEGMA), and PECs prepared with [NR+4]/[COO-] ratios from 0.1 to 50, acquired from the broth microdilution method.</p><!><p>In broth microdilution assays, PAA did not show any antibacterial activity against E. coli in the given concentration range. On the other hand, poly(QVP-co-OEGMA) had MIC and MBC values of 125 μg/mL and 250 μg/mL, respectively, which confirms the antibacterial activity of the copolymer against E. coli .</p><p>When poly(QVP-co-OEGMA) was mixed with PAA in varying concentrations, the antibacterial activity of the copolymer changed with [NR+4]/[COO−] ratio (Figure 7). It is noteworthy that antibacterial activity was observed, although the PEC is negatively charged at [NR+4]/[COO−] ratios of 0.1 and 0.5. The mechanism behind this phenomenon can be explained by the fact that polyelectrolytes can migrate from one surface to another under certain conditions [30]. The dissociation of PECs by the migration of one of the components of PEC to another oppositely charged competitive polyelectrolyte was also studied in vivo by Mustafaev and coworkers [31]. In their studies, when a competitive polyanion was injected into mice just after the administration of an antigen containing PEC, PEC did not exhibit its function due to the dissociation of PEC by the migration of polycation of PEC to the competitive polyelectrolyte surface. Therefore, in broth medium, poly(QVP-co-OEGMA) can transit form PAA chains to a much larger (0.5–0.7 μm width and 1–3 μm length [32]) and negatively charged surface (–18.7 mV [33]) of E. coli and exhibit its antibacterial activity. The question arises when antibacterial activity of [NR+4]/[COO−] = 1 is not observed against E. coli . At [NR+4]/[COO−] = 1, more intermolecular attractions between PAA and poly(QVP-co-OEGMA) occur due to the almost stoichiometric complex formation. The higher number of intermolecular attractions causes stronger cooperative binding of polymers that prevented the transition of poly(QVP-co-OEGMA) to the bacterial cell membrane. At higher [NR+4]/[COO−] ratios from 2 to 50, formed PECs are positively charged and these positive charges are free quaternized 4VP groups that are not interacting with PAA due to insufficient number of acrylic acid groups. Therefore, the antibacterial activity of these complexes depends on these free groups and also on the migration of poly(QVP-co-OEGMA) to the bacterial surface. By increasing the [NR+4]/[COO−] ratio, the zeta potential of the complex becomes closer to free poly(QVP-co-OEGMA) and also the size of PEC becomes smaller, close to free poly(QVP-co-OEGMA) again. Therefore, PEC's characteristics become similar to free polycation, which causes a similar antibacterial activity.</p><!><p>MIC values of poly(QVP-co-OEGMA) and PECs depending on the [NR+4]/[COO-] ratio.</p><!><p>PEC formation and the ratio of components of formed PECs affected the antibacterial activity of poly(QVPco-OEGMA) against E. coli. It is significant that both negatively and positively charged PECs exhibited MIC values, which can be related to the transition of the cationic copolymer from PAA to the cell membrane of bacteria. Only the [NR+4]/[COO-] ratio of 1 inhibited the antibacterial activity of the copolymer, which can be due to an increased number of intermolecular electrostatic interactions between poly(QVP-co-OEGMA) and PAA, preventing the interaction between poly(QVP-co-OEGMA) and the cell membrane of E. coli.</p><p>It has been shown in previous studies that copolymers of QVP and OEGMA can be both antibacterial and biocompatible, which allows these polymers to be used in vivo. Therefore, PEC complexes of QVP-OEGMA copolymers can be used to designate supramolecular structures with augmented antibacterial activity by delivering active pharmaceuticals or to adjust the biological activity of copolymer for balancing the antibacterial activity and biocompatibility. Also, PECs formed between QVP-OEGMA copolymers and genetic materials, such as antisense oligonucleotides, can exhibit dual effects in cancer patients by treatment of cancer and prevention of infectious diseases.</p>

PubMed Open Access

Amelogenin and Enamel Biomimetics

Mature tooth enamel is acellular and does not regenerate itself. Developing technologies that rebuild tooth enamel and preserve tooth structure is therefore of great interest. Considering the importance of amelogenin protein in dental enamel formation, its ability to control apatite mineralization in vitro, and its potential to be applied in fabrication of future bio-inspired dental material this review focuses on two major subjects: amelogenin and enamel biomimetics. We review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets including other enamel proteins, apatite mineral, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human enamel.

amelogenin_and_enamel_biomimetics

1 Introduction<!>2.1 Amelogenin structural adaptability<!>2.2 Self-assembly of amelogenin<!>2.3 Amelogenin-mineral interactions<!>2.4 Amelogenin-enamelin interactions<!>2.5 Amelogenin-ameloblastin interactions<!>2.6 Amelogenin-phospholipid interactions<!>2.7 Amelogenin-MMP-20 interactions<!>3 Enamel and its biomimetics<!>3.1 Enamel microstructure and mechanical properties<!>3.2 Biomimetic systems for enamel reconstruction<!>3.2.1 Biomimetic systems based on calcium phosphate nanoparticles<!>3.2.2 Biomimetic systems based on peptides<!>3.2.3 Biomimetic systems based on amelogenin-inspired dendrimers<!>3.2.4 Amelogenin-containing hydrogels for human enamel regrowth<!>3.2.5 Other biomimetic systems<!>4 Concluding remarks and future challenges<!>

<p>The protein-mediated biomineralization in biological tissue is a great source of inspiration for the engineering of advanced materials.1–7 The biomaterials in mineralized tissues are generally optimized for their function through precise control over the structure, size, shape, and hierarchical assembly of the component parts and can be superior to many synthetic materials.3, 4 Enamel is a highly mineralized tissue that protects the mammalian tooth from external physical and chemical damage. Mature enamel is acellular and composed of 95–97% mineral by weight with less than 1% organic material.2, 8 The fluoridated carbonate-apatite nanocrystals found in enamel are tightly packed and arranged into an intricate interwoven structure. This organized hierarchical microstructure provides enamel with increased hardness and resistance to fracture compared to monolithic hydroxyapatite.9 The high degree of mineralization makes enamel a fascinating model for understanding fundamental mechanisms of protein-mediated mineralization, which could be utilized by scientists for development and design of biomimetic materials with potential for application in biomedicine, dentistry and industry.2</p><p>The enamel formation (amelogenesis) involves a series of highly regulated cellular activities and protein-controlled mineralization processes. The generally recognized stages of enamel development are the presecretory, secretory, transitional, and maturation stages which are defined by the morphology and function of ameloblasts.10, 11 The ameloblasts are a single cell layer that covers the developing enamel and is responsible for the enamel composition and hierarchical structure. The dynamic process of enamel biomineralization occurs in the extracellular space between the presecretory ameloblasts and the mineralized dentin.8 Enamel crystallites initiate at the dentino-enamel junction (DEJ) immediately following fenestration and removal of the basement membrane beneath fully differentiated pre-ameloblasts.12 After establishing the DEJ and mineralizing a thin layer of aprismatic enamel, ameloblasts develop a secretory specialization, or Tomes' process.12 The extracellular protein matrix is continuously secreted and processed in a stepwise and controlled manner during the secretory stage, when enamel crystals grow primarily in length and the enamel layer thickens. Once the full thickness of the enamel layer is achieved the transition and maturation stages begin. The protein matrix is proteolytically degraded during the maturation stage as crystals grow mainly in width and thickness, and is eventually removed from the extracellular space to allow completion of mineralization.13 These orchestrated cellular and biochemical activities result in the formation of a highly mineralized and hierarchically structured biological material.</p><p>The enamel matrix proteins play vital roles in the regulation of mineralization and crystal organization during enamel development. Amelogenin is the most abundant protein in the forming enamel, constituting more than 90% of the extracellular organic matrix.14 This protein is required for proper enamel development, as indicated by genetically engineered amelogenin-null mice, which display distinctly abnormal teeth with disorganized hypoplastic enamel.15 The amelogenin-null enamel phenotype reveals that amelogenin is essential for the organization of the prismatic pattern, control of crystal size and regulation of elongated crystal growth and enamel thickness.15, 16</p><p>Proposed molecular mechanisms for the amelogenin-regulated apatite mineralization in enamel formation include two different theories. The classical theory postulates that amelogenin and its isoforms bind specifically and selectively to the crystal sides, inhibiting ion deposition on these facets and permitting crystal growth only in length during the secretory stage and in thickness during the maturation stage.14, 17, 18 In the non-classical theory, a pathway of crystallization was suggested in which amelogenin interacts with noncrystalline calcium phosphate to form, stabilize and assemble intermediate pre-nucleation clusters that further transform into organized apatite crystals.19–21 In addition to amelogenin, lesser amounts of other enamel proteins, such as enamelin, ameloblastin and proteinases, have also shown to be critical for normal enamel formation. These enamel matrix proteins control the initiation, habit, orientation and organization of enamel crystals in a cooperative manner and then are gradually degraded and removed during enamel maturation.8</p><p>Mature enamel is acellular and does not resorb or remodel. As a result, following failure enamel regeneration cannot occur in vivo and is therefore an attractive target for future biomimetic therapeutic approaches. Understanding these protein-mediated enamel mineralization processes can provide the knowledge and scientific tools needed for the design of biomimetic systems for promotion of enamel regrowth in situ.</p><p>Numerous recent reviews and book chapters summarize the function of extracellular matrix proteins in the process of enamel formation.8, 22, 23 Excellent reviews of the historical and chronological advancement of amelogenin biochemistry and genetics are available.14, 24, 25 The concept of enamel biomimetics was introduced in 1997 5 and different aspects of enamel bio-inspired material synthesis have been reviewed by various investigators.2, 4, 26 Considering the importance of amelogenin in enamel formation, its ability to control apatite mineralization in vitro 18, 20, 21 and its potential to be applied in fabrication of future bio-inspired material,4 this review focuses on two major subjects: amelogenin and enamel biomimetics. We first review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets such as other enamel proteins, minerals, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human tooth enamel and the application of amelogenin protein in preparing such materials.</p><!><p>The most recent structural studies highlight a unique characteristic of amelogenin protein, namely the tendency to adopt a structure that fits its environment (Table 1, Fig. 1). The primary structure of amelogenin has a hydrophobic-hydrophilic polarity that might determine its ability to assemble in different modes depending upon the conditions surrounding the macromolecule.8, 14, 27, 28</p><p>The sequence of amelogenin is typically divided into three prominent amino acid domains: a hydrophobic tyrosine-rich N-terminal domain, called the tyrosine-rich amelogenin peptide (TRAP);29 the central proline-rich region, which is hydrophobic and primarily composed of X-Y-proline (where X and Y are often glutamine) repeat motifs; and the hydrophilic C-terminal domain. The N- and C-terminal region amino acid sequences are highly conserved among mammalian species, suggesting that these segments play important roles in enamel development and mineralization.30 In the following sections, we will demonstrate how amelogenin interacts with different targets mainly via these N- and C-termini (Table 1). The native amelogenin contains a single phosphate group on serine-16 that is presumed to be involved in amelogenin-calcium phosphate interactions and contribute to the ability of amelogenin to stabilize the precursor amorphous calcium phosphate.31</p><p>The primary sequence is enriched with disorder-promoting residues, such as Pro (P), Gln (Q), Glu (E), Arg (R), and Lys (K), leading to the intrinsically labile nature of the protein.32, 33 Bioinformatics and experimental studies in our laboratory have demonstrated that amelogenin belongs to the class of intrinsically disordered or unstructured proteins (IDPs).27 Unlike folded proteins, IDPs lack regular secondary or tertiary structure but are capable of transforming into a folded state following interactions with their targets and as part of their overall function.34 Structural studies through circular dichroism (CD) and solution NMR have revealed that recombinant porcine amelogenin (rP172) exists in an extended, unfolded state in the monomeric form under acidic aqueous conditions.27 Although the protein was reported to be globally unfolded, the presence of several short structure regions (α-helix, extended β-strand, turn/loop, and polyproline type II (PPII) conformation) was detected, hinting at the potential of this protein to recognize different interacting targets (Table 1, Fig. 1). Such interactions were proposed to serve several functional roles within the enamel extracellular matrix during the dynamic process of enamel biomineralization.27 For example, the unfolded N-terminal TRAP domain (self-assembly A domain) offers sufficient molecular contacts for assembly within the protein-protein interaction process.35, 36 The tri-tyrosine motif (PYPSYGYEPMGGW) in this N-terminal region has also been reported to have lectin-like properties (high affinity to N-acetyl D-Glugosamine), increasing the ability of amelogenin to interact with other enamel matrix glycoproteins or the cell surface glycocalyx.37, 38 The randomly coiled structure of the C-terminal domain would provide enhanced multiple charged contacts with the mineral surface.27 Regions close to the C-terminal have also been identified as being involved in amelogenin self-assembly (self-assembly B-domain).39</p><p>We have proposed that the extended and flexible structure is an important feature that facilitates the assembly of amelogenin into different quaternary structures as well as its interactions with other matrix proteins or biominerals.27 The labile conformation provides amelogenin with structural adaptability in response to various potential enamel matrix targets. Our most recent NMR study using "solvent engineering" techniques confirmed that both the N- and C- termini of amelogenin are conformationally responsive to the structure-inducing solvent 2,2,2-trifluoroehanol (TFE) and represent potential sites for amelogenin-target interaction during enamel matrix mineralization. As it is evident from the change in CD spectra TFE induced significant increase in alpha-helical content of recombinant amelogenin rP172 (Fig. 2).40 Based on the PPII propensity scale for individual amino acid residues, we previously reported that PPII is the dominant structure in the central region of amelogenin33 and our solvent engineering studies confirmed that the Pro, Gln central domain is resistant to folding.40 The rigidity in the central region and flexibility at the N- and C- termini may have important functional significance for amelogenin in vivo.40</p><p>To further evaluate structural adaptability of amelogenin we have recently analyzed the structural behavior of amelogenin in the presence of sodium dodecyl sulfate (SDS), which has the ability to mimic biological cell membranes by forming amphiphilic micelles.41 NMR spectroscopy and structural refinement calculations using CS-Rosetta modeling confirmed that the highly conserved N-terminal domain of amelogenin was prone to forming helical structure when bound to SDS micelles (Fig. 3). These findings revealed that significant changes in the secondary structure of amelogenin occurred upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence-specific helical propensity, which might enable it to structurally adapt when bound to targets.</p><!><p>The strong tendency of amelogenin to aggregate has been known since 1960's.42 We now have increasing evidence that amelogenin self-assembles into quaternary structures under a variety of conditions in vitro. These include nanospheres (Fig. 4A), nanochains (Fig. 4B), oligomers (Fig. 4C), microribbons and nanoribbons (Fig. 4D).43–46 Self-assembly of amelogenin is sensitive to pH, protein concentration, temperature and ionic strength,31 and also can be affected by the presence of proteases,47 other enamel proteins, calcium phosphate ions,45 solvent hydrophobicity and charged surfaces such as apatite minerals.48 A single phosphorylated serine present in the sequence might have a subtle influence on amelogenin self-assembly.49</p><p>Earlier studies showed that the full-length recombinant amelogenin molecules can spontaneously self-assemble into nanospheres under pH = 8 conditions in the absence of calcium-phosphate.36, 46, 50–53 We have suggested that the nanospheres were formed through intermolecular hydrophobic interactions when the hydrophilic segment of each molecule was exposed on the surface of the nanospheres.8 The nanospheres were then proposed to be the basic structural entities of the developing enamel extracellular matrix and to play a crucial function in enamel biomineralization.52</p><p>The original AFM images of amelogenin nanospheres were based on samples that were processed using Karnovsky fixative 46 to prevent them from collapsing or disintegrating (Fig. 4A). While the size distribution was heterogeneous at low protein concentrations, at higher concentrations (100–300 µg/ml) particles ranging from 10 to 25 nm in diameter were detected under the AFM. It is now acceptable that nanospheres are not rigid structures and can disassemble if adsorbed onto surfaces with different hydrophobicity or different charges.48 The formation of do-decamers as basic subunits of nanospheres at pH 8 was illustrated by single-particle reconstruction of cryo-TEM images (Fig. 4 C).44</p><p>Our analyses of the particle size distribution of amelogenin assemblies in solution have suggested the existence of substructures such as monomers and discrete oligomers prior to nanosphere formation in solution (Fig. 5).54 The formation of oligomers as the basic subunits of the typical nanospheres was also observed by transmission electron microscopy.43 Dissecting the nanospheres by reducing the pH to 3.5 or 5.5 allowed us to better understand the chemical interactions responsible for amelogenin assembly/aggregation in the absence of calcium phosphate ions. Partial deprotonation of histidine residues at pH 5.5 resulted in the formation of oligomers, via N-terminally mediated intermolecular interactions. At pH 8, when the histidine residues are completely deprotonated, hydrophobic forces are enhanced to bind the oligomers together in the form of a nanosphere. Our recent CD analysis has revealed that while oligomer formation accompanies conformational changes in amelogenin, little structural change occurs as a result of nanosphere formation.54 In their NMR studies, Lu et al. 55 reported a lack of conformational changes in amelogenin nanospheres during gel formation. We have reported that amelogenin oligomer formation in vitro is mediated by N-terminal protein-protein interactions and assembly, while nanosphere formation is mainly regulated by hydrophobic interactions via the histidine-rich central portion.54 The question is whether the amelogenin oligomers (RH = 7.5 nm) are the functional entities in enamel formation and that the nanospheres (RH = 14nm) are simply the result of aggregation of oligomers. Particles of 15–20 nm in diameter detected by TEM are the oligomeric structures that were detected in between enamel crystallites.52, 56 Oligomeric structures can also be stabilized under neutral conditions and in the presence of an acidic protein, namely 32 kD enamelin (section 2.4). Fluorescence experiments with single-tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp161) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp25 and Trp45 is involved in protein-protein interaction.</p><p>Amelogenin has shown a tendency to further assemble into higher-order structures in vitro. Nanochain assemblies of amelogenin were observed in a number of studies (Fig. 4B).20, 43, 57, 58 It was suggested that the nanochain structures were formed through further association of the amelogenin nanospheres (or association of amelogenin oligomers).43 The bipolar nature of the molecule can facilitate the formation and/or reorganization of the chain structures.43 Li et al.57 studied the kinetics of amelogenin nanochain formation via a Brownian dynamics simulation of both translational and rotational motions. Their simulations showed a hierarchical self-assembly process in which the molecules aggregate to form oligomers and nanospheres, and the assembly of the nanospheres then leads to the formation of nanochains, in agreement with experimental findings.</p><p>The idea that oligomeric, dimeric, or even monomeric structures might be functional components during enamel formation was proposed by Shaw et al.,59 who demonstrated that nanospheres can disassemble following their adsorption onto surfaces. Chen et al. 48 then reported that the self-assembly of amelogenin can be dramatically different on substrates and depends upon the charge of the interacting surfaces. These studies have important implications because amelogenin may be in contact with charged surfaces such as mineral and cell membranes in vivo.</p><p>As opposed to the previously proposed nanochain model, He et al.58 reported the formation of amelogenin nanoribbons in an emulsion oil-water system. They proposed an alternative model for the protein-mediated enamel biomineralization based on parallel alignment and extension of ribbons. Nanoribbons also form in the presence of calcium phosphate under low pH conditions (pH 4–6) and stay stable for three days when exposed to neutral pH (Fig. 4D).45, 58, 60 Histidine protonation was proposed to play an important role in providing the binding sites for phosphate bridging and formation of elongated dimers.45</p><p>While the ability of amelogenin to self-assemble to a variety of quaternary structures has been well documented in vitro, a clear model for in vivo functional entities of amelogenin is still lacking and requires further investigation. It is noteworthy that amelogenin molecules do not occur in isolation in vivo, so the presence of other components such as mineral, other proteins, cell surface and lipoid particles needs to be considered.</p><!><p>Experimental evidence that amelogenin interacts with apatite crystals was documented in studies of seeded crystal growth and analytical evaluation of adsorption affinity.61–63 Removal of the hydrophilic C-terminal significantly decreased amelogenin affinity to apatite crystals.64, 65 Direct evidence for the orientation of the charged C-terminal region near the apatite crystal surface was provided by solid state NMR and neutron scattering as well as computational methods.66, 67 The C-terminal portion has a significantly higher affinity for binding to the (100) side face of apatite compared to the cross section (001) face, a finding that might explain the c-axial elongated growth of enamel apatite crystals.68 Amelogenin can also interact with the apatite surface through the N-terminal region, as isolated peptides from the N-terminal were detected to be bound after proteolytic digestion of full-length amelogenin in the presence of apatite.69 Although serine phosphorylation is not required for the binding of amelogenin LRAP (Leucine Rich Amelogenin Polypeptide) to apatite, the N-terminal binding was stronger when the 16Ser was phosphorylated.70–72 Lu et al.55 found that regions of amelogenin that appear to be primarily random coils in the nanosphere-gel adopt a β-strand structure and are less mobile after HAP binding, indicative of a structural switch upon binding that may be important to the role of amelogenin in enamel development (Table 1). The binding of LRAP to apatite promotes folding of domains at both the C- and N-terminal regions, promoting a conformational transition from random coil to extended beta strand at the C-terminal, and partial alpha helix at the N-terminal.55, 70, 73 LRAP-bound apatite is mostly extended, thereby covering the apatite surface efficiently (Fig. 6).</p><p>Specific roles for amelogenin in nucleation, growth, regulation of crystal size and shape, and control of crystal-crystal aggregation have been proposed.26, 74–76 Studies of the kinetics of crystal nucleation in situ and in real time using a quartz crystal microbalance (QCM) showed that recombinant amelogenin reduced the induction time for nucleation compared to solutions without protein.74 Wang et al. 76 reported that amelogenin dramatically accelerates the nucleation kinetics by decreasing the induction time in a dose-dependent manner in a controlled constant-composition in vitro crystallization system. An interfacial structural match between amelogenin assemblies (mainly oligomers) and Ca-P nanoclusters was proposed. Considering the ability of amelogenin assemblies to reduce the interfacial energy needed for nucleation of hydroxyapatite, it is not surprising that apatite nucleation is promoted by this protein in in vitro model systems. However, there is no direct evidence that enamel mineralization occurs in vivo via amelogenin-mediated heterogeneous nucleation. In particular, the observation that the enamel mineralization still occurs in amelogenin null mice does not support a direct nucleating function for amelogenin in vivo.15</p><p>The classical theory of crystallization in amelogenesis postulates that extracellular matrix proteins shape crystallites by specifically inhibiting ion deposition on the crystal sides, orient them by binding multiple crystallites and establish higher levels of crystal organization. Numerous in vitro experiments have been conducted to support the classical theory and to investigate the amelogenin-crystal interaction.17, 18, 77–82 These studies suggested that amelogenin specifically and selectively adsorbs to crystal faces to direct growth only in the c-axis direction. For example, amelogenin was found to interact most strongly with the (010) face, followed by the (001) and then (100) faces of OCP, explaining the elongated growth and increase of the thickness-to-width ratio. Nevertheless, it is still questionable whether the classical theory can explain how enamel forms naturally.83</p><p>In the last decade an increasing number of studies have proposed a non-classical pathway of crystallization for enamel mineralization, which involves a co-assembly of amelogenin and the transient mineral phases.20 The presence of transient mineral phases in developing enamel has been suggested in several in vivo studies.84–86 Beniash et al. 19 characterized the forming enamel mineral during the early secretory stage as amorphous calcium phosphate, which eventually transforms into apatite crystals. It has been further suggested that the mineral morphology and organization in enamel are determined prior to its crystallization.19 While this might be a reasonable explanation for the early stage of enamel crystal formation, the eventual ribbon-shaped morphology of enamel crystals cannot be easily explained due to the dynamic processing of enamel matrix during the maturation stage when the crystals mainly grow in thickness and width.</p><p>It is speculated that amelogenin transiently stabilizes amorphous calcium phosphate (ACP) and regulates the formation of parallel arrays of mineral crystals. The native phosphorylated amelogenin has been found to stabilize ACP for extended periods of time. By investigating the amelogenin-mediated crystallization in a constant composition (CC) crystallization system, Yang et al. 21 suggested a nucleation model in which amelogenin was proposed to stabilize the pre-nucleation Ca-P clusters and mediate their aggregation to form the oriented and elongated organized crystals (Fig. 7). In this model, two stages are proposed in the process of amelogenin-mediated apatite mineralization: (i) controlled aggregation of the Ca-P nanoclusters and (ii) organized postnucleation crystal growth involving a stepwise hierarchical co-assembly of Amel−Ca−P nanoclusters. Hierarchical co-assembly of Amel−ACP particles gives rise to a remarkably high degree of cooperativity at low driving force. Under cooperative kinetic control, the co-assembly of Amel−Ca−P clusters plays an explicit role in directing Amel/ACP nanoparticles toward the final elongated crystalline structure.21</p><p>The formation of the primary building blocks (nanocluster composites) probably imparts kinetic and thermodynamic stability to the system, which may lower the free-energy barrier to formation of secondary structures of intermediate phases (Amel/ACP nanosphere chains and nanorods), which ultimately undergo phase transformation to form the final crystalline phase of mature enamel (elongated HAP). Numerous in vitro experiments provide evidence for the function of amelogenin in the non-classical crystallization of enamel crystals;44 however, additional studies are needed to identify the pre-nucleation clusters in vivo and clarify their role in enamel mineralization.</p><!><p>Another potential target for amelogenin is the least abundant (3–5%) acidic phosphorylated glycoprotein enamelin. It is believed that interactions between amelogenin and enamelin play a vital role in controlling enamel crystal formation.8 The importance of enamelin in dental enamel formation has been demonstrated unequivocally by various in vivo studies.87–90 In enamelin null mice, a true enamel layer was not formed.12, 89, 90 Among the different enamelin cleavage products, the 32 kDa enamelin is the most stable fragment and is highly conserved among species, suggesting that it plays a critical functional role in enamel formation.91, 92</p><p>Enamelin in cooperation with amelogenin promoted the kinetics of apatite nucleation in a dose-dependent manner.93 We have also demonstrated the cooperative regulatory action of the 32 kDa enamelin and amelogenin on the growth morphology of octacalcium phosphate (OCP) crystals.94, 95 Adding enamelin to the amelogenin "gel-like matrix" resulted in an obvious increase in the length-to-width ratio (aspect ratio) of OCP crystals in a dose-dependent manner. Moreover, the presence of enamelin in the amelogenin matrix enhanced the stability of the transient amorphous calcium phosphate (ACP) phase.94 It was proposed that the cooperative effect of enamelin and amelogenin was attained through co-assembling of enamelin and amelogenin.</p><p>Enamelin has been shown to interact directly with amelogenin, changing its conformation (Fig. 8), stabilizing the oligomers, and partially dissociating amelogenin nanospheres.96,97 The 32 kDa enamelin has the potential to interact with both full-length and truncated amelogenin lacking the C-terminal through the tyrosyl motif at the N-terminal.</p><p>We recently analyzed the co-localization between enamelin and amelogenin in postnatal day 1–8 mandibular mouse molars using dual-color confocal microscopy.98 The results showed that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion of both proteins continues to at least day 8. At day 8 enamelin and amelogenin co-localize near the secretory face of the ameloblasts. The degree of co-localization decreases as the enamel matures, both along the secretory faces of the ameloblasts and throughout the entire thickness of the enamel. The finding that enamelin and amelogenin co-localize in vivo further supports our hypothesis that they cooperate to control crystal formation, particularly at the beginning of enamel formation.</p><!><p>Ameloblastin is considered critical for proper enamel formation because a true enamel layer fails to appear on the teeth of ameloblastin mutant mice.99–101.</p><p>A recent study showed that amelogenin-ameloblastin double knock-out mice have additional enamel defects not observed in either amelogenin knock-out or ameloblastin knock-out mice,102 lending support to the notion that amelogenin and ameloblastin interact and have synergistic roles in enamel development. The co-distribution of amelogenin and ameloblastin in the majority of the secretory granules in Tomes' processes during appositional growth of the enamel layer may reflect a form of functional association between these two distinct proteins.101 Ameloblastin is therefore another potential target for amelogenin.</p><p>Ameloblastin is the second most abundant enamel matrix protein after amelogenin.91, 103, 104 It is secreted together with amelogenin and rapidly processed after secretion. The hydrophobic N-terminal cleavage products accumulate in the "sheath" space throughout the enamel layer while the calcium-binding C-terminal cleavage products are on the rods and are not detectable beyond a depth of 50 µm from the surface of the newly formed enamel.13, 105</p><p>The first in vitro evidence of interactions between amelogenin and ameloblastin was provided a decade ago and revealed that these interactions may occur via the lectin-like binding domain of amelogenin.106 In our most recent CD spectra analysis of ameloblastin in the presence of equimolar amelogenin we demonstrated that amelogenin induced changes in the secondary structure of ameloblastin, increasing alpha helical context (Fig. 9). The structural changes in ameloblastin at this low amelogenin concentration was a strong support for the notion that the proteins formed hetero-assemblies.</p><p>In vivo evidence of co-localization of ameloblastin with amelogenin was provided using immune histochemical methods.107 Quantitative co-localization analysis along the secretory faces of ameloblasts using antibodies against the N-terminal and C-terminal of ameloblastin revealed that at day 1, very high percentages of both the ameloblastin and amelogenin co-localized. Analysis of the entire thickness on day 8 revealed no significant co-localization of amelogenin with the C-terminal of ameloblastin in the bulk of enamel, but a low level of co-localization was detected with the N-terminal of ameloblastin. With the progress of amelogenesis and as ameloblastin and amelogenin degradation progressed, co-localization pattern changed as following: (i) there was a segregation in distribution of ameloblastin C- and N-terminal, (ii) co-localization of amelogenin with the C-terminal of ameloblastin decreases while co-localization of amelogenin with the ameloblastin N-terminal did not change. Amelogenin and N-terminal ameloblastin co-localized in the "sheath" space.107 (P.Mazumder, S.Parapajari and J.Moradian-Oldak, in preparation.) Our data suggest that amelogenin-ameloblastin complexes may be the functional entities not only at the early stage of enamel mineralization but also later during maturation.</p><!><p>We recently explored interactions between amelogenin and liposomes in order to shed light on the mechanisms of amelogenin-cell interactions during amelogenesis. Amelogenin proteins are synthesized by the ameloblasts and secreted via matrix secretory vesicles. Moreover, interactions between enamel extracellular matrix components and ameloblasts might be of great importance for polarization, differentiation or migration of ameloblasts during the dynamic process of amelogenesis.108</p><p>We applied fluorescence spectroscopy, CD, NMR and DLS to investigate binding between recombinant amelogenin rP172 with negatively-charged (POPG)1 and zwitterionic (POPC)2 small unilamellar vesicles as model membranes. We prepared a mixture of different lipids to mimic the apparent lipid composition of the ameloblast membrane, called ACML3. We demonstrated that amelogenin has the ability to interact with zwitterionic and negatively charged liposomes via electrostatic as well as hydrophobic interaction.109</p><p>Adding negatively-charged small unilamellar vesicles to monomeric amelogenin at pH 3.5 resulted in greater burial of the Trp residues of rP172, and the hydrophobic membrane environment of the phospholipids induced a structural transition of rP172 from random coil to alpha helix. NMR studies revealed conformational changes and alterations in backbone dynamics within the amelogenin molecule, and suggested that such changes may be concentrated at the N- and C-termini (Table 1).</p><p>Under more physiologically relevant pH conditions (pH 8.0), where amelogenin forms nanospheres, the wavelength of the maximal intrinsic fluorescence emission is 15 nm shorter than at pH 3.5 because the Trp residues are in a more hydrophobic environment. At pH 8.00, amelogenin interactions with negatively charged lipid vesicles were weak and did not show a blue shift in the fluorescence spectra. However, quenching experiments and drawing the Stern-Volmer plots indicated that rP172 interacted with lipid vesicles. Interestingly, the ANS fluorescence decreased upon interaction of rP172 with anionic lipid vesicles, confirming that nanospheres disassembled upon interaction with the lipids. A systematic analysis of four mutant amelogenins, in each of which only one Trp residue was present and behaved precisely in the same way as the wild-type, we were able to show that amelogenin possesses membrane-binding ability mainly via its N-terminal close to residues W25 and W45 (S. Bekshe Lokappa, K. Balakrishna Chandrababu, and J. Moradian-Oldak, in preparation). A disordered-to-ordered conformational change was observed based on CD and NMR studies (Table 1).</p><!><p>During enamel formation, amelogenin and other enamel proteins are cleaved by proteinases after they are secreted and further degraded during the early maturation phase, allowing the enamel layer to achieve a high degree of mineralization.13 Two major proteinases, matrix metalloproteinase-20 (MMP-20, also known as enamelysin) and serine proteinase kallikrein-4 (KLK-4), have been described. Generally, during the secretory stage, MMP-20 cleaves the amelogenin and other proteins into a number of stable intermediate products, while during the maturation stage, KLK-4 degrades and eventually removes the protein matrix in a specific and timely manner.</p><p>The full-length amelogenin is first cleaved by MMP-20 at the hydrophilic C-terminus, followed by the N-terminus. The interaction of amelogenin with MMP-20 is of a particular interest as the most prominent interacting domains are gradually removed by MMP-20. The action of MMP-20 on amelogenin therefore can affect its interactions with all of the aforementioned targets (sections 2.2–2.6).</p><p>In light of these amelogenin-MMP-20 interactions, MMP-20 was proposed to: (i) control amelogenin self-assembly, (ii) decrease amelogenin-apatite binding affinity, (iii) control ACP to apatite phase transformation by amelogenin,110 and/or (iv) prevent unwanted protein occlusion inside apatite crystals.111</p><p>It has been proposed that control of the protein self-assembly process by MMP-20 allows the programmed and elongated growth of apatite crystals in a hierarchically organized manner. Nanorod structures can be formed in vitro by means of co-assembly of amelogenin and its cleavage products during a comparatively slow proteolysis process.47 The proteolytic activities of MMP-20 also affect amelogenin-apatite interactions by producing intermediate products that have less affinity for apatite and can affect OCP crystal morphology in different ways.17, 65, 69</p><p>The idea that MMP-20 prevents unwanted amelogenin occlusion inside growing crystals was recently tested utilizing calcite as a mineral system.111 It was found that recombinant porcine amelogenin (rP172) could alter the shape of calcite crystals and became occluded inside the crystals. In contrast, the occlusion of amelogenin into the calcite crystals was drastically decreased in rP172-rhMMP-20 samples. Truncated amelogenin lacking the hydrophilic C-terminal and the 25-residue C-terminal domain alone produced crystals with regular shapes and less occluded organic material. Based on these in vitro observations, we suggested that removal of the C-terminus by MMP-20 diminishes the affinity of amelogenin to the crystals, and therefore prevents occlusion of amelogenin into them. The concept that MMP-20 prevents occlusion of amelogenin in calcium phosphate crystals was been examined and demonstrated using brushite crystals as a model system (Ren et al., in preparation). Systematic analysis of occluded proteins in MMP-20 knockout and wild-type mice is currently ongoing to unequivocally confirm this hypothesis (Parjapati et al., in preparation).</p><!><p>Dental enamel is a masterpiece among bioceramics and the hardest material found in mammals. The unique mechanical properties of enamel enable it to perform the functions of incision, laceration, and grinding of food during mastication.112 It also faces the lifelong challenge of maintaining robust mechanical performance in a bacteria-filled environment.2 Additionally, mature enamel is acellular and does not regenerate after substantial mineral loss, which often occurs as dental caries or erosion as well as due to congenital malformation, trauma or mastication.</p><p>The established way to treat initial carious lesions and submicrometer erosive is the application of remineralizing agents. Oral healthcare products containing fluoride or casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) are effective in re-mineralizing enamel but none of these commercially available products have the potential to promote the formation of organized apatite crystals.113, 114 The conventional treatments for deep enamel cavities involve mechanical drilling and subsequent filling with artificial materials such as amalgam, ceramic or composite resins. However, even after those treatments secondary caries often arises at the interface between the original enamel and the filling materials due to weakening adhesion over time. As a result, a synthetic enamel-like material with a robust adhesion to the enamel is an attractive target for future biomimetic and therapeutic approaches.</p><p>Biomimetic strategy for enamel repair may offer an ideal solution when organized enamel apatite crystals with robust attachment to the enamel surface can be grown.115 Such strategy will lead to development of a strong material and will eliminate the problem of secondary caries. As discussed in the previous section, amelogenin plays a critical role in enamel formation and has a great potential to develop the biomimetic systems for enamel repair. Therefore, following a brief description of enamel microstructure and mechanical properties, this section of the review focuses on the biomimetic reconstruction of human tooth enamel with an emphasis on the amelogenin-containing system.</p><!><p>It is generally recognized that the mechanical response of enamel depends upon its unique architecture and mineral/organic composition. Understanding the enamel microstructure and associated mechanical properties could therefore motivate engineering of more robust dental materials as well as inspire fabrication of non-biological materials.</p><p>Enamel contains structures at different hierarchical levels from the nanoscale to microscale (Fig.10).2, 116 On the nanoscale level, the basic elements of mature enamel are highly organized, hydroxyapatite crystallites that are parallel to their c-axis with dimensions of 50–70 nm in width, 20–35 nm thickness and with an aspect ratio greater than 1000 (Fig. 10A–C).8 The thickness of the enamel crystallites increases from the DEJ towards the outer layer.117, 118 On the microscale, these crystallites are grouped into more complex, micrometer-sized structures known as rods (prisms) and interrods (interprismatic substance), which are regarded as the fundamental organizational units of mammalian enamel (Fig. 10D). At the boundary between the rod and interrod enamel is a narrow space containing organic material so-called "rod sheath" material. The sizes of the rods and interrods vary depending on the depth of the enamel. It has been reported that the outer surface of the enamel has smaller rods (∼3 um) and wider interrod regions (∼2 um).119 On a higher level, the rods and interrods further assemble into a distinct structural pattern (Fig. 10E), which presents differing arrangements across the thickness of the enamel layer in the enamel. In a superlayer of human molar enamel, the rods are oriented radially and intercept the occlusal surface perpendicularly.120 In the inner two-thirds of the enamel, the enamel rods deviate from the long axis in an undulating or weaving pattern, which is generally referred to as "decussation".121</p><p>The complex hierarchical microstructure is believed to be a key factor responsible for the unique anisotropic mechanical properties of enamel.9, 112, 122–126 Because of the changing arrangements of the enamel rods, the hardness and elastic modulus decrease gradually from the occlusal surface of the enamel to the DEJ. Besides distinct declines in hardness and modulus upon moving away from the occlusal surface, the mechanical properties of the enamel also differ from the lingual to the buccal side of the molar.127</p><p>Besides the enamel structure, the chemical composition also plays an important role in determining the mechanical behavior of enamel. Xie et al. 128 found that an increase of protein content is the primary factor that causes the deterioration of stiffness or elastic modulus of hypomineralized enamel. Due to their higher protein content, the nanohardness and elastic modulus of the tested enamel sheaths were about 73.6 % and 52.7 % lower than those of the enamel rods, respectively.129 The organic matter in the rod sheaths contributes significantly to the strengthening and toughening of the enamel, which is reflected by increasing crack growth resistance with crack extension from the outer to the inner enamel.119, 123, 130 In addition, it was also suggested that the mechanical properties of enamel were also dependent on its magnesium (Mg), sodium (Na) and carbonate (CO32-) contents.125, 127</p><!><p>In the last few decades, various biomimetic systems have been developed for the synthesis of biomaterials with the enamel-like structure at the nanoscale level (Table 2). Here, we review progress in the development of biomimetic systems for enamel restoration containing calcium phosphate nanoparticles, peptide, amelogenin-inspired polymers, and other organic additives. We will summarize our latest efforts to utilize amelogenin in the biomimetic reconstruction of enamel.</p><!><p>In recent years, biomimetic treatment of caries lesions by the application of calcium phosphate (CaP) materials has received considerable attention. Various types of biomimetic systems containing nanoparticles of amorphous calcium phosphate (ACP) or hydroxyapatite (HAP) have been developed for enamel regrowth.131–138</p><p>ACP has been proposed to be an essential precursor phase during the formation of mineralized tissue. The unique role of ACP makes it a potential remineralizing agent for the preservation and repair of tooth structures.139,140 The effectiveness of a nanocomposite containing nanoparticles of amorphous calcium phosphate (NACP) on enamel remineralization was evaluated in vitro.131 Quantitative microradiography showed that the NACP nanocomposite promoted significantly more enamel remineralization (21.8 ± 3.7%) than a fluoride-releasing composite control (5.7 ± 6.9%). This result indicated the ability of ACP nanocomposite in the remineralization of demineralized tooth structures; however, the metastable nature of ACP tremendously limits its application in clinic. ACP is more soluble than the crystalline polymorphs of calcium phosphate, so it readily converts to HAP in aqueous solution. To tackle this limitation, several systems were developed to stabilize and carry the ACP for enamel repair. For example, phosphorylated chitosan was used to stabilize ACP in a calcification solution to remineralize enamel subsurface lesions.132 An electrospun hydrogel mat of ACP/PVP (poly(vinylpyrrolidone)) nanofibers was also developed for the in vitro remineralization of dental enamel.133 The application of the ACP/PVP hydrogel mat resulted in transformation of the spherical ACP phase in situ at the surface of the enamel to produce a contiguous overlayer of crystalline fluoridated hydroxyapatite with a approximately 500 nm thickness. While the lesions were remineralized, no enamel-like structure formed on the remineralized enamel surface. Instead, the surface was textured with filament-like structures approximately 1 µm in length, along with small spherical particles around 250 nm in diameter (Fig. 11A). These in vitro studies demonstrate the potential of ACP-based materials in the repair and prevention of initial enamel lesions; however, these effects have not yet been confirmed in a clinical trial.</p><p>Beside ACP, synthetic apatite is also considered as a promising agent for biomimetic regrowth of human enamel because of the chemical similarity to tooth enamel. Yamagishi et al. 134, 135 have prepared a white crystalline paste of modified hydroxyapatite (HAP), which chemically and structurally resembles natural enamel, and used it to repair an early caries lesion in a lower premolar tooth.134,135 The artificially formed enamel-like layer was about 10 µm thick and was formed seamlessly on the enamel within 15 minutes. Unfortunately, the paste is highly acidic (pH 3.5) and contains high concentrations of hydrogen peroxide. In another biomimetic approach, nano-sized HAP particles were used to repair initial submicrometer enamel erosions.136 It was suggested that repair at the enamel surface could be greatly improved if the size of the apatite particles are adapted to the scale of the nano-defects caused by erosive demineralization of the natural apatite crystallites. In vitro experimental results revealed that the HAP with a size of 20 nm adsorbed strongly to the enamel surface and further reinforced the acid-etched enamel. In contrast, these outcomes have not been observed when the large-sized HAP (> 100 nm) and ACP are applied to the erosive enamel surface. Interestingly, a repaired layer with enamel-like structure was formed under physiological conditions when glumatic acid (Glu) was introduced in this system (Fig. 11B).137 It was proposed that the nano-apatite particles absorbed onto the enamel substrate were the building blocks, while the Glu selectively adsorbed onto the apatite (001) faces and induced oriented aggregation using the end carboxylate groups.</p><!><p>Inspired by the functions of proteins in tooth formation, various peptides have been synthesized to repair enamel defects. For instant, an anionic peptide (P11-4, Ace-Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Gln-Gln-NH2) was synthesized and shown a potential to introduce apatite mineralization to caries-like lesions in human dental enamel.141 Recently, Li et al.142 fabricated an anionic oligopeptide amphiphile (OPA, C18H35O-Thr-Lys-Arg-Glu-Glu-Val-Asp) that contains the hydrophilic functional domain of amelogenin to initialize hydroxyapatite nucleation and promote biomimetic mineralization of demineralized enamel. It was shown that apatite crystals were formed on the etched enamel after treated with OPA peptide.</p><p>Some other researchers have focused on biomimetic approaches for enamel remineralization based on the peptide derived from dentin phosphoprotein (DPP),141, 143–148 which is the most abundant non-collagenous extracellular matrix protein in dentin. Human DPP contains numerous repetitive nucleotide sequences of aspartate-serine-serine (DSS) that are believed to promote the formation of hydroxyapatite. Several small peptides consisting of multiple repeats of the tripeptide DSS have been designed based on the DPP sequence.143 Of the multiple-DSS peptides tested so far, a peptide carrying 8 repeats (8DSS) has been shown to promote mineral deposition onto human enamel and improve the surface properties of demineralized enamel in in vitro studies.144 Mineral loss after 12 days of pH cycling was significantly lower in samples treated with 8DSS than in the control buffer-only samples, and lesions in the 8DSS samples were significantly less deep. In another study, samples treated with 8DSS had significantly higher mineral content than buffer-only samples in the region extending from the surface layer (30 µm) to the average lesion depth (110 µm).145 Moreover, high-magnification SEM revealed a definitive change in surface morphology, from elongated hydroxyapatite nanorods in the demineralized enamel to nanoscale flakes.144 (Fig. 11C)</p><!><p>Poly(amido amine) (PAMAM) dendrimers have been used as "artificial proteins" and investigated as a biomineralized material, especially in the crystallization process of HAP. It has been reported that PAMAM-type dentrimers or dendrons have a self-assembly behavior similar to that of amelogenin. For example, an amphiphilic PAMAM dendron was observed to initially self-assemble into nanospheres and further translated to linear chains in aqueous solution.149 In another study, Yang et al. 150 demonstrated that carboxyl terminated PAMAM dendrimers had a strong tendency to self-assemble into hierarchical structures with the morphology of nanospheres, subsequent nanochains and microfibers, and finally macroscopic aggregates consisting of microribbons, which is similar to that of amelogenin. Furthermore, these dendrimer assemblies exhibited a function similar to amelogenin in controlling the oriented growth of HAP.149 It was found that the apatite crystals formed in the presence of the linear assemblies resembled some of the features of the lowest level of the hierarchical structure of enamel, such as the preferential orientation of the c-axis of the HAP crystals along the amelogenin aggregates (Fig. 10).</p><p>Accordingly, several PAMAM-based dendrimers have been synthesized as the amelogenin analogs on the remineralization process of acid-etched human tooth enamel.151–153 Wu et al.152 shown that alendronate-conjugated PAMAM dendrimer (ALN-PAMAM-COOH) could induce in situ remineralization of tooth enamel, attributed to the combined effect of the HA-anchored property of the ALN moiety and the remineralization capability of the –COOH moiety. In addition, the newly formed crystals had nanorod-like structure similar to that of human tooth enamel (Fig. 11D). Most recently, a phosphate-terminated dendrimer (PAMAM-PO3H2) was synthesized and assessed for the ability to remineralize acid-etched human tooth enamel.153 After being incubated in artificial saliva for 3 weeks, a newly generated HAP layer of 11.23 µm thickness was found on acid-etched tooth enamel treated with PAMAM-PO3H2.</p><!><p>A very promising route to achieve oriented enamel-like materials would be the in situ remineralization of enamel in the presence of amelogenin and other enamel matrix proteins.8 We have used several strategies to prepare enamel-like materials that contain nano- and microstructures using amelogenin to control the crystallization of biomimetic calcium and phosphate.8, 79, 154–157 For example, using an electrolytic deposition (ELD) technique, we have synthesized an enamel-mimicking composite coating from a mineralization solution containing soluble recombinant amelogenin proteins at near-physiological pH and ionic strength.154 A modified biomimetic approach in the presence of mineralization-modulating amelogenin was implemented to rebuild enamel structure on an acid-etched enamel surface as a model for demineralized enamel.155 In another study, an amelogenin-releasing agar hydrogel containing calcium, phosphate, and fluoride was prepared to remineralize etched enamel in a cyclic treatment model and multispecies oral biofilm model.156 Repetitive application of this hydrogel significantly improved enamel hardness continuously over time. These results have opened up the promising possibility of remodeling complex enamel minerals in an amelogenin-containing system.</p><p>Most recently, taking advantage of the potential of amelogenin to control the organized growth of apatite crystals and the potential antimicrobial activity of chitosan, we have developed a new amelogenin-containing chitosan (CS-AMEL) hydrogel for superficial enamel reconstruction.158–160 It was suggested that amelogenin assemblies carried in chitosan hydrogel could stabilize Ca-P clusters and arrange them into linear chains, which fuse with enamel crystals and then develop into enamel-like co-aligned crystals.158 After treatment with CS-AMEL hydrogel for 7 days, an enamel-like layer with a thickness of 15 µm was formed on an etched enamel surface. The newly grown layer was made of highly ordered arrays of crystals with a diameter of ∼50 nm, growing preferentially along the c-axis, perpendicular to the surface. (Fig. 12A) It is noteworthy that these needle-like crystals were organized into bundles, which are similar to the fundamental units of natural enamel within the prisms. The organized enamel-like layer formed in the CS-AMEL hydrogel significantly improved the hardness and elastic modulus of the etched enamel.158 Importantly, this biomimetic in situ regrowth of apatite crystals generated a robust enamel-restoration interface, which is important for ensuring the efficacy and durability of restorations (Fig. 12B–C).158 In a follow-up study, we optimized the conditions to produce organized enamel-like crystals in a CS-AMEL hydrogel (Fig. 12D). 159</p><p>Compared with other biomimetic treatments, CS-AMEL hydrogel is easier to prepare for clinical use. Besides its biocompatibility and biodegradability, it has unique antimicrobial and adhesion properties that are important for dental applications. Another advantage is that the robust interface between the synthetic and natural enamel crystals promotes strong bonding between the newly grown layer and the tooth surface.</p><p>However the CS-AMEL technology still has the following limitations: (i) the hardness and modulus still do not meet the level of natural healthy enamel due to the presence of organic material and lack of hierarchical prismatic-interprismatic structure; and (ii) the extended amount of time (3–7 days) needed for the hydrogel to dry and mineralization to complete could be a challenge in a clinical setting.160 Further studies are needed to overcome these limitations. One possible strategy to improve the mechanical properties will be the repeated application of CS-AMEL hydrogel to obtain a thicker repaired layer. Digestion of the organic material during the mineralization process is another strategy for improving the mechanical properties. In addition, to test biomimetic approaches like this one properly, it is necessary to develop a caries model system that accounts for the effects of salivary proteins on crystal growth.</p><p>Beside the full-length amelogenin, the leucine-rich amelogenin peptide (LRAP) is another candidate for biomimetic approaches for enamel reconstruction. LRAP is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length amelogenin. In vitro studies have shown that LRAP has striking similarities with full-length amelogenin in respects of assembly and protein-mineral interaction.66, 161 Furthermore, LRAP could stabilize ACP and guide ACP transformation into ordered bundles of apatite crystals.161 Base on these evidences, it is reasonable to propose that LRAP, like full-length amelogenin, also has a great potential for biomimetic regrowth of tooth enamel.</p><!><p>Other biomimetic systems have been developed to repair enamel defects, including liquids and hydrogels that contain different organic additives.162–166 A glycerine-enriched gelatin system has been used to form dense fluorapatite layers on human enamel.162, 163 Reconstructed layers containing ordered enamel-like structures of fluoride-substituted hydroxyapatite microcrystals were synthesized on a human enamel surface using ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) as the mediating agent under near-physiological conditions.164, 165 It was also reported that a regrown layer with prism-like hydroxyapatite can be formed on an enamel surface by an agarose hydrogel in the presence of calcium ions and a high concentration of fluoride (Fig. 13).166</p><!><p>Enormous progress has been made over the last few decades in identification of the gene products involved in dental enamel formation and elucidation of their function. With advances in nanoscience and molecular biology, we now have acquired more knowledge about the unique characteristic of amelogenin and its specific interaction with different targets such as mineral, non-amelogenin proteins, cell surfaces, and proteinases.</p><p>We learned that the extended and flexible structure of amelogenin may provide the structural adaptability that facilitates the assembly of amelogenin into different quaternary structures as well as facilitates interaction with various potential targets in the enamel extracellular matrix. Amelogenin may be functional in vivo in oligomeric, dimeric, or even monomeric forms depending on the surface that amelogenin interacts with. A clear model for in vivo functional units of amelogenin however is still lacking. Amelogenin molecules do not occur in isolation in vivo, so the presence of other components such as mineral, other proteins, cell surface and lipid particles needs to be considered. While apatite nucleation is promoted by amelogenin in vitro, there is no direct evidence that enamel mineralization occurs in vivo via amelogenin-mediated heterogeneous nucleation. This is because enamel mineralization still occurs in amelogenin null mice and this observation does not support a direct nucleating function for amelogenin in vivo. A classical theory of enamel biomineralization in which the organic matrix controls the shape of crystallites by specifically inhibiting ion deposition on the crystal sides, and orient them by binding multiple crystallites is supported by many in vitro studies. However, observations from loss of function studies using knock- out animal models do not support such mechanisms. The non-classical mineralization pathway which involves co-assembly of the organic matrix and the inorganic transient phase to result in elongated crystals may well explain enamel mineral formation at the early stage, but growth of the final ribbon-shaped morphology of enamel crystals cannot be easily explained by the non-classical mineralization pathway. In the maturation stage the apatite crystals mainly grow in thickness and width with concomitant and dynamic processing of the enamel matrix, and the non-classical theory cannot explain this.</p><p>In summary, understanding of the detailed underlying mechanisms of enamel formation is far from complete. In particular, extended in vitro and in vivo studies are still needed to achieve a deeper understanding of how amelogenin, associated with the non-amelogenin protein components, interact with each other and with the ameloblast cell surface as well as mineral phase, and finally produce a highly mineralized and hierarchically structured biological material.</p><p>Understanding mechanisms of protein-mediated enamel biomineralization provides a valuable foundation for development of biomaterials with composition and structures similar to enamel. In the last decade, various biomimetic systems have been investigated to mimic the enamel-like microstructures in the presence of calcium phosphate nanoparticles, peptides, amelogenin-inspired polymers, and other organic additives. Despite all these promising studies, the biomimetic strategies still face ongoing challenges in the fields of dentistry and material sciences. To date, a material that can completely take the place of human dental enamel with similar biological and mechanical properties has not yet been fabricated. Human teeth have a more complicated structure, better mechanical properties and better biocompatibility than any enamel-mimic material mentioned in this review. All the existing biomimetic materials are limited to mimicking the enamel structure on the nanoscale level. Fabrication of the complex hierarchical rod-and-interrod structures of enamel is still a major challenge for the materials scientists of today.</p><p>Additional studies are required to evaluate the clinical applicability of the biomimetic materials mentioned. The clinical application of the existing biomimetic approaches for the treatment of larger visible cavities in the enamel is not yet conceivable. The growth of a repaired enamel layer usually takes an extended amount of time (from several hours to days, sometimes even few weeks) in the classical biomimetic strategies, which will dramatically limit the application of these materials in the clinical setting.</p><p>The CS-AMEL hydrogel we recently developed has shown a great potential for biomimetic reconstruction of enamel because of its antimicrobial property and the robust interface between the synthetic and natural enamel crystals. However the amelogenin-containing system still does not fully replicated the entire process to produce materials identical to natural enamel. While it has been established that the non- amelogenin proteins (enamelin, ameloblastin, proteinases) are also critical for controlling enamel mineralization, their application in the development of biomimetic systems remains to be explored.</p><!><p>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol</p><p>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</p><p>ACML, ameloblast cell membrane-mimicking lipid vesicles</p>

PubMed Author Manuscript

Kinetic resolution of sulfur-stereogenic sulfoximines by Pd(<scp>ii</scp>)–MPAA catalyzed C–H arylation and olefination

A direct Pd(II)-catalyzed kinetic resolution of heteroaryl-enabled sulfoximines through an ortho-C-H alkenylation/arylation of arenes has been developed. The coordination of the sulfoximine pyridyl-motif and the chiral amino acid MPAA ligand to the Pd(II)-catalyst controls the enantio-discriminating C(aryl)-H activation. This method provides access to a wide range of enantiomerically enriched unreacted arylpyridyl-sulfoximine precursors and C(aryl)-H alkenylation/arylation products in good yields with high enantioselectivity (up to >99% ee), and selectivity factor up to >200. The coordination preference of the directing group, ligand effect, geometry constraints, and the transient six-membered concertedmetalation-deprotonation species dictate the stereoselectivity; DFT studies validate this hypothesis.

kinetic_resolution_of_sulfur-stereogenic_sulfoximines_by_pd(<scp>ii</scp>)–mpaa_catalyzed_c–h_arylat

Introduction<!>Results and discussion<!>Conclusions

<p>The directing group (DG) assisted desymmetrization of prochiral C-H bonds provides a suitable way to construct carbon, phosphorus, silicon, and sulfur centered functionalized chiral molecules. [1][2][3] However, this approach requires achiral precursors with two identical enantiotopic groups, which prevents its application for broad synthetic benets. On the other hand, kinetic resolution (KR) of C-H bonds offers booming advantages for making functionalized enantioenriched molecules. In this regard, Yu's pioneering work on DG assisted chiral amino acid (MPAA) enabled Pd-catalyzed carbon centered KR of arene C-H bonds through alkenylation, arylation, and/or iodination is undoubtedly a breakthrough (Fig. 1A). 4 In spite of this success, the related strategy of Pd-catalyzed heteroatom centered KR of arenes remains to be explored, although exceedingly appealing.</p><p>Sulfoximines, which are congurationally stable motifs with S-stereogenecity, are found in molecules of medicinal importance and agrochemicals. 5 Notably, sulfoximines have emerged as chiral auxiliaries and DG for C-H functionalizations. 6 The syntheses of enantioenriched sulfoximines have invariably relied on resolution techniques, stereoselective imination, and oxidation processes. 7,8 Elegant enantioselective and KR routes to sulfoximines have been independently developed by Cramer, Li, Shi, and others, but all these approaches rely on Rh/Ru-catalyzed [4 + 2] annulation of diazoesters/sulfoxonium ylides and aryl-sulfoximines in the presence of specially designed ligands. 9 On our side, we have devised an expedient Pd-catalyzed C-H functionalization method for KR of 2-pyridylaryl sulfoximines, using Pd(II) catalyst and MPAA ligand, via C(aryl)-H arylation and olenation (Fig. 1B). The concept relies on kinetically regulated concertedmetalation-deprotonation (CMD) step of C(aryl)-H activation (k 1 >> k 2 , Fig. 1B) through preferred coordination of pyridine over imine to Pd-MPAA (Fig. 1C-I) 10 and ligand geometry CMD Pyr-DS over CMD Pyr-DR (Fig. 1C-II). The transformation is general, constructing a wide array of enantiomerically enriched C-olenated/arylated aryl-pyridyl-S-sulfoximines.</p><!><p>The study was initiated with the non-substituted N-Boc-phenyl-2-pyridyl sulfoximine rac-1a-1 and ethyl acrylate (2a; 0.6 equiv.) in presence of Pd(OAc) 2 (10 mol%), Boc-L-Phe-OH (L1; 30 mol%), Ag 2 CO 3 (2.0 equiv.) in ClCH 2 CH 2 Cl (1,2-DCE) at 75 C ee c (%) ee (%) a Reaction conditions: rac-1a-1 (0.1 mmol), ethyl acrylate 2a (0.6 equiv.), Pd(OAc) 2 (10 mol%), ligand (30 mol%), Ag 2 CO 3 (2.0 equiv.), 2-Cl-BQ (0. (Table 1a). The desired C2-alkenylation product (S)-3a-1 (18%, conversion aer 3 days) along with precursor (R)-1a-1 were obtained in 75% ee and 17% ee, respectively, exhibiting a low selectivity factor (s) of 8. This encouraging result unfolded our curiosity about examining the effect of other ligands. None of the N-Boc, N-acetyl-, and N-imide-protected commercially available a-amino acid ligands (L2-L6) with distinct side chains were effective. Assuming the additional coordination ability of the easily modiable OH group in threonine, various N,Oprotected threonine ligands were tested. The reaction s factor was improved a little for (S)-3a-1 to 12 and 11 when Boc-L-Thr(t-Bu)-OH (L7) and Boc-L-Thr(Bn)-OH (L8) were used, respectively. Electronic perturbation in the O-benzyl moiety did not have any impact on the enantioselectivity (L9 and L10). The use of 2a (2.0 equiv.) in presence of ligand L8 improved the conversion (50%) with (S)-3a-1 (70% ee) (entry 13).</p><p>To enhance sulfoximine resolution efficiency while maintaining conversion ($50%; Table 1a), we scrutinized the cooxidant effect (Table 1b). 11,12 2-Chlorobenzoquinone (2-Cl-BQ) was found to be the best, providing (S)-3a-1 in 77% ee with 39% conversion (s factor of 13; entry-2, Table 1b). Next, sulfoximines having various N-protecting groups (PG) were screened; the results are shown in Table 2a. None of the N-Me/Piv/Cbzprotected sulfoximines were found effective.</p><p>Next, we studied the DG effect (Table 2b). Thus, various substituted 2-pyridyl containing sulfoximines were independently subjected to 2a. Aer several trials, the 3-methyl pyridyl DG was found superior, affording the alkenylation resolution species (S)-3a in 96% ee with s factor of 58, although conversion was limited to 17% (entry 1). On the other hand, 3-Cl/Br substituted pyridyl DG were unsuccessful (entries 2 and 3). While trace of desired olenation product (S)-3s with 96% ee was noticed from the reaction of 3-phenyl-pyridyl (DG 5 ) bearing a Reaction conditions: rac-1 (0.25 mmol), olen (2.0 equiv.), Pd(OAc) 2 (10 mol%), L8 (30 mol%), Ag 2 CO 3 (2.0 equiv.), 2-Cl-BQ (0.5 equiv.), 1,2-DCE (2.5 mL), 75 C, 3 days. b Yield of the isolated olenated product. c Olen (1.8 equiv.) was used and reaction was performed for 1.5 days. Calculated conversion, C ¼ eeSM/(eeSM + eePR). Selectivity (s</p><p>phenyl sulfoximine (1a-4) with methyl acrylate (2b, 0.6 equiv.; entry 4). The 4-Me/5-Cl substituted pyridyl DG were ineffective, providing a lower selectivity factor (s) of 12 and 11, respectively (entries 5 and 6). No desired olenation product was obtained when 3-F substituted (DG 8 ) pyridyl group was used (entry 7). The reaction conversion was improved to 22% when 2a (2.0 equiv.) was employed under the reaction shown in entry 1 (entry 8). The identical transformation with 2b (2.0 equiv.) could enhance the conversion to 27% (entry 9). Finally, a 50 mol% loading of 2-Cl-BQ led to (S)-3b (96% ee, factor of 85 with 34% conversion; entry 10), which was found optimum.</p><p>The generality of the Pd-catalyzed C-H alkenylative KR of sulfoximines was then surveyed (Table 3). Compound 3b (98.2 : 1.8 er) was isolated in 26% yield. The alkenylation occurred at the less-hindered arene C-H bond and the chiral sulfoximines 3c and 3d were obtained with s factors of 162 and 44, respectively. The catalytic system was compatible with common functional groups, such as ketone, sulfone, and phosphate in the alkene, providing access to 3e (95.6 : 4.4 er), 3f (94.6 : 5.4 er), and 3g (97.6 : 2.4 er). Notably, the reaction of methyl vinyl sulfone with 1b displayed an exceptional s factor of >200 for compound 3h. The reaction of p-(Me/ t Bu/ i Pr)substituted aryl sulfoximines with 2b/vinyl-ketone (2c)/vinylsulfone (2f) smoothly delivered 3i-m in excellent enantioselectivity and s factor of 56 to >200. The m-substituted electron donating (OEt, Me) and chloro-bearing aryl-sulfoximines underwent olenation with 2b to give the desired products 3n-p with s factor of 24 to 111. Even the sterically hindered m,m 0 -dimethyl substituted aryl sulfoximine 1i reacted well, yielding 3q (36%, 97.9 : 2.1 er, s factor of 107). The reaction of heteroaryl bearing 2-thiophenyl-2-pyridylsulfoximines (1m) with 2b afforded the olenation product 3r (32%, 96.2 : 3.8 er, s factor of 51). 13 Next, we investigated the feasibility of Pd-catalyzed C-H arylative KR of sulfoximines (Table 4). 14 The reaction of N-Boc-3-methoxyphenyl-2-(3-methylpyridyl) sulfoximine ( 1b) with (4-CF 3 )Ph-Bpin (4a; 2.0 equiv.) was performed under the catalytic conditions of entry 10, Table 2. Pleasingly, the desired product (S)-5a was obtained in 94% ee with s factor of 39 along with the recovery of (R)-1b in 20% ee and 18% conversion (entry 1). The oxidant Ag 2 O played a vital role; the conversion was increased to 51% (entry 2). Carrying out the reaction at 60 C enhanced the s factor to 50 (entry 3). The s factor was raised to 64 with reaction conversion 41% and 94% ee of (S)-5a, when triuorotoluene (TFT) was used (entry 4). Performing the reaction with 20 mol% L8 improved the outcome (entry 5). Importantly, reaction concentration from 0.1 M to 0.067 M led to (S)-5a (94% ee) and (R)-1b (88% ee) with 48% conversion and s factor of 95 (entry 6); this catalytic system was thus able to provide a balanced outcome.</p><p>We next probed sulfoximines KR via enantioselective C-H arylation with arylpinacol boronate esters (Table 5). The reaction of 1b with various arylpinacol boronate esters having electron withdrawing groups [p-CF 3 (4a), m-CF 3 (4b), m-COMe (4c), and p-F (4d)], electron donating groups [p-Me (4e) and p-OMe-m-OEt ( 4f))] at the aryl motif independently led to the arylative resolution products 5a (96.1 : 3.9 er, 42%), 5b (96.5 : 3.5 er, 43%), 5c (97.5 : 2.5 er, 41%), 5d (98.4 : 1.6 er, 40%), 5e (97.2 : 2.8 er, 41%), and 5f (96.4 : 3.6 er, 44%), respectively, with s factor of 70-168 and conversion 46-49%. Moreover, the precursor (R)-1b was isolated in 41-46% yield with good enantioselectivity. The labile -Cl group was tolerated under the Pd-catalytic system, making 5g (97.8 : 2.2 er, 39%) with an s factor of 117. Notably, p-conjugated naphthyl-enabled sulfoximine resolution product 5h (99.0 : 1.0 er, s factor of >200) was reliably accessed. Next, the arylation of m-OEt-phenyl bearing sulfoximine 1f with 4a provided 5i (>99% ee) with s factor of >200. Likewise, 5j (97.2 : 2.8 er, s factor of 110) was made from the arylation of 2-naphthyl containing sulfoximine 1j with 4e. The sterically bulky o-tolyl enabled sulfoximines 1k</p><p>and 1l were successful in undergoing arylation with 4a/4c/4e to afford 5k-n in good enantioselectivity; the moderate s factors of 19-24 and conversions (c ¼ 29-38%) are considered suitable.</p><p>We performed a theoretical study to unveil the reaction mechanism (Fig. 2 and 3). [14][15][16] The MPAA ligand coordination to the metal center lowers the energy barrier of the CMD step, forming a semiplanar ve membered ring. 15 We believe the CMD step could be the main responsible for the kinetic resolution. This hypothesis has been previously validated by Wu et al., who also focused their study on the CMD as determining step. 15 Based on their ndings, and considering the plane dened by the coordination of MPAA to the Pd, the bulky a-side chain of the ligand (above the plane) pushes the N-Boc moiety down to avoid steric hindrance (Fig. 2 and 1-C-II). Thus, sulfoximine phenyl group coordination complex with Pd-MPAA can point upward (U) or downward (D) on the plane, with R or S congurations. This translates to four possible CMDs: CMD Pyr-UR , CMDPyr-US, CMDPyr-DR, and CMD Pyr-DS . The CMDs adopt a 6-membered palladacycle with twisted boat conformation.</p><p>In case of upward phenyl group linkage (CMDPyr-UR and CMDPyr-US), the sulfur atom and its substituents are located above the plane; while these substituents are below the plane for CMDPyr-DR and CMDPyr-DS. In agreement with Wu's observations, 15 the C1-N2-Pd-O3 dihedral angle for CMDPyr-UR and CMDPyr-US is ca. 170 , which generates a high steric interaction when compared with the ca. 140 for CMDPyr-DR and CMDPyr-DS. These latter are favored by hydrogen bond interactions, making the combination of steric and electronic effects accounting for a difference of nearly 10 kcal mol À1 in each enantiomer (Fig. 2).</p><p>The preference for the S conguration by $2.5 kcal mol À1 over the R isomer, lies in a steric clash of the NBoc group with the methyl group from the pyridine moiety and in consequence with the phenyl group causing an energetically demanding a Reaction conditions: rac-1 (0.2 mmol), 4 (2.0 equiv.), Pd(OAc) 2 (10 mol%), L8 (20 mol%), Ag 2 O (2.0 equiv.), 2-Cl-BQ (0.5 equiv.), TFT (3.0 mL), 60 C, 3 days. b Yield of the isolated arylation product. Calculated conversion, C ¼ eeSM/(eeSM + eePR). Selectivity (s arrangement. The coordination of both 'N' atoms in sulfoximine 1a forms int-0 with the displacement of acetic acid, where the S-conguration at sulfur is 1.0 kcal mol À1 more stable than the R one (Fig. 3). Prior to deprotonation, a cis coordination of aryl group to the N-protected moiety of the MPAA-ligated intermediate occurs. This assists the CMD process by establishing the absolute conguration of the sulfur motif. This calculation fully complies with the experimental observations of the resolution selectivity (calc. 98 : 2, exp. 98 : 2; Fig. 3-III). Notably, the experimentally observed S-int-2Pyr is thermodynamically favored over R-int-2Pyr isomer by 6 kcal mol À1 . In retrospect, the CMD transition states of int-1S]N (Fig. 3-I) and int-1S¼O (Fig. 3-II) lie much higher than int-1Pyr (Fig. 3-III), and their respective DGs do not coincide with the experimental ndings. Of note, the CMD process through int-1pyr is endergonic (Fig. 3-III); thus, the calculated R/S ratio is relevant if the next steps display lower free energies of activation than the CMD Pyr transition states. However, the system becomes too large to study the insertion step; simplication is therefore needed. Since we aim to distinguish the absolute conguration at the sulfur atom, a monodentate ligand for example, acetyl-Lalanine instead of bulky mono-protected threonine moiety was used for modelling purposes. 11 The olen insertion with metalated sulfoximine (made by the coordination of S]N and Pyr) is next considered (Fig. 4). The corresponding S]N coordination with R conguration Int-3 S]N is found most stable (Fig. 4). The detailed analysis of transition states (INS) occurred in the CMD revealed that the pyridine directed insertion (INS Pyr ) involves lowest energy barriers (17.41 kcal mol À1 for the S isomer and 21.80 for the R isomer); see Fig. 4. This results a nal selectivity >99 : 1 (Fig. 4). This exergonic step, thus, funnels the reaction without affecting the ratio earlier dictated by the CMD. Interestingly, both INS Pyr and INS s]N structures are same (ignoring conguration); since both DGs (S]N and Pyr) are coordinated to the metal center in their corresponding products (int-4; Fig. 4).</p><p>The synthetic potential of chiral sulfoximine was next probed (Scheme 1). The triuoroacetic acid (TFA) mediated N-Boc deprotection of (R)-1b provided chiral sulfoximine (R)-6 (>88% ee). Next, reduction of (R)-6 led to chiral sulfoxide (R)-7 (90% ee) when exposed to t-BuNO 2 at rt for 2 h. The N-Boc deprotection and intramolecular Michael cyclization to the activated olenmoiety of (S)-3c smoothly delivered 8 (as a single diastereomer) in 95% ee. A TFA assisted N-Boc deprotection and oxidative intramolecular C-N bond formation of (S)-5d furnished (S)-9 (93% ee, 62% yield).</p><!><p>In summary, a Pd(II)-catalyzed pyridyl substituted KR of sulfoximines through C(aryl)-H alkenylation and arylation has been revealed. The transformation addresses the inherent challenges in the KR of coordinatively active pyridyl-enabled sulfoximines (highly susceptible to TM-catalyst quenching) with no prochiral center in the presence of chiral amino acid MPAA ligands and Pd(II)-catalyst. The common functional groups were tolerated under Pd-catalysis exhibiting good substrate scope for C-H alkenylative and arylative sulfoximines KR products in high enantioselectivity with s factor up to >200. In-depth DFT studies uncover the salient features of coordination selectivity of pyridyl-group over sulfoximine imine.</p>

Royal Society of Chemistry (RSC)

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays

Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid-based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments we found the depth of the polymer matrix to be 1\xe2\x80\x932 \xc2\xb5m. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2 \xc2\xb5m. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering (SERS) is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform.

properties_of_coatings_on_rfid_p-chips_that_support_plasmonic_fluorescence_enhancement_in_bioassays

1. Introduction<!>2.1. p-Chips<!>2.2. Coating p-Chips with polymer<!>2.3. Deposition of SIF on p-Chips<!>2.4. IL-6 immunoassay<!>2.5. SEM<!>2.6. Elemental analysis in SEM<!>2.7. Fluorescence microscopy: FLIM and intensity data<!>2.8. Fluorescence lifetime measurements<!>2.9. Raman scattering measurements<!>3.1. SEM<!>3.2. Fluorescence enhancement and lifetime change<!>3.3. Raman measurements<!>4. Conclusions

<p>Fluorescence-based techniques have become an indispensable tool for the modern biologist. They are the ideal methodologies for medical diagnostics, DNA sequencing, genetic analysis, forensics, and cellular/molecular imaging. Many approaches involve the use of a solid phase to facilitate manipulations and data acquisition. The solid phase that has been recently successfully implemented in multiplex bioassays [1,2] is an ultra-small RFID silicon p-Chip. These light-activated microtransponders are 500 µm × 500 µm × 100 µm integrated circuits that have electronic elements on one side of the chip. The electronic circuits on the p-Chip include photocells, read-only memory (ROM), a loop antenna, and control elements. Their role is to transmit the ID of the chip when the chip is illuminated by laser light during the assay to reveal the type of biological material carried on the chip. In the assays, biological material (oligonucleotides, antibodies, antigens) is conjugated to the p-Chip, the chip is exposed to the sample and fluorescently stained, and the chip's fluorescence intensity is quantified in a flow-based custom analyzer [3] revealing concentrations of analytes in the sample. The benefits of p-Chips in bioassays are clear, as they allow a high-level multiplexing to obtain concentration of a large number of analytes in the sample [1–3]. The chips have been also used to tag small laboratory animals [4–6].</p><p>Aside from their obvious advantages in multiplex bioassays, the p-Chips also provide a means of fluorescence enhancement, improving the sensitivity of assays for low-abundance biomolecules. In traditional fluorescence assays, fluorophores are examined in the free-space condition, i.e., the fluorophore may be modeled as an oscillating dipole radiating energy into a transparent, homogeneous medium. The free-space spectral properties are not modified by local polarity, quenching, or energy transfer. In the case of p-Chip-based assays, it is possible to place the fluorophore in close proximity to a conducting metal surface, thereby altering the free-space properties through surface plasmon resonance [7–10]. This refers to the interaction between excited state fluorophores and mobile electrons on the surface of the metal, and it has been shown to increase quantum yields, increase photostability, and decrease lifetimes [11–24].</p><p>Metal-enhanced fluorescence (MEF) has been extensively studied over the last two decades, and these studies often revolved around the morphology of the metal particles providing the enhancement. Great success has been seen from not only silver island film (SIF) substrates [25–31] but also silver colloids [32–33], nanorods [34], nanotriangles [35–36], and silver fractal surfaces [10]. However, these systems usually involve nanoparticles resting on a substrate, while the flow-based p-Chip assays require that silver particles be attached to the surface in a robust manner and on a mass scale. One such method, in which p-Chips are coated with a SIF and the film is held in place by a coating of polymer above and below, has been previously demonstrated [2]. The polymer prevents the SIF from being removed during an assay as a result of mechanical forces acting on the polymer. It also provides a bed for the conjugation of biological molecules, e.g., DNA or protein. The polymer provides another added benefit in that the fluorophore is separated from the silver and located at optimal distances for fluorescence enhancement. The optimal distance for enhancement between a fluorophore and a metal surface is ~100 Å, while a fluorophore is severely quenched within 50 Å [11].</p><p>It is known that the size and shape of silver particles have a direct effect on plasmon resonance and the subsequent fluorescent spectral properties [37]. It is also known that the separation between the fluorophore and the metal particles will affect the enhancement [23]. The structure and geometrical properties of the SIF and the polymer are thus the focus of the present study. We also investigated fluctuations of both fluorescent intensity and fluorescent lifetimes across the chip's surface to better understand the significance of silver particle clusters within the polymer on fluorescence enhancement.</p><!><p>p-Chips were provided by PharmaSeq, Inc. (Monmouth Junction, NJ, www.pharmaseq.com). The properties of the p-Chips and two types of p-Chip readers (flow-based bench-top analyzer and hand-held ID reader) have been described [1–4]. The 10-bit p-Chips used in the work described allowed a maximum 1,024 different IDs.</p><!><p>The p-Chips were coated by aminopropyltriethoxysilane (APTS) and 3-glycidoxypropyl-trimethoxysilane (GPTS) as previously reported [2] with several modifications. Briefly, p-Chips were washed with 99.5% methyl alcohol at room temperature (RT) for 5 min three times. The p-Chips were then rinsed with toluene/dimethylformamide (DMF) mixture with 0.01% distilled water and 0.9% APTS at RT four times. After rinsing, p-Chips were immediately treated with a coating solution (mixture of toluene and DMF with 0.01% distilled water, 0.9% APTS, and 0.3% GPTS) at 80°C for 20 min. After the coating reaction, p-Chips were washed once with toluene, three times with DMF, and three times with acetonitrile at RT, followed by air drying. The procedure places both amino and hydroxyl groups within the polymer and on the surface of p-Chips.</p><p>To introduce the carboxyl groups, the derivatized p-Chips were treated with 10% succinic anhydride in dry pyridine:DMF (1:9) and placed on a tissue culture rotator at RT for 30 min. This step was repeated once using fresh reagents. After the reaction, the carboxylated p-Chips were washed with DMF four times and acetonitrile twice, followed by air drying.</p><p>All chemicals used for coating and carboxyl conversion were purchased from Sigma-Aldrich, St. Louis, MO.</p><!><p>In order to make SIF-coated p-Chips, the chips were coated with polymer first as described above. Then SIF was deposited on the surface of polymer-coated p-Chips as reported previously [2] with several modifications. Briefly, 60 µL of 5% NaOH was slowly added to 18 mL of 0.83% AgNO3 solution with intensive stirring at RT in a 50 mL reaction tube. Then 400 µL of 30% NH4OH was added with intensive stirring at RT. The clear solution was incubated in an ice bath for 10 min, followed by the addition of 4.5 mL of a fresh 4.8% glucose solution with intensive stirring. Polymer-coated p-Chips were incubated in this solution in a 1.5 mL Eppendorf tube on a tissue culture rotator at RT for 50 min. After the silver deposition, the p-Chips were immediately washed with distilled water three times followed by air drying. The dried SIF-deposited p-Chips were coated with another polymer layer and carboxylated as described above to seal the SIF layer.</p><!><p>We performed a standard Il-6 assay on polymer coated p-Chips and SIF/polymer coated p-Chips in order to test the two platforms under identical conditions. To conjugate an antibody, the carboxylated p-Chips were incubated with 100 µL 261 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 230 mM N-hydroxysulfosuccinimide (NHSS) in 0.1M HEPES buffer (pH 7.5) on a rotator for 30 min at RT. The p-Chips were then washed with 200 µl PBS three times and incubated with 30 µL 200 µg/mL monoclonal anti-human IL-6 antibody (R&D Systems, Minneapolis, MN) for 2 hrs at RT on a tissue culture rotator. The p-Chips were washed with PBS three times and blocked with SuperBlock solution (Thermo Fisher Scientific, Waltham, MA) for 5 min at RT on a rotator three times. The p-Chips were then washed with PBS three times and stored in TBS with 3% BSA at 4 °C.</p><p>Anti-IL-6-conjugated p-Chips were incubated with 50 µL 100ng/mL recombinant human IL-6 protein standard (R&D Systems) in TBS with 3% BSA for 1 hour at RT on a rotator. After incubation, the p-Chips were washed with 200 µL of Tris-buffered saline with 0.05% Tween-20 (TBST) three times.</p><p>The detection antibody solution was prepared by diluting biotinylated anti-human IL-6 antibody (R&D System) to 5.0 µg/mL with PBS. The p-Chips were then incubated with 50 µL of this solution for 1 hour, followed by washing with TBST three times. The p-Chips were pooled and incubated with 50 µL of 8 µg/mL streptavidin-Alexa Fluor 555 conjugate in TBST for 30 min at RT in the dark. After incubation, the p-Chips were washed with TBST three times and with distilled water twice and were stored in PBS at 4°C between measurements. All measurements except SEM were conducted with the p-Chips immersed in PBS.</p><!><p>Samples were imaged with an FEI model XL30 (Hillsboro, OR) scanning electron microscope (SEM). The SEM is equipped with a tungsten filament operating at accelerating voltages up to 30 kV. The samples were fixed to aluminum sample stubs via carbon conductive tape, which provided a continuous, conductive path from the sample through the stage. When p-Chip cross-sections were imaged, the p-Chip was first cut in half with a razor blade before being attached to the carbon tape, cut side up towards the primary electron beam. The chips were then placed into the sample chamber, where they were measured under vacuum with pressures always below 1 microtorr. The working distance between the bottom of the column and the sample was 10 mm. The spot size and accelerating voltage were varied as needed, and these values are indicated on each particular image. As is typical for SEM imaging, all images reported here were collected from the detection of secondary electrons, emitted as the primary electron beam rasters across the sample surface.</p><!><p>The SEM was equipped for energy dispersive spectroscopy (EDS) with a detector manufactured by EDAX (Mahwah, NJ). Because semi-conductive and nonconductive materials exhibit very low signals in EDS, the accelerating voltage used for EDS was always 30 kV so as to provide the highest possible count rate.</p><!><p>Fluorescence lifetime imaging (FLIM) data were collected by the MT200 system (PicoQuant GmbH, Berlin, Germany). This system was coupled to an Olympus (Shinjuku, Tokyo, Japan) IX71 confocal microscope with a 100× oil objective, unless it was noted that a 60× water objective was used. A single photon avalanche detector (SPAD) manufactured by Micro Photon Devices (Bolzano, Italy), model PD1CTC, was chosen for its sensitivity. A 470 nm pulsed laser diode with a 20 MHz repetition rate was utilized for excitation. The emission was filtered by a 470 long pass liquid filter as well as a 560/40 band pass filter. For reflection images, the laser power was reduced and a 470/10 band pass filter was used. The system is capable of picosecond time resolution, and the piezo stage is accurate to within 1 nm.</p><p>Symphotime (version 5.2.4) software by Picoquant was used to process all FLIM data and to extract intensity information from the FLIM data. FLIM images are produced with grayscale intensity data and false color overlaid to indicate the lifetime calculated for every pixel. The enhancement in fluorescence intensity was measured with the MT200 simultaneously with FLIM data.</p><!><p>Fluorescence lifetime was measured with a Fluorotime 200 fluorometer, also from Picoquant. The FT200 was equipped with a 470 nm pulsed laser diode, a microchannel plate photomultiplier ultrafast detector, and a monochromator. The p-Chips were placed between two coverslips, a drop of buffer was added, and the coverslips were taped together. The sample was then arranged in a front face attachment with a 495 nm long pass filter on emission. The monochromator was set to 565 nm. The response was measured with the monochromator set to 470 nm along with a 470/10 band pass filter.</p><!><p>Raman scattering measurements were collected on a custom-built system consisting of a dispersive spectrometer from Kaiser Optical Systems, Inc. (Ann Arbor, MI) with 4.0 cm−1 resolution and a front-illuminated CCD detector from Roper Scientific (Sarasota, FL). The p-Chips were illuminated with an argon-ion laser (514 nm) emitting 220 mW of power. The light was focused by an Olympus BH-2 microscope with a 40× objective, resulting in a 1.5 µm collection area. The system was capable of measuring Raman shifts up to 2000 cm−1, corresponding to the 572 nm wavelength. The experiments were conducted on p-Chips coated with SIF and polymer in the manner described above but using a different assay. Briefly, the polymer coating the SIF and non-SIF p-Chips was carboxylated and further derivatized by conjugating streptavidin to the carboxyl groups (see sections 2.3 and 2.4 for the conjugation methods). The streptavidin-conjugated p-Chips were then incubated in 50 µL of 10 µg/ml Cy3-labeled biotinylated oligonucleotide at RT in a dark enclosure for 30 min. The p-Chips were washed with TBST three times and air dried. The sequence of the oligo was 5'-Cy3-TTT TTT TTT T-biotin-3'.</p><!><p>To avoid the suspected complex effects of electronic circuitry on the front of the p-Chips, the back of the p-Chips were viewed under SEM at high magnification in order to evaluate the morphology of the SIF and polymer coatings. Further, p-Chip cross-sections were viewed to measure the thickness of the coatings. When investigated via SEM (Figure 1), the surfaces of the p-Chips derived with SIF and polymer coatings were not smooth, as we expected them to be. Instead, they consisted of irregularly shaped nanoparticles as well as dispersed aggregates on top of nanoparticle layers. The sizes of the nanoparticles were measured according to their shortest axis, and their average was found to be around 270 nm when a log-normal distribution was fitted to 216 measurements (Figure 2). To determine the thickness of the SIF and polymer layer, we cut the SIF/polymer coated p-Chip in half and imaged the cross-section by SEM. Figure 3 shows that the particles were deposited in a 1–2 µm thick layer. This layer was confirmed by EDS mapping to be silver, with traces of carbon and oxygen likely originating from the polymer coating (Figure 4). Surprisingly, we were unable to detect an expected smooth, polymer coating on top of the silver particles. We expected to see this layer because the SEM images were formed from low-energy secondary electrons produced from the bombardment by the primary electron beam. These electrons are capable of traveling around 100 nm in insulators [30]. Secondary electrons originating at the silver would be lost as they traveled through the polymer. We hypothesize that when this polymer coating is placed into the high vacuum environment of the SEM (10−5 bar), the moisture was removed, leaving only a very thin and undetectable coating surrounding the silver islands.</p><p>In another independent measurement, we determined that the polymer coating is around 1.0–1.5 µm thick. This was done by stacking several polymer coated p-Chips, measuring the thickness, and then comparing that thickness to a stack of uncoated p-Chips (data not shown). These measurements were conducted immediately after removing the p-Chips from PBS buffer solution before the polymer had dried. The results are consistent with the SEM-based measurements for SIF/polymer p-Chips.</p><!><p>To evaluate the fluorescence enhancement by the silver nanoparticles in bioassays, we performed IL-6 ELISA assays on both SIF and non-SIF p-Chips. IL-6 is a pleiotropic cytokine involved in inflammation, hematopoiesis, and immune response [38]. Aberrant IL-6 levels have been observed in many autoimmune disorders and cancers [39]. The IL-6 ELISA assays are widely used in cancer studies and basic biological research and have been demonstrated to work well on the p-Chip platform [2]. In the assays, Alexa Fluor 555 was used as the fluorescent dye. We used an IL-6 standard of known concentration to ensure the conditions of the assay were identical for both platforms. After the complete assay was performed (please see section 2.4), fluorescence intensity was determined by averaging the count rate into the SPAD during collection of the corresponding FLIM image in Figure 5. To measure the enhancement, the laser intensity had to be reduced 15.6-fold while imaging the SIF p-Chip to avoid detector damage, and so the average count rate was scaled accordingly for comparison with the non-SIF p-Chip. We observed an 8.7-fold enhancement from the SIF-derived p-Chips over chips without SIF, which is consistent with previous findings [2].</p><p>FLIM images were recorded from both the SIF-polymer coated p-Chips from the IL-6 assay and from p-Chips covered with polymer only. The z-distance was adjusted for the brightest focal plane before the image was collected, and the reflection image was recorded at the same plane. The color seen in Figure 5 reflects the fluorescence lifetime. The results indicate that the lifetime was reduced from 0.62 ns in the polymer-only p-Chip to 0.36 ns in the polymer/SIF p-Chip. The sensitivity required for FLIM imaging necesitated the use of detectors that, when coupled with the electronics of our FLIM system, unfortunately had responses around 100 ps in FWHM, thereby making it impossible to resolve the full extent of the plasmonic effect on radiative decay. However, the system's temporal resolution was adequate for microscopic mapping of the enhancement, and we direct the reader to a previous study for more information regarding the radiative decay rates on SIF-enhanced p-Chips [2]. The 10-fold reduction was confirmed in the present study as well with a Fluotime 200 system from PicoQuant.</p><p>While the increase is well understood and has been quantified to a high degree of precision previously [2], the FLIM images show that the fluorescence enhancement is, surprisingly, relatively uniform across the p-Chip surface, both with and without the SIF layer. We had expected to see a pattern of "hot spots" following the morphology of the SIF where intensity and lifetime were dramatically different. The deposition of the SIF produces a very rough surface (Figures. 1, 3, and 5b), and enhancement hot spots were seen previously [40] and found to be correlated with morphological features. Instead, we found that the intensity and lifetime do not follow the morphology of the SIF and that there are no obvious "hot spots" present on the SIF surface. This uniform enhancement is presumably due to the dense packing of silver particles on the surface. The gaps between silver particles (Figure 1) are much smaller than the lateral resolution of the microscope system, which is around 300 nm. Therefore the diffraction-limited optics lead to a spreading out of the fluorescence across the p-Chip.</p><p>Fluctuations in the intensity of the reflection image (Figure 5) result from the varying height of the silver particles, as evidenced by Figure 3, so the depth of the fluorescence emission was investigated as well. While adjusting the focus, it was obvious that the depth of the fluorescence signal extended over 1 µm. To quantify this thickness, the p-Chip was scanned in the X and Z directions (see Figure 6). The photon counts were averaged along the X axis and plotted as a function of the axial position. Assuming a Gaussian distrubtion, the full width at half maximum was ~1.9 µm. As a calibration of this approach, the same procedure performed on 20 nm fluorescent nanobeads showed that the axial resolution of our system in this configuration was ~700 nm. Thus we may conclude that the thickness of the polymer was at least 1.2 µm. Even more interestingly, the enhancement from the SIF extended throughout this 1.2 µm layer. Given that the metal-enhanced fluorescence is limited in range to hundreds of angstroms, this evidence suggests that the polymer and dye molecules are wrapped around the SIF particles in a three-dimensional matrix above the surface of the p-Chip. This structure is very beneficial to the fluorescence enhancement because the three-dimensional morphology increases surface area, thereby maximizing the number of dye molecules that can bind to the chip and experience enhancement.</p><!><p>While the polymer coating itself did not contribute appreciably to the fluorescent signal observed on the coated p-Chips, a Raman scattering effect introduced by the polymer would add complications to the type of simple bioassay for which the p-Chips are intended. Raman scattering occurs when the excitation light is inelastically scattered, and thus a weak, red-shifted signal is produced that may be confused with fluorescence in the absence of spectral decomposition. Raman scattering has been previously observed and studied in silane-based polymers [41]. The concern is that, when in contact with the SIF, the well-known surface-enhanced Raman scattering effect [42] would greatly increase the signal. Therefore Raman scattering measurements were conducted at several places on the back side of a SIF/polymer coated p-Chip to determine if such an effect would interfere in bioassay experiments; the data from an example spot is shown in Figure 7. The intensity data was measured in terms of the shift with respect to the 514 nm excitation source. The signal observed was not typical of Raman scattering—the signal was broad and corresponded to the emission from the fluorophore used in the assay. After several minutes, the fluorophore was bleached (bleaching is not seen in Raman scattering), and no signal remained. It may be noted that the pileup of intensity on the low end of the x-axis is due to the bandwidth of the intense 514 nm laser. Raman scattering would be independent of the assay performed on the p-Chip, thus we can conclude that it will not present any problems for broad use of the derivatized p-Chips.</p><!><p>In this study, we explored the morphology and radiative properties of our SIF and polymer coatings enveloping p-Chips. In SEM experiments we found that the SIF layer itself consisted of silver particles averaging 270 nm in diameter. Direct measurement of the polymer coating on top of the SIF via SEM was not feasible, as the high vacuum likely collapsed the polymer by the removing moisture. However, FLIM images showed that the enhancement was extended to a depth of 1.2 µm, thus the actual depth of the polymer layer must have been greater than or equal to this. This polymer depth was also supported by direct measurements of the thickness of several p-Chips in a stack.</p><p>We observed a significant, 8.7-fold plasmonic fluorescence enhancement as evidenced by the increased intensity and the decrease in lifetime. We expected that silver particles would create "hotspots" of plasmonic enhancement when an ELISA assay (IL-6 assay with Alexa Fluor 555) was performed. Therefore, the fluorescence intensity and lifetime were expected to vary greatly with the relief of the SIF layer. FLIM images showed that fluorescence enhancement was relatively uniform over the surface. All this lends evidence to the idea that the SIF/polymer layer exists as a three-dimensional matrix over the p-Chip surface. This matrix is accessible to solvent and allows fluorophores to surround the silver particles, resulting in an increased number of dye molecules that are exposed to the plasmonic enhancement than would be the case if the polymer surface were flat and impenetrable to solvent and dyes.</p><p>This work has shown that a robust platform may be created for immunoassays. The polymer layer protects the silver island film from being scratched off, while together, the SIF and polymer layer create a volume accessible to a greater number of dye molecules for plasmonic enhancement. Future improvements of the SIF/polymer system on p-Chips may involve increasing the relief of the SIF layer or alterations to the layering process.</p>

PubMed Author Manuscript

Vinyl Sulfone Analogs of Lysophosphatidylcholine Irreversibly Inhibit Autotaxin and Prevent Angiogenesis in Melanoma

Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 \xe2\x80\x93 0.39 \xce\xbcM, consistent with a Ki of 3.50 \xce\xbcM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.

vinyl_sulfone_analogs_of_lysophosphatidylcholine_irreversibly_inhibit_autotaxin_and_prevent_angiogen

INTRODUCTION<!>General chemicals and spectroscopy<!>Chemical synthesis of intermediates and final products<!>ATX assay<!>Dilution/dialysis for reaction of 10b (CVS-16) or 21 (PVS-16) with ATX.<!>Cell culture<!>Cell viability assay<!>Wound healing assay<!>Animal model of melanoma<!>Tumor specimen analysis<!>Serum analysis<!>Statistics<!>Chemical synthesis of vinyl sulfone analogs<!>Kinetic inhibition of ATX<!>In vitro biological activity<!>Inhibition of melanoma progression and angiogenesis<!>DISCUSSION

<p>The ectonucleotide pyrophosphatase/phosphodiesterase 2 or its more common designation autotaxin (ATX), is an enzyme with lysophospholipase D activity that converts lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) 1 by hydrolysis of the choline head group from LPC. Although it is central to the biosynthesis of LPA, ATX also hydrolyzes p-nitrophenyl thymidine-5'-monophosphate, which is a type I phosphodiesterase substrate 2. Thus, it is a multi-faceted enzyme with more than one important function.</p><p>ATX was isolated from conditioned medium of melanoma cells 3, and was discovered to play a major role in the development of both the vascular 4 and nervous systems 5 as well as malignancy 6. In particular, ATX promotes cancer progression, since it can increase growth factor signaling, cell survival, proliferation and migration in many cancers, including pancreatic cancer 7, cutaneous and uveal melanoma 8, breast cancer 6b, follicular lymphoma 9, glioblastoma multiforme 10 and gynecologic malignancies 11. In addition, ATX also protects cancer cells from chemotherapy-induced apoptosis 12, which could significantly compromise cancer treatment and result in worsened outcomes when ATX is present in the tumor microenvironment.</p><p>Another occurrence affecting cancer treatment is angiogenesis. This is the process whereby tumors develop blood vessels to access nutrients and oxygen from circulation and is necessary for tumor growth beyond 1 mm, at which point necrosis occurs in otherwise hypoxic tissues. To state succinctly – angiogenesis is required for tumor progression. Without angiogenesis, the growing tumor will not thrive and will remain small. Because of this dependency, anticancer therapeutics targeting angiogenesis exploit tumors by inhibiting growth factors required for the formation of new blood vessels, such as the vascular endothelial growth factor (VEGF). This and other growth factors are secreted by the tumor when it exceeds a certain distance from its primary blood supply and senses hypoxia. On the other hand, since the circulatory system is exploited by intravenous anticancer therapeutics to reach the insides of tumors, timing is critical to properly treat patients with angiogenesis inhibitors.</p><p>Interestingly, ATX is a direct and indirect angiogenic factor that stimulates human endothelial cells to form tubules and tumors to become more hyperemic 13. It is thus not surprising that the outcome of knocking out ATX on vasculogenesis results in embryonic lethal mutations in mice embryos which display aberrant blood vessel formation upon death 4. Mechanistically, an increase in VEGF increases ATX expression and secretion among (at least) ovarian cancer cells. This resultant increase in ATX then results in more lysophosphatidic acid and also drives cells to produce more of the receptors LPA3, LPA4 and VEGFR2 14. This represents a positive feedback loop between ATX and growth factors involved in angiogenesis, especially since lysophosphatidic acid also stimulates VEGF production. In other words, ATX plays a central role in angiogenesis and thus, tumor progression.</p><p>Because of the critical role ATX has in angiogenesis and various malignancies, extensive research is devoted to the design, synthesis and evaluation of novel inhibitors of ATX 8a, 8b, 15 as well as the evaluation of natural substances with inhibitory activity 16. Some recently designed chemical inhibitors have not possessed the necessary characteristics to proceed into clinical development due to a lack of bioavailability or large millimolar concentrations required for activity. Interestingly, ATX is product-inhibited by both sphingosine 1-phosphate and lysophosphatidic acid 17. This suggests that these bioactive lipids are capable of regulating their own synthesis in the microenvironment and this knowledge bestows a scheme to chemically exploit their abundance. Other studies have elucidated the crystal structure of ATX 18 and further described how the enzyme discriminates substrates 19, which resulted in novel ideas for the design of additional ATX inhibitors.</p><p>Herein we report the synthesis and biological testing of a series of alkyl vinyl sulfone analogs of LPC. These analogs feature a quaternary ammonium derivative, a reactive vinyl sulfone, and alkyl chains varying in length from C6 to C18. We also prepared a control compound, a phenyl vinyl sulfone, which lacked the targeting by the quaternary ammonium derivative but retained the long alkyl chain. Vinyl sulfone derivatives are widely known as irreversible inhibitors of cysteine proteases 20. The mechanism of this irreversible inhibition depends on the vinyl sulfone acting as a Michael acceptor covalently reacting with the soft nucleophiles, such as thiols 21. Studies indicate that peptide vinyl sulfone inhibitors also modify the active site threonine (Thr) of the Escherichia coli HsIV homologue and Rhodococcus proteasome 21b, 22. Similar to the inhibition of cysteine protease, the inhibition of proteasome by peptide vinyl sulfone also involves covalent modification of the N-terminal Thr of the catalytic β subunits, via a Michael addition 21. Taken together, this suggested that vinyl sulfone might be an appropriate electrophile for targeting the active site Thr of ATX.</p><p>We hypothesized that the choline headgroup of ATX was an important recognition element for ATX, and would position the vinyl sulfone moiety in the active site in the vicinity of the Thr residue. A straight chain alkyl group was positioned in the 2-position of the vinyl sulfone, leaving a reactive, unsubstituted 1-position available to interact with an active site nucleophile. Several alkyl chains varying in length from C6 to C18 were employed to identify the optimal analog. We proposed that the metal-activated hydroxyl group of the catalytic-site threonine could attack the vinyl sulfone and form a covalent bond that would irreversibly inactivate the enzyme.</p><p>Indeed, we observed that vinyl sulfone analogs could reduce in vitro cell viability, cell motility, wound closure and melanoma growth in vivo. We previously synthesized ATX inhibitors and examined their biological activity against melanoma 8a, 8b, 23 and other malignancies 15d, 15e, 24. Since the in vitro activity observed by ATX inhibitors on melanoma cells is striking, we hypothesized that the mechanism of action is a directly-targeted, cellular consequence. However, we did not observe a direct effect against proliferation of melanoma models in vivo. Instead, the animal tumors treated with the highest compound concentration displayed significantly smaller tumors than controls, which resulted from an inhibition of angiogenesis, not mitogenesis. Herein we report the synthesis of novel vinyl sulfones and their action against the progression of melanoma via angiogenesis inhibition.</p><!><p>All synthetic reagent chemicals and solvents were purchased from Aldrich, Alfa Aesar or Acros Chemical Corporation and used without prior purification. Thin-layer chromatography was performed on pre-coated silica gel aluminum sheets (EM SCIENCE silica gel 60F254). Purification of compounds was carried out by normal phase chromatography (ISCO Combiflash, Silica gel 230~400 mesh). NMR spectra were recorded using a Varian Mercury 400 at 400 MHz (1H), 101 MHz (13C), 162 MHz (31P), 376 MHz (19F) at 25°C. Chemical shifts are given in ppm with TMS as internal standard (δ = 0.00); 31P, 85% H3PO4 (δ = 0.00). NMR peaks were assigned by MestRe-C (4.9.9.9). Low resolution mass spectra were obtained on HP5971A MSD double focusing mass spectrometer instrument. For biological studies, oleoyl (18:1) lysophosphatidic acid was purchased from Avanti Polar Lipids (Alabaster, AL) and reconstituted in chloroform and methanol. Prior to cell incubation, organic solutions were removed and ATX was added in 0.1% charcoal-stripped BSA.</p><!><p>Ethyl 2-bromopropionate 2 (5 g, 36.0 mmol) was dissolved in acetone (200 mL), K2CO3 (7.45 g, 54.0 mmol, 1.5 equiv.) was added, followed by of methyl-2 mercaptoacetate 1 (4.2 g, 39.5 mmol, 1.1 equiv.) and heated at reflux for 2 h. After cooling to room temperature the mixture was filtered out, and then the white cake was washed with acetone (25 mL × 2). All combined organic solvents were evaporated under reduced pressure. The residue was purified on silica column (ethyl acetate-hexanes, 15 to 40%) to yield the pure product methyl 2-(3-hydroxypropylthio)acetate 3 (5.78 g, 35.3 mmol, 98%).</p><p> </p><p>1H NMR (CDCl3) δ 3.67 (s, 3H), 3.66 (t, J = 6.0 Hz, 2H), 3.19 (s, 2H), 2.68 (t, J = 7.2 Hz, 2H), 2.54 (s, 1H), 1.78 (m, 2H); 13C NMR (CDCl3) δ 171.1 (s), 61.0 (s), 52.4 (s), 33.4 (s), 31.5 (s), 29.2 (s); MS (ESI) m/z 165.2 (M++1).</p><p>Trityl chloride (3.34g, 12mmol), triethylamine (2.5mL, 25mmol) and the alcohol 3 (1.64g, 10 mmol) was added to CH2Cl2 (100mL). The reaction mixture was stirred at room temperature (rt) overnight. The reaction mixture was then washed twice with 5% NaHCO3 (60 mL), followed by saturated NaCl solution (50 mL). The organic layer was dried over Na2SO4, solvents were removed by evaporation, and the residue was purified on silica column (ethyl acetate-hexanes, 0 to 10%) to yield the pure product, methyl 2-(3-(trityloxy)propylthio)acetate 4 (3.82 g, 9.4 mmol, 94%).</p><p> </p><p>1H NMR (CDCl3) δ 7.41-7.45 (m, 6H), 7.20-7.33 (m, 9H), 3.71 (s, 3H), 3.20 (s, 2H), 3.16 (t, J = 6.0 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 1.89 (m, 2H); 13C NMR (CDCl3) δ 171.0 (s), 144.2 (s), 128.6 (s), 127.8 (s), 126.9 (s), 86.5 (s), 62.0 (s), 52.4 (s), 33.4 (s), 29.7 (d); MS (ESI) m/z 429.3 (M+Na+).</p><p>The sulfide 4 (24.2 g, 59.7 mmol, 1 equiv.) was dissolved in CH2Cl2 (300 mL) and m-CPBA (25.7 g, 149 mmol, 2.5 equiv.) was added at 0 °C. The reaction mixture was allowed warm up to rt and stirred for 8 h at rt. Volatiles were evaporated under reduced pressure. The residue was taken up in ethyl acetate (200 mL) and washed with 10% Na2SO3, saturated NaHCO3, and brine. The organic layer was then dried over anhydrous Na2SO4. The volatiles were evaporated under reduced pressure to yield the crude mixture that was purified on silica column (ethyl acetate-hexanes, 0 to 50%) to yield the pure product, methyl 2-(3-(trityloxy)propylsulfonyl)acetate 5 (24.8 g, 56.7 mmol, 95%).</p><p> </p><p>1H NMR (CDCl3) δ 7.41-7.51 (m, 6H), 7.20-7.38 (m, 9H), 3.97 (s, 2H), 3.81 (s, 3H), 3.38-3.46 (m, 2H), 2.29 (t, J = 5.6 Hz, 2H), 3.20 (s, 2H), 2.05-2.21 (m, 2H); 13C NMR (CDCl3) δ 163.5 (s), 143.9 (s), 128.6 (s), 128.0 (d), 127.9 (s), 127.2 (s), 86.9 (s), 61.3 (s), 57.2 (s), 53.4 (s), 51.3 (s), 22.9 (s); MS (ESI) m/z 439.3 (M++H).</p><p>To a solution of 3.7 g (6.41 mmol, 1 equiv.) of intermediate 5 in 20 mL of DMF was added sodium hydride (60% in mineral oil, 400 mg, 9.7 mmol, 1.15 equiv.). The reaction mixture was then stirred at rt for 1 h before 3.82 g (10.2 mmol, 1.2 equiv.) oleyl iodide in 10 mL DMF was added. The mixture was stirred additional 4 h before 1 N HCl (100 mL) was added to quench the reaction, and the mixture was extracted with 100 mL of ethyl ether. The combined organic extracts were washed with saturated NaHCO3 solution, followed by a saturated NaCl solution. Solvents were removed at reduced pressure and the crude mixture was purified on silica column (ethyl acetate-hexanes, 0 to 20%) to give clear oil (Z)-methyl 2-(3-(trityloxy)propylsulfonyl)eicos-11-enoate 6a. (3.18 g, 4.61 mmol, 72%).</p><p> </p><p>1H NMR (CDCl3) δ 7.41-7.51 (m, 6H), 7.20-7.38 (m, 9H), 5.31-5.39 (m, 2H), 3.83 (s, 3H), 3.76-3.81 (m, 1H), 3.15-3.32 (m, 4H), 1.99-2.18 (m, 6H), 1.20-1.40 (m, 26H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 167.0 (s), 143.8 (s), 130.0 (s), 129.8 (s), 128.6 (s), 127.9 (d), 127.1 (s), 86.8 (s), 68.7 (s), 61.5 (s), 53.2 (s), 48.6 (s), 31.9 (s), 29.1-29.8 (m), 27.2 (m), 26.2 (s), 22.7 (s), 22.1 (s), 14.2 (s); MS (ESI) m/z 711.6 (M+Na+).</p><p>A similiar procedure for 6a was followed, using hexadecyl iodide, to give methyl 2-(3-(trityloxy)propylsulfonyl)octadecanoate 6b (6.1 g, 9.2 mmol, 88%).</p><p> </p><p>1H NMR (CDCl3) δ 7.40-7.51 (m, 6H), 7.21-7.39 (m, 9H), 3.81 (s, 3H), 3.76-3.81 (m, 1H), 3.21-3.46 (m, 4H), 2.14-2.23 (m, 4H), 1.60-1.67 (m, 2H), 1.20-1.40 (m, 26H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 167.0 (s), 143.8 (s), 128.6 (s), 127.8 (d), 127.1 (s), 86.8 (s), 68.7 (s), 61.5 (s), 53.2 (s), 48.6 (s), 31.9 (s), 29.1-29.8 (m), 27.2 (m), 26.2 (s), 22.7 (s), 22.1 (s), 14.2 (s); MS (ESI) m/z 685.3 (M+Na+).</p><p>To an ice-cooled solution of 800 mg (1.16 mmol, 1 equiv.) of 6a in 15 mL of THF was added 44 mg (1.16 mmol, 1.0 equiv.) of lithium aluminium hydride. The reaction mixture was then stirred at 0 °C for 10 min. Next, 20 mL water was added dropwise to quench the reaction, and the mixture was extracted twice with 50 mL of ethyl acetate. The combined organic extracts were washed with 1 N HCl, saturated NaHCO3 solution, and saturated NaCl solution. Solvents were removed at reduced pressure and the crude mixture was purified on silica column (ethyl acetate-hexanes, 5 to 25%) to give the product (Z)-2-(3-(trityloxy)propylsulfonyl)eicos-11-en-1-ol 7a (390 mg, 0.59 mmol, 51%).</p><p> </p><p>1H NMR (CDCl3) δ 7.41-7.51 (m, 6H), 7.20-7.38 (m, 9H), 5.31-5.39 (m, 2H), 3.93-4.06 (m, 2H), 3.24 (t, J = 6.0 Hz, 2H), 3.11-3.17 (m, 2H), 2.92-3.00 (m, 1H), 2.57 (t, J = 6.0 Hz, 2H), 2.16-2.20 (m, 2H), 1.95-2.04 (m, 4H), 1.82-1.92 (m, 1H), 1.61-1.71 (m, 1H), 1.45-1.54 (m, 1H), 1.20-1.40 (m, 22H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 143.8 (s), 130.0 (s), 129.8 (s), 128.5 (s), 127.9 (s), 127.1 (s), 86.8 (s), 63.8 (s), 61.5 (s), 59.5 (s), 49.5 (s), 31.9 (s), 29.1-29.8 (m), 27.2 (d), 26.9 (s), 24.3 (s), 22.7 (s), 22.3 (s), 14.1 (s); MS (ESI) m/z 683.6 (M+Na+).</p><p>Similarly, 6b was reduced, worked up, and purified to give (trityloxy)propylsulfonyl)octadecan-1-ol 7b (2.45 g, 3.86 mmol, 34%).</p><p> </p><p>1H NMR (CDCl3) δ 7.40-7.51 (m, 6H), 7.21-7.38 (m, 9H), 3.93-4.06 (m, 2H), 3.24 (t, J = 6.0 Hz, 2H), 3.11-3.17 (m, 2H), 2.92-3.00 (m, 2H), 2.57 (t, J = 6.0 Hz, 2H), 2.16-2.20 (m, 2H), 1.82-1.92 (m, 1H), 1.61-1.71 (m, 1H), 1.20-1.40 (m, 26H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 143.8 (s), 128.5 (s), 127.9 (s), 127.1 (s), 86.8 (s), 63.8 (s), 61.5 (s), 59.5 (s), 49.5 (s), 31.9 (s), 29.1-29.8 (m), 27.2 (d), 26.9 (s), 24.3 (s), 22.7 (s), 22.3 (s), 14.1 (s); MS (ESI) m/z 657.5 (M+Na+).</p><p>To a solution of 330 mg (0.6 mmol, 1 equiv.) of 7a in 5 mL of CH2Cl2 was added 0.4 mL of trifluoroacetic acid. Next, the reaction mixture was stirred at rt for 30 min; 30 mL saturated NaHCO3 solution was added to quench the reaction, and the mixture was extracted with three portions of 50 mL of ethyl acetate. The organic solvents were removed at reduced pressure and the crude mixture was purified on silica column (ethyl acetate-hexanes, 50 to 100%) to give the product (Z)-2-(3-hydroxypropylsulfonyl)eicos-11-en-1-ol 8a (203 mg, 0.49 mmol, 81%).</p><p> </p><p>1H NMR (CDCl3) δ 5.31-5.39 (m, 2H), 3.93-4.06 (m, 2H), 3.81 (t, J = 6.0 Hz, 2H), 3.22 (t, J = 7.2 Hz, 2H), 2.95-3.03 (m, 1H), 2.07-2.17 (m, 2H), 1.88-2.12 (m, 6H), 1.61-1.71 (m, 1H), 1.45-1.54 (m, 1H), 1.20-1.40 (m, 24H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 130.0 (s), 129.8 (s), 64.5 (s), 60.7 (s), 59.6 (s), 49.0 (s), 31.9 (s), 29.1-29.8 (m), 27.2 (d), 26.9 (s), 24.3 (s), 22.7 (s), 14.1 (s); MS (ESI) m/z 419.4 (M++1).</p><p>Similarly, 7b was deprotected was to give 2-(3-hydroxypropylthio)octadecan-1-ol 8b (580 mg, 1.61 mmol, 66%).</p><p> </p><p>1H NMR (CDCl3) δ 3.93-4.06 (m, 2H), 3.80 (t, J = 6.0 Hz, 2H), 3.21 (t, J = 7.6 Hz, 2H), 2.95-3.03 (m, 1H), 2.07-2.17 (m, 2H), 1.88-2.12 (m, 2H), 1.61-1.71 (m, 1H), 1.45-1.54 (m, 1H), 1.20-1.40 (m, 26H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 64.5 (s), 60.7 (s), 59.6 (s), 49.0 (s), 31.9 (s), 29.1-29.8 (m), 27.2 (d), 26.9 (s), 24.3 (s), 22.7 (s), 14.1 (s); MS (ESI) m/z 361.4 (M++1).</p><p>The next protocols involve reaction of both primary alcohols and selective elimination to afford a vinyl sulfone in two steps. To a solution of 3.31 g (7.92 mmol, 1 equiv.) of 8a and diisopropyl ethylamine (5.1 g, 39.4 mmol, 5 equiv.) in 40 mL of CH2Cl2 was added mesyl chloride (2.74 g, 19.0 mmol, 2.4 equiv.). The reaction mixture was then stirred at rt for 2 h. The precipiate was removed by filtration, and the filtrate was washed with saturated NaHCO3 solution and dried over sodium sulfate. The organic solvent was concentrated and the residue was dissolved into acetone (30 mL) followed by NaI (3.6 g, 16 mmol, 3 equiv.) and NaHCO3 (1.0 g, 11.9 mmol, 2.2 equiv.). The reaction mixture was stirred at rt overnight, filtered out and concentrated by evaporation; then, 50 mL water and 50 mL ethyl acetate was added to the residue. The organic layer was collected, the aqueous layer was re-extracted twice with ethyl acetate, and the combined organics were dried over sodium sulfate and then removed at reduced pressure. Next, 100 mL ethyl acetate was added, followed by diisopropylethylamine (5 mL). The reaction mixture was stirred at rt for 3 days. The organic solvent was removed at reduced pressure and the crude mixture was purified on silica column (ethyl acetate-hexanes, 0 to 15%) to give the product (Z)-2-(3-iodopropylsulfonyl)eicosa-1,11-diene 9a (3.27 g, 6.42 mmol, 81%).</p><p> </p><p>1H NMR (CDCl3) δ 6.25 (s, 1H), 5.83 (s, 1H), 5.31-5.40 (m, 2H), 3.28 (t, J = 6.4 Hz, 2H), 3.07 (t, J = 7.6 Hz, 2H), 2.41 (t, J = 7.6 Hz, 2H), 2.22-2.30 (m, 2H), 1.94-2.06 (m, 4H), 1.56-1.66 (m, 2H), 1.20-1.40 (m, 22H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 146.0 (s), 126.9 (s), 126.6 (s), 122.0 (s), 64.5 (s), 49.7 (s), 28.8 (s), 26.1-26.6 (m), 25.9 (s), 24.6 (s), 24.1 (d), 23.0 (s), 19.6 (s), 11.0 (s); MS (ESI) m/z 533.3 (M+Na+).</p><p>A similiar two-step procedure was employed using 8b as the starting material to give 2-(3-iodopropylsulfonyl)octadec-1-ene 9b (198 mg, 0.41 mmol, 45%).</p><p> </p><p>1H NMR (CDCl3) δ 6.18 (s, 1H), 5.77 (s, 1H), 3.22 (t, J = 6.4 Hz, 2H), 3.02 (m, 2H), 2.34 (t, J = 7.6 Hz, 2H), 2.14-2.23 (m, 2H), 1.50-1.58 (m, 2H), 1.56-1.66 (m, 2H), 1.20-1.40 (m, 24H), 0.81 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 145.6 (s), 122.0 (s), 49.5 (s), 28.7 (s), 26.0-26.4 (m), 25.8 (s), 24.4 (s), 22.9 (d), 19.5 (s), 10.9 (s); MS (ESI) m/z 507.3 (M+Na+).</p><p>Next, the quaternary ammonium derivatives were introduced. To a solution of 81 mg (0.16 mmol, 1 equiv.) of 9a in 5 mL ethanol + 1 mL methylene chloride was added trimethylamine in ethanol. Then the reaction mixture was stirred at rt for 4 days. The resulting mixture was concentrated and then dissolved into 0.5 mL ethanol followed by adding 10 mL diethyl ether. The white solid precipiated and the white solid was collected by filtration to give the final product, which was passed through a chloride ion exchange resin to convert to (Z)-3-(eicosa-1,11-dien-2-ylsulfonyl)-N,N,N-trimethylpropan-1-aminium chloride CVS-18 (51 mg, 0.11 mmol, 71%).</p><p> </p><p>1H NMR (CDCl3) δ 6.26 (s, 1H), 5.88 (s, 1H), 5.85 (s, 1H), 5.31-5.40 (m, 2H), 3.98-4.40 (m, 2H), 3.59 (s, 3H), 3.45 (s, 9H), 3.18 (t, J = 6.8 Hz, 2H), 2.84 (d, J = 6.8 Hz, 2H), 2.34-2.43 (m, 4H), 1.57-1.62 (m, 2H), 1.32-1.42 (m, 22H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 149.1 (s), 130.0 (s), 129.7 (s), 125.6 (s), 64.5 (s), 53.7 (s), 52.0 (s), 48.6 (s), 44.9 (s), 31.9 (s), 29.0-29.7 (m), 27.7 (s), 27.2 (d), 22.6 (d), 16.5 (s), 14.1 (s); MS (ESI) m/z 442.5 (M+).</p><p>A similiar procedure for CVS-18 was used to give N,N,N-trimethyl-3-(octadec-1-en-2-ylsulfonyl)propan-1-aminium chloride CVS-16 (21 mg, 0.05 mmol, 85%).</p><p> </p><p>1H NMR (CD3OD) δ 6.25 (s, 1H), 5.98 (s, 1H), 3.48-4.55 (m, 2H), 3.26 (s, 2H), 3.16-3.22 (m, 11H), 2.45 (t, J = 7.6 Hz, 2H), 2.18-2.28 (m, 2H), 1.60-1.67 (m, 2H), 1.32-1.42 (m, 24H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR (CD3OD) δ 149.2 (s), 125.1 (s), 64.3 (s), 52.2 (m), 48.4 (s), 31.7 (s), 29.0-29.4 (m), 28.8 (s), 27.6 (s), 22.3 (d), 16.2 (s), 13.0 (s); MS (ESI) m/z 416.5 (M+).</p><p>Modified protocols were required to prepare the two shorter chain analogs. Thus, bromo ester 12 (12.06 g, 36.0 mmol) was dissolved in acetone (200 mL), K2CO3 (7.45 g, 54.0 mmol, 1.5 equiv.) was added, followed by of methyl 2-mercaptoacetate 11 (4.2 g, 39.5 mmol, 1.1 equiv.) and heated at reflux for 2 h. After cooling to room temperature the mixture was filtered and the white solid was washed twice with 25 mL acetone. Combined organic solvents were evaporated under reduced pressure, and the residue was purified on silica column (ethyl acetate-hexanes, 15 to 40%) to yield the pure product ethyl 2-(3-methoxy-3-oxopropylthio)tetradecanoate 13a (12.0 g,32.0 mmol, 89%).</p><p> </p><p>1H NMR (CDCl3) δ 4.14-4.25 (m, 2H), 3.69 (s, 3H), 3.20-3.26 (m, 1H), 2.81-2.92 (m, 2H), 2.56-2.64 (m, 2H), 1.81-1.92 (m, 2H), 1.60-1.68 (m, 2H), 1.21-1.48 (m, 21H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 172.7 (s), 172.1 (s), 61.1 (s), 51.7 (s), 46.8 (s), 34.4 (s), 31.9 (s), 31.4 (s), 29.2-29.6 (m), 27.3 (s), 26.2 (s), 22.7 (s), 14.2 (s), 14.1 (s); MS (ESI) m/z 375.4 (M++1).</p><p>A similiar procedure was followed with the shorter bromo ester 12 to give ethyl 2-(3-methoxy-3-oxopropylthio)octanoate 13b (3.51 g, 12.1 mmol, 93%).</p><p> </p><p>1H NMR (CDCl3) δ 4.14-4.25 (m, 2H), 3.69 (s, 3H), 3.20-3.26 (m, 1H), 2.80-2.92 (m, 2H), 2.56-2.64 (m, 2H), 1.81-1.92 (m, 2H), 1.60-1.68 (m, 2H), 1.21-1.48 (m, 9H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 172.7 (s), 172.0 (s), 61.1 (s), 51.7 (s), 46.8 (s), 34.4 (s), 31.5 (s), 31.3 (s), 28.8 (s), 27.3 (s), 26.2 (s), 22.5 (s), 14.2 (s), 14.0 (s); MS (ESI) m/z 291.3 (M++1).</p><p>As for the two long chain analogs, the bis primary alcohols were prepared, in this case by reducing both esters simultaneously. Thus, to an ice-cooled solution of 3.8 g (13.1 mmol, 1 equiv.) of 13a in 100 mL of THF was added 2.0 g (52.6 mmol, 4.0 equiv.) of lithium aluminum hydride. Then the reaction mixture was stirred at 0 °C for 10 min. Then the reaction temperature was raised to 60 °C overnight, then cooled down to rt. The resulting mixture was filtered, and concentrated before 100 mL ethyl acetate was added. The organic layer was washed with 1 N HCl, saturated NaHCO3 solution, followed by a saturated NaCl solution. Solvent was removed at reduced pressure and the crude mixture was purified on silica column (ethyl acetate-hexanes, 25 to 50%) to give the product 2-(3-hydroxypropylthio)tetradecan-1-ol 14a (2.6 g, 11.8 mmol, 90%).</p><p> </p><p>1H NMR (CDCl3) δ 3.76 (t, J = 6.0 Hz, 2H), 3.55-3.60 (m, 1H), 3.46-3.52 (m, 1H), 2.58-2.76 (m, 3H), 2.00 (s, 2H), 1.81-1.89 (m, 2H), 1.45-1.62 (m, 3H), 1.21-1.44 (m, 19H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 63.9 (s), 61.5 (s), 49.6 (s), 32.4 (s), 31.9 (s), 31.8 (s), 29.3-29.6 (m), 27.1 (s), 26.7 (s), 22.7 (s), 14.1 (s); MS (ESI) m/z 327.3 (M+Na+).</p><p>A similiar procedure to reduce 13b to give 2-(3-hydroxypropylthio)octan-1-ol 14b (1.87 g, 8.51 mmol, 88%).</p><p> </p><p>1H NMR (CDCl3) δ 3.76 (t, J = 6.0 Hz, 2H), 3.46-3.52 (m, 1H), 2.58-2.76 (m, 3H), 2.03 (s, 2H), 1.81-1.89 (m, 2H), 1.45-1.62 (m, 3H), 1.21-1.44 (m, 8H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 63.9 (s), 61.5 (s), 49.6 (s), 32.4 (s), 31.8 (s), 31.7 (s), 29.1 (s), 27.1 (s), 26.7 (s), 22.6 (s), 14.0 (s); MS (ESI) m/z 203.3 (M+-OH).</p><p>The corresponding sulfide 14a (10.6 g, 34.8 mmol, 1 equiv.) was dissolved in CH2Cl2 (180 ml) and m-CPBA (15.0 g, 87.0 mmol, 2.5 equiv.) was added to it at 0 °C (ice bath). The reaction mixture was allowed warm to rt and stirred for 8 h at rt. Volatiles were evaporated under reduced pressure. The residue was taken up in ethyl acetate (150 mL) and washed with 10% Na2SO3, saturated NaHCO3, and brine. The organic layer was then dried over anhydrous Na2SO4. The volatiles were evaporated under reduced pressure to yield the crude mixture that was purified on silica column (ethyl acetate-hexanes, 30 to 80%) to yield the pure product 2-(3-hydroxypropylsulfonyl)tetradecan-1-ol 15a. (9.0 g, 26.8 mmol, 77%)</p><p> </p><p>1H NMR (CDCl3) δ 3.90-4.05 (m, 2H), 3.79 (t, J = 6.0 Hz, 2H), 3.22 (t, J = 7.6 Hz, 2H), 2.94-3.02 (m, 1H), 2.49 (s, 2H), 2.06-2.15 (m, 2H), 1.84-1.95 (m, 1H), 1.52-1.72 (m, 1H), 1.45-1.55 (m, 1H), 1.22-1.40 (m, 19H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 64.6 (s), 60.6 (s), 59.6 (s), 49.4 (s), 31.4 (s), 29.3-29.6 (m), 26.9 (s), 24.3 (s), 24.2 (s), 22.5 (s), 14.0 (s); MS (ESI) m/z 337.3 (M++H).</p><p>Sulfide 14b was oxidized as described above for sulfide 14a to give methyl 2-(3-hydroxypropylsulfonyl)octan-1-ol 15b (1.79 g, 7.1 mmol, 81%).</p><p> </p><p>1H NMR (CDCl3) δ 3.90-4.05 (m, 2H), 3.75 (t, J = 6.0 Hz, 2H), 3.21 (t, J = 7.6 Hz, 2H), 2.94-3.02 (m, 1H), 2.82 (s, 2H), 2.03-2.11 (m, 2H), 1.84-1.95 (m, 1H), 1.58-1.69 (m, 1H), 1.42-1.52 (m, 1H), 1.22-1.40 (m, 7H), 0.86 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 64.6 (s), 60.6 (s), 59.6 (s), 49.4 (s), 31.4 (s), 29.0 (s), 26.9 (s), 24.3 (s), 24.2 (s), 22.5 (s), 14.0 (s); MS (ESI) m/z 253.3 (M++H).</p><p>As above, the vinyl sulfones were formed and primary iodide introduced in a two-step protocol. Thus, to a solution of 2.0 g (7.92 mmol, 1 equiv.) of 15b and diisopropyl ethylamine (5.1g, 39.4 mmol, 5 equiv.) in 40 mL of CH2Cl2 was added mesyl chloride (2.74 g, 19.0 mmol, 2.4 equiv.). Then the reaction mixture was stirred at rt for 2 h. The mixture was filtered, and the filtrate was washed with saturated NaHCO3 solution and then dried over sodium sulfate. The organic solvent was concentrated. The residue was dissolved into acetone (30 mL) followed by NaI (3.6 g, 16 mmol, 3 equiv.) and NaHCO3 (1.0 g, 11.9 mmol, 2.2 equiv.). The reaction mixture was stirred at rt overnight, filtered, and concentrated. Then, 50 mL water and 50 mL ethyl acetate were added to the residue, and the organic layer was collected and the aqueous layer re-extracted twice with ethyl acetate. The combined organics were dried over sodium sulfate and then removed at reduced pressure. Next, 100 mL ethyl acetate was added followed by diisopropylethylamine (5 mL). The reaction mixture was stirred at room temperature for 3 days. The organic solvent was removed at reduced pressure and the crude mixture was purified on silica column (ethyl acetate-hexanes, 0 to 15%) to give the product 2-(3-iodopropylsulfonyl)oct-1-ene 16b (2.23 g, 6.49 mmol, 82%).</p><p> </p><p>1H NMR (CDCl3) δ 6.25 (s, 1H), 5.83 (s, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2H), 2.41 (t, J = 7.6 Hz, 2H), 2.21-2.30 (m, 2H), 1.52-1.63 (m, 2H), 1.25-1.42 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 145.9 (s), 122.0 (s), 49.7 (s), 28.3 (s), 26.6 (s), 25.6 (s), 24.6 (s), 23.1 (s), 19.4 (s), 10.9 (s); MS (ESI) m/z 367.2 (M+Na+)</p><p>A similiar procedure to convert 15a to 2-(3-iodopropylsulfonyl)tetradec-1-ene 16a (346 mg, 0.81 mmol, 69%).</p><p> </p><p>1H NMR (CDCl3) δ 6.23 (s, 1H), 5.81 (s, 1H), 3.28 (t, J = 6.4 Hz, 2H), 3.07 (t, J = 7.6 Hz, 2H), 2.40 (t, J = 7.6 Hz, 2H), 2.21-2.30 (m, 2H), 1.52-1.63 (m, 2H), 1.25-1.42 (m, 18H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 145.9 (s), 122.0 (s), 49.7 (s), 28.7 (s), 26.1-26.6 (m), 25.9 (s), 24.6 (s), 23.0 (s), 19.5 (s), 11.0 (s); MS (ESI) m/z 451.3 (M+Na+).</p><p>A procedure similar to that described above for CVS-18 was used to obtain N,N,N-trimethyl-3-(tetradec-1-en-2-ylsulfonyl)propan-1-aminium chloride CVS-12 (17a, 23 mg, 0.058 mmol, 58%).</p><p> </p><p>1H NMR (CD3OD) δ 6.25 (s, 1H), 5.98 (s, 1H), 3.48-3.56 (m, 2H), 3.27 (s, 2H), 3.16-3.22 (m, 11H), 2.45 (t, J = 7.6 Hz, 2H), 2.20-2.28 (m, 2H), 1.60-1.66 (m, 2H), 1.26-1.44 (m, 16H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR (CD3OD) δ 149.2 (s), 125.1 (s), 64.3 (s), 52.2 (m), 31.4 (s), 28.8-29.3 (m), 27.7 (s), 26.7 (s), 22.3 (s), 16.2 (s), 13.0 (s); MS (ESI) m/z 360.3 (M+).</p><p>A procedure similar to that described above for CVS-18 was used to provide N,N,N-trimethyl-3-(oct-1-en-2-ylsulfonyl)propan-1-aminium chloride CVS-6 (17b, 11 mg, 0.035 mmol, 72%).</p><p> </p><p>1H NMR (CDCl3) δ 6.28 (s, 1H), 5.88 (s, 1H), 4.05-4.13 (m, 2H), 3.43 (s, 9H), 3.22 (t, J = 6.8 Hz, 2H), 2.38-2.46 (m, 4H), 1.55-1.65 (m, 2H), 1.28-1.42 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 149.1 (s), 125.8 (s), 54.1 (s), 48.3 (s), 31.5 (s), 29.6 (s), 28.6 (s), 27.7 (s), 22.5 (s), 16.6 (s), 14.0 (s); MS (ESI) m/z 276.4 (M+).</p><!><p>ATX inhibition was performed using an ATX Inhibitor Screening Kit (K-4200, Echelon Biosciences, Inc. Salt Lake City, UT). Stock solutions (1 mM) of each compound were made in DMSO and then diluted with water to the appropriate experimental concentration. Fourteen final inhibitor concentrations from 0.1 to 10,000 nM were employed to calculate Ki values. DMSO was spiked into each reaction mixture to equalize vehicle concentrations. The assay employs the fluorescence-quenched, ATX analogue FS-3 as the ATX substrate 25 and recombinant, human ATX purified from Sf9 insect cells as the enzyme source. Compounds were pre-incubated with the enzyme at 25° C for 10 minutes, after which FS-3 was added. The rate of fluorescence increase was measured between 5 and 25 minutes after substrate addition. In all circumstances fluorescence increase was linear during this time window. Rates were normalized to control reactions that contained all reaction components except test compound.</p><!><p>ATX was incubated with compound 10b or 21 (0, 1 or 10 µM) for two minutes in 50 mM Tris-Cl pH 8.0, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 140 mM NaCl, 1 mg/ml Fatty Acid Free BSA. Half of each reaction was placed in a centrifugal filtration device (Millipore) with a molecular weight cutoff of 30 kDa and subjected to repeated rounds of concentration and dilution using buffer absent of test compound. ATX activity was then assessed in each sample by monitoring FS-3 (1 µM) fluorescence increase over time and compared to the activity of ATX not incubated with test compound or subjected to dialysis.</p><!><p>Cell lines were acquired from the American Type Culture Collection or ATCC (Manassas, VA). Human cancer cell lines SKOV-3, OVCAR-3, PC-3 and MDA-MB-231 cells were maintained in RPMI (Mediatech Inc., Manassas, VA) supplemented with 10% fetal bovine serum or FBS (Sigma, St. Louis, MO). MeWo fibroblast malignant melanoma cells were cultured similarly, but supplemented with only 5% FBS. HT-29 human colon cancer cells and SB-2 human non-metastatic melanoma cells were maintained in DMEM (Mediatech) supplemented with 10% fetal bovine serum.</p><!><p>Approximately 5,000 cells were grown in each well of a 96-well plate for 24 h prior to the addition of CVS-16 (10b) compound at the indicated concentrations for 48 h. The assay was then performed by removing medium from the 96-well plate and replacing it with serum free media containing CellTiter-Blue® reagent (Promega Corporation, Madison, WI) and incubating for 4-6 h at 37 °C. Then the fluorescent absorbance was measured with a microplate reader, the SpectraMax M2 model (Molecular Devices, Sunnyvale, CA).</p><!><p>Cells were seeded in triplicates in 24-well plates and then grown to confluence. 'Wounds' were then created using a pipette tip. The wells were then repeatedly rinsed using serum-free medium followed by the addition of 18:1 LPA (Avanti Polar Lipids, Alabaster, AL) or CVS-16 (10b) to the wells and incubated for 24 h. Photomicrographs were taken of the wells, at least six per well, and treatments were performed in triplicate. The distance between confluent monolayers was measured.</p><!><p>Six-week old female athymic nude mice acclimated to the animal facility for one week prior to the study commencement. Animals were anesthetized before tumor cell injection into their right flank with Glycosan Extracel® (BioTime, Inc, Alameda, CA) containing approximately 1×106 MeWo cells per 0.15 mL injection. Extracel® was used in this study over traditional Matrigel to eliminate potential interference of exogenous mouse growth factors which could have affected our in vivo study. In addition, Glycosan Extracel® allows us to conserve resources through the achievement of a 100% tumor efficiency rate, whereby this rate is typically unachievable otherwise. Injected mice were measured for tumor formation, tumor volume, body weight and body conditioning scores. After three weeks, 100% of mice displayed tumor formation at which time they were randomized into treatment groups; PBS control (n=10), CVS-16 (10b) at 20 mg/kg (n=5) or 50 mg/kg (n=5), DMF control (n=10), HA-130 at 30 mg/kg (n=5) and PF-8380 at 30 mg/kg (n=5) dose per 0.1 mL injection. Mice in each treatment group were anesthetized (2-4% isofluorane) before i.p. treatment injections three times a week over the course of the 65-day study. The animals were euthanized according to the animal use protocol approved by the University of Georgia IACUC committee. The tumor volume (mm3) was calculated using the equation: tumor volume = (width)2 × length/2, and then graphed using GraphPad Prism (La Jolla, CA). The tumor volume for each group and overall significance was plotted.</p><!><p>Melanoma tumors were dissected from the right flank of athymic nude mice and flash-frozen with cryomatrix (Thermo Fisher Scientific Inc, Waltham, MA) in 2-methylbutane (Sigma) and cooled to -140°C. Cryopreserved tumors were cut in 9 μm sections using a cryostat (Thermo Fisher Scientific) and then mounted on microscope slides. Hematoxylin and eosin staining was performed according to standard protocols and processed for pathological evaluation.</p><p>For tissues stained with antibodies specific for immunofluorescence, tissue sections first were blocked for 30 min in 5% donkey serum (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) diluted in phosphate-buffered saline (PBS) for 30 min at room temperature. The tumor sections were then incubated in primary antibodies at 4°C overnight using a humidity chamber. The following day slides were washed with PBS and the primary antibody was detected using a fluorescent secondary antibody. After washing the slides again with PBS, they were mounted with Permount (Thermo Fisher Scientific) and imaged using an X71 inverted microscope (Olympus, Center Valley, PA) at 20x magnification. Overlapping pictures were aligned to generate an image of an entire tumor cryosection. The tumor area was automatically calculated using the polygon tool in Image-Pro Precision software (Media Cybernetics, Inc., Rockville, MD), then the Ki-67 pixel intensity was automatically determined. The average pixel staining intensity per area measurement was plotted.</p><!><p>Whole blood was collected from animals at necropsy and placed into a Becton Dickinson Microtainer® tube with serum separator additive and allowed to clot for at least 20 min in a vertical position at room temperature. The tubes were then inverted five times prior to centrifugation at 2000 RCF for 10 min at room temperature. The serum was transferred to a glass vial and stored in −80°C until use. Approximately 200 μl of serum was obtained from each mouse. From that, 100 μl of serum was analyzed for cytokines, chemokines, growth factors and interleukins using the mouse Bio-Plex, 23-Plex panel (Bio-Rad, Hercules, CA) and following the instructions provided by the manufacturer. The assay plates were measured using the Bio-Plex Multiplex Suspension Array system (Bio-Rad). Serum samples were measured via comparison to an 8-point standard dilution series included as an integral component of the high-throughput assay. For analysis of ATX, the remaining 100 μl of serum was used processed using the mouse ectonucleotide pyrophosphatase/phosphodiesterase family member 2 (ENPP2) ELISA kit according to the instructions provided by the manufacturer (MyBioSource.com, San Diego, CA).</p><!><p>The statistical differences were analyzed using an analysis of variance (ANOVA) test, followed by Bonferroni's multiple comparison test between groups using GraphPad Prism. When comparing only two groups, the Student's t-test was used. Where it is indicated in the figures, *p < 0.05 **p < 0.01 and ***p < 0.001 indicate the levels of significance.</p><!><p>Although palladium or copper-promoted reaction of sulfinic acids with alkyl halides or triflates can provide vinyl sulfones in good yield 26, the harsh conditions (80 °C) were not appropriate for these analogs. Our strategy for the synthesis of desired targets involves conversion of the key intermediate β-hydroxy sulfones 8 to vinyl sulfones 9. In addition, the quaternary ammonium salts 10 and 17 were prepared in the final step to avoid difficulties in purification of reactive electrophilic species.</p><p>Two different synthetic routes were carried out, based on the availability of the starting materials. The vinyl sulfone analogues with the 16:0 and 18:1 alkyl chains were prepared as shown in Figure 1. We selected these chain lengths based on the known bioactivity of LPA agonists for the LPARs, with the expectation that these would be the best probes for the ATX active site. The formation of the thioether 3 could be performed by displacement of the bromide anion 1 with bromide in good yield (98%). The primary hydroxyl group was then tritylated (TrCl, Et3N) to give compound 4. Conversion of sulfide 4 to sulfone 5 was accomplished by oxidation with m-CPBA in dichloromethane. In order to alkylate compound 5 regioselectively, we chose sodium hydride as the base so that the reaction could be performed at room temperature in moderate yield; the regioisomer was not detected.</p><p>Next, the reduction of ester and removal of the trityl group was carried out to give the diol 8. However, preliminary attempts to reduce the ester were unsuccessful. An unexpected over-reduction of hydroxyl to methyl was observed using either excess lithium aluminum hydride or sodium borohydride. Possible reasons for the over-reduction include excess reductant, long reaction time, reaction temperature, or the proximity of the β-sulfone moiety, but this was not further explored. After optimization, moderate yields of reduction (34-51%) could be obtained by using 1 equiv. of lithium aluminum hydride in THF at 0 °C for 10 min. The conversion of diols 8 to vinyl sulfone 9 involved a three-step sequence, although no intermediates need to be isolated. Conversion of 8 to the corresponding mesylate and subsequent iodination afforded the iodo intermediates. Next, the β-iodo sulfones were β-dehydroiodinated by treatment with diisopropylethylamine (DIPEA) in EtOAc to give the desired vinyl sulfones. importantly, γ-iodoalkyl groups were stable in the presence of DIPEA, which allowed us to install the quaternary ammonium derivative in a subsequent step. Thus, the corresponding quaternary ammonium salts could be obtained by treating the remaining iodoalkyl group with trimethylamine in a mixture of ethanol and dichloromethane for 4 days. Recrystallization of crude salts from CH3OH-Et2O provided the final targets 10 (CVS-18 and CVS-16) as white solids. (Figure 1)</p><p>In an effort to synthesize the shorter chain vinyl sulfone, which we hypothesized would be more water-soluble and still retain ATX inhibitory activity, we took the advantage of commercially available α-bromo esters 12, which reacted with thiol directly to give corresponding sulfide (Figure 2). Excess lithium aluminum hydride was employed to reduce ester groups to diols 14. Oxidation with m-CPBA in dichloromethane at 0 °C converted the sulfides 14a,b to sulfones 15a,b in good yield. Then final targets 17a and 17b (CVS-6 and CVS-12) were prepared from sulfones 15a, b in an analogous fashion described previously for the structurally-similar analogue 10a. (Figure 2)</p><p>A non-choline-like head vinyl sulfone 21 (PVS-16) that retained the 16:0 alkyl chain was synthesized as a negative control. Alkylation of commercially available compound 18 by using sodium hydride (60 % in mineral oil) as base proceeded in high yield (95 %). After the hydrophobic chain was installed, phenyl vinyl sulfone compound 21 could be achieved by mesylation/iodination and elimination as employed above. (Figure 3)</p><!><p>The analysis of the kinetic inhibition of ATX demonstrated that the enzymatic activity decreased in a time-dependent manner. However, the time course of inhibition is very rapid since pre-incubating ATX with the compound CVS-16 (10b) for just 2 minutes results in complete inhibition. Different hydrophobic chain lengths were synthesized to study the structure-activity relationship of the compound. The inhibition of ATX by the vinyl sulfone ATX analogues CVS-18 (10a) and CVS-16 (10b) was tested using an ATX inhibitor screening kit (Echelon Biosciences, Inc.) in a dose-response mode (Table 1). ATX activity was measured by the hydrolysis of the fluorogenic ATX analogue FS-3, which has a KM value of 6.3 μM 25. The results showed that all analogues except the shorter-chain analogue CVS-6 (17b) inhibited ATX effectively, with C18:1 ATX showing the greatest inhibition 27. Non-c quaternary ammonium head group vinyl sulfone PVS-16 (21) showed no binding effect at the highest test concentration, which reveals the importance of quaternary ammonium head group for achieving the binding affinity. (Table 1)</p><p>We tested the irreversible nature of compound CVS-16 (10b) by performing a washout experiment (Supplementary Figure 1). ATX was pre-incubated with compound CVS-16 (10b) or buffer for two minutes, after which time half of each reaction mixture was placed in a centrifugal filtration device. Over approximately one hour, samples were subjected to multiple rounds of concentration and dilution, resulting in over 1000-fold dilution of compounds CVS-16 (10b) and PVS-16 (21). ATX activity was then assessed by monitoring FS-3 hydrolysis. Attempted wash out of CVS-16 (10b) did not decrease the ATX inhibition, a finding consistent with CVS-16 (10b) irreversibly binding to the enzyme. PVS-16 showed no evidence of inhibiting ATX activity whether or not the compound was subjected to the washout experiment.</p><!><p>In order to determine whether CVS-16 (10b) possessed in vitro biological activity, studies were conducted to explore the functional potency of CVS-16 (10b) against cell viability. For these studies, we used multiple cancer cell lines to represent a wide range of subtypes including HT-29 (colon), PC-3 (prostate), MDA-MB-231 (breast), MeWo (melanoma), SB-2 (melanoma), OVCAR-3 (ovarian) and SKOV-3 (ovarian). In all cell lines, the CVS-16 (10b) compound reduced cell viability to some extent (Figure 4A) with significant activity of CVS-16 (10b) observed between 0.5-5 μM (Figure 4B). The IC50 values of CVS-16 (10b) are listed in Table 2 and the IC50 values of PVS-16 are in Supplementary Figure 2). Interestingly, CVS-16 (10b) had strong activity against MDA-MB-231 cells (IC50 = 0.39 μM) in comparison to the other cell types, but it was effective against all cell lines tested (*p > 0.05). As a control, we also examined the effect of PVS-16 (21) cell against viability and observed that most cell types were unaffected by its presence (Figure 4C), especially at relevant concentrations (Figure 4D).</p><p>Next we assessed confluent monolayer wounding and measured subsequent closure using MDA-MB-231, OVCAR-3, SKOV-3 and MeWo cells in the presence or absence of LPA (18:1, 1 μM) and/or CVS-16 (10b, 5 μM). For all cell lines examined, we observed that CVS-16 (10b) significantly (*p<0.05 vs. untreated) inhibited wound healing, which encompasses cell proliferation and migration from a denuded front that eventually closes the gap between confluent cell monolayers on either side (Supplementary Figure 3). In addition, the CVS-16 (10b) analog (5 μM) displays a signification reduction (over 75%) in both OVCAR-3 and MDA-MB-231 cell lines. Unexpectedly in most cases, LPA was not able to overcome the inhibition of CVS-16 (10b) on wound closure when these two reagents were combined.</p><p>Since the in vitro data thus far suggested that CVS-16 (10b) has a significant impact against MeWo cells, we wanted to compare the functional activity of this compound against other state-of-the-art ATX inhibitors; for this comparison, we selected: HA-130 15a and PF-8380 15b. Since melanoma cells are notoriously and intrinsically resistant to traditional chemotherapy, we also included several negative controls: dacarbazine (DTIC), paclitaxel (PTX/TAX), vincristine (VCR) and cisplatin (CDDP). Indeed, the chemotherapy had little impact on MeWo cells – never dropping below 50% cell viability (Figure 5). In contrast, the ATX inhibitors CVS-16 (10b) and HA-130 were able to reduce cell viability below 25%. Although there was no significant difference in activity between HA-130 with either of the other ATX inhibitors, CVS-16 (10b) was significantly different from PF-8380 (*p<0.05) and chemotherapy (**p<0.01).</p><!><p>In light of the fact that the data thus far suggested CVS-16 (10b) had significant biological activity in vitro, we wanted to next assess whether it also had in vivo activity. Since there is a dearth of clinical therapeutics to treat melanomas lacking BRAF mutations, we examined CVS-16 (10b) in an animal model of melanoma. After establishing small pigmented melanoma tumors using MeWo cells injected with Glycosan Extracel®, the mice were randomized into groups and then treated every-other-day with either diluent (control), CVS-16 (10b) at 20 mg/kg or CVS-16 (10b) at 50 mg/kg, which began 21 days post tumor cell injection. Prior to day 45, the tumor sizes in all groups appeared identical, but then began to diverge with the control groups displaying a linear rate of growth (Figure 6A). The group of mice treated with 20 mg/kg of CVS-16 (10b) also showed linear growth around day 50. On day 57, the tumor sizes between groups achieved statistical significance (Figure 6B, *p<0.05, 50-40 mg/kg CVS-16 (10b) vs. control; day 65 -***p<0.001, 50-40 mg/kg CVS-16 (10b) vs. control) and this trend continued through the conclusion of the study, whereby control groups reached maximum allowable tumor volume. Mouse weight data was also collected (Supplementary figure 4).</p><p>Of note is that on the 46th day of the study, the group of mice treated with 50 mg/kg of CVS-16 (10b) looked severely dehydrated and required medical intervention. Unfortunately, one mouse in the 50 mg/kg group (with a very small tumor and no signs of ascites upon necropsy) rapidly declined in health and then died the following day, even after veterinarians helped treat the mouse for this condition. Thus, on the 48th day, we reduced the concentration of CVS-16 (10b) to 40 mg/kg (marked with an arrow) so that the experiment could continue without additional mice succumbing to a possible unspecified side effects of CVS-16 (10b) at 50 mg/kg. Subsequently, all of the mice in this group were then fed a special diet to curb dehydration, in addition to their regular chew pellets and no others died before the date of necropsy.</p><p>Upon necropsy, we collected tissues and serum from all mice remaining in the study. We then measured the mouse serum for 25 different secreted factors, including cytokines, chemokines, interleukins, etc. Understanding that many interleukins would only be expressed by animals with immune-intact systems, we also coupled this study to another whereby we treated C57/Bl6 animals (n=20) with CVS-16 (10b) and measured their serum (data not shown). Interestingly, the most significant decrease in chemokines among treated animal serum from the present study shown herein was the keratinocyte chemoattractant (KC, also referred to as Chemokine C-X-C Motif Ligand 1 (CXCL1) or the Melanoma Growth Stimulating Activity Alpha protein) (Figure 6C, *p<0.05, ***p<0.001). The KC/CXCL-1 chemokine is homologous to the human growth regulated oncogene alpha (GRO-alpha), which is regulated by ATX 28 and associated with cancer progression29. Since chemokines are often significantly upregulated during tumorigenesis and melanoma tumor cells secrete KC/CXCL-1 30, which exerts signaling effects on endothelial cells, the significant reduction of KC/CXCL-1 suggests inhibition of tumor progression.</p><p>We then sectioned and stained tumor specimens with hematoxylin and eosin for pathology analysis of the tissues. Intriguingly, the percent of necrotic tissue within the tumor of the animals treated at the highest concentration of CVS-16 (10b) (40-50 mg/kg) had significantly more necrosis than control (**p<0.01) and 20 mg/kg treated animals (*p<0.05) (Figure 6D). In addition, the endothelial cells and state of the tumor specimens was examined. One hundred percent of the control specimens contained viable endothelial cells. This was in contrast to 20 mg/kg treated animals, of which 50% contained only viable endothelial cells present and the other 50% of specimens displayed mixed areas with viable endothelial cells and also necrotic endothelial cells (Figure 6E). Most interesting was that 100% of specimens from animals treated with 40-50 mg/kg of CVS-16 (10b) contained necrotic endothelial cells in close proximity to necrotic malignant cells. Taken together, the data suggests an anti-angiogenesis mechanism of action for CVS-16 (10b). In support of this idea, we stained tumor sections with Ki-67 and detected no significant differences between specimens (data not shown).</p><p>In order to understand whether CVS-16 (10b) had biological activity in vivo, we isolated the mouse serum at necropsy for analysis of circulating ATX. We detected a significant reduction in the ATX of treated animals, compared to control (Figure 6F, *p<0.05 vs. control). This suggests that CVS-16 (10b)-treated tumors manifested significant reduction in the expression and production of ATX. Taken together with the previous data, this indicates that the vinyl sulfone inhibits ATX which affects angiogenesis and melanoma progression in vivo.</p><p>Using the same in vivo model of melanoma progression, we also tested the efficacy of HA-130 and PF-8380. Surprisingly, neither of these ATX inhibitors reached statistical significance against the continuous growth of tumors from MeWo cell inoculation in mice (Figure 7A and 7B). Upon necropsy, we collected the serum to analyze circulating ATX levels and observed no significant difference between the treated animals and the control group (Figure 7C). This corroborated the results we observed with solid tumor growth, suggesting the compounds were not able to have a significant impact on ATX. Taken together, this further suggests that the vinyl sulfone CVS-16 (10b) has superior functional activity in comparison to these other ATX inhibitors.</p><!><p>Herein we report the synthesis and biological activity of a vinyl sulfone analog of LPC, which acts as an active-site targeted irreversible inhibitor of ATX in vitro, and also shows activity against melanoma in vitro and in vivo. In addition to enzymatic inhibition of ATX by the vinyl sulfone analogs in vitro, we observed the reduction of cell viability and migration among multiple cancer cell types in vitro. Most importantly, we observed the inhibition of tumor progression using an in vivo model of melanoma and measured a reduction of ATX in the serum of treated animals. Taken together, our data suggests that the vinyl sulfone CVS-16 (10b) is a potent small molecule inhibitor of ATX with biological activity against its target, which reduces viability of cancer cells and inhibits angiogenesis necessary for tumor progression.</p><p>Our data also suggests that the vinyl sulfone CVS-16 (10b) compares very favorably against other compounds synthesized to inhibit ATX, like HA-130 and PF-8380. The first known inhibitor of ATX was L-histidine and it was limited in application due to the millimolar concentrations of L-histidine required to inhibit the lysophospholipase D activity of ATX 15g. Later work identified a-halophosphonate analogs of LPA as potent ATX inhibitors 31, as well as aromatic phosphonates 15i, 32. This report was the first to provide a proof-of-concept study that led to further innovation, as reviewed by Parrill, Baker and coworkers 33. For example, using a chemical library screening approach and ~40,000 drug-like small molecules, thiazolidinediones were identified as ATX inhibitors that could be enhanced by the addition of a boronic acid moiety to achieve lower IC50 levels 15a. Another screen of a small-molecule library yielded several ATX inhibitors, which lead to the synthesis of analogues based on their structural motifs and further validated ATX as a target for melanoma 34.</p><p>We, and other groups, have also taken advantage of cyclic phosphatidic acid, a naturally occurring molecule that inhibits ATX and is an analogue of ATX. Manipulating the compound structure to yield 3-carba analogues of cyclic phosphatidic acid produced potent inhibitors of ATX that were effective in vivo 8a. Similarly, a phosphonothionate analogue of carba cyclic phosphatidic acid also yielded a compound that inhibited the lysophospholipase D activity of ATX, the viability of melanoma cells and reduced melanoma metastasis in vivo 8b. A recent study reported the evaluation of the stereoisomers of 3-carba cyclic phosphatidic acid, which are agonists of the LPA5 receptor, yet inhibits melanoma metastasis in vivo 35.</p><p>There are several differences between these reports and the current inhibitor. First and foremost among these differences is the use of a vinyl sulfone moiety as a mild electrophile that is targeted to the active site by an alkyl chain and a quaternary ammonium derivative that recapitulate key recognition elements of the LPC substrate structure. As a result, CVS-16 (10b) is an irreversible, "suicide" inhibitor of ATX. There is only one other reported irreversible inhibitor of ATX, a series of monofluoro- and difluoromethyl phenyl alkyl phosphodiesters that liberate a reactive quinone methide upon hydrolysis by ATX 36. This irreversible inhibitor lacks the quaternary ammonium derivative for targeting, which we show is crucial for activity of the LPC-mimicking vinyl sulfones; lacking the quaternary ammonium derivative, analog PVS-16 (21) is essentially inactive. In addition, the reactive quinone methide is released as a diffusible highly reactive electrophile, rather that the more selective Thr-targeted vinyl sulfone.</p><p>Secondly, in previous studies we focused exclusively on advanced, metastatic models of melanoma using an allograft system 8a, 8b. Herein, we established a solid xenograft melanoma tumor at one primary site, which is ideal for assessing the effects of angiogenesis. In this melanoma model, the tumors are highly pigmented, allowing for ease of visualization and measurements on nude mice. Our shift away from the highly metastatic allograft model is due to its poor reflection of human disease along with dubious conclusions that might be drawn from biological data derived by using it 37.</p><p>Through the solid xenograft melanoma model, we discovered that the mechanism of action of CVS-16 (10b) in vivo is the reduction of angiogenesis, which is not what we had predicted based on the data gleaned from our in vitro studies. Since we detected reduced viability in the presence of CVS-16 (10b), we hypothesized that CVS-16 (10b) had a direct effect against tumor cells, possibly an inhibition of mitogenesis. However, mitogenesis inhibition is not what we observed in animals, rather it was angiogenesis inhibition. Interestingly, the angiogenic response of ATX was previously described as "comparable to that elicited by VEGF" 13a. Thus, our data is consistent with the known properties of ATX. Although we cannot completely rule out all other molecular mechanisms that may also contribute to this phenomenon, we can state that angiogenesis does not proceed in vivo, in the presence of 40-50 mg/kg CVS-16 (10b).</p><p>It is highly likely that 50 mg/kg (or very close to) is the MTD for CVS-16 (10b), certainly without pre-emptive supportive therapy for the animals. One treated mouse (50 mg/kg) died, presumably to the unspecified side effects of CVS-16 (10b) since it had only a miniscule-sized tumor and no obvious signs of metastases, even at necropsy. However, it is not uncommon to have 1 or 2 otherwise healthy, untreated mice in our animal colony die from dehydration for no obvious reason deduced by our veterinary staff. Nevertheless, the fact that a mouse possibly succumbed to side effects is consistent with the harsh adverse drug events manifested by angiogenesis inhibitors. For example, bevacizumab contains several black-box warnings, which includes: fatal hemorrhage, increased arterial thromboembolic events (myocardial infarction and stroke), gastrointestinal perforation and complications to wound healing which requires discontinuation of the drug at least 28 days before surgery 38. These black-box warnings are in addition to the other known drug toxicities of bevacizumab including hypertension, proteinuria and central nervous system events (e.g. dizziness, depression, headaches, seizure, lethargy, visual disturbances, etc.) 39.</p><p>Although adverse events are a concern for further pre-clinical development of ATX inhibitors, most anticancer therapeutics, in particular traditional, cytotoxic chemotherapy, produces harsh side effects for patients. However, this regrettable actuality is tolerated because of favorable therapeutic indexes and the potential for ameliorating a life-threatening illness. Even the newer classes of targeted biologics possess severe unwanted side effects, some even serving as a measure of therapeutic activity (e.g. an acne-like rash). Thus, there is extreme variability among patient compliance and tolerability of anticancer therapeutics, along with patients' willingness to continue therapy.</p><p>Although CVS-16 (10b) (40-50 mg/kg) was capable of sustaining an inhibition on tumor volume for approximately 53 days after the injection of tumor cells, the sizes of tumors in the highest treated group of animals started to increase on day 54. This was 6 days after we had reduced the dosage in this group from 50 mg/kg to 40 mg/kg. We cannot be certain whether this shift from static to growing tumors was due to the dosage reduction, chemoresistance or another factor. We did not observe any change in the amount of circulating VEGF among the treated mice in comparison to the controls (data not shown). This argues against a shift to VEGF or chemoresistance using this mechanism, which is what we had predicted since ATX works similarly to VEGF, and inhibiting both might be logically necessary to sustain a long-term response against tumor angiogenesis. Therefore, our supposition is that 50 mg/kg of CVS-16 (10b) is required for a sustained inhibition of ATX-dependent angiogenesis and tumor progression and that supportive care is required at this dosage.</p><p>Nevertheless, it was very exciting to observe the ability of CVS-16 (10b) to prevent melanoma tumor progression in animals. Advanced melanoma is a particularly difficult type of cancer to treat because it is unresponsive to traditional chemotherapy; therefore, immunotherapy is typically administered even though responses are achieved in less than 20% of patients. In the past few years, several new therapeutics for melanoma were approved for the first time in over a decade and these included trametinib, dabrafenib and vemurafenib. The drugs are intended for patients with activating BRAF mutations, which occurs in a majority of melanomas, but not all cases. Thus, more research is desperately needed to uncover drugs that can treat melanoma, especially since the incidence of this disease is rising. Besides melanoma, the compound was also effective against the viability and migration of MDA-MB-231 breast cells in vitro. This is very intriguing considering this cell line was isolated from the pleural effusion of a patient and represents a highly invasive triple-negative breast cancer, which is a clinically challenging subtype to treat. Taken together, our data supports further pre-clinical testing of the vinyl sulfone CVS-16 (10b) as an ATX inhibitor in a combination approach with other anticancer therapeutics against the progression of melanoma.</p>

PubMed Author Manuscript

Evaluation of neurological effects of cerium dioxide nanoparticles doped with different amounts of zirconium following inhalation exposure in mouse models of Alzheimer's and vascular disease

Increasing evidence from toxicological and epidemiological studies indicates that the brain is an important target for ambient (ultrafine) particles. Disturbance of redox-homeostasis and inflammation in the brain are proposed as possible mechanisms that can contribute to neurotoxic and neurodegenerative effects. Whether and how engineered nanoparticles (NPs) may cause neurotoxicity and promote neurodegenerative diseases such as Alzheimer's disease (AD) is largely unstudied.We have assessed the neurological effects of subacute inhalation exposures (4 mg/m3 for 3 h/day, 5 days/week for 4 weeks) to cerium dioxide (CeO2) NPs doped with different amounts of zirconium (Zr, 0%, 27% and 78%), to address the influence of particle redox-activity in the 5xFAD transgenic mouse model of AD. Four weeks post-exposure, effects on behaviour were evaluated and brain tissues were analysed for amyloid-β plaque formation and reactive microglia (Iba-1 staining). Behaviour was also evaluated in concurrently exposed non-transgenic C57BL/6J littermates, as well as in Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice as a model of vascular disease. Markers of inflammation and oxidative stress were evaluated in brain cortex.The brains of the NP-exposed 5xFAD mice revealed no accelerated amyloid-β plaque formation. No significant treatment-related behaviour impairments were observed in the healthy C57BL/6J mice. In the 5xFAD and ApoE-/- models, the NP inhalation exposures did not affect the alternation score in the X-maze indicating absence of spatial working memory deficits. However, following inhalation exposure to the 78% Zr-doped CeO2 NPs changes in forced motor performance (string suspension) and exploratory motor activity (X-maze) were observed in ApoE-/- and 5xFAD mice, respectively. Exposure to the 78% doped NPs also caused increased cortical expression of glial fibrillary acidic protein (GFAP) in the C57BL/6J mice. No significant treatment-related changes neuroinflammation and oxidative stress were observed in the 5xFAD and ApoE-/- mice.Our study findings reveal that subacute inhalation exposure to CeO2 NPs does not accelerate the AD-like phenotype of the 5xFAD model. Further investigation is warranted to unravel whether the redox-activity dependent effects on motor activity as observed in the mouse models of AD and vascular disease result from specific neurotoxic effects of these NPs.

evaluation_of_neurological_effects_of_cerium_dioxide_nanoparticles_doped_with_different_amounts_of_z

<!>Introduction<!>Animals<!>Inhalation study design<!>Nanomaterial production, characterization and inhalation exposure<!>Behaviour tests<!>Necropsy and immunohistochemical analyses of paraffin embedded slices<!>Western blot analyses<!>Statistical analyses<!>Exposure conditions<!><!>Effects on motor activity and cognitive function<!><!>Effects on motor activity and cognitive function<!><!>Discussion<!>Conclusions<!>Funding<!>Declaration of competing interest

<p>4-week mouse inhalation study with 0%, 27% and 78% Zr-doped CeO2 nanoparticles.</p><p>No acceleration of Alzheimer-related features in 5xFAD mouse model.</p><p>Motor performance changes in 78% Zr-doped CeO2 exposed ApoE-/- and 5xFAD mice.</p><p>Increased GFAP levels in 78% Zr-doped CeO2 exposed C57BL/6J mice.</p><!><p>Several research groups have postulated that ultrafine air pollution particles are an important environmental risk factor for neurotoxicity and, more specifically, may potentiate the risk of neurodegenerative disorders, like Alzheimer's Disease (AD) (reviewed in (Heusinkveld et al. 2016). In relation to this, concerns have been raised about the potential neurotoxic and neurodegenerative effects of engineered nanoparticles (NPs). However, despite great progress in nanotechnologies, comparatively little is known to date on the potential adverse effects that exposure to manufactured NPs may have on the human brain, including the potential induction of pathways leading to neurodegeneration (Cupaioli et al. 2014). Indeed, NPs can enter the human body through several routes, e.g. via inhalation, absorption from the digestive tract, or following injection into the blood in nanomedical applications. With regard to potential adverse impacts on the brain, uptake and retrograde axonal transport of NPs via the olfactory nerve has been demonstrated in rodent inhalation studies (Oberdorster et al. 2004; Elder et al. 2006; Elder and Oberdorster, 2006). Besides, NPs may reach the central nervous system via the blood–brain barrier (BBB), where they have been suspected to impair several molecular pathways and contribute to neurodegeneration (Iqbal et al. 2013; Cupaioli et al. 2014). The ability to generate reactive oxygen species and associated inflammation is considered one of the key mechanisms of nanomaterials' toxicity to the respiratory tract and cardiovascular system (Unfried et al. 2008; Miller et al., 2012; Stone et al. 2017) and thus could also play a major role in their neurotoxic and neurodegenerative effects. Indeed, oxidative stress and neuroinflammation have long been recognised in neurotoxicity and neurodegenerative diseases including AD (Heneka et al. 2015; Zhao and Zhao, 2013).</p><p>Among the various types of NPs, cerium oxide NPs (CeO2 NPs) have been subjected to various toxicological investigations in relation to inhalation exposure (Cassee et al. 2011; Demokritou et al. 2013). CeO2 NPs are widely used as catalysts in industrial applications. They are used as additive to diesel fuels in order to reduce the amount of emitted pollutants after their combustion. Because of their radical-scavenging properties, CeO2 NPs have gained strong interest in the field of nanomedicine (reviewed in (Das et al. 2013)). The antioxidant properties of CeO2 NPs are accomplished through its ability to switch from the 3+ to the 4+ valence state (Hirst et al. 2009). It has been shown that the antioxidant efficacy of CeO2 NPs can be affected by incorporation of zirconium (Zr) in the CeO2 lattice (Tsai et al. 2008). However, whilst research has been devoted since many years to elaborate on neuroprotective and potential anti-neurodegenerative effects of CeO2 (Singh et al., 2007), adverse effects on the brain should also be considered for this type of nanoparticles as indicated e.g. from intravenous application studies in rats (Hardas et al. 2010, 2014) and in vitro neuronal activity experiments with primary rat cortex cultures (Strickland et al. 2016).</p><p>Given that free radicals play a prominent role in the pathology of many neurological diseases, we explored the neurotoxicity of CeO2 NPs doped with varying amounts of Zr following inhalation exposure in three different mouse models, i.e. C57BL/6J, 5xFAD and ApoE-/- mice. The 5xFAD transgenic mice were used in this study as a model for AD. The 5xFAD mouse model was used in a previous study, in which we have demonstrated that inhalation exposure to diesel engine exhaust results in an accelerated formation of Aβ-plaques as well as motor function impairment (Hullmann et al. 2017). Diesel engine exhaust represents a major source of unintentionally generated NPs in most urban environments and therefore supports the selection of the 5xFAD model for the investigation of the neurological effects of engineered NPs after inhalation. The nontransgenic littermate controls of the 5xFAD mice (C57BL/6J background) were used as a healthy mouse model. Finally, apolipoprotein E-deficient (ApoE-/-) mice, subjected to a high-fat diet, were included in the present study. ApoE-/- mice represent a well-established model for the study of atherosclerosis, a disease characterized by the build-up of lipid- and inflammatory cell-rich plaques within arteries, which underlies the majority of cardiovascular diseases (Cassee et al. 2012; Miller et al. 2013). Since this ApoE deficiency compromises the blood brain barrier (Methia et al. 2001) this model could also be useful to study the susceptibility to NP-induced neurological effects. The adverse cardiovascular effects of diesel exhaust particles as well as specific types of engineered NPs have been clearly demonstrated in ApoE-/- mice in several studies (Hansen et al. 2007; Kang et al. 2011; Miller et al. 2013). Interestingly, a comparative inhalation study with engine exhausts generated using fuels with or without added CeO2 NPs in ApoE-/- mice revealed differences in atherosclerotic plaque formation but also in pro-inflammatory responses in (sub)cortical brain regions (Cassee et al. 2012; Lung et al. 2014), which could reflect a direct effect of these redox active NPs on the central nervous system.</p><p>The aim of the current study was to evaluate the potential neurotoxic and neurodegenerative effects of CeO2 NPs in mice following a four-week inhalation exposure and to assess the influence of redox activity by the concurrent evaluation of CeO2 NPs with different Zr-doping grades. The investigations formed part of a large study conducted in to explore the (patho)physiological effects of NP exposure on multiple organ systems in various mouse models (Dekkers et al. 2017, 2018).</p><!><p>In this study, three different mouse models were used. The 5xFAD transgenic mice were used as a model for AD. Only the female mice were used for the study in view of the reported sex-specific differences in age- and treatment related Aβ development (Devi et al. 2010). The 5xFAD mice overexpress the 695 amino acid isoform of the human amyloid precursor protein (APP695) carrying Swedish (K670N), London (V717I) and Florida (I716V) mutations as well as the human PS1 (M146L; L286V) mutations (Oakley et al. 2006; Ohno et al. 2004). The mice develop a specific phenotype that includes high APP expression levels, amyloid deposition (beginning at two months of age) and memory impairments and motor deficits (Oakley et al. 2006; Jawhar et al. 2012). The breeding was performed by mating heterozygote transgenic founders with C57BL/6J wild-type mice. The nontransgenic female littermates were used as model of healthy mice in this study. The 5xFAD and C57BL/6J mice originated from Jackson Laboratories. For the study, female 5xFAD mice (n = 64) and female cross bred C57BL/6J littermates (n = 40) were used at the age of 8–11 weeks. As a third model, ApoE-/- mice were used. Female ApoE-/- mice (n = 32) were obtained from Taconic, Denmark at age 10–12 weeks at the beginning of the study. The four-week inhalation exposure protocol in the ApoE-/- mice was integrated into an 8-week high-fat (Western diet) feeding regime (Purified Diet Western 4021.06, ABdiets, Woerden, The Netherlands), which has been shown to generate complex atherosclerotic plaques with many of the hallmarks of the human disease in specific arterial locations (Cassee et al. 2012; Miller et al. 2013; Dekkers et al. 2017). All mice were barrier maintained and housed in a single room in macrolon cages. Temperature and relative humidity were controlled at 22 ± 2 °C and at 40–70%, respectively. Lighting was artificial with a sequence of 12 h light (during daytime) and 12 h dark (at night). Feed and drinking water were provided ad libitum from the arrival of the mice until the end of the study, except during exposure. The study was conducted at Intravacc (Bilthoven, The Netherlands) under a protocol approved by the Ethics Committee for Animal Experiments of the RIVM and performed according to applicable national and EU regulations.</p><!><p>The mice were exposed via nose only inhalation to CeO2 NPs with varying amounts of Zr-doping (0%, 27% or 78% Zr) or clean air, respectively, over a four-week period (4 mg/m3 for 3 h/day, 5 days/week). The number of animals per treatment group designed for the present study was n = 10 for the C57BL/6J mice, n = 16 for the 5xFAD mice and n = 8 for the ApoE-/- mice. For three mice data could not be obtained because of their early removal from the study for humane reasons not related to the toxicity of the NP exposure. Combined with the genotyping verification, this resulted in the following animal numbers per group: ApoE-/-: control (n = 8); CeO2 (n = 7); 27% ZrO2-doped CeO2 (n = 8); 78% ZrO2-doped CeO2 (n = 8). 5xFAD: control (n = 16); CeO2 (n = 14); 27% ZrO2-doped CeO2 (n = 16); 78% ZrO2-doped CeO2 (n = 16). C57BL/6J: control (n = 10); CeO2 (n = 10); 27% ZrO2-doped CeO2 (n = 10); 78% ZrO2-doped CeO2 (n = 10). On day 52 and day 53 after the first exposure day behaviour tests were performed with the mice to assess for exposure-related neurotoxic effects. The animals were killed on day 57. The 4-week post-exposure period was included in the study design to address persistency of the effects and for the compromised mouse models to develop their respective disease phenotypes, i.e. Aβ formation in the brains of the 5xFAD mice and the atherosclerotic plaques in the ApoE-/- mice.</p><!><p>Production and detailed characterization of CeO2 NPs doped with different amounts of ZrO2 (ZrO2 contents in the doped NPs were 0 mol%, 27 mol% and 78 mol%) is described elsewhere (Dekkers et al. 2017). Approximately one week before the four-week exposure period, 20 samples of each NP (one for each day) were prepared at a concentration of 1 mg/mL from the stock dispersions (20, 20 or 29 mg/mL for 0%, 27% and 78% ZrO2-doped CeO2 NPs, respectively) by diluting with ultrapure water. Before use, stock and sample dispersions were sonicated for 5 min in an ultrasonic bath (Branson CPX2800, 40 kHz, 110W) before use to re-disperse any possible agglomerates. Aerosols of NPs were freshly generated using a spray nozzle technique, diluted with pressurized, clean and particle-free air, and heated to 24–25 °C (for detailed description see (Dekkers et al. 2017). Control animals were exposed to 3-h filtered air under the same exposure conditions (i.e. nose-only tubes) for the same amount of time. Prior to the day of exposure start all animals were trained to get used to the nose-only inhalation tubes.</p><!><p>At day 52 and 53 (i.e. 24 and 25 days after the last exposure day) the mice were examined by means of behavioural tests. At least 1 h before behavioural testing, mice were placed in the test room for acclimatisation. All tests were performed in dim red light. All test equipment and mazes were cleaned with 70% ethanol prior to each test to avoid odour recognition. On day 52, a string suspension test was performed: as a test of agility and grip strength (Miquel and Blasco, 1978), a 3 mm thick, 35 cm long cotton string was stretched between two escape platforms on top of two vertical poles. The mice were permitted to grasp the central part of the string by their forepaws, released immediately thereafter and allowed to escape to one of the platforms. A rating system from 0 to 7 was used during a single 60 s trial to assess each animals' performance (Moran et al. 1995) with the following modifications. Score: 0, unable to hang on the string; score 1, hangs only by forepaws; score 2, attempting to climb the string; score 3, climbing the string with four paws successfully; score 4, moving laterally along the string; score 5, escaping to the end of the string; score 6, falls while trying to climb the platform, score 7, reaches the platform.</p><p>On day 53, the X-maze task was performed to reflect activity and spatial working memory of mice by spontaneous alternation. Spontaneous alternation in rodents is based on the willingness to explore; a mouse tends to rotate in their entries between the four arms arranged in 90° position extending from a central space, which makes it more discriminative (arm sizes: 30 cm length, 8 cm width and 15 cm height). During 5 min test sessions, each mouse was placed in one arm and was allowed to move freely through the maze. The total number of arm entries was recorded using an infrared beam video camera during the 5 min interval to evaluate exploratory motor activity and this was then combined with the alternation to assess spatial working memory. Alternation was defined as successive entries into the four arms in overlapping quadruple sets (for example 1, 2, 3, 4 or 2, 3, 4, 1 but not 3, 2, 1, 3). Mice with impaired working memory will not remember visited arms leading to a decrease in spontaneous alternation (Holcomb et al. 1999). A successful entry was defined as a mouse entering one arm with all four paws. The alternation percentage was calculated as % of the actual alternations to the possible arm entries.</p><!><p>At day 57, the mice were anesthetized with a mixture of ketamine and xylazine. The right brain hemispheres of 5xFAD mice were stored in 4% PFA for later processing for immunohistochemistry. The left brain hemispheres of C57BL/6J, ApoE-/- and 5xFAD animals were rapidly dissected into cortex, olfactory bulb, cerebellum and midbrain. All brain regions were immediately transferred in liquid nitrogen and stored at −80 °C until further processing for Western blotting (see below). Dehydration was performed in a series of ethanol concentrations, followed by a transfer into xylene. Subsequently, the brains were embedded in paraffin. Four μm thick paraffin sections were cut using a sliding microtome and transferred on Superfrost Ultra Plus object slides (Thermo Scientific) and dried over night at 40 °C. Sections were deparaffinised in xylene, followed by rehydration in a series of ethanol (100%, 96%, 70%) and blocking of endogenous peroxidase by treatment with 0.3% H2O2 in PBS. Antigen retrieval was performed by boiling sections in 10 mM citrate buffer, pH 6.0 followed by incubation for 3 min in 88% formic acid. Non-specific antibody binding was blocked via incubation in 10% fetal calf serum (FCS) and 4% skimmed milk in 0.01 M PBS. Thereafter, slides were incubated overnight with primary anti human Aβ 42 antibody (clone G2-11, Cat.N0. MABN12, Merck Millipore, Darmstadt, Germany diluted 1:1000 in 0.01 M PBS and 10% FCS in a humid chamber at room temperature. After washing slices were incubated with a biotinylated anti-mouse secondary antibody (dilution 1:200 in 0.01M PBS and 10% FCS), and the signal was visualized avidin-biotin-complex-method (ABC) by a Vectastain kit (Vectorlabs, Burlingame, USA) using diaminobenzidine (DAB, Sigma-Aldrich, Deisenhofen, Germany)) as chromogen and Hematoxylin for nuclear counterstaining. Light microscope images from cortex and hippocampus were taken with 100x or 50x magnification, respectively, using a Zeiss Axiophot microscope equipped with AxioCam MRc (Carl Zeiss, Jena, Germany). Quantitative Aβ42 plaque analyses were performed via calculation of the percentage of total amyloid plaque load in the analysed area of the section. Plaque load was determined using ZEN 2011 image processing software (Zeiss) after a fixed adjustment of contrast threshold for stained Aβ42 plaques. Plaque load was interactively determined in the whole hippocampal area as well as in a defined cortex region. From each animal, three brain slides with an interspace of approximately 30 μm were analysed. For the immunostaining of ionized calcium-binding adapter molecule 1 (Iba-1), brain sections were incubated overnight with Iba-1 antibody (Cat No. GTX100042, GeneTex; dilution 1:1000 in 0.01 M PBS and 10% FCS) at 4 °C. The next day, slides were washed and incubated for 45 min at RT with biotinylated secondary antibody (dilution 1:200 in 0.01M PBS and 10% FCS. Staining was visualized using the ABC Vectastain kit (Vectorlabs, Burlingame, USA) and diaminobenzidine (DAB, Sigma-Aldrich, Deisenhofen, Germany)) as chromogen and Hematoxylin for nuclear counterstaining. Light microscope images from cortex and hippocampus were taken with 200x magnification using a Zeiss Axiophot microscope equipped with AxioCam MRc (Carl Zeiss, Jena, Germany). Iba-1 area (%) was quantified using ZEN 2011 image processing software (Zeiss) via calculation of the positive stained microglia (brown colour) in the defined cortical and hippocampal area.</p><!><p>Protein expression of ionized calcium-binding adapter molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was evaluated by Western blot to address whether the exposures to the CeO2 NPs resulted in neuroinflammation and oxidative stress. Iba-1 and GFAP represent well-established markers of activated microglia (Kovacs, 2017; Sasaki et al. 2001) and mature astrocytes in neuroinflammation (Li et al. 2020; Sofroniew and Vinters, 2010), respectively. The transcription factor Nrf2 is a master regulator of cellular responses to oxidants via its activation of oxidative stress response genes including HO-1. Both Nrf2 and HO-1 are implicated in neurotoxicity and neurodegenerative diseases including AD (Kanninen et al. 2009; Sandberg et al. 2014; Schipper et al. 2019). For the analysis of these markers, cortex brain tissues were homogenized in ~5 vol of ice-cold RIPA buffer for 2 h in a potter tissue grinder. The total protein level was evaluated with the BCA kit (Thermo) according to the manufactures protocol. Equal amounts of protein (50 μg) were loaded on a 4–12% precast NUPAGE gel (Invitrogen) and separated at 180 V in a Mini-PROTEAN II tank (BIO-RAD). The proteins were blotted at 250 mA for 45 min in a Mini Trans-Blot tank (BIO-RAD) on a 0.45 μm pore diameter nitrocellulose transfer membrane (Whatman, Schleicher & Schuell). With 5% milk in PBS-T (0.01 M PBS and 0.05% Tween-20) unspecific protein binding was blocked for 60 min. After the blocking, the membrane was incubated with the primary antibody: GFAP (Cat No. ab7260, Abcam, 1:5000), Iba-1 (Cat No. GTX100042, Gentex, 1:1000), HO-1 (Cat No. AB1284, Merck, 1:1000), Nrf2 (C-20) (Cat No. sc-722, Santa Cruz,1:500) overnight at 4 °C. Next day, secondary hrp-conjugated antibody and β-Actin-hrp (AC-15) (Cat No. A384, Sigma,1:50000) was incubated for 1 h at room temperature. Detection of proteins was performed with ECL solution (GE Healthcare) and visualized with CHEMI Premium Imager (VWR). With the use of ImageJ software (National Institutes of Health, Bethesda, USA) quantification of protein expression was evaluated relative to β-actin protein level.</p><!><p>Data were analysed using IBM-SPSS (version 22) and are expressed as mean ± SEM unless stated otherwise. Data were evaluated by one-way analysis of variance (ANOVA) with Dunnett post-hoc analysis using the air exposed animals as statistical control group. Differences were considered statistically significant at p < 0.05.</p><!><p>Detailed characteristics of NPs and their particle size distributions, mass and number exposure concentrations as well as lung deposited dose estimations for the inhalations are described in detail elsewhere (Dekkers et al. 2017, 2018). Briefly, the different CeO2 particles had a primary particle size of 4.7 ± 1.4 nm. The gravimetric mass concentrations and size distribution of the aerosols were almost identical for the exposures to the CeO2, 27% Zr-doped CeO2 and 78% Zr-doped CeO2 NPs.</p><!><p>Effects of redox-modified CeO2on performance in the string suspension task.</p><p>Female C57BL/6J (A), ApoE-/- (B) and 5xFAD (C) mice were exposed to clean air (control) or CeO2 and 27% ZrO2-doped CeO2 or 78% ZrO2-doped CeO2 NPs via inhalation. The ability of the mice to escape to a platform within 60 s was measured and transferred to a rating system from 0 to 7 whereby a higher score represents a better performance. Data are expressed in mean ± SEM, *statistical significantly different from the respective control in Dunnet post-hoc test following one-way ANOVA with p < 0.05. Number of animals per group: ApoE-/-: control (n = 8); CeO2 (n = 7); 27% ZrO2-doped CeO2 (n = 8); 78% ZrO2-doped CeO2 (n = 8). 5xFAD: control (n = 16); CeO2 (n = 14); 27% ZrO2-doped CeO2 (n = 16); 78% ZrO2-doped CeO2 (n = 16). C57BL/6J: control (n = 10); CeO2 (n = 10); 27% ZrO2-doped CeO2 (n = 10); 78% ZrO2-doped CeO2 (n = 10).</p><!><p>There was no significant difference in test performance between the controls (clean air exposed mice) of the three different strains. Exposure of the mice with the distinct CeO2 NPs did not affect the performance of the C57BL/6J mice and the 5xFAD transgenic mice in the string suspension task. However, among the ApoE-/- mice the string suspension test performance was diminished in the group that was exposed to the 78% Zr-doped CeO2 NPs compared to controls, indicative of an adverse impact on the motor function (Fig. 1). The inhalation exposures to the CeO2 NPs that contained less (27%) or no (0%) Zr did not significantly alter the behaviour of the ApoE-/- mice in the string suspension test in comparison to the clean air exposed animals.</p><!><p>Effects of redox-modified CeO2 on performance in the X-maze task.</p><p>Female C57BL/6J (A, D), ApoE-/- (B, E) and 5xFAD (C, F) mice were exposed to clean air (control) or CeO2 and 27% ZrO2-doped CeO2 or 78% ZrO2-doped CeO2 NPs via inhalation. After this treatment, the differently exposed groups were subjected to the X-maze task. Mice were place in the maze for 5 min. The behavioural parameters analysed were total arm entries (A, B, C) and alternation (D, E, F) and expressed in mean ± SEM. *Statistical significance different from the respective control in Dunnet post-hoc test following one-way ANOVA with p < 0.05. Number of animals per group: ApoE-/-: control (n = 8); CeO2 (n = 7); 27% ZrO2-doped CeO2 (n = 8); 78% ZrO2-doped CeO2 (n = 8). 5xFAD: control (n = 16); CeO2 (n = 15); 27% ZrO2-doped CeO2 (n = 16); 78% ZrO2-doped CeO2 (n = 16). C57BL/6J: control (n = 10); CeO2 (n = 10); 27% ZrO2-doped CeO2 (n = 10); 78% ZrO2-doped CeO2 (n = 10).</p><!><p>In concordance with the string suspension task, the X-maze test also revealed a significant effect on behaviour following inhalation exposure to the 78% Zr-doped CeO2 NPs, whereas the other types of NPs showed no effects. In this case, however, the effect was seen in the 5xFAD mouse model: The 5xFAD mice that had been exposed to the 78% Zr-doped CeO2 NPs showed a significantly reduced number of total arm entries compared to the control 5xFAD mice, indicative of a decreased exploratory motor activity for this treatment group (Fig. 2). However, the alternation in the X-maze task, which is an indicator of the spatial working memory of mice, did not differ between these groups. In fact, the alternation percentage among the 5xFAD groups tended to be highest in the 78% Zr-doped CeO2 NPs. In the ApoE-/- and C57BL/6J mice, no significant treatment-related effects on locomotor activity and spatial working memory were found with the X-maze testing.</p><!><p>Effect of redox-modified CeO2NPs inhalation on β-Amyloid pathology in 5xFAD mice.</p><p>Aβ plaque load was determined in parasagittal brain slices of 5xFAD mice after exposure to clean air (control n = 16), CeO2 (n = 15), 27% ZrO2-doped CeO2 (n = 16) or 78% ZrO2-doped CeO2 (n = 16) NPs. Aβ42 was visualized by IHC in 4 μm sections of paraffin-embedded brain hemispheres (Representative pictures are shown in A). For quantification, plaque load was determined in the hippocampus (B) and in the cortex (C) using image analysis software and calculated as the percentage area occupied by Aβ immunostaining expressed in mean ± SEM. For determination of plaques in the cortex, whole image sections were evaluated while the hippocampus regions were defined by hand to evaluate only the hippocampus. A trend was observed of reduced Aβ plaques in the brains of mice exposed to the 78% Zr-doped CeO2 NPs, but this effect was not statistically significant.</p><p>Effect of redox-modified CeO2NP inhalation on Iba-1 immunostaining in hippocampus and cortex of 5xFAD mice.</p><p>Parasagittal brain slices of 5xFAD mice exposed to clean air of CeO2 NPs with different doping of Zr (n = 6 per group), were stained with an antibody against Iba-1 to detect activated microglia (representative pictures are shown in A). For quantification, Iba-1 stain was determined in (B) CA1/subiculum of the hippocampus (200-fold microscopic magnification) and (C) cortex layer 5 (200-fold microscopic magnification) using image analysis software and calculated as the percentage area occupied by Iba-1 immunostaining and expressed in mean ± SEM.</p><p>Effect of redox-modified CeO2NP inhalation on Iba-1 and GFAP protein levels.</p><p>Levels of Iba-1 (B, C, D) and GFAP (E, F, G) were assessed by Western blot analysis in lysates of the cortex of female C57BL/6J (B, E), ApoE-/- (C, F) and 5xFAD (D, G) exposed to clean air or CeO2 NPs with different doping of Zr (representative blots are shown in A). Data were normalized to the level of β-actin and expressed in mean ± SEM. * Statistical significance different from the respective control in Dunnet post-hoc test following one-way ANOVA with p < 0.05. Number of animals per group: ApoE-/-: control (n = 5); CeO2 (n = 5); 27% ZrO2-doped CeO2 (n = 5); 78% ZrO2-doped CeO2 (n = 4). 5xFAD: control (n = 4); CeO2 (n = 4); 27% ZrO2-doped CeO2 (n = 4); 78% ZrO2-doped CeO2 (n = 4). C57BL/6J: control (n = 5); CeO2 (n = 5); 27% ZrO2-doped CeO2 (n = 5); 78% ZrO2-doped CeO2 (n = 5).</p><p>Effect of redox-modified CeO2NP inhalation on Nrf2 and HO-1 protein levels.</p><p>Lysates of the cortex of C57BL/6J (B, E), ApoE-/- (C, F) and 5xFAD (D, G) mice exposed to clean air or CeO2 NPs with different doping of Zr were subjected to Western blot analysis. Levels of Nrf2 (B, C, D) and HO-1 (E, F, G) were normalized to the level of β-actin and expressed in mean ± SEM. Representative blots are shown in A. Number of animals per group: ApoE-/-: control (n = 5); CeO2 (n = 5); 27% ZrO2-doped CeO2 (n = 5); 78% ZrO2-doped CeO2 (n = 4). 5xFAD: control (n = 4); CeO2 (n = 4); 27% ZrO2-doped CeO2 (n = 4); 78% ZrO2-doped CeO2 (n = 4). C57BL/6J: control (n = 5); CeO2 (n = 5); 27% ZrO2-doped CeO2 (n = 5); 78% ZrO2-doped CeO2 (n = 5).</p><!><p>The experiments performed in this study formed part of a large study to assess the influence of redox activity on the toxicity of inhaled CeO2 NPs in mice, by comparison of the effects of different quantities of Zr-doping. Detailed physicochemical and exposure characteristics of the NPs as well as the pulmonary and cardiovascular findings in the exposed mice have been published in a separate paper (Dekkers et al. 2017). In all three mouse models (C57BL/6J, 5xFAD, ApoE-/-) the four-week inhalation exposures were without any major toxicological effects in the lungs. In the ApoE-/- mouse model of vascular disease, the inhalation exposures to the NPs did not cause a statistically significant change in the overall size of atherosclerotic plaques. However, there was a trend towards an increased inflammatory cell content (i.e. macrophage-derived foam cells) in the plaques with the inhalation of CeO2 NPs with increasing ZrO2 content (Dekkers et al. 2017).</p><p>In the present study, we evaluated whether inhalation exposure to these NPs could also cause neurotoxicity and promote AD. Therefore, mouse behaviour tests were performed in all three mouse models to explore effects on motor activity and cognitive function. Brain tissue protein levels of HO-1, Nrf2, Iba-1 and GFAP were measured to address the role of oxidative stress and neuroinflammation. The potential effects of the inhaled CeO2 NPs on amyloid-β plaque formation were assed in the 5xFAD mouse model. In this study, we could observe specific effects that were dependent on the mouse model as well as the NP modification. While the behaviour effects were observed in both compromised mouse models, increased protein levels of GFAP were found only in the healthy C57BL/6J mice. These significant effects were observed exclusively for the CeO2 NPs that were doped with the highest amount of Zr (78%). In the ApoE-/- mice, the four-week inhalation exposure to these specific NPs resulted in a significantly diminished performance in the string suspension test. Such effect could be an indication of a greater susceptibility to an impaired forced motor performance in this mouse model of vascular disease. In the 5xFAD mice, the exposure to the 78% Zr-doped CeO2 NPs resulted in a significant reduction of the total of arm entries in the X-maze task. This latter effect suggests a possible reduction in explorative locomotor activity for this mouse model of AD. However, alternation behaviour in the X-maze test, which is an indicator of cognitive performance, was not impaired in the same treatment group.</p><p>Interestingly, while the motor performance effects on behaviour were observed with the two disease models, no behavioural effects were seen in the healthy (C57BL/6J) mice. Rodent models of susceptibility and disease are being increasingly used in toxicological studies exploring air pollution to better understand the underlying mechanisms (Oberdorster et al., 2005; Stone et al. 2017). In line with our present findings with the Zr-doped CeO2 NPs, impaired motor performance was observed following diesel engine exhaust inhalation exposure in 5xFAD mice but not in their wildtype littermates (Hullmann et al. 2017). Studies with diesel exhaust particles in high-fat fed ApoE deficient mice have also demonstrated the value of this susceptibility model over wildtype mice to support the epidemiological evidence that links exposure to airborne particles to cardiovascular disease (Miller et al. 2013). Interestingly, the behaviour changes in the two compromised mouse models were exclusively seen with the highest Zr-doped CeO2, indicating that these effects appear to depend on the redox-activity of the inhaled NPs. The introduction of Zr into the crystalline structure of CeO2 NPs is considered to enhance their antioxidant properties (Tsai et al. 2008). As such, one would have expected a possibly protective effect for the undoped CeO2 NPs. However, our present findings are in line with the previously reported effects of the inhalation exposures on the inflammatory content of atherosclerotic plaques in the ApoE-/- mice, which revealed an increased presence of macrophage-derived foam cells for CeO2 NPs with increasing Zr content (Dekkers et al. 2017).</p><p>Behaviour tests form an important component of neurotoxicity testing (Moser, 2011; OECD, 1997). It is therefore tempting to speculate that the observed motor performance effects in the ApoE-/- and 5xFAD mice result from a direct neurotoxic effect of the high Zr-doped CeO2 NPs. Indeed, several studies that have explored the pulmonary toxicity of NPs, including CeO2, indicate that their adverse effects are driven by oxidative stress and inflammation (Unfried et al. 2008; Morimoto et al. 2016; Stone et al. 2017; Schwotzer et al. 2018). However, in our present inhalation study we found no significant treatment related changes in HO-1 or Nrf2 for all three mouse models. In contrast, Hardas and colleagues observed increased HO-1 in rat brain upon intravenous administration of CeO2 NPs (Hardas et al. 2014). The fundamental differences in exposure route and dose offer a plausible explanation for these contrasts. The brains of ApoE-/- and 5xFAD mice in our inhalation study also did not display significant treatment related changes in protein levels of Iba-1 and GFAP, even for the groups that were exposed to the 78% Zr-doped CeO2 NPs. Taken together, this suggests that the motor function effects which we observed in both compromised mouse models were not mediated by local oxidative stress and neuroinflammation.</p><p>Surprisingly, however, increased protein levels of GFAP were observed in the cortex of the healthy C57BL/6J mice, the only mouse model that did not show significant changes in (motor function) behaviour. On the one hand, this adds further support to the absence of a mechanistic link between neuroinflammation and motor activity changes for inhaled CeO2 NPs. On the other hand, the finding again indicates the importance of the redox-properties of CeO2 NPs, as the effect on GFAP was only seen with the particles that were doped with the highest amount of Zr (78%). Increased GFAP levels were previously also found in rat brain following repeated inhalation exposures to steel welding fumes (Antonini et al. 2009). In contrast to our findings, increased GFAP levels were observed in ApoE-/- mice after long term inhalation of ambient ultrafine particles (Kleinman et al. 2008). In another study with in C57BL/6 mice, the long term inhalation of fine (μm size mode) ambient particulate matter (PM2.5) did not cause significant changes in brain levels of GFAP and Iba-1 (Bhatt et al. 2015).</p><p>The ApoE-/- mice were selected a priori for the investigation of cardiovascular effects following NP inhalation exposure, however, due to the logistical requirements of the extensive tissue collection, we were unable the further evaluate the brain tissue from these mice by immunohistochemistry (Dekkers et al. 2017). However, the brains of the 5xFAD mice were prioritised to address the potential impact of (undoped and Zr-doped) CeO2 NPs on the development of the neurodegenerative processes. Previously, we demonstrated an accelerated amyloid plaque load formation (whole brain Aβ42 protein levels) in 15 week old female 5xFAD mice following a three-week diesel engine exhaust inhalation exposure (0.95 mg/m3, 6 h/day, 5 days/week) (Hullmann et al. 2017). In the present study, however, we did not observe a significant alteration in the β-amyloid pathology in the brains of 5xFAD animals following four-week inhalation exposure to the (Zr-doped) CeO2 NPs (4 mg/m3 for 3 h/day, 5 days/week). Moreover, in alignment with the Western blot findings, the brains of the 5xFAD mice did not reveal significant differences in immunostaining of Iba-1. Combined with the observed absence of (cognitive) behaviour changes in the 5xFAD mice, these data argue against the hypothesis that CeO2 NPs may promote AD pathology in association with their redox-activity. The motor performance changes observed to NPs in the ApoE-/- and 5xFAD mice may not necessarily be related to a direct neurotoxic effect in isolation, but instead due to indirect effects, or alternatively, a result of an interaction between the exposure and increased susceptibility of both disease models. Age related changes in motor performance are well-described in the 5xFAD mouse model (Jawhar et al. 2012; O'Leary et al. 2018) and have also been reported for the ApoE-/- mice (Raber et al. 2000; Zerbi et al. 2014). Importantly, however, we did not observe statistically significant differences in behaviour test performance of the (clean air exposed) control mice between the three different mouse modes. This indicates that there was no major behaviour impairment per se in the two mouse disease models, and also suggests that it is unlikely that the effects of NPs on ApoE-/- mice were principally due to the high-fat diet fed to the mice. Further research is needed to verify the potential adverse impact of inhaled CeO2 NPs on motor function and to unravel the mechanism that could explain the redox-involvement for these metal oxide NPs.</p><p>Up to now, there is only very limited data about the potential neurotoxic effects of CeO2 NPs in association with inhalation exposure. A recent study showed that female ICR mice exposed to CeO2 particles (intranasal instillation, daily dose of 40 mg/kg body weight) of varying sizes (i.e. 35 nm, 300 nm and >1 μm) displayed significantly increased GFAP expression in the hippocampus and olfactory bulb. The authors claim that intranasal instillation of CeO2 particles induced damage within the olfactory bulb and hippocampus, but that particle size does not play a major role in the observed adverse responses (Liu et al. 2016). Nemmar and colleagues reported increased levels of the inflammatory cytokine Tumor Necrosis Factor-α, reactive oxygen species and DNA damage in the brains of mice by CeO2 NPs, 24 h after a single intratracheal instillation (0.5 mg/kg) (Nemmar et al. 2017). However, for the aforementioned studies the observed effects require perspective on the method and site of administration in the respiratory tract for the CeO2 particles, when compared to the outcomes of our present controlled inhalation exposure study. This relates to the obvious differences in dose and dose-rate of the NPs (i.e. bolus application versus inhalation) as well as to the regional deposition in the respiratory tract organ (i.e. nasal versus alveolar).</p><!><p>We have investigated the neurological effects of redox-modified CeO2 NPs using varying levels of Zr-doping (0%, 27% and 78%), after four-week inhalation exposures in three different mouse models. Our study findings reveal that the subacute inhalation exposure to CeO2 NPs did not cause major cognitive behavioural impairments in mice or promote amyloid-β plaque formation and neuroinflammation in the 5xFAD transgenic mouse model of AD. However, motor performance changes were observed both in the 5xFAD and ApoE-/- mice for the CeO2 NPs that were doped with the highest amount of Zr. In healthy C57BL/6J mice, the same particles caused increased GFAP levels in the absence of behaviour changes. The observed behavioural effects in the two compromised models were not substantiated further by changes in markers of neuroinflammation and oxidative stress. Therefore, further investigations are warranted to unravel the mechanism whereby inhaled CeO2 NPs can affect motor activity in a redox activity dependent manner.</p><!><p>The work leading to these results has received funding from the European Union Seventh Framework Programme for research, technology development and demonstration [grant agreement no. 310451 (NanoMILE)] and the Netherlands Food and Consumer Product Safety Authority (NVWA) [V090016]. MRM is supported by the 10.13039/501100000274British Heart Foundation [SP/15/8/31575; CH/09/002].</p><!><p>The authors declare that they have no competing interests.</p>

PubMed Open Access

Kinetics of cooperative CO2 adsorption in diamine-appended variants of the metal–organic framework Mg2(dobpdc)†

Carbon capture and sequestration is a key element of global initiatives to minimize anthropogenic greenhouse gas emissions. Although many investigations of new candidate CO2 capture materials focus on equilibrium adsorption properties, it is also critical to consider adsorption/desorption kinetics when evaluating adsorbent performance. Diamine-appended variants of the metal–organic framework Mg2(dobpdc) (dobpdc4− = 4,4′-dioxidobiphenyl-3,3′-dicarboxylate) are promising materials for CO2 capture because of their cooperative chemisorption mechanism and associated step-shaped equilibrium isotherms, which enable large working capacities to be accessed with small temperature swings. However, the adsorption/desorption kinetics of these unique materials remain understudied. More generally, despite the necessity of kinetics characterization to advance adsorbents toward commercial separations, detailed kinetic studies of metal–organic framework-based gas separations remain rare. Here, we systematically investigate the CO2 adsorption kinetics of diamine-appended Mg2(dobpdc) variants using a thermogravimetric analysis (TGA) assay. In particular, we examine the effects of diamine structure, temperature, and partial pressure on CO2 adsorption and desorption kinetics. Importantly, most diamine-appended Mg2(dobpdc) variants exhibit an induction period prior to reaching the maximum rate of CO2 adsorption, which we attribute to their unique cooperative chemisorption mechanism. In addition, these materials exhibit inverse Arrhenius behavior, displaying faster adsorption kinetics and shorter induction periods at lower temperatures. Using the Avrami model for nucleation and growth kinetics, we determine rate constants for CO2 adsorption and quantitatively compare rate constants among different diamine-appended variants. Overall, these results provide guidelines for optimizing adsorbent design to facilitate CO2 capture from diverse target streams and highlight kinetic phenomena relevant for other materials in which cooperative chemisorption mechanisms are operative.

kinetics_of_cooperative_co2_adsorption_in_diamine-appended_variants_of_the_metal–organic_framework_m

Introduction<!>Experimental setup for a thermogravimetric assay to study CO2 adsorption kinetics<!>Adsorption kinetics and temperature dependence for m-2-m–Mg2(dobpdc)<!>Avrami model of the adsorption kinetics of m-2-m–Mg2(dobpdc)<!>Inverse Arrhenius behavior<!>A structure–property kinetics relationship using a panel of ethylenediamine analogues<!>Adsorption kinetics of 2,2-dimethyl-1,3-diaminopropane–Mg2(dobpdc)<!>Desorption kinetics<!>Conclusions<!>Conflicts of interest

<p>Steadily rising atmospheric CO2 levels and the associated increase in average global temperatures have created an urgent need to curb anthropogenic CO2 emissions.1 While long-term solutions to this challenge necessitate a shift to renewable energy sources, fossil fuels will continue to supply a major portion of global energy in the near future.2 One proposed strategy to mitigate atmospheric CO2 emissions in the short term is carbon capture and sequestration from major point sources, such as power plants.3 To realize this strategy, carbon capture materials are needed that possess high selectivities and CO2 capacities, as well as minimal energy requirements for CO2 desorption. Rapid kinetics to bind and release CO2 are also critical, because they can dictate bed utilization and thus impact the cost and efficiency of a carbon capture process.4</p><p>Aqueous solutions of organic amines are a mature CO2 capture technology, but they suffer from numerous drawbacks. For example, solutions with relatively low amine concentrations are necessary to minimize corrosive effects, thereby decreasing the solution CO2 absorption capacities and increasing the energy required to heat the absorbent during temperature-swing cycling.5 As a result, the implementation of aqueous amine scrubbers in a power plant places a parasitic load of 25–30% on the net power output.6 Furthermore, aqueous amine solutions are prone to thermal and oxidative degradation.3 As an alternative, porous solid adsorbents have been proposed as capture materials, owing to their lower heat capacities and high surface areas, which create the potential for high adsorption capacities and more efficient adsorption–desorption cycling. Nevertheless, many of these solid adsorbents fail to capture CO2 selectively from humid gas streams.7</p><p>Amine-functionalized solid adsorbents combine the advantages of aqueous amine solutions and porous solid adsorbents. Examples of these materials include amine-functionalized silicas,8–11 porous polymers,12,13 zeolites,14,15 and metal–organic frameworks.16–20 Owing to their high crystallinity and chemical adjustability, metal–organic frameworks possess ordered structures that can be tailored with respect to pore size, shape, and chemical environment. In particular, amine functionalities can be incorporated within the organic linkers of these materials, both during framework synthesis21 or through post-synthetic modification.16–20,22 Additionally, the high internal surface areas accessible with metal–organic frameworks can allow for rapid diffusion of CO2 through the pores.23 Diamine-appended variants of the metal–organic framework Mg2(dobpdc)24–31 (dobpdc4− = 4,4′-dioxidobiphenyl-3,3′-dicarboxylate) represent a particularly promising class of amine-functionalized frameworks, as they readily adsorb CO2 through a chemically-specific, cooperative mechanism (Fig. 1).25 Here, the metal-bound amine reacts covalently with CO2 to generate a carbamate while the pendent amine is concomitantly protonated. This process propagates through the material to yield chains of ammonium carbamate stabilized through ionic interactions along the pore axis. As a result of this unique capture mechanism, diamine-appended variants of Mg2(dobpdc) exhibit step-shaped CO2 adsorption profiles, which give rise to large CO2 cycling capacities that are accessible with relatively small temperature swings.25 Importantly, by varying the metal cation,25 diamine,26–28 or organic linker,28 the adsorption step position can be tuned in pressure by over five orders of magnitude (from ∼10−5 to ∼1 bar at 40 °C) to enable the precise targeting of specific CO2 separation conditions. Moreover, these materials have also been shown to maintain high CO2 working capacities after 1000 adsorption/desorption cycles under humid gas streams.27</p><p>While the thermodynamics of CO2 capture in diamine-appended variants of Mg2(dobpdc) are promising for numerous carbon capture applications, the kinetics also play a crucial role in the practical application of these materials. For porous solid adsorbents, small-scale breakthrough experiments are often used to simulate a fixed-bed adsorption process. In these experiments, shaped particles of the adsorbent are packed into a column, a CO2-containing gas stream is fed through the inlet, and the outlet composition and flow rate are measured as a function of time until CO2 "breaks through." Multiple kinetic parameters can influence the performance of an adsorbent in a fixed bed, including interparticle, intraparticle, and micropore diffusional resistances, as well as potential reaction limitations for amine-based chemisorption of CO2. Critically, the overall CO2 adsorption kinetics must be sufficiently fast to maximize bed utilization in the process. Promising initial results have been obtained for diamine-appended Mg2(dobpdc) variants in gram-scale breakthrough experiments for CO2 capture from simulated coal flue gas (15% CO2 in N2) under both dry and humid conditions.27 In addition, rapid cycle times have been employed for diamine-appended Mg2(dobpdc) variants in simulated temperature-swing experiments under 15% CO2 with a pure CO2 purge, and here cycle times were limited only by the temperature ramp rate of the thermogravimetric analyzer.27,28</p><p>Given the promise of diamine-appended Mg2(dobpdc) for CO2 capture applications, a detailed analysis of the kinetics of CO2 adsorption in these materials is necessary for optimal process implementation. Toward this end, we herein utilize thermogravimetric analysis (TGA) to systematically investigate the dry CO2 adsorption kinetics in diamine-appended variants of Mg2(dobpdc) under a range of adsorption conditions. Our results demonstrate the influence of adsorption temperature, CO2 concentration, and diamine structure on the rate of adsorption. On the basis of these correlations, we conclude with guidelines for the optimization of adsorbent structure and process parameters in CO2 capture applications.</p><!><p>Diamine-appended variants of Mg2(dobpdc) were synthesized using our previously reported procedure.26 Specifically, methanol-solvated Mg2(dobpdc) was soaked in a 20% (v/v) toluene solution of the diamine of interest, and subsequent filtration, washing with toluene, and activation yielded adsorbents with one diamine per metal site. To ensure internal consistency for this study, all experiments were performed with the same batch of Mg2(dobpdc). The rod-shaped crystallites in this batch exhibited an aspect ratio of ∼10 and were heterogeneous in size, with widths ranging from ∼60 nm to >1 μm (Fig. S1†). Measurements in this study were carried out on as-synthesized diamine–Mg2(dobpdc) samples in powder form; future studies will explore adsorption/desorption kinetics in structured forms of these materials.</p><p>We developed a TGA assay at atmospheric pressure to evaluate the CO2 adsorption kinetics of diamine-appended Mg2(dobpdc). In this assay, samples were first activated in the TGA furnace under flowing N2 to remove any captured CO2, solvent, or excess diamine present in the pores. Next, samples were cooled to a temperature of interest, the gas flow was switched to a CO2-containing stream, and the change in mass was monitored as a function of time (Fig. 2). Because N2 adsorption is negligible at and above room temperature in these materials,26,27 we approximated that all mass increase was due to CO2 adsorption. In addition, mass changes due to buoyancy effects upon switching the gas stream from N2 to CO2 were negligible compared to the mass of CO2 adsorbed by the standard sample size of 3 mg of diamine–Mg2(dobpdc) used in this work (Fig. S2†).</p><p>This TGA-based assay, which has been reported for fundamental kinetics characterization of other solid CO2 adsorbents,11,32–35 is advantageous given its simple setup, the small sample mass requirement, and the fact that adsorbents can be activated in situ and rapidly tested under many temperatures and partial pressures of CO2. However, this assay also has some limitations. For example, because TGA detects gas adsorption through a change in sample mass, it cannot discriminate between CO2, H2O, and N2 adsorption (although, as mentioned above, N2 adsorption is negligible for diamine-appended Mg2(dobpdc) variants under the conditions relevant for CO2 chemisorption). As a result, determining CO2 adsorption kinetics under humid conditions remains challenging, and here we discuss only dry adsorption/desorption kinetics profiles. In addition, the first few points of kinetics data from a TGA experiment are obscured by exchange of the initial gas in the furnace with the CO2-containing stream in adsorption experiments or the N2 purge stream in desorption experiments.</p><p>As has been previously noted,11 differences in flow rate and sample mass can also greatly affect TGA adsorption kinetics profiles. For example, in the case of m-2-m–Mg2(dobpdc) (m-2-m = N,N′-dimethylethylenediamine), we found that the sample mass has a substantial impact on adsorption kinetics from a simulated coal flue gas stream of 15% CO2 in N2,36 with larger samples displaying slower overall adsorption kinetics (Fig. S3†). Therefore, to maintain consistency across all samples in this study, we used a sample mass of 3 mg and ensured the powder was evenly distributed across the surface of the TGA pan. Also consistent with previous reports, faster adsorption kinetics were observed with faster flow rates (Fig. S3†). This flow rate effect could be due to the time required to completely exchange the initial gas in the furnace and/or to the observed kinetics being influenced by a mass transfer resistance related to diffusion. As a result, the kinetics of adsorption presented in this work likely represent lower bounds on the intrinsic adsorption kinetics of these materials. For the fastest flow rates tested (>100 mL min−1), adsorption was essentially complete within the first few data points collected on the TGA, making it difficult to quantitatively compare the kinetics among different adsorbents under these conditions. We therefore used a consistent flow rate of 25 mL min−1 for all experiments to facilitate quantitative comparisons among the diamine-appended variants. Under these conditions, a consistent delay of 19 s was observed before the sample mass increased, after switching the TGA valving to the analysis gas. This delay time corresponds to the time required for CO2 to reach the sample in the TGA furnace. Additionally, given a flow rate of 25 mL min−1, we approximate that at least 14–23 s are required at temperatures ranging from 120 to 30 °C for complete exchange of gases in the TGA furnace, beyond the initial 19 s delay (Table S1†). By accounting for these considerations in the measurements detailed below, we were able to compare the effects of temperature, CO2 partial pressure, and diamine structure on the CO2 adsorption kinetics of these materials.</p><!><p>We utilized our optimized TGA assay to characterize the CO2 adsorption kinetics of m-2-m–Mg2(dobpdc), the first reported diamine-appended variant of Mg2(dobpdc),24 at selected temperatures. Extensive gas adsorption, structural, and spectroscopic data have been previously reported for this material,24–26 and an overview of the adsorption mechanism is shown in Fig. 3a. We first investigated the CO2 adsorption kinetics of m-2-m–Mg2(dobpdc) from a pure CO2 stream at atmospheric pressure over a range of temperatures below the adsorption step temperature (Tstep) of 127 °C,26 defined here as the onset of the step-shaped adsorption isobar (Fig. 3b). Rapid uptake of CO2 was observed below Tstep, as is evident in a plot of fraction of diamine sites occupied (Qt) vs. time (Fig. 3c). Interestingly, adsorption of CO2 is faster at lower temperatures and follows an inverse Arrhenius behavior (Fig. 3d). Such inverse Arrhenius behavior has been observed previously for a polyethylenimine-appended mesoporous silica11 and amine-functionalized carbon nanotubes,37 whereas normal Arrhenius behavior has been observed for other amine-appended solid adsorbents.34,35,38 Our results suggest that although the equilibrium capacity of m-2-m–Mg2(dobpdc) is relatively insensitive to temperature below Tstep,26 lower adsorption temperatures promote more rapid saturation with CO2. A more in-depth discussion of this phenomenon is provided below (see "Inverse Arrhenius behavior").</p><p>In addition to this inverse Arrhenius behavior, we also observed an induction period between the time at which the mass begins to increase, which corresponds to CO2 first entering the furnace, and the time associated with the fastest rate of CO2 uptake (Fig. 3d). This effect is particularly pronounced at temperatures close to Tstep. To elucidate whether the observed induction period is intrinsic to the CO2 adsorption kinetics of m-2-m–Mg2(dobpdc) or is an artifact of the experimental setup, we investigated CO2 adsorption in the bare framework material with no appended diamines, Mg2(dobpdc) (Fig. 3e and f). In Mg2(dobpdc), CO2 binds to open metal coordination sites exposed upon solvent removal from the framework, leading to a typical Langmuir adsorption profile (Fig. 3e).24 Importantly, because CO2 adsorption in Mg2(dobpdc) does not involve a chemical reaction, the adsorption kinetics are likely diffusion-limited. Consistently, Mg2(dobpdc) reaches its equilibrium CO2 adsorption capacity within a similarly short time for all temperatures examined (Fig. 3g and h), with the maximum rate of adsorption occurring at earlier times as the temperature is increased, in stark contrast to the behavior exhibited by m-2-m–Mg2(dobpdc). For example, at 120 °C, approximately 51 s elapses before the maximum rate of adsorption is achieved in m-2-m–Mg2(dobpdc), whereas the maximum rate is reached in only 14 s for bare Mg2(dobpdc) under the same conditions (see asterisks in Fig. 3d and h). Note that comparison of the absolute rate of adsorption is complicated by the lower equilibrium capacity of Mg2(dobpdc) at the low partial pressures present in the TGA furnace during the initial mixing period (Fig. S4†). Furthermore, the apparent kinetics of Mg2(dobpdc) include competition of N2 and CO2 for the same binding sites, and thus CO2 adsorption requires displacement of any adsorbed N2 molecules. Nevertheless, only a small fraction of metal sites (∼2–10%) is expected to be occupied by N2 under the conditions examined here, based on previously reported N2 adsorption isotherms for the isoreticular smaller-pore framework material Mg2(dobdc).39</p><p>In addition to the marked differences in the kinetics of CO2 adsorption, the bare framework Mg2(dobpdc) and amine-appended m-2-m–Mg2(dobpdc) also exhibit distinct trends in their variable–temperature equilibrium CO2 adsorption capacities (Qe). Whereas the CO2 saturation capacity of m-2-m–Mg2(dobpdc) is similar across nearly all temperatures investigated, decreasing only near Tstep (Fig. 3b), the capacity of Mg2(dobpdc) varies substantially with temperature (Fig. 3f). This difference arises due to the different adsorption profiles of these two materials and is best demonstrated through a van't Hoff plot (Fig. 4a). To quantify the kinetics in these two materials while accounting for variations in equilibrium capacity, we also analyzed the percent of adsorption complete (Qt/Qe) vs. time (Fig. 4b and c). This analysis again reveals that adsorption is faster at lower temperatures for m-2-m–Mg2(dobpdc), with induction periods observed at temperatures close to Tstep. In contrast, the adsorption profiles are similar at all temperatures for Mg2(dobpdc), with the highest temperatures exhibiting the fastest initial progress toward equilibrium. Overall, these findings corroborate that the chemisorptive mechanism operational in m-2-m–Mg2(dobpdc) leads to an unusual induction period that is not observed in the physisorptive mechanism in Mg2(dobpdc).</p><p>To further understand the induction period exhibited by m-2-m–Mg2(dobpdc), we characterized the adsorption kinetics from a 15% CO2 stream (Fig. 5), corresponding to the approximate partial pressure of CO2 in coal flue gas.36 In this case, the investigated temperature range (45–100 °C) is lower than that used in the experiments with 100% CO2 (75–120 °C), reflecting the lower adsorption step temperature under 15% CO2 (102 °C) compared to 100% CO2 (127 °C) (Fig. S8†). At temperatures below 75 °C, adsorption was nearly complete in less than 1 min. At temperatures near Tstep, however, the induction period was even more pronounced than in the analogous experiments with 100% CO2 (Fig. 5bvs.Fig. 3d). The effect was particularly dramatic at 100 °C (Fig. 5c)—a small amount of CO2 was rapidly adsorbed (6% of the diamine sites occupied within 1 min), followed by a period of 4 min during which almost no additional adsorption occurred. After 5 min, the rate of CO2 adsorption accelerated, and a substantial CO2 occupancy was ultimately reached, corresponding to occupation of approximately half of the diamine sites. The fast capture of CO2 at ∼6% of the m-2-m sites, as shown in Fig. 5c, is consistent with the previous finding that m-2-m–Mg2(dobpdc) adsorbs a small amount of CO2 even at pressures below the step.24,25 Spectroscopic studies indicate that this pre-step chemisorption arises due to ammonium carbamate species that form without metal–amine insertion.7 We hypothesize that the pre-step ammonium carbamate species forms rapidly, followed by slower cooperative adsorption of additional CO2via the formation of metal-bound ammonium carbamate chains. Qualitatively similar results were obtained using a 5% CO2 stream, for which the induction period is even more pronounced (Fig. S6†).</p><p>Taken together, these results suggest that the sigmoidal kinetics profile of m-2-m–Mg2(dobpdc) is directly related to its cooperative CO2 adsorption mechanism. This material exhibits a high degree of cooperativity in equilibrium gas adsorption isotherms, with its Hill coefficient of ∼11 indicating that, from a thermodynamic standpoint, capturing one CO2 molecule facilitates the adsorption of subsequent CO2 molecules.25 The pronounced sigmoidal kinetics profile exhibited by m-2-m–Mg2(dobpdc) likewise suggests that initial capture of a CO2 molecule also enhances the adsorption kinetics of subsequent CO2 molecules. Similar sigmoidal kinetic profiles have been reported previously for autocatalytic40 and autoinductive41 chemical reactions. While a recent TGA-based assay previously revealed that a polyethylenimine-appended mesoporous silica also exhibited sigmoidal CO2 adsorption kinetics,11 amine-impregnated clays did not exhibit sigmoidal uptake kinetics when evaluated using comparable TGA equipment and procedures.34 These precedents establish that not all amine-based CO2 adsorbents exhibit sigmoidal kinetics. As noted in a recent study,42 a long induction period is undesirable for implementation in a practical process because it can lead to lower bed utilization. Specifically, in breakthrough experiments simulating direct air capture with m-2-m–Mg2(dobpdc), it was found that after rapid partial breakthrough of CO2, the CO2 concentration at the outlet decreased before full breakthrough eventually occurred, consistent with a delayed onset of adsorption.42 Hence, it is critical to identify appropriate diamine variants and/or CO2 adsorption conditions that minimize this induction period (see below).</p><!><p>We next sought to model the CO2 adsorption kinetics of m-2-m–Mg2(dobpdc) from a gas stream of 15% CO2 in N2. The fraction of sites occupied at time t (Qt) was fit at each temperature using either a pseudo-first order model (eqn (1)) or Avrami's kinetics model (eqn (2)), which was originally developed to model nucleation-growth kinetics43 and has recently found application as a model for chemisorption in an amine-appended mesoporous silica.1112</p><p>Note that in both models, the constant partial pressure of CO2 (P/P0 = 0.15) is embedded in the rate constant k1 or kA, respectively, and that nA in the Avrami model is also likely a function of P/P0. As expected, a pseudo-first order model using rate constant k1 (eqn (1)) failed to capture the induction period at temperatures near Tstep (Fig. 6a). In contrast, the Avrami model fits the data well, particularly for temperatures just below the step. We attribute the deviation from the model in the first 1.5 min to incomplete gas mixing in the furnace and to pre-step chemisorption, as discussed above. This model incorporates the Avrami parameter, nA, as well as the Avrami rate constant, kA, to produce sigmoidal kinetics profiles, with larger nA values leading to longer induction periods. For example, nA = 2 corresponds to the sigmoidal kinetics profile of the growth of a one-dimensional crystal.44,45</p><p>A plot of the Avrami rate constant kAvs. T, which is useful for visualizing trends in these data, is presented in Fig. 6b. This plot indicates that the Avrami rate constant becomes progressively larger with decreasing temperature, reflecting the inverse Arrhenius behavior of m-2-m–Mg2(dobpdc) (Fig. 6b). At temperatures below 70 °C, the slope of kAvs. T substantially decreases, possibly due to the intrinsic properties of the material or to instrumentation limitations associated with the high rate of adsorption at these colder temperatures. Unexpectedly, kAvs. T follows a linear trend from 70 to 95 °C, inconsistent with standard Arrhenius behavior (see "Inverse Arrhenius Behavior" below). Notably, the x-intercept of this plot (kA = 0) should correspond to Tstep; indeed, the x-intercept of a linear fit to the high-temperature data (70–95 °C) of 100 °C is close to the step temperature determined by cooling a sample of m-2-m–Mg2(dobpdc) under 15% CO2 (102 °C; see Fig. S8 and Table S2†). In addition, the parameter nA progressively decreases from an initial value of 1.7 at 95 °C to a value close to 1 at the lowest temperature of 55 °C, reflecting the longer induction periods near Tstep. Therefore, the parameters from these Avrami fits successfully reflect the experimental observations of slower CO2 adsorption kinetics (smaller kA) and longer induction periods (larger nA) at high temperatures close to the step temperature in m-2-m–Mg2(dobpdc). The smaller values of nA at lower temperatures reflect shorter induction periods and demonstrate an advantage of maintaining a buffer between the adsorption temperature and the step temperature.</p><!><p>The inverse Arrhenius behavior observed here can be rationalized using a reaction coordinate diagram derived from several previous investigations of reactions with apparent negative activation energies (Fig. 7).40,46–49 In general, inverse Arrhenius behavior requires the reversible formation of an intermediate species with equilibrium constant Keq that (i) is lower in energy than the reactant(s), with energy difference Δgint; and (ii) proceeds to product formation through a transition state, with kinetic barrier Δg‡rxn, that is also lower in free energy than the reactant(s), such that |Δg‡rxn| < |Δgint| (see Fig. 7). In addition, the product formation must be an effectively irreversible process, with negligible conversion back to the intermediate species under the reaction conditions. This scenario leads to inverse Arrhenius behavior because the rate of product formation is dependent on the concentration of the intermediate species. Critically, for an adsorption process, this key intermediate species is thermodynamically disfavored at higher temperatures due to the greater entropic penalty associated with removing CO2 from the gas phase. In other words, increasing the temperature drives the intermediate species back toward the reactant(s) more than it promotes overcoming the barrier to form the product. In m-2-m–Mg2(dobpdc), reversible adsorption of CO2 to form a labile intermediate, such as a physisorbed or weakly chemisorbed species, likely precedes an exothermic rate-determining chemisorption step. The final product of chemisorption is an ammonium carbamate chain,26 which forms with a thermodynamic free energy change of Δgads. However, C–N bond formation, proton transfer, and metal–oxygen bond formation must all occur between the starting material and the final ammonium carbamate chains; the number of steps in this reaction pathway and the rate-determining step remain points of ongoing investigation. Nevertheless, the temperature dependence of the kinetics of CO2 adsorption in m-2-m–Mg2(dobpdc) is consistent with an overall mechanistic model involving the reversible formation of an entropically disfavored, CO2-bound intermediate followed by a rate-determining chemisorption step. A similar model can be invoked to explain the observed inverse Arrhenius behavior of CO2 adsorption in other amine-functionalized adsorbents.11,37</p><p>To further understand the behavior of m-2-m–Mg2(dobpdc), we constructed an Arrhenius plot using the Avrami rate constants given in Fig. 6b. As expected, ln(kA) increases with 1/T (Fig. 8), and the sharp curvature occurring near 1/Tstep is consistent with the unusually linear behavior of kAvs. T near Tstep (Fig. 6b). The data can be fit well using a logarithmic function with a vertical asymptote corresponding to ∼97 °C, close to the Tstep of 102 °C (see Fig. S8†). The steeper slope of the Arrhenius plot near 1/Tstep indicates that the temperature dependence of the rate of adsorption in m-2-m–Mg2(dobpdc) is greatest close to Tstep.</p><p>While the reaction coordinate diagram in Fig. 7 accounts for the inverse Arrhenius behavior of CO2 adsorption in m-2-m–Mg2(dobpdc), it does not necessarily predict the extreme curvature of the Arrhenius plot near 1/Tstep. Because cooperative CO2 adsorption does not occur above Tstep, the Arrhenius plot for the cooperative adsorption process should deviate to −∞ upon approaching Tstep from low to high temperature. Discontinuities have previously been observed in linear Arrhenius plots when a phase change occurs at a specific temperature in an enzyme.50 However, the curvature exhibited in the Arrhenius plot of m-2-m–Mg2(dobpdc) is unusual and suggests a progressive decrease in the adsorption kinetics as T → Tstep. The curved Arrhenius plot can potentially be explained by considering the entropy change in the reaction coordinate diagram in Fig. 7, as has been previously described.51 Moving from left to right along the reaction coordinate in Fig. 7 corresponds to a significant decrease in degrees of freedom as gaseous CO2 is immobilized and the initially dynamic diamines are locked into ammonium carbamate chains. As the temperature increases, the entropic penalty for immobilizing CO2 increases and ultimately outweighs the enthalpic favorability of adsorption when T exceeds Tstep. Accordingly, the kinetic barrier of the rate-determining chemisorption step should become increasingly large at higher temperatures, consequently slowing the reaction kinetics. Therefore, increasing the temperature towards Tstep imposes two compounding deleterious entropic effects on the rate of adsorption in m-2-m–Mg2(dobpdc): it thermodynamically disfavors the formation of the intermediate species (as discussed above), and it increases the magnitude of the rate-limiting kinetic barrier. Together, these effects can potentially explain the observed progressively slower adsorption kinetics and resulting curved Arrhenius plot as T approaches Tstep. Determining the identity of the intermediate species and elucidating the mechanism of the rate-limiting chemisorption process remain active areas of investigation.</p><!><p>To determine the generalizability of these characteristics for m-2-m–Mg2(dobpdc) to other diamine-appended frameworks, we evaluated the kinetics of CO2 adsorption from a gas stream of 15% CO2 in N2 by a panel of Mg2(dobpdc) variants appended with structurally-diverse alkylethylenediamines. Notably, frameworks with 1°,3° alkylethylenediamines26 displayed minimal adsorption at temperatures ≥40 °C (Fig. S12†) and thus were not investigated further. In contrast, rate constants could be determined for frameworks appended with 1°,1°; 1°,2°; and 2°,2° amines (see Fig. 9). We note that the diamines N-isopropylethylenediamine (i-2) and N,N′-diethylethylenediamine (e-2-e) exhibit two-step adsorption behavior,28 and thus their kinetic profiles are complex at temperatures at which both steps are operative. As a result, Fig. 9 depicts only the temperatures under which the less thermodynamically favorable step is not operative for these two diamine-appended frameworks.</p><p>A similar overall sigmoidal kinetic profile was observed for all ethylenediamine variants in Fig. 9, consistent with our previous findings that these materials capture CO2 by the same mechanism.26,52 In each case, kA increases with decreasing temperature, indicative of inverse Arrhenius behavior, as observed for m-2-m–Mg2(dobpdc) (Fig. S13–S18†). Furthermore, all of the variants exhibit linear plots of kAvs. T (Fig. 9b) and curved Arrhenius plots that could be fit by logarithmic functions (Fig. S13–S18†), suggesting that the complex behavior characterized for m-2-m–Mg2(dobpdc) represents a general feature of these materials. In Fig. 9b, the linear fits to the kAvs. T data yield different x intercepts for each diamine-appended framework, reflecting their different adsorption step temperatures. The x-intercepts all match closely to step temperatures determined by cooling the frameworks under 15% CO2 (Fig. S8–S11 and Table S2†), except in the case of (±)-trans-dach–Mg2(dobpdc), for which the kAvs. T plot deviates from linearity at high temperatures (Fig. S20†).</p><p>The differences in adsorption step temperatures complicate comparisons between materials because the free energy change associated with CO2 adsorption (Δgads) is different for each material at a constant temperature. To account for this difference, we also plotted kAvs. Tstep − T to scale the x-intercepts of the linear fits to zero (Fig. 9c). Note that we do not consider Tstep − T to be a physically meaningful metric of chemical potential—rather, this plot is only intended to help visualize differences among diamine-appended frameworks. Importantly, the linear fits to the data in the corresponding plots all exhibit distinct slopes, reflecting variability in the extent to which decreasing the temperature below Tstep increases the CO2 adsorption kinetics. For practical applications, a steep slope in Fig. 9c is desirable to enable fast adsorption kinetics even at temperatures just below the step. In the plot of nAvs. Tstep − T (Fig. 9d), nA generally becomes smaller with decreasing temperature. Most of the materials exhibit values of nA between 1.3 and 1.8 at temperatures close to the step, decreasing to ∼1 at the lowest temperatures. Materials with smaller values of nA show less pronounced induction periods and are therefore preferable for implementation in a process.</p><p>Interestingly, the steepest slopes were observed for frameworks appended with the 2°,2° diamines m-2-m and e-2-e.24–26 These variants are notable for having weak metal–amine bonds, which serve to increase |Δhads| for CO2 and facilitate CO2 insertion, thus likely contributing to their fast adsorption kinetics.26 Despite these desirable kinetics properties, the weak metal–amine bonds lead to diamine volatilization under humid conditions, particularly at the high temperatures necessary for desorption under a pure CO2 stream.53,54 The second-fastest kinetics were found for variants of Mg2(dobpdc) functionalized with the 1°,2° diamines N-methylethylenediamine (m-2), N-ethylethylenediamine (e-2), and i-2.26,28,55 We previously determined that the primary amine preferentially binds to the metal site and reacts with CO2, which may account for the slower adsorption kinetics in these materials compared to those functionalized with 2°,2° diamines.26 However, this effect also bestows 1°,2° diamine-appended variants with enhanced stability toward diamine loss.26,28 The compounds with the least sterically-encumbered 1°,2° diamines exhibit slightly faster kinetics, as indicated by steeper slopes in the plots of kAvs. T (Fig. 9c). Overall, these results suggest a tradeoff between stability and adsorption kinetics for frameworks appended with 1°,2° diamines compared to those bearing 2°,2° diamines.</p><p>Finally, we studied the behavior of Mg2(dobpdc) variants appended with the 1°,1° diamines ethylenediamine (en) and (±)-trans-diaminocyclohexane (dach),26,29,31,56 which have previously been shown to exhibit significant adsorption/desorption hysteresis and unit cell contraction upon CO2 adsorption.56 Interestingly, the CO2 adsorption kinetics of en–Mg2(dobpdc) are extremely similar to those of i-2–Mg2(dobpdc)—the 1°,2° variant with the slowest CO2 adsorption kinetics—while (±)-trans-dach–Mg2(dobpdc) exhibits much slower kinetics. In addition, (±)-trans-dach–Mg2(dobpdc) displays an unusually high nA of ∼3 at temperatures close to the step, corresponding to a very long induction period (Fig. S19–S21†). As with the other diamines, the adsorption kinetics in (±)-trans-dach–Mg2(dobpdc) become much faster and the induction periods become much shorter at lower temperatures (nA = 1.3 at 60 °C; see Fig. S21†), and use of a fast flow rate (100 mL min−1) also shortens the induction period for this material (Fig. S22†). One possible explanation for the large nA value and slower overall kinetics of (±)-trans-dach–Mg2(dobpdc) is the unit cell contraction that occurs upon CO2 adsorption in conjunction with crystallographically characterized ion-pairing interactions between neighboring ammonium carbamate chains in the ab plane.56 Intriguingly, nA = 3 for the Avrami model corresponds to the growth of a crystal in two dimensions, whereas nA = 2 corresponds to growth in one dimension.44,45 Accordingly, cooperative CO2 adsorption in (±)-trans-dach–Mg2(dobpdc) may be akin to two-dimensional sheet growth, whereas cooperative CO2 capture by the other variants may be more akin to one-dimensional chain growth. Elucidating the potential correlation between ab plane contraction and the CO2 adsorption kinetics for 1°,1° diamine–Mg2(dobpdc) variants remains a subject of investigation.</p><!><p>In addition to ethylenediamines, Mg2(dobpdc) has also been appended with diaminopropanes to yield cooperative adsorbents.27 For example, dmpn–Mg2(dobpdc) (dmpn = 2,2-dimethyl-1,3-diaminopropane) has a favorable adsorption step temperature for CO2 capture from coal flue gas and maintains a high working capacity over 1000 cycles under humid conditions.27 Notably, the complex CO2 chemisorption mechanism in dmpn–Mg2(dobpdc) ultimately leads to the formation of carbamic acids interacting with ammonium carbamates, and this material exhibits a mixture of ∼4 chemisorbed species in the early stages of CO2 adsorption.52 Given this complexity and the promising attributes of dmpn–Mg2(dobpdc) for implementation in practical processes, we sought to investigate its adsorption kinetics from a gas stream of 15% CO2 in N2.</p><p>Consistent with previous findings, the 15% CO2 adsorption isobar for this material exhibits a step around 60 °C that is more broad than those of other diamine–appended Mg2(dobpdc) variants, reflecting its complex adsorption mechanism (Fig. 10a).27,52 Similarly, the kinetics profile of dmpn–Mg2(dobpdc) is distinct from that of the other diamine-appended variants (Fig. 10b and c), with no induction period observed even at temperatures close to the adsorption step. At all temperatures below 55 °C, dmpn–Mg2(dobpdc) exhibits a very rapid initial uptake, followed by slower uptake until reaching equilibrium. This unusual curve shape results in Avrami fits with small nA (∼0.4–0.7) and kA (∼0.003–0.006 s−1) values (Fig. S23†). The fastest initial uptake in dmpn–Mg2(dobpdc) occurs at 35 °C—the lowest temperature investigated—but only up to an occupancy of ∼0.6, after which the kinetics slow (Fig. S24†). We note that this slower adsorption at low temperatures and high occupancies was not observed in a comparable temperature range for the other diamine-appended variants, but a similar effect was reported previously in "molecular basket" polyamine-appended mesoporous silicas.10 Overall, the lack of induction period for dmpn–Mg2(dobpdc) is promising for implementation in a CO2 capture process.</p><!><p>Desorption kinetics are also a critical consideration for the practical use of an adsorbent. In a typical fixed-bed process, adsorption and desorption stages are operated simultaneously using a minimum of two beds. As a consequence, fast desorption kinetics are desirable to minimize cycle times. In a simulation of the desorption conditions typical of a temperature swing adsorption process, we previously demonstrated that CO2 can be desorbed from diamine-appended Mg2(dobpdc) under a humidified stream of pure CO2 within less than 10 min, as limited by the heating rate of the TGA furnace.27,28 Alternatively, the introduction of dry N2 as a purge gas can be used to simulate the application of a concentration- or vacuum-swing process. Because diamine-appended Mg2(dobpdc) variants can maintain high CO2 capacities under pure CO2 even at relatively high temperatures, these materials can be heated under CO2 to a temperature of interest and allowed to equilibrate, after which the gas stream can be switched to dry N2 and the decrease in mass can be monitored.</p><p>We utilized this dry N2 desorption assay to compare the desorption kinetics of dmpn–Mg2(dobpdc) and m-2-m–Mg2(dobpdc) (Fig. 11). At the highest temperature investigated for each material (70 and 110 °C for dmpn and m-2-m, respectively), nearly all of the CO2 was desorbed within 1.5 min, and decreasing the desorption temperature progressively decreased the rate of desorption. For each adsorbent, the initial Qt values varied with temperature, consistent with the isobaric adsorption profiles, and the larger Qt values at colder temperatures can be attributed to CO2 physisorption. Overall, desorption from dmpn–Mg2(dobpdc) occurs in a lower temperature range compared to desorption from m-2-m–Mg2(dobpdc), reflecting the lower desorption step temperature for the dmpn variant of 93 °C 27 compared to 134 °C for m-2-m,26 as determined from dry 100% CO2 desorption isobars collected using a ramp rate of 1 °C min−1.</p><p>As shown in Fig. S25,† the desorption kinetics for dmpn–Mg2(dobpdc) and m-2-m–Mg2(dobpdc) fit well to the Avrami model across multiple temperatures. For both materials, desorption is initially slow, but is accelerated after some CO2 has been desorbed. Interestingly, the desorption kinetics behavior of dmpn–Mg2(dobpdc) differs from its adsorption kinetics behavior. While this material exhibits no induction period for adsorption (nA ∼0.5), a substantial induction period was observed for desorption (nA ∼2 for many of the conditions tested; see Fig. S25†). These dissimilar kinetics profiles may result from dmpn–Mg2(dobpdc) exhibiting a mixture of approximately four chemisorbed species in the early stages of CO2 adsorption, while a 1 : 1 mixture of ammonium carbamates:carbamic acids is formed following saturation with CO2 and long equilibration times.52</p><!><p>The foregoing results describe a systematic investigation into the CO2 adsorption kinetics of diamine-appended variants of the metal–organic framework Mg2(dobpdc) using a TGA-based assay. Sigmoidal kinetics profiles were observed for all of the materials that exclusively form ammonium carbamate chains upon CO2 adsorption, which we attribute to their cooperative adsorption mechanism. While these adsorbents exhibit long induction periods at temperatures near the adsorption step, the kinetics of adsorption can be accelerated and the length of the induction period minimized by decreasing the adsorption temperature. Additionally, the diamine structure can be optimized to yield adsorption thermodynamics and kinetics that are most favorable for a target gas separation involving CO2. Finally, no induction period was observed for the material dmpn–Mg2(dobpdc), which adsorbs CO2via a mixed ammonium carbamate/carbamic acid mechanism. Thus, accessing new chemisorption products within the diamine-appended materials may give rise to advantageous changes in kinetics as well as thermodynamics. Overall, this study provides key guidelines for utilizing diamine-appended variants of Mg2(dobpdc) in CO2 separations and should prove more broadly useful for the design of new chemisorptive adsorbents for carbon capture applications.</p><!><p>The authors declare the following competing financial interest: J. R. L. has a financial interest in Mosaic Materials, Inc., a start-up company working to commercialize metal–organic frameworks for gas separations, including CO2 capture applications. The University of California, Berkeley has applied for a patent on some of the materials discussed herein, on which J. R. L., P. J. M., and R. L. S. are listed as inventors.</p>

PubMed Open Access

Methamphetamine Craving Induced in an Online Virtual Reality Environment

The main aim of this study was to assess self-reported craving and physiological reactivity in a methamphetamine virtual reality (METH-VR) cue model created using Second Life, a freely available online gaming platform. Seventeen, non-treatment seeking, individuals that abuse methamphetamine (METH) completed this one-day, outpatient, within-subjects study. Participants completed four test sessions: 1) METH-VR 2) neutral-VR 3) METH-video 4) neutral-video in a counterbalanced (latin square) fashion. The participants provided subjective ratings of urges to use METH, mood, and physical state throughout each cue presentation. Measures of physiological reactivity (heart rate variability) were also collected during each cue presentation and at rest. The METH-VR condition elicited the greatest change in subjective reports of \xe2\x80\x9ccrave METH\xe2\x80\x9d, \xe2\x80\x9cdesire METH\xe2\x80\x9d, and \xe2\x80\x9cwant METH\xe2\x80\x9d at all time points. The \xe2\x80\x9chigh craving\xe2\x80\x9d participants displayed more high frequency cardiovascular activity while the \xe2\x80\x9clow craving\xe2\x80\x9d participants displayed more low frequency cardiovascular activity during the cue conditions, with the greatest difference seen during the METH-VR and METH-video cues. These findings reveal a physiological divergence between high and low craving METH abusers using heart rate variability, and demonstrate the usefulness of VR cues for eliciting subjective craving in METH abusers, as well as the effectiveness of a novel VR drug cue model created within an online virtual world.

methamphetamine_craving_induced_in_an_online_virtual_reality_environment

1. Introduction<!>2.1 Participants<!>2.2 Cue Presentation Procedures<!>2.3 Virtual Reality Cues<!>2.4 Video & In Vivo Cues<!>2.5 Subjective Response Monitoring<!>2.6 Heart Rate Variability Recording<!>2.7 Subjective Response Analysis<!>2.8 Heart Rate Variability Analysis<!>2.9 High and Low Craving Participant Analysis<!>3.1 Demographics and Drug Use Characteristics<!>3.2 Subjective Craving and Mood<!>3.3 Heart Rate Variability<!>4. Discussion<!>

<p>Drug craving represents a key component of addiction and serves to propagate drug-taking behavior, and to elicit relapse in abstinent individuals (Galloway and Singleton, 2009; Hartz et al., 2001; McKay et al., 1999; Rohsenow et al., 2007). Craving represents a complex condition that includes emotional and cognitive aspects along with behavioral and physiological states (Merikle, 1999; Tiffany, 1990; Tiffany and Conklin, 2000). Craving has been extensively studied in an effort to standardize methods of quantifying this multifaceted condition (Rosenberg, 2009). Much of this work has focused on cue-induced craving, measured by self-reports and/or physiological reactivity to environmental stimuli previously associated with drug use (Carter and Tiffany, 1999; Stewart et al., 1984). Traditionally, visual (photographs and videos), in vivo (drug and/or paraphernalia) and imagery (individualized drug scripts) cues have been used to elicit craving in the laboratory (Carter and Tiffany, 1999). However, these methods typically elicit modest subjective craving (Avants et al., 1995), do not reliably induce physiological reactivity (Dudish-Poulsen and Hatsukami, 1997; Ooteman et al., 2006) and inconsistently predict subsequent drug use (Galloway and Singleton, 2009). For these reasons, more realistic and individualized cue exposure models are required to assess craving in drug abusers.</p><p>Addiction researchers have recently created and validated tobacco (Baumann and Sayette, 2006; Bordnick et al., 2004; Bordnick et al., 2005a; Bordnick et al., 2005b; Carter et al., 2008; Lee et al., 2003; Lee et al., 2005; Traylor et al., 2008), alcohol (Bordnick et al., 2008; Cho et al., 2008), cannabis (Bordnick et al., 2009), heroin (Kuntze et al., 2001) and cocaine (Saladin et al., 2006) VR drug cue systems. These systems incorporate multimodal drug cues into computer simulated environments using real-time graphics, 3-D visual displays, movement tracking devices, surround-sound audio, and tactile stimulators to create a fully immersive experience. VR drug cue models have elicit significantly more craving than traditional methods of cue exposure (Kuntze et al., 2001; Lee et al., 2003) and have also been applied to behavioral therapies for addiction (Lee et al., 2007). One pilot study applied VR cues to a stimulant abusing population and demonstrated that VR crack-cocaine cues elicit greater craving and physiological reactivity than neutral VR cues (Saladin et al., 2006). To our knowledge, no study has been conducted to assess VR cues in METH abusers.</p><p>The current study aimed to assess the effectiveness of a newly developed METH-VR cue model, compared to METH video cues, previously validated by our group (Newton et al., 2006). The METH-VR model was created using a freely available online VR platform, and included animate, inanimate, contextual and auditory cues. METH abusing participants provided subjective ratings of urges to use METH using multi-item craving questionnaire. Physiological reactivity was monitored via an electricardiogram (ECG) and analyzed for fluctuation in heart rate, or heart rate variability (HRV), that correspond with autonomic nervous system functions (1996; Allen et al., 2007; Ori et al., 1992). We hypothesized that METH abusers would exhibit greater increases in self-reported craving and display larger fluctuations of heart rate variability during the METH-VR cue exposure compared to traditional METH cues and neutral cues.</p><!><p>Otherwise healthy, non-treatment seeking METH users were recruited through local newspaper, radio and Internet advertisements. All participants underwent an initial telephone screening and provided information on their medical, psychiatric and drug use histories. Potential participants that successfully completed the telephone screen were invited to an in-person screening that included questions regarding demographics and drug use, and a urine toxicology test. Eligible participants were also required to provide self-reports of mood and recent drug use, and a urine toxicology test on the test day. All participants received a detailed verbal and written description of the study procedures before giving written informed consent, as approved by the University of California Los Angeles Institutional Review Board.</p><p>Exclusion criteria included 1) history of any self-reported Axis I psychiatric diagnosis (other than METH or nicotine dependence), 2) vision or hearing impairments, 3) use of any medication or medical condition that may significantly effect cardiovascular function, 4) illicit drug use (cocaine, heroin, hallucinogens, etc), other than METH, in the past 30 days. Any subject reporting recreational alcohol (≤ 1 drink per day) or marijuana (≤ 3 use per week) use, not meeting criteria for abuse/dependence, was allowed to participate, but instructed to abstain prior to testing (confirmed with self-report, urine toxicology test and alcohol breathalyzer during the in-person screen and on the test day).</p><!><p>Participants were permitted to smoke cigarettes ad libitum prior to the start of the study, but were required to abstain from smoking during the study (approximately 2 hours). Participants first completed questionnaires (30 min) before starting the cue sessions. This procedure standardized the time since the last cigarette (30 minutes), and allowed for modest cigarette craving (Schuh and Stitzer, 1995), while avoiding the possibility of heavy cigarette craving caused by prolonged abstinence. The cue condition presentation order was counterbalanced between participants (Latin Square). The participants viewed each cue condition for 10 min, with a ten minute break between each cue condition, in a sensory-attenuated setting on a Sharp Aquos 32″ LCD HDTV. The VR and video cues were run from a Dell Dimension DXP061 desktop containing a NVIDIA GeForce 8800 GTX graphics card and 768 MB of graphics memory. The participants navigated the VR environments using a Logitech Dual Action controller. Movement within the VR environments was limited to forward/backward walking (using the D-pad) and 360-degree head directional movements (using the right analog stick). Following completion of the last cue condition, participants were debriefed to ensure that their participation would not alter future drug taking behavior. Participants remained under supervision until their self-reported craving reached baseline levels, at which point they were discharged from the study session.</p><!><p>Photographs of the real apartment were taken (using a 6.3 MegaPixel Digital Canon EOS camera) from multiple angles under two conditions: "METH-house" (Figure 1) and "neutral-house" (Figure 2) to capture realistic light and textures in digital form. The photographs were then visually manipulated using Adobe Photoshop ® (version 7.0) to include additional METH paraphernalia and to enhance the overall realism. The finished images were applied to a 3-D mock up of the real apartment created in Second Life (www.secondlife.com). Finally, virtual avatars and drug-use animations (e.g. smoking, injecting, snorting) were created using Second Life tools and Poser Version 6, respectively, and placed into the VR environments.</p><p>The METH-VR environment was developed on the basis of self-reports from METH abusers' to represent a "METH-house" (i.e. a location where drug transactions and use occurs). This VR environment included animate (avatars administering METH), inanimate (drug paraphernalia), contextual ("METH-house" characteristics) and auditory (music reported by each subject to be associated with METH use) cues (Figure 1). The neutral-VR environment was modeled after a modern apartment (devoid of any drug cues) and includes neutral auditory stimuli (Latin jazz) (Figure 2). The participants were instructed to explore each VR environment freely, but were restricted from leaving by natural barriers, such as doors and walls.</p><!><p>The METH-video included professional actors/actresses administering METH via multiple routes (smoking, snorting, injecting) in a variety of settings with a set soundtrack. The participants were also provided with in vivo mock METH paraphernalia (e.g. glass pipe, mock syringe, medical tubing, and a small plastic bag containing a substance that appears to be METH) to examine during METH-video. The neutral-video contained footage of tropical fish swimming in a tank and included neutral music (classical guitar). The participants were also provided with in vivo neutral objects (e.g. feather, pinecone, pencil) to examine during the neutral-video. The participants were instructed to "imagine yourself in the situation while you are watching" at the start of each video.</p><!><p>All subjective responses were recorded on a visual analogue scale (VAS) from 0 to 100 ("none" to "very much"). The VAS form incorporated the nine following questions: four regarding urges to use METH ("How much do you crave/desire/want METH right now?", and "If you had access to METH right now, how likely would you be to use it right away?") two questions regarding mood ("How depressed/anxious do you feel right now?") and three questions regarding physical state ("Do you feel any drug effect right now?", "How high are you right now?", and "How stimulated do you feel right now?"). Desire was specifically defined in subtext next to the question as "to want (a feeling)" and crave as "strong or intense need (an internal force)".</p><!><p>Electrocardiogram (ECG) activity was recorded over 10-min intervals using two active EL126 snap leads and EL204 electrodes placed on the left pectoral and sternum with a third grounded lead/electrode placed on the left lateral rib cage. ECG data was filtered through a PSYLAB Isolation BioAmplifier and measured using a PSYLAB Stand Alone Monitor (Contact Precision Instruments, Cambridge, MA). The ECG data was recorded at 500 Hz and stored in PSYLAB Measurement acquisition software on a Latitude D600 Dell laptop.</p><!><p>Subjective reports provided prior to (time = 0), during (time = 5), after (time = 10) and following (time = 15) each cue condition served as the dependent measure of greatest interest. Change in craving for each cue condition, rather than raw craving score, was analyzed in order to measure acute cue-induced craving, to eliminate baseline variability between participants, and to account for carry-over effects between cue conditions. The craving change score was calculated by subtracting the baseline rating (time = 0) for each cue condition from the ratings at later time points (time = 5, 10, 15). A within-subjects general linear model (GLM) for repeated measures was used to assess the effect of cue condition and time on each subjective measure independently. In the case that sphericity could not be assumed (Mauchly's Test of Sphericity), the statistics reported were adjusted via the Greenhouse-Geisser method. A one-way ANOVA, including post hoc analysis (Tukey), was used to compare cue conditions at each time point (time = 5, 10, 15). A Pearson bivariate correlation was also applied to assess the relationship between recent drug use and subjective responding. Statistical analysis was performed using SPSS 11 for Mac OS X.</p><!><p>The ECG data was transferred to QRS Tool where the inter-beat interval (IBI) was manually extracted from an 8-min segment of each 10-min recording (excluding first and last minute to reduce artifacts) (Allen et al., 2007). Successive R-waves in each QRS complex were marked using individualized amplitude thresholds and the average heart beat period as guiding factors. The IBI series was then transferred to Kubios HRV 2.0 analysis software where time-domain and frequency-domain (parametric AR modeling) analyses were conducted (1996). The three frequency bands extracted for analysis included a very low frequency band (VLF, 0–0.04 Hz), a low frequency band (LF, 0.04–0.15 Hz), and a high frequency band (HF, 0.15–0.4 Hz). The measures of interest extracted from each frequency band included 1) the relative powers of VLF, LF, and HF bands, 2) the normalized LF and HF band powers and 3) the LF/HF power ratio. The time domain and frequency domain measures were analyzed separately using two multivariate GLMs for repeated measures to assess for an overall effect of cue condition on each set of HRV measures. A Pearson bivariate correlation was also applied to assess the relationship between recent drug use and heart rate variability. Statistical analysis was performed using SPSS 11 for Mac OS X.</p><!><p>The participants were characterized as "high craving" and "low craving" participants using a median split on baseline "crave METH" scores. The two groups were assessed for differences in demographic and drug use characteristics, and heart rate variability measures. A one-way ANOVA was used to compare all demographic and drug use characteristics, except gender and ethnicity, which were assessed using a Chi-Square test. A multivariate GLM for repeated measures was used to assess an effect of craving group on time domain and frequency domain HRV measures separately. A one-way ANOVA was then used to assess group differences in each time domain and frequency domain HRV measure independently. Lastly, a Pearson bivariate correlation was applied to each craving group separately to assess the relationship between change in subjective responses and HRV measures during each cue condition.</p><!><p>The study sample consisted of 17 (14 men, 3 women) adults (mean ± SD age 39.5 ± 8.8 yr) with, on average, a high school education (mean ± SD 12.0 ± 2.0 yr). The participants used METH for 10.9 ± 5.9 (mean ± SD) years and reported 13.2 ± 11.3 (mean ± SD) days of METH use in the month prior to participation in this study. The majority of participants smoked cigarettes (82%), drank alcohol (71%) and used marijuana (59%) in the last 30 days. At baseline, the participants reported a median score of 20 out of 100 on ratings of "crave METH" (9 "high craving" participants > 20; 8 "low craving" participants ≤ 20). No significant differences were observed between the "high craving" and "low craving" participants on demographic or drug use characteristics (Table 1).</p><!><p>A within-subjects GLM for repeated measures assessing change in each subjective response demonstrated a significant effect of cue condition on reports of "crave METH" (F3, 13 = 8.08, P = 0.001), "desire METH" (F3, 14 = 6.50, P = 0.001), "want METH" (F3, 13 = 6.40, P = 0.005), "use METH right away" (F3, 14 = 7.10, P = 0.006), and feeling "anxious" (F3, 14 = 3.85, P = 0.036), and "high" (F3, 14 = 3.42, P = 0.049) (Figure 3). No effect of time was observed on subjective responses.</p><p>A one-way ANOVA assessing change in subjective responses at each time point separately revealed a significant effect of cue condition during (time = 5 min), after (time = 10 min) and following (time = 15 min) cue presentation for "crave METH", "desire METH", "want METH", and "use METH right away" (P < 0.01 for all). A significant effect of cue condition was also observed for feeling "anxious" during and after cue presentation (P < 0.02 for both) and for feeling "high" following cue presentation (P < 0.03). Post hoc (Tukey) analysis revealed that the METH-VR cue condition elicited significantly greater increases in "crave METH", "desire METH", "want METH", and "use METH right away" compared to both neutral conditions at almost all time points (Table 2). No correlations were observed between recent drug use and subjective responding.</p><!><p>A within-subjects GLM for repeated measures revealed no effect of cue condition on time domain or frequency domain HRV measures. A multivariate GLM for repeated measures revealed a significant effect of craving group on frequency domain HRV measures (F3, 14 = 12.37, P = 0.003). A one-way ANOVA further revealed that the "high craving" participants exhibited a significantly greater amount of relative and normalized HF power during the METH-VR (P < 0.001 for both), METH-video (P < 0.001 for both), and the neutral-video (P < 0.02) cues compared to the "low craving" subjects. Conversely, the "low craving" participants exhibited a significantly greater amount of relative and normalized LF power during the METH-VR (P < 0.001 for both), METH-video (P < 0.01 for both) and neutral-video (P < 0.03 for both) cues compared to the "high craving" participants. The "low craving" participants also displayed a significantly higher LF/HF ratio during the METH-VR and METH-video (P < 0.003 for both) cues, and during the neutral-VR and neutral-video cues (P < 0.03 for both), compared to the "high craving" participants. No significant differences were observed between the craving groups at rest (Table 3). No correlations were observed between recent drug use and time domain or frequency domain HRV measures.</p><p>A bivariate correlation revealed a positive association between LF/HF ratio and change in self-reported "crave METH" (R7 = 0.85, p = 0.016), "want METH" (R8 = 0.85, p = 0.015), and "use METH right away"(R8 = 0.87, p = 0.006), and a trend towards an association with "desire METH" (R8 = 0.65, p = 0.08) during the METH-video in the "low craving" participants.</p><!><p>METH abusers demonstrated increases in subjective craving (measured as "crave METH", "desire METH", "want METH" and "use METH right away") when presented with METH-VR cues compared to the neutral-VR or neutral-video cues. The participants reported approximately twice as much subjective craving during the METH-VR cues compared to the METH-video cues, although this finding did not reach significance. The participants also report a greater increase in "anxiety" to the METH-VR cues compared to the neutral-VR cues. These findings parallel those seen in other drug abusing populations (Baumann and Sayette, 2006; Bordnick et al., 2009; Bordnick et al., 2004; Bordnick et al., 2005a; Bordnick et al., 2008; Bordnick et al., 2005b; Carter et al., 2008; Cho et al., 2008; Kuntze et al., 2001; Lee et al., 2003; Lee et al., 2007; Lee et al., 2005; Saladin et al., 2006; Traylor et al., 2008), and demonstrate the usefulness of VR cues for eliciting subjective craving in METH abusers, as well as the effectiveness of a novel VR drug cue model created within an online virtual world.</p><p>The METH-VR system was created with the goal of enhancing realism, accessibility and adaptability while reducing complexity and cost. Existing VR drug cue models incorporate proprietary software and expensive hardware to create drug specific experiences (Bordnick et al., 2009; Bordnick et al., 2005a; Bordnick et al., 2008; Carter et al., 2008; Lee et al., 2003; Saladin et al., 2006). Recently, user-created online gaming platforms have emerged to provide a forum for creating personalized virtual environments and an alternative to private VR software packages. As seen here and in previous studies, visual presentation of VR environments elicits significant levels of craving (Baumann and Sayette, 2006). These findings do not detract from the importance of presenting an immersive environment, but rather demonstrate the viability of creating such environments using freely available online software and minimal hardware (i.e. computer and monitor). Online virtual worlds have become home to a wide range of health related activities ranging from patient education to scientific experimentation (Beard et al., 2009; Boulos et al., 2007). These user-created 3-D worlds offer a number of benefits over previous VR systems including adaptability, accessibility and cost. These systems contain a variety of tools that allow users to modify and specify all aspects of the environment, such as contextual, animate and inanimate drug cues, in real time. Furthermore, all of this data from these environments is stored onto external servers, thus allowing the user to access their VR world through a high-speed Internet connection. Anyone can download and access these systems for free, and the cost of development remains relatively very low compared to alternative VR systems. The results reported here support future applications of virtual worlds in addiction research and present a viable opportunity to create and share standardized drug cue environments in a collaborative effort.</p><p>Contrary to previous research in alcoholics (Ingjaldsson et al., 2003), the "high craving" and "low craving" participants reported here demonstrated an inverse relationship between baseline craving and physiological reactivity. The "high craving" participants exhibited a greater parasympathetic response while the "low craving" participants exhibited a greater sympathetic response to all of the cue conditions, with the greatest difference during the METH-VR and METH-video cues (1996; Ori et al., 1992). The "low craving" participants demonstrated a mild increase in sympathetic activity from rest to all of the cue conditions, and displayed a positive association between subjective craving and sympathetic activity during the METH-video, while the "high craving" participants exhibited little change from rest. The results concerning the "low craving" participants support the traditional theory of cue reactivity, which proposes that psychological craving coincides with a sympathetic or stress response (Sinha, 2009), while the results from "high craving" participants support research suggesting that a disconnect exists between subjective craving and physiological reactivity (Dudish-Poulsen and Hatsukami, 1997; Ooteman et al., 2006). Taken together, these findings present an intriguing dichotomy between "high craving" and "low craving" METH abusers and provide insight into the effect of baseline carving on subsequent physiological cue-reactivity.</p><p>This study has some limitations. The HRV results did not reveal any differences in autonomic response between the four cue conditions. A number of factors specific to this population including cardiovascular deficits (Kaye et al., 2007; Thayer et al., 2009), a blunted or disconnected physiological response to psychological stimuli (Dudish-Poulsen and Hatsukami, 1997; Ooteman et al., 2006), or general heterogeneity may account for this lack of observable difference (Newton et al., 2009). The study design used here (i.e. one-day outpatient) limited experimenter control over the participants' behavior prior to testing. To account for this lack of control, a number of measures (demographics and drug use history) were collected and included in the analyses to explore for associations between these factors and craving. Although on par with previous studies of VR craving (Baumann and Sayette, 2006; Bordnick et al., 2009; Bordnick et al., 2005a; Saladin et al., 2006), the number of participants included in the present study, particularly female participants, was low. Future studies with a higher number of women could provide insight into gender differences in cue reactivity. The participants included in this study had a wide variety of METH, nicotine, alcohol and cannabis use characteristics, which probably accentuated variability. However, we felt it was important to include a diversely representative sample of METH abusers to obtain results that are generalizable to the overall population of METH abusers. Due to the repetitive nature of measuring acute craving, a multi-item VAS craving questionnaire was substituted for the more comprehensive multidimensional craving questionnaire (Rosenberg, 2009). The numerical limitations of the VAS craving questionnaire (1–100) may have resulted in ceiling effects in the "high craving" participants and flooring effect in the "low craving" participants. In light of these limitations, the consistency of the results presented here only further support the central finding of this study that the METH-VR model serves as a powerful clinical tool for manipulating acute craving in METH abusers.</p><p>VR technology continues to rapidly advance with commercial demands from the gaming entertainment community. Researchers now have access to unprecedented tools for creating realistic VR environments to monitor and manipulate human behavior in the laboratory. The results presented here provide evidence for the applicability of these VR tools. Future research and development of VR drug cue environments should focus on enhancing realism and specificity to complement the complexity of individuals with substance-use disorders. Improved VR drug cue models will allow researchers to better assess cue-induced craving, as well as drug self-administration, neurocognitive abilities and patterns of locomotor behavior (e.g. drug seeking, conditioned place preference, approach avoidance) in a realistic and interactive setting. Integrating these investigative methods into VR environments will provide insight into connectivity between factors that underlie drug craving and provide an optimum paradigm for designing and testing treatments for drug addiction.</p><!><p>Screenshots of the methamphetamine virtual reality (METH-VR) cue environment</p><p>Screenshots of the neutral virtual reality (neutral-VR) cue environment</p><p>Change in subjective reports of "crave METH" (A), "desire METH" (B), "want METH" (C), "use METH right away" (D), feeling "anxious" (E), and feeling "high" for all participants (N=17) during, after and following each cue condition (values represent the mean change in subjective responses (−100 – 100) ± standard error mean).</p><p>Demographic and Drug Use Characteristics for "High Craving" and "Low Craving" Participants</p><p>Self-Reported Subjective Responses During, After, and Following Each Cue Condition</p><p>Values represent mean ± S.E.M.,</p><p>p<0.01;</p><p>p<0.05 compared to Meth-VR</p><p>p<0.01;</p><p>p<0.05 compared to Meth-Video</p><p>Between Group Comparisons of Heart Rate Variability Frequency Domain Measures During Each Cue Condition</p><p>Values represent mean ± S.E.M.,</p><p>p<0.01;</p><p>p<0.05</p>

PubMed Author Manuscript

Perylenequinone Natural Products: Enantioselective Synthesis of the Oxidized Pentacyclic Core\xe2\x80\xa1

An enantioselective approach to the perylenequinone core found in the mold perylenequinone natural products is outlined. Specifically, the first asymmetric syntheses of helical chiral perylenequinones absent any additional stereogenic centers are described. Key elements of the synthetic venture include a catalytic enantioselective biaryl coupling, a PIFA-induced naphthalene hydroxylation, and a palladium-mediated aromatic decarboxylation. Transfer of the binaphthalene axial stereochemistry to the perylenequinone helical stereochemistry proceeded with good fidelity. Furthermore, the resultant perylenequinones were shown to possess sufficient atropisomeric stability to be viable intermediates in the biogenesis of the perylenequinone natural products. This stability supports the use of the helical axis as a stereochemical relay in synthesis of the natural products containing additional stereochemical centers.

perylenequinone_natural_products:_enantioselective_synthesis_of_the_oxidized_pentacyclic_core\xe2\x8

Introduction<!>Retrosynthetic Analysis<!>Monomer Synthesis and Asymmetric Oxidative Coupling<!>Perylenequinone via a bis-ortho-Quinone<!>Perylenequinone via an Oxidized Binaphthalene<!>Decarboxylation and Atropisomeric Stability<!>Summary<!>Conclusions<!>2-(4-Methoxy-3-propylphenyl)acetic acid (49)<!>Methyl 1,3-dihydroxy-7-methoxy-6-propyl-2-naphthoate (50)<!>Methyl 1-acetoxy-3-hydroxy-7-methoxy-6-propyl-2-naphthoate (45d)<!>General Procedure for the Oxidative Biaryl Coupling<!>(M)-Dimethyl 4,4\'-diacetoxy-2,2\'-dihydroxy-6,6\'-dimethoxy-7,7\'-dipropyl-1,1\'-binaphthyl-3,3\'- dicarboxylate (52d)<!>Dimethyl 2,2\'-bis(benzyloxy)-4,4\'-dihydroxy-7,7\'-dimethoxy-5,5\',6,6\'-tetraoxo-5,5\',6,6\'- tetrahydro-1,1\'-binaphthyl-3,3\'-dicarboxylate (54)<!>Dimethyl 2,2\',4,4\'-tetramethoxy-5,5\',6,6\'-tetraoxo-7,7\'-dipropyl-5,5\',6,6\'-tetrahydro-1,1\'-binaphthyl-3,3\'-dicarboxylate (61)<!>2,2\',4,4\'-Tetramethoxy-7,7\'-dipropyl-1,1\'-binaphthyl-5,5\',6,6\'-tetraone (40)<!>2,2\xe2\x80\x99,6,6\xe2\x80\x99-Tetramethoxy-4,4\xe2\x80\x99-diox-7,7\xe2\x80\x99-dipropyl-4H,4\xe2\x80\x99H-[1,1\xe2\x80\x99]binaphthalenylidene-3,3\xe2\x80\x99- dicarboxylic acid dimethyl ester (69)<!>(M)-Dimethyl 2,2\',4,4\',6,6\'-hexamethoxy-7,7\'-dipropyl-1,1\'-binaphthyl-3,3\'-dicarboxylate (82)<!>(M)-Dimethyl 5,5\'-dihydroxy-2,2\',4,4\',6,6\'-hexamethoxy-7,7\'-dipropyl-1,1\'-binaphthyl-3,3\'- dicarboxylate (87)<!>(M)-5,5\'-Bis(benzyloxy)-2,2\',4,4\',6,6\'-hexamethoxy-7,7\'-dipropyl-1,1\'-binaphthyl-3,3\'- dicarboxylic acid (90)<!>(M)-5,5\'-Bis(benzyloxy)-2,2\',4,4\',6,6\'-hexamethoxy-7,7\'-dipropyl-1,1\'-binaphthyl (42)<!>4,9-Dihydroxy-2,6,7,11-tetramethoxy-1,12-dipropylperylene-3,10-dione (39)<!>

<p>The parent perylenequinone 1 was first prepared by Zinke in 1929,1 but it was not until 1954 that the correct structure was elucidated (Figure 1).2 This compound was subsequently isolated from the fungus, Daldinia concentrica as blue-black crystals and is the simplest of the naturally occurring perylenequinones.3 Most members of this family of natural products fall into 3 classes (examples of each in Figure 1): (a) C20 compounds without carbon substituents,4 (b) mold perylenequinones containing carbon substituents, and (c) perylenequinones from aphids. Of the three, class B is the most prevalent. Due to their diversity, interesting stereochemistries, and anticancer activities we have undertaken the syntheses of these compounds via dimerization of achiral naphthols.</p><p>The archetypical cercosporin 6 (Figure 2) was among the first of the mold perylenequinones to be isolated in 1957.5 The unusual features of this molecule hindered a complete structural determination until the 1970s.6 The majority of the structural details were determined by classical redox quinone chemistry, but the most significant finding was the transformation involving the thermal atropisomerization of (M)-cercosporin into (P)-epi-cercosporin. The opposite helical configuration of the perylenequinone epimers was confirmed by circular dichroism (CD) analysis,6b and the helical nature along with the absolute and relative configuration of 6 was established crystallographically.7 Since the discovery of 6, more than 20 perylenequinones have been isolated as dark red pigments from a variety of fungi within the Ascomycota phylum (Figure 2).8 Correlation with the CD and NMR spectra of 6 has allowed determination of the helical configurations and relative stereochemistries of most of the remaining natural products.</p><p>Notably, all of the mold perylenequinones (Figure 2) are derivatives of 1 (Figure 1). With the exception of hypomycin B (15), the C4,C4'- or C 5,C5'-phenols are not alkylated. These acidic phenols (pKa ~7.3–8.3)9 are characteristic of the structures and form strong internal hydrogen bonds with the quinone carbonyls.10 Many of the mold perylenequinones are C2-symmetric, having C7,C7'-substitution with centrochiral stereocenters of the same absolute configuration. The remainder (hypocrellin, hypocrellin A–B, shiraiachrome A, elsinochrome B1-B2, hypomycin B11) likely originated from C2-symmetric intermediates.</p><p>Regardless of the varied C7,C7'-substitution, all class B perylenequinones contain a conjugated pentacyclic core in a highly oxidized state. Furthermore, the distinct oxygenation pattern at the C2,C2',C4,C4',C5,C5',C6,C6'-centers (Figure 2) is conserved throughout the series. In the past two decades, the construction of this highly oxygenated core en route to the perylenequinone natural products has been examined by several groups.12,13,14,15,16,17,18,19 Prior to these achievements, there had been relatively few synthetic efforts devoted to this family of natural products. As mentioned above, 1 (class A, Figure 1) was first unknowingly prepared by Zinke and coworkers (ca. 1929) from tetranitroperylene 18 upon exposure to sulfuric acid at 140 °C under air.1 Due to the atypical stability of the proposed product 19, Calderbank and coworkers repeated the procedure (Scheme 1),2 and the structure of 4,4',5,5'-tetraquinone 19 reported by Zinke was correctly reassigned as the parent perylenequinone 1.</p><p>Surprisingly, it was not until 1972 that there was further progress when Weisgraber and Weiss attempted to synthesize the oxygenation pattern present in the mold perylenequinones (Figure 2). The initial assignments of both the coupling product 21 and the subsequent perylenequinone 22 were discovered to be incorrect,20,21 and the final cyclized product was reassigned as dinaphthofuranedione 24 (Scheme 2).</p><p>In work by Dallacker and Leidig, a different approach was used in the synthesis of 2,2',4,4'-tetramethoxyperylenequinone, commencing with formation of bisphenyl substrate 25. Unfortunately, the synthesis was lengthy and lacked the necessary C6,C6'-oxygenation.22</p><p>Prior to the report by Broka on the calphostins,14 syntheses of the highly oxidized perylenequinone core were either racemic or yielded optically inactive products. Thus, these early efforts did not address a key architectural element of the perylenequinones – the chiral helical pentacyclic core. Due to steric interactions of the C7,C7'- and C2,C2'-substitution, the pentacyclic core is forced to adopt a chiral helical structure (dihedral angle of 20°). With larger C7,C7'- and C2,C2'-groups, the rate of interconversion of the two possible enantiomeric conformational isomers is slow leading to stable atropisomers. As discussed above, the helicity of most perylenequinones was determined by correlation to cercosporin. In the cases of hypocrellin B 4 and scutiaquinone B 17, there is an absence of helicity in the CD spectra. However, calculations indicate that the lowest energy ground state structures are in fact helical (Scheme 4). Presumably, the double bond of hypocrellin B 4 and the lack of substituents at C2,C7'-positions of scutiaquinone B 17 lower the atropisomerization barrier such that the two helical forms rapid interconvert.23,24</p><p>The helicity of the perylenequinone natural products is not constant. Rather compounds exhibiting both (M)- and (P)-helicity have been isolated, leaving the origin of the helical stereochemistry an interesting and unsolved issue. Since different helical stereochemistries arise from different organisms (hypocrellin from Hypocrella bambusae25 and hypocrellin A from Shiraia bambusicola23), it seems likely that an enzyme-mediated enantioselective dimerization occurs in the biosynthetic routes. The fact that horseradish peroxidase, a metalloporphyrin enzyme, has been found to elicit asymmetric naphthol couplings with good yields and modest enantioselectivities indicates that enzymes are capable of catalyzing this transformation (Eq 1).26 Presumably, the peroxidase catalyzes the production of a stabilized radical to enable the homocoupling.27 It is entirely probable, therefore, that helical chiral perylenequinones, such as 30, absent any centrochiral elements (i.e. R2 not containing further stereocenters) can be produced enzymatically. Such production likely occurs via an axial chiral binaphthalene with transfer of axial stereochemistry to helical stereochemistry in the perylenequinone forming step (Scheme 5).</p><p> (1)</p><p>In an examination of prior approaches, the installation of the helical chirality has been the most difficult aspect and thus the focal point of the syntheses.14–17 Poor diastereocontrol in the couplings of chiral C7,C7'-substituted naphthalenes also points away from such a pathway in the biosynthesis (i.e. 38 to 8a–d, Scheme 6).14–17 Thus, we formulated a plan revolving around diketone perylenequinone 33. Notably, perylenequinone 33 is a retrosynthetic intermediate to four different natural product classes: the calphostins (8a–d) and cercosporins (6) via reduction, the hypocrellins (2) via aldol cyclization, and the elsinochromes (10) via oxidative enol coupling. In contrast, there is no direct route from monomer 38 to the hypocrellins (2) and elsinochromes (10); oxidation and loss of the two alcohol stereocenters would be required first. We proposed that helical perylenequinone 33 could be produced from cyclization of axial chiral binaphthalene 34 which would in turn be produced by facile enantioselective oxidative coupling of the corresponding monomer 35. The illustrated monomer 35 is generated in a few simple steps from phenylacetic acid 37, in contrast to the laborious preparations of enantiopure monomers 38.</p><p>A central element of this synthetic plan is the atropisomeric stability of the key intermediate 33. The calphostins (8a–d) exhibit good atropisomeric stability, but the simple change of introducing a bridging methylidene as in cercosporin (6) abrogates much of this stability with atropisomerization occurring at 37 °C.7 Due to the sterically larger C7,C7'-hydroxypropyl groups in the calphostins (8a–d) compared to the 2-oxopropyl groups of 33, we anticipated that 33 would exhibit lower atropisomeric stability. Since no enantiopure perylenequinones with only the helical stereochemistry as a chiral element had been reported previously, we set out to establish the degree of configurational stability of such species. In this paper, we detail the synthesis of the enantiopure perylenequinone core, addressing installation of the necessary oxygenation pattern as well as the formation and stability of the helical stereochemistry in the absence of other stereochemical substitution.28</p><!><p>With an initial goal of producing an enantiopure perylenequinone possessing only the helical stereochemical component, we elected to undertake the synthesis of analog 39 possessing simple n-propyl groups at the C7, C7'-positions. Based on prior reports, we identified two intermediates (40 and 42) that could lead to the desired perylenequinone 39, both devolving to common chiral biaryl intermediate 41(Scheme 7). Merlic has reported diastereoselective oxidative couplings to either provide the calphostin perylenequinone directly or via a bis-ortho-quinone intermediate,17,29 such as 40 (path a). Similarly, Broka14 and Coleman16 utilized a diastereoselective biaryl coupling to yield an axial chiral biaryl as an intermediate in their calphostin syntheses. The perylenequinone arose from the cyclization of the corresponding 5,5'-bisnaphthol, similar to debenzylated 42 (path b). For both paths a and b, effective stereochemistry transfer of the axial stereochemistry to the ultimate helical stereochemistry was observed.14–17 However, the role of the bulky protected C7,C7'-hydoxypropyl groups in stabilizing this stereochemistry transfer was unknown.</p><p>Due to both the reported success of Merlic29 and also the precedent for the oxidation of naphthalenes to bis-ortho-quinones,30 we initially chose to pursue path a. We commenced this effort by exploring the ability of our catalyst system in the enantioselective coupling of a series of highly substituted naphthols to provide the required axial chiral precursor 41.28</p><!><p>Based on prior methods for the formation of highly substituted naphthols,20a we developed a protocol to generate naphthols 45a–d with high efficiency (Scheme 8, Scheme 9).28 Commencing with simple commercially available phenylacetic acids (43a–c), the requisite naphthols were generated rapidly via acid chloride formation, malonate acylation, and cationic cyclization. After diacetylation, the less hindered C2-acetate was hydrolyzed with NaOMe to provide the desired coupling substrates 45a–c (Scheme 8). The flexibility of this route to quickly generate substrates with a variety of substitution patterns has proven invaluable in identifying optimal substrates for the enantioselective oxidative biaryl coupling and for the perylenequinone syntheses.</p><p>In the formation of biaryl intermediate 41(Scheme 7), the first naphthol proposed was C7-allyl 48 (Scheme 9). The allyl group would provide a platform both to intermediate 41 (Scheme 7) and also to introduce functional groups present in the natural products. The synthesis began with an allylation of the hydroxyl and carboxyl groups of commercially available 46 followed by a Claisen rearrangement and methylation of the revealed phenol (Scheme 9). Unfortunately, all attempts to form the desired 48 from 47 were unsuccessful, most likely due to incompatibility of the allyl group with the strongly acidic cyclization conditions. In light of this result, the hydrogenation of 47 was performed to yield the inert propyl derivative. After saponification to supply 49, the naphthol 45d was successfully prepared using the optimized conditions for 45a–c.</p><p>In previous efforts, we have assessed the versatility of our diaza-cis-decalin catalyst 51 in catalytic asymmetric oxidative naphthol couplings.28,18,19,31 Table 1 outlines the significant results pertinent to this study.32 Notably, an electron-withdrawing, coordinating group at C3 enhances selectivity,31 presumably by via coordination to the copper complex together with the C2-phenol. The effects of the substrate oxidation potential are illustrated in entries 1–3 (Table 1),28 where the electron rich nature of the trismethoxy substrate (entry 2) led to lower optical purities due to rapid atropisomerization of the product 52e via a further oxidation. Replacement of the donating C4-methoxy group with an electron-withdrawing acetoxy group (entry 3) reduced the oxidation potential and furnished the pentasubstituted naphthalene 52b in high enantioselectivity. The introduction of an additional electron-donating group (C5-methoxy) counteracted the effect of the C4-acetoxy group and facilitated enantiomerization of 52c (entry 4).28 Additionally, the steric gearing effects prevented the substrate from adopting the conformation needed for catalyst coordination, explaining the slow conversion as well as the low enantioselectivity. With these results in hand, it was straightforward to design substrates with a suitable oxidation potential (sufficiently electron rich for the substrate to oxidize, but not too electron rich to allow product oxidation) to obtain high reactivity and selectivity. For example, 52a,b,d (entries 1, 3, 5) furnish functionalized binaphthols in high yield and enantioselectivity. The M-helicity of these products was assigned based on trends observed with related substrates; in the over 30 cases where the stereochemistry has been assigned via correlation, crystallization, or calculation, the S,S-catalyst provides the M-helicity and the R,R-catalyst the P-helicity.31 Significantly, the enantioenriched naphthol 52d for this investigation is generated with the necessary functionalization at the C2,C3,C4,C7-centers.</p><!><p>Following the success of the enantioselective biaryl couplings, investigations begun into transforming biaryl 52d, arising from 45d, to perylenequinone 39 via bis-ortho-quinone 40 (Scheme 7). As previously mentioned, there is precedent for oxidation of naphthalenes with a phenol at the position peri to the reaction center using various oxidants.30 Our efforts commenced with the synthesis of model biaryl 53, containing the necessary C4,C4'-phenols, but possessing C7,C7'-methoxy groups instead of propyl groups. Direct benzylation of the C2,C2'-phenols to generate 53 was unsuccessful, a trend that was observed throughout the series. Apparently, the C2,C2'-positions are relatively hindered, which slows alkylation significantly. On the other hand, Mitsunobu reaction generally proceeded well on these centers and was used here to install the C2,C2'-benzyl ethers. Subsequent hydrolysis of the C4,C4'-acetates afforded 53 (Scheme 10). After screening a number of oxidants, the hypervalent iodine reagent bis(trifluoroacetoxy)iodobenzene [PIFA = PhI(O2CCF3)2] following McKillop's protocol was found to generate the desired bis-ortho-quinone 54 as confirmed by the crystal structure.</p><p>With the model bis-ortho-quinone 54 in hand, we next examined formation of the perylenequinone core. Merlic has reported the transformation of a similar bis-ortho-quinone 55 to perylenequinone 56 using trifluoroacetic acid (TFA) under O2 (Eq 2).17 When applied to methylated 57, this procedure yielded only decomposition products (Eq 3). We attributed this result to the differences between the two ortho-quinones (55 vs. 57), specifically the presence of C2,C2'-benzyl ether, C3,C3'-ester, and C7,C7'-methyl ether groups in 57.</p><p> (2)</p><p> (3)</p><p>Reasoning that the C7,C7'-methoxy groups of 57 (Eq 3) changed the electronic characteristics of the ortho-quinone relative to 55 (Eq 2), we moved to biaryl 59 (Scheme 11) with C7,C7-n-propyl groups as well as C2,C2'-methoxy groups to more closely approximate Merlic's system (Eq 2) in formation of the pentacyclic core. Coupling of 45d with CuCl(OH)TMEDA under O2 provided large quantities of the racemic biaryl 52d, which we employed to develop a viable route to 39. Biaryl 52d was treated with NaH and MeI to methylate the C2,C2'-phenols. As has been seen throughout the series, alkylation of the hindered C2,C2'-phenols is difficult and this transformation required the use of anhydrous DMF33 to succeed. Subsequent hydrolysis of C4,C4'-acetates efficiently afforded 59 (Scheme 11). Oxidation utilizing PIFA yielded the desired bis-ortho-quinone 60 in quantitative yield, but this compound was unstable to chromatography or prolonged exposure to air. Immediate methylation of the C4,C4'-phenols with n-Bu4NF and MeI provided the stable 61, which could be purified. Surprisingly, 61 could not be isomerized to the perylenequinone 62 and only decomposition occurred following the procedure developed by Merlic. Presumably, the isomerization requires one of the ortho-quinone moieties to reduce so that a nucleophilic phenol can undergo a transannular addition to the remaining electrophilic ortho-quinone. Subsequent tautomerization and air oxidation would then furnish the perylenequinone. In an attempt to reproduce this manifold, bis-ortho-quinone 61 was treated under conditions to induce reduction (NaBH4 and O2 or DBU and O2); however, only decomposition was observed.</p><p>The presence of the C3,C3'-methyl ester groups accounts for the last major difference between the previously isomerized bis-ortho-quinone 55 (Eq 2) and the structures 57 (Eq 3) and 61 (Scheme 11). The electron withdrawing nature of the methyl esters may destabilize the perylenequinone 62 and/or interfere with the isomerization. To test the hypothesis, decarboxylation of the biaryl was examined. To generate a biaryl that would be stable to saponification and decarboxylation, the previously formed bis-ortho-quinone 61 (Scheme 11) was reduced with sodium dithionite (Na2S2O4) and methylated (Scheme 12). Unfortunately, mixtures of the desired octamethyl ether 63, partially-reduced hexamethyl ether 63a and starting material 61 were obtained with the highest yield of 46% for 63. Saponification of 63 proceeded smoothly with aqueous LiOH in refluxing dioxane, to afford the bisacid. Subsequent decarboxylation with Cu-quinoline at 180 °C furnished 64 in a modest 30% yield over two steps (Scheme 12). Thereafter, treatment of 64 with BCl3 in an attempt to selectively cleave the C5,C5'-methyl ethers resulted in a mixture of demethylated compounds, which upon treatment with K3Fe(CN)6 yielded only a complex mixture from which none of the desired 65 was isolated. Fortunately, removal of the C5,C5'-methyl ethers was not necessary for oxidation and direct exposure of 64 to ceric ammonium nitrate (CAN) afforded the oxidized bis-ortho-quinone 40 (Scheme 12). Longer reaction times in the CAN oxidation did not afford the desired perylenequinone 65, and all attempts to isomerize 40 to 65 led only to decomposition. This result was surprising due to the close structural similarity of 40 (Scheme 12) to 55 (eq 2).</p><p>Since an examination of the transformation proposed by Hauser and Merlic15,17 indicated that a direct isomerization of 40 to 65 (Scheme 12) is not viable without a reducing agent, protocols involving a reduction were examined. Strong precedent for such an approach is seen in the work of Lown13 and Zhang12 on the syntheses of a perylenequinones from bis-ortho-quinones via a two-step reduction-oxidation sequence (FeCl2, then FeCl3; SO2-H2O, then FeCl3). Due to the length of the synthetic route needed to obtain large quantities of bis-ortho-quinone 40, 61 was used to examine these new isomerization procedures. Treatment of 61 with the reducing agent Na2S2O4 followed by FeCl3 afforded a complex mixture that was determined to contain the undesired isomer 61 as the major product as well as the desired product 62(Scheme 13).34</p><p>With confidence that the synthesis of perylenequinone 62 via this route could be optimized the reactions were examined with enantioenriched 52d to verify the optical purity of the synthetic intermediates. Formation of enantioenriched 59 (70% ee) following the racemic synthesis seen in Scheme 11, permitted the PIFA-mediated oxidation of the optically active intermediate to bis-ortho-quinone 61 in 70% yield (Eq 4). Disappointingly, when evaluated by both optical rotation and HPLC, compound 61 was found to be racemic.</p><p> (4)</p><p>The mechanism of the oxidation reaction was studied to provide an explanation of the atropisomerization. The use of PIFA and phenyliodonium diacetate [PIDA = PhI(OAc)2] for phenolic oxidation has been well reviewed,35 and notably, biphenol 67, upon oxidation leads to the extended quinone 68 (Eq 5).36 When only two equivalents of PIFA (7.5 equiv used in Eq 4) were used in the oxidation of 59, a binaphthone (69, Figure 3) was isolated as a dark green material from the reaction mixture. In our assignment of 69, the propyl groups are placed anti to each other because the 1H NMR spectrum does not indicate any restricted rotation about the propyl groups (i.e., diastereotopic protons), which is seen in the 1H NMR spectra of perylenequinones where the propyl groups are in close proximity. Similar to 69, the highly strained bianthrone 70 has been reported in the literature.37,38 Interestingly, bianthrone 70 was found to equilibrate between twisted and stack conformations (Figure 3). Analogously, it is clear that 69 is a chiral compound, but the facile interconversion of the twisted and stacked conformations provides a ready racemization pathway (Figure 4).</p><p> (5)</p><p>The use of the less reactive PIDA allowed a full evaluation of the oxidation pathway of 59 (Scheme 14). While all efforts to optimize formation of binaphthone 69 with PIFA as the oxidant proved to be unsuccessful, PIDA allows 69 to be isolated in a 91% yield (Scheme 14) with minimal amounts of decomposition during the reaction. The treatment of 69 with two equivalents of PIFA yielded racemic 60, strongly supporting the intermediacy of 69 during the PIFA-oxidation of 59 to bis-ortho-quinone 61 (Eq 4).</p><p>Based upon the above evidence, an oxidative mechanism for conversion of binaphthol 59 to ortho-quinone 60 is proposed in Scheme 15. One equivalent of PIFA oxidizes 59 to the binaphthone 69 via adduct 71. The trifluoroacetic acid liberated from the PIFA protonates the C4-carbonyl, promoting conjugate addition of H2O to the C5-center. After tautomerization, another oxidation forms the binaphthone 74. A second conjugate addition of H2O at the C5'-position is followed by tautomerization to intermediate 75 that can then be oxidized twice further. Final hydrolysis with the excess H2O present of the alkylated carbonyls of 76 furnishes bis-ortho-quinone 60. This mechanism requires four equivalents of an oxidant to form 60 from 59, which is consistent with the amount employed in Eq 4.</p><!><p>Due to the racemization of biaryl 59 during the formation of bis-ortho-quinone 60, path a (Scheme 7) was not a viable route to enantiopure 39. Shifting to path b, we examined the formation of 39 via an advanced biaryl intermediate 42, containing C5,C5'- benzyl ethers (Scheme 7). The first undertaking was the hydroxylation of the C5,C5'-centers to provide 42 from available precursor 41. Based upon the above results (Scheme 15), the C4,C4'-phenols needed to be masked as ethers during this oxidation to prevent formation of binaphthone 69. Thus, an oxidation was required that did not rely on hydroxyl activation in oxidizing the C5,C5'-centers. One such method is the oxygenation of aryl lithiums with electrophilic oxygen sources. Since the requisite binaphthalene (see 82 below) is very electron rich and the C5,C5'-centers are hindered, a naphthalene model system 78 was utilized to explore the stability and reactivity of the corresponding C5-lithium species. Model 78 was prepared by C5-bromination of 77 followed by hydrolysis of the C2,C4-acetates and methylation to provide trimethyl ether 78. With tert-butyllithium, lithium halogen exchange provided intermediate 79 as confirmed by quenching with D2O which generated the corresponding C5-deuterated arene and minimal amounts of C3-tert-butyl ketone. Unfortunately, reaction with a series of oxidants and electrophiles (oxygen gas, Davis oxaziridine, lithium salt of tert-butyl hydroperoxide, and B(OMe)3)39 was unsuccessful, which was attributed to the hindered nature of the C5 center. Given this challenge, an alternate aryl hydroxylation reaction was sought.</p><p>Notably, Kita has utilized PIFA to induce the nucleophilic substitution of phenyl ethers to afford functionalized arenes.40,41 His studies indicate that the reaction proceeds by a single-electron transfer (SET) resulting in the formation of a radical cation [ArH•+] as the reactive intermediate (81, Eq 6).40 This mechanism contrasts with PIFA-oxidations described above (Scheme 10, Scheme 11, Scheme 14, and Scheme 15; eq 4), which proceeded via phenolic C4,C4'-hydroxyl activation (Scheme 15). Based on the reported success of the acetoxy group as a nucleophile in the PIFA-induced SET pathway, we set out to synthesize an intermediate containing the necessary perylenequinone oxygenation in this manner. The greatest concern in this transformation was the regioselection in the addition to the radical cation. In considering the radical cation, many resonance structures are possible, but the two that are pertinent (cations at unsubstituted positions where reaction will allow regeneration of aromaticity via deprotonation) are illustrated in Scheme 17. The stabilization of the cations is expected to be similar at C5 and C8. However, the radical would need to reside at C6 or C7 to maintain the greatest amount of conjugation in the system. The C6-methoxy would be expected to stabilize radical character at the C6-position to a greater degree. Thus, resonance structure 82b is expected to predominate leading to reaction at the C5-center. In addition, the C5-position is less hindered by the neighboring C4,C6-methoxys than the C8-position with larger flanking groups (C1-aryl group and C7-propyl group).</p><p> (6)</p><p>The requisite substrate to test this hypothesis was prepared from 52d using reagent grade DMF during methylation to conveniently prepare hexamethyl ether 82 in one step.33 After initial C2,C2'-methylation, the small amount of water in the DMF was converted to NaOH, cleaving the C4,C4'-acetates; subsequent methylation afforded 82 (Scheme 18). Treatment of 82 with PIFA and TMSOAc in (CF3)2CHOH afforded a mixture of compounds, from which bisphenol 87 and the C5,C5'-bistrifluoroacetate 86 were isolated. The reaction also proceeded well without TMSOAc; presumably, trifluoroacetate from the PIFA is sufficiently nucleophilic to trap the radical cation. When the reaction mixture (without TMSOAc) was treated with aqueous NaOH, the bistrifluoroacetate intermediate 86 was efficiently cleaved furnishing 87 in 89% yield (Scheme 18). However, there was erosion in the optical purity, from 86% ee in 82 to 68–70% ee in bisnaphthol 87. We propose that this attrition arises from racemization of radical cation or cation intermediates where the atropisomerization barrier is lower.28 To mitigate this effect, hexafluoroisopropanol was replaced with the less polar trifluoroethanol, which should be sufficiently polar to permit the initial SET to the radical cation but will reduce its lifetime. Pleasingly, bisnaphthol 87 was obtained with improved enantiopurity (82% ee) but in a diminished 51% yield.42</p><p>The oxidative cyclization of 87 with MnO2 proceeded smoothly to yield perylenequinone 88,16 representing the first enantioselective synthesis of a perylenequinone with the helical chirality as the only stereochemical element. Since the optical purity of 88 could not be measured directly, the C4,C4'-methyl ethers were selectively removed using MgI2 to provide 89.15,17 An HPLC assay verified the stereochemical integrity of the axial to helical chirality transfer (Scheme 19).</p><!><p>This initial result on the atropisomeric stability of a perylenequinone with no stereogenic elements aside from the helical axis was encouraging. However, it was unclear that the corresponding compound lacking the C3,C3'-ester groups, which were needed as outlined in the retrosynthesis (see 33 in Scheme 6), would possess the same degree of atropisomeric stability. Removal of the C3,C3'-ester groups alleviates steric gearing interactions thereby decreasing the effective size of the C2,C2'-groups so that they might more easily slide by one another, thereby lower the atropisomerization barrier.</p><p>Thus, the synthesis of 39, which contains the necessary substitution pattern of the natural products perylenequinone core, was our next goal (Scheme 20). To accomplish this goal, 87 was treated with benzyl bromide and NaH to mask the reactive C5,C5'-phenols. Hydrolysis was affected by LiOH to supply the diacid 90. Disappointingly, the high temperatures of conventional aromatic decarboxylation protocols, such as Cu-quinoline, caused not only atropisomerization of the biaryl but also significant decomposition resulting in modest yields of 42 (31% after optimization). As a consequence we developed a palladium decarboxylation protocol involving stoichiometric palladium(II) trifluoroacetate and silver carbonate followed by the reduction of the resultant aryl palladium species. The described procedure worked well on these systems and supplied the key intermediate 42 in 87% yield without racemization.19,43 Further examination of this strategy lead to the development of a single-step decarboxylation using catalytic palladium and excess of trifluoroacetic acid as the proton source to provide a variety arenes in good yield.43 After hydrogenolysis of bisbenzyl ether 42, the axial chiral bisphenol was oxidized by MnO2 to afford the helical chiral perylenequinone as a bright red resin (Scheme 20). The directed, selective cleavage of the C4,C4'-methyl ethers was accomplished by MgI2 to complete the first synthesis of optically active perylenequinone 39.44,45 Pleasingly, perylenequinone 39 was also found to exhibit considerable atropisomeric stability with an atropisomerization half-life of 4 d at 60 °C in benzene.19</p><!><p>As summarized in Scheme 21, the final route to 39 proceeded in 18 steps and a 5.4% overall yield (92% average yield per step). The key steps in this synthesis were an enantioselective biaryl coupling, a PIFA-induced phenol formation, and a palladium-mediated aromatic decarboxylation. The development and optimization of these transformations was crucial not only to this investigation but also for the syntheses of the perylenequinone natural products detailed in the subsequent papers in this series.</p><!><p>The synthesis of 39 provided the first helical chiral perylenequinone containing the natural product substitution pattern but absent the conventional stereogenic C7,C7'-substitution. The central issues addressed here including generation of the necessary oxygenation pattern in a complex binaphthol and formation of the helical stereochemistry. Importantly, such perylenequinones were shown to possess sufficient atropisomeric stability to be viable intermediates in the biogenesis of the perylenequinone natural products. Furthermore, this stability supports their use as key intermediates in biomimetic total synthesis ventures (see 33 in the retrosynthesis in Scheme 6). Thus, these discoveries laid the groundwork for the total syntheses of (+)-calphostin D (8d) and (+)-phleichrome (9)19 and the first total syntheses of cercosporin (6)19 and hypocrellin A (ent-2)18, which are described in the succeeding papers in this series. Importantly, the versatility of this strategy enabled selective and convergent synthesis of all stereoisomeric combinations permitting comparison of their biological effects for the first time.19</p><!><p>To a solution of 47 (1.0 g, 4.0 mmol) in MeOH (50 mL) was added 10% Pd/C (106 mg) and the mixture was stirred under H2 (1 atm). After 2 d, the reaction was filtered through Celite, rinsing with CH2Cl2. This solution was filtered through SiO2 and the solvent was evaporated to yield the dipropyl product as an oil (1.0 g, 100%): 1H NMR (360 MHz, CDCl3) δ 0.90–0.97 (m, 6H), 1.57–1.67 (m, 4H), 2.57 (t, J = 7.5 Hz, 2H), 3.54 (s, 2H), 3.80 (s, 3H), 4.05 (t, J = 6.5 Hz, 2H), 6.78 (d, J = 8.1 Hz, 1H), 7.06 (m, 2H).</p><p>A mixture of propyl ester (329 mg, 1.31 mmol) and 1 N NaOH (5 mL, 5 mmol) in THF (5 mL) was stirred 16 h. The reaction was diluted with 1 N NaOH and hexanes, and the aqueous phase was extracted with hexanes. The aqueous phase was then acidified with 1 N HCl and extracted with EtOAc. The organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield 49 as a white solid (250 mg, 92%): mp 58–60 °C; IR (thin film) 3076 (br), 3003, 2961, 1710, 1251 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.94 (t, J = 7.4 Hz, 3H), 1.56–1.62 (m, 2H), 2.56 (t, J = 7.6 Hz, 2H), 3.57 (s, 2H), 3.80 (s, 3H), 6.79 (d, J = 8.2 Hz, 1H), 7.03 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 11.0 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 178.5, 157.0, 131.8, 131.3, 128.0, 125.3, 110.8, 55.8, 40.7, 32.6, 23.3, 14.5; HRMS (CI): [M+] calcd for C12H16O3, 208.1099; found, 208.1104.</p><!><p>To a solution of acid 49 (6.4 g, 30.7 mmol) in PhH (30 mL) was added SOCl2 (9.0 mL, 123 mmol). The reaction was heated to reflux under Ar for 2 h. The solvent was evaporated in vacuo to yield a brown oil. To a suspension of NaH (60 % in mineral oil, 3.7 g, 92.1 mmol) in THF (200 mL) under Ar was added dimethyl malonate (10.5 mL, 92.1 mmol). After stirring for 30 min, the mixture was chilled in an ice-H2O bath and a solution of the above acid chloride in THF (50 mL) was added. The reaction was stirred for 3 h with warming to room temperature. The mixture was poured into a mixture of ice and 1 N HCl and extracted into EtOAc. The organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated. The oil was filtered through SiO2, rinsing with 0–20% EtOAc/hexanes. The solvent was evaporated and the oil was dissolved in MeSO3H (55 mL), and P2O5 (5.0 g) was added. After stirring under Ar for 3 h, the mixture was poured over ice-H2O and filtered. The yellow solid was dissolved in CH2Cl2 and was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield 50 (6.9 g, 77%) that was carried on to the next step without further purification: IR (thin film) 3443, 2957, 1670, 1644, 1252, 1217, 1158 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.99 (t, J = 7.3 Hz, 3H), 1.64–1.71 (m, 2H), 2.69 (t, J = 7.4 Hz, 2H), 3.92 (s, 3H), 4.10 (s, 3H), 6.71 (s, 1H), 7.29 (s, 1H), 7.43 (s, 1H), 8.63 (br s, 1H), 11.32 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 171.0, 160.4, 155.2, 152.4, 138.5, 133.7, 126.7, 118.9, 102.4, 101.2, 97.2, 55.7, 53.3, 33.3, 23.0, 14.5; HRMS (CI): [M+] calcd for C16H18O5, 290.1154; found, 290.1156.</p><!><p>A solution of naphthol 50 (6.0 g, 20.7 mmol), Ac2O (60 mL) and pyridine (60 mL) was stirred for 3 h. The reaction mixture was poured over ice-1 N HCl and stirred 30 min. The solid was filtered, rinsing with H2O, and dried by pulling air through the filter cake to yield the diacetate (6.9 g, 89%): IR (thin film) 2955, 1776, 1724, 1316, 1195 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.97 (t, J = 7.3 Hz, 3H), 1.64–1.69 (m, 2H), 2.32 (s, 3H), 2.44 (s, 3H), 2.72 (t, J = 7.4 Hz, 2H), 3.90 (s, 3H), 3.91 (s, 3H), 7.01 (s, 1H), 7.37 (s, 1H), 7.52 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 169.2, 164.6, 158.0, 146.4, 143.8, 137.1, 130.6, 128.2, 125.6, 118.4, 116.5, 99.5, 55.7, 52.8, 33.1, 22.9, 21.3, 21.2, 14.4; HRMS (ESI) calcd for C20H22O7 (M+) 374.1366, found 374.1364.</p><p>To a solution of this acetate (2.5 g, 6.7 mmol) in MeOH (150 mL) and CH2Cl2 (15 mL) under Ar was added K2CO3 (913 mg, 6.6 mmol). After stirring for 15 min, the green reaction mixture was poured over ice-1 N HCl. The aqueous phase was extracted with CH2Cl2 and the organic extracts were washed with brine, dried (Na2SO4), and the solvent was evaporated. Purification was accomplished with chromatography (10–20% EtOAc/hexanes) to yield product 45d as a yellow solid (1.7 g, 76%): mp 121.5–125.5 °C; IR (thin film) 3155, 2957, 1773,46 1675, 1223, 1197 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.98 (t. J = 7.4 Hz, 3H), 1.65–1.69 (m, 2H), 2.48 (s, 3H), 2.69 (t, J = 7.5 Hz, 2H), 3.89 (s, 3H), 4.00 (s, 3H), 6.91 (s, 1H), 7.17 (s, 1H), 7.39 (s, 1H), 10.48 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 169.9, 169.6, 156.3, 155.2, 147.6, 138.5, 133.8, 127.0, 121.6, 110.1, 107.2, 99.1, 55.5, 53.4, 33.2, 22.9, 21.3, 14.4; HRMS (CI) calcd for C18H20O6 (M+) 332.1260, found 332.1250.</p><!><p>To a solution of the 2-naphthol substrate dissolved in the appropriate solvent was added the copper catalyst (10 mol%). The mixture was sonicated to yield a clear green, blue, purple, or red solution, which was stirred under O2 (1 atm) at the indicated temperature and time. After cooling, the reaction mixture was diluted with CH2Cl2 and washed with 1 N HCl. The aqueous phase was back extracted with CH2Cl2 and the combined organic solutions were dried over Na2SO4. Filtration and concentration afforded the crude product. Purification was accomplished by SiO2 chromatography.</p><!><p>A mixture of naphthol 45d (103 mg, 0.309 mmol), 4 Å molecular sieves (86 mg) and CuI-(S,S-51) (11.4 mg, 0.0329 mmol) in MeCN (1.5 mL) was stirred at room temperature under an O2 atmosphere for 24 h. The reaction was quenched with 1 N HCl, and the aqueous phase was extracted with EtOAc. The organic extracts were washed with H2O and brine, dried (Na2SO4), and filtered through SiO2 with 10% MeOH/CH2Cl2. Purification was accomplished via chromatography to yield the product as a resin (86.7 mg, 85%) in 87% ee: [α]D20 +20.9 (c 0.23, MeOH, 87% ee); IR (thin film) 3130, 2958, 1770, 1672, 1234, 1195 cm−1; 1H NMR (300 MHz, CDCl3) δ 0.79 (t, J = 7.4 Hz, 6H), 1.39–1.44 (m, 4H), 2.44–2.55 (m, 10H), 3.90 (s, 6H), 4.01 (s, 6H), 6.91 (s, 2H), 7.05 (s, 2H), 10.64 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 170.1, 169.7, 156.3, 152.9, 147.8, 138.7, 133.2, 125.8, 121.8, 115.4, 107.3, 99.7, 55.5, 53.4, 33.5, 23.2, 21.9, 14.3; HRMS (ESI) calcd for C36H38O12Na (MNa+) 685.2261, found 685.2276; CSP HPLC (Chiralpak AD, 1.0 mL/min, 70:30 hexanes:iPrOH) tR(S) = 10.5 min, tR(R) = 13.3 min.</p><!><p>Bisphenol 53(51 mg, 0.07 mmol, 74% ee) was dissolved in MeCN:CH2Cl2:H2O (6:1:1, 8 mL) and PhI(OCOCF3)2 (300 mg, 0.7 mmol) was added. After 2 h at room temperature, the deep red solution was diluted with CH2Cl2 and washed with H2O. Concentration afforded an oil which was purified by SiO2 chromatography (2–3.5% MeOH/ CH2Cl2) to provide 54 as a red solid (45 mg, 88%): IR (thin film) 3683, 2954, 1733, 1693, 1636, 1608, 1274, 1261, 1227, 1115 cm−1; 1H NMR (500 MHz, CD2Cl2/d6-DMSO) δ 3.60 (s, 6H), 3.99 (s, 6H), 4.84 (d, J = 9.0 Hz, 2H), 5.08 (d, J = 9.0 Hz, 2H), 5.84 (s, 2OH), 7.09 (m, 4H), 7.24 (m, 6H), 12.9 (s, 2OH); 13C NMR (125 MHz, CD2Cl2/d6-DMSO) δ 179.8, 175.3, 165.4, 163.6, 152.8, 137.1, 135.6, 128.5, 128.4, 128.1, 127.7, 126.7, 119.7, 115.3, 109.2, 109.1, 76.2, 56.0, 53.0; HRMS (ESI) cald for C40H30O14Na (MNa+) 757.1558, found 757.1533.</p><!><p>To a solution of binaphthol 59 (230 mg, 0.379 mmol) in MeCN (50 mL), CH2Cl2 (20 mL), and H2O (10 mL) was added PhI(OC(O)CF3)2 (1.2 g, 2.8 mmol). The dark blue reaction mixture was stirred for 45 min, then diluted with H2O and EtOAc. The phases were separated and the aqueous phase was washed with EtOAc. The organic phase was extracted with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield 60 as an orange solid that was carried on to the next step without further purification: 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 6H), 1.39–1.44 (m, 4H), 2.32 (t, J = 6.7 Hz, 4H), 3.80 (s, 6H), 3.99 (s, 6H), 6.60 (s, 2H), 12.8 (br s, 2H); CSP HPLC (Chiralpak AD, 1.0 mL/min, 70:30 hexanes:iPrOH) tR(S) = 12.3 min, tR(R) = 22.2 min.</p><p>To a solution of ortho-quinone (0.379 mmol) and MeI (0.20 mL, 3.2 mmol) in THF (30 mL) was added TBAF (1 M in THF, 1.5 mL, 1.5 mmol). The black reaction mixture was stirred for 16 h, then diluted with EtOAc and H2O. The phases were separated and the aqueous phase was washed with EtOAc. The organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield an orange solid. Purification by chromatography (30% EtOAc/hexanes) yielded product 61 as an orange solid (168 mg, 70%): mp 95–97 °C; IR (thin film) 2955, 1737, 1667, 1224 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 6H), 1.41 (m, 4H), 2.31 (m, 4H), 3.71 (s, 6H), 3.94 (s, 6H), 3.97 (s, 6H), 6.71 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 180.8, 177.3, 165.7, 163.6, 161.1, 143.0, 138.0, 137.0, 122.9, 122.7, 118.9, 63.8, 60.8, 53.5, 32.0, 21.9, 14.0; HRMS (ESI) calcd for C34H34O12Na (MNa+) 657.1948, found 657.1936; CSP HPLC (Chiralpak AD, 1.0 mL/min, 70:30 hexanes:iPrOH) tR(S) = 14.7 min, tR(R) = 36.3 min.</p><!><p>To a solution of 64 (3.0 mg, 0.0052 mmol) in MeCN (0.5 mL) was added H2O (1 drop) and CAN (31 mg, 0.057 mmol). The red reaction mixture was stirred for 10 min, then diluted with H2O and extracted with EtOAc. The organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield 40 as an orange oil that was not stable to further purification: 1H NMR (500 MHz, CDCl3) δ 0.83 (t, J = 7.4 Hz, 6H), 1.35–1.40 (m, 4H), 2.26 (t, J = 6.4 Hz, 4H), 3.83 (s, 6H), 4.08 (s, 6H), 6.52 (s, 2H), 6.69 (s, 2H).</p><!><p>A mixture of MeCN (7 mL), CH2Cl2 (3 mL), and H2O (1 mL) was degassed with Ar for 15 min. Naphthol 59 (32.3 mg, 0.0532 mmol) was added followed by PIDA (44.2 mg, 0.118 mmol). The reaction mixture turned a dark blue color and was stirred for 1 h. The reaction mixture was diluted with EtOAc and H2O, the phases were partitioned, and the organic layer was washed with H2O and brine. After drying (Na2SO4) the solvent was evaporated. Purification was accomplished with chromatography to yield 29 mg (91%) of a dark green resin that was not stable in solution over extended periods of time: IR (thin film) 2957, 1737, 1586, 1227 cm−1; 1H NMR (500 MHz, CD2Cl2) δ 0.86 (t, J = 7.4 Hz, 6H), 1.47–1.50 (m, 2H), 2.52 (m, 4H), 3.58 (s, 6H), 3.90 (s, 6H), 3.97 (s, 6H), 7.34, (s, 2H), 7.50 (s, 2H); HRMS (ES) calcd for C34H37O10 (MH+) 605.2387, found 605.2412. Bianthrone 69 was unstable in solution over time precluding 13C NMR characterization.</p><!><p>To a solution of binaphthol (M)-52d (70 mg, 0.11 mmol) in DMF (2 mL) under an Ar atmosphere was added NaH (60%, 82 mg, 2.1 mmol). The mixture was stirred for 10 min, then CH3I (0.2 mL, 3.2 mmol) was added. The reaction was stirred at room temperature for 18 h. The yellow mixture was quenched with H2O and the aqueous phase was extracted with EtOAc. The organics were washed with brine, dried (MgSO4), and the solvent was evaporated in vacuo to yield a yellow oil. Purification was accomplished via chromatography (25% EtOAc/hexanes) to yield 82 as a resin (52 mg, 75%): [α]D20 – 26.4 (c 0.22, MeOH, 86% ee); IR (thin film) 2956, 1731, 1592, 1493, 1454, 1224, 1085, 1015, 733 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.74 (t, J = 7.3 Hz, 6H), 1.37–1.41 (m, 4H), 2.44–2.49 (m, 4H), 3.31 (s, 6H), 3.95 (s, 6H), 3.97 (s, 6H), 4.13 (s, 6H), 6.90 (s, 2H), 7.40 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 167.9, 156.7, 153.6, 151.8, 135.5, 130.8, 127.1, 124.9, 120.4, 120.2, 100.2, 63.1, 62.3, 55.8, 52.9, 33.2, 23.2, 14.2; HRMS (ESI) calcd for C36H42O10Na (MNa+) 657.2676, found 657.2666.</p><!><p>To a solution of binaphthalene 82 (131 mg, 0.269 mmol) in CF3CH2OH (10 mL) under an Ar atmosphere was added PhI(OC(O)CF3)2 (199 mg, 0.463 mmol). After stirring the purple mixture for 30 min, the solvent was evaporated in vacuo. The resultant mixture was treated with a mixture of 1 N NaOH (0.1 mL, 0.1 mmol) in H2O/THF/EtOH (1:1:1, v:v:v) for 30 min, followed by acidification with 1 N HCl. The aqueous phase was extracted with EtOAc, the organics were washed with brine, dried (Na2SO4), and the solvent was evaporated. Purification was accomplished by chromatography (25% EtOAc/hexanes) to yield product 87 as a yellow resin (70 mg, 51%, 82% ee): [α]D20 –12.1 (c 0.22, MeOH, 76% ee); IR (film) 3369, 2957, 1732, 1311 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.74 (t, J = 7.2 Hz, 6H), 1.35–1.42 (m, 4H), 2.42–2.51 (m, 4H), 3.32 (s, 6H), 3.93 (s, 6H), 3.98 (s, 6H), 4.16 (s, 6H), 6.42 (s, 2H), 9.24 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 167.1, 154.4, 152.6, 146.0, 142.6, 139.8, 132.9, 120.8, 119.2, 117.6, 114.2, 64.6, 62.3, 60.9, 53.2, 33.2, 23.9, 14.2; HRMS (ESI) calcd for C36H42O12Na (MNa+) 689.2574, found 689.2599; CSP HPLC (Chiralpak AD, 1.0 mL/min, 98:2 hexanes:i-PrOH) tR(S) = 12.3 min, tR(R) = 15.6 min.</p><!><p>To a solution of 68% ee binaphthol 87 (37 mg, 0.055 mmol) and benzyl bromide (46 µL, 0.38 mmol) in DMF (1.0 mL) under an Ar atmosphere was added 60% NaH (12.5 mg, 0.312 mmol). The dark green mixture was stirred for 15 h, quenched with H2O, and washed with EtOAc. The organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield an orange oil. Purification was accomplished by chromatography (15% EtOAc/hexanes) to yield the bisbenzyl ether (36 mg, 78%) as a yellow resin: [α]D20 –24.7 (c 1.0, CHCl3, 68% ee); IR (thin film) 2939, 1735, 1330, 1094 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.75 (t, J = 7.4 Hz, 6H), 1.39–1.43 (m, 4H), 2.49–2.54 (m, 4H), 3.39 (s, 6H), 3.95 (s, 6H), 3.98 (s, 6H), 4.00 (s, 6H), 5.09 (m, 4H), 6.76 (s, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.44 (t, J = 7.3 Hz, 4H), 7.63 (d, J = 7.0 Hz, 4H); 13C NMR (125 MHz, CDCl3) δ 167.8, 154.4, 152.6, 146.8, 138.9, 138.4, 133.8, 129.2, 128.8, 128.3, 127.4, 123.1, 120.7, 120.3, 114.2, 77.1, 64.6, 62.3, 60.9, 53.2, 33.2, 23.9, 14.2; HRMS (ESI) calcd for C50H54O12Na (MNa+) 869.3513, found 869.3541.</p><p>To a suspension of the bisbenzyl ether (163 mg, 0.192 mmol) in dioxane (8 mL) and H2O (9 mL) was added LiOH·H2O (402 mg, 9.57 mmol), and the mixture was heated to reflux for 24 h. The brown mixture was cooled to room temperature and diluted with H2O. The aqueous phase was washed with EtOAc, and this organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to recover 73 mg (0.086 mmol, 45%) of starting material. The aqueous phase was acidified with 1 N HCl, extracted with EtOAc, and the organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield the diacid 90 (81 mg, 93% based on recovered starting material) as an orange resin that was used for the next step without further purification: [α]D20 – 29.1° (c 1.0, CHCl3, 82% ee); IR (thin film) 2937, 1736, 1707, 1327, 1107 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.76 (t, J = 7.3 Hz, 6H), 1.40–1.44 (m, 4H), 2.50–2.54 (m, 4H), 3.44 (s, 6H), 3.72 (s, 6H), 3.98 (s, 6H), 3.99 (s, 6H), 5.11 (dd, J = 9.8, 14.9 Hz, 4H), 6.80 (s, 2H), 7.36 (t, J = 7.3 Hz, 2H), 7.43 (t, J = 7.3 Hz, 4H), 7.64 (d, J = 7.0 Hz, 4H); 13C NMR (125 MHz, CDCl3) δ 172.5, 155.2, 152.4, 150.7, 146.9, 139.3, 138.2, 134.5, 129.3, 128.8, 128.3, 122.9, 122.1, 120.6, 120.1, 67.5, 65.2, 62.4, 61.7, 33.2, 23.9, 14.2; HRMS (ESI) calcd for C48H50O12Na (MNa+) 841.3200, found 841.3240.</p><!><p>To a solution of diacid 90 (26.7 mg, 0.0326 mmol) in DMSO-DMF (0.3 mL) was added Pd(OC(O)CF3)2 (22.8 mg, 0.0739 mmol). The mixture was heated in a 90 °C oil bath for 1 h. After cooling the brown reaction mixture to room temperature, 1 N HCl was added, and the mixture was extracted with EtOAc. The organic phase was washed with H2O and brine, dried (Na2SO4), and the solvent was evaporated to yield a brown oil. This material was dissolved in THF (1.0 mL) and stirred under a H2 atmosphere for 5 min. After filtration through Celite, purification was accomplished via chromatography (20% EtOAc/heanes) to yield 42 (20.7 mg, 87%) as a resin: [α]D20 0 (c 1.0, CHCl3, 70% ee)*; IR (thin film) 2933, 1338 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.75 (t, J = 7.2 Hz, 6H), 1.35–1.42 (m, 4H), 2.46–2.53 (m, 4H), 3.74 (s, 6H), 3.92 (s, 6H), 3.99 (s, 6H), 5.10 (m, 4H), 6.64 (s, 2H), 6.76 (s, 2H), 7.37 (t, J = 7.3 Hz, 2H), 7.43 (t, J = 7.3 Hz, 4H), 7.62 (d, J = 7.0 Hz, 4H); 13C NMR (125 MHz, CDCl3) δ 157.1, 154.9, 148.8, 147.2, 138.9, 137.0, 133.7, 129.0, 128.5, 127.8, 122.1, 116.5, 112.5, 95.9, 76.2, 61.4, 57.5, 56.1, 32.8, 23.6, 14.0; HRMS (ESI) calcd for C46H50O8Na (MNa+) 753.3403, found 753.3427.</p><p>*This compound was re-synthesized but the optical rotation was still 0. The compound was carried on to the next step and enantioselectivity confirmed by CSP HPLC (69% ee).</p><!><p>To a solution of binaphthalene 42 (6.4 mg, 0.0088 mmol) in MeOH (0.3 mL) and THF (0.3 mL) was added 10% Pd/C (2 mg). The mixture was stirred under an atmosphere of H2 for 3 h. The reaction was filtered through Celite, rinsing with MeOH and CH2Cl2. The solvents were evaporated to yield an unstable yellow oil (69% ee): 1H NMR (500 MHz, CDCl3) δ 0.76 (t, J = 7.3 Hz, 6H), 1.38–1.43 (m, 4H), 2.43–2.47 (m, 4H), 3.69 (s, 6H), 3.89 (s, 6H), 4.16 (s, 6H), 6.37 (s, 2H), 6.70 (s, 2H), 9.28 (s, 2H); CSP HPLC (Chiralpak AD, 1.0 mL/min, 98:2 hexanes:i-PrOH) tR(S) = 37.9 min, tR(R) = 45.2 min.</p><p>To a solution of bisnaphthol (11 mg, 0.020 mmol) in Et2O (1 mL) was added MnO2 (60 mg, 0.69 mmol). The mixture was stirred for 30 min, filtered through Celite, and the solvent was evaporated to yield a red resin, which was chromatographed (2.5% MeOH/CH2Cl2) to yield the perylenequinone as a red resin: [α]D20 –410 (c 0.021, MeOH, 69% ee); 1H NMR (500 MHz, CDCl3) δ 0.54 (t, J = 7.3 Hz, 6H), 0.96–0.99 (m, 2H), 1.18–1.22 (m, 2H), 2.48–2.51 (m, 2H), 3.08–3.11 (m, 2H), 4.05 (s, 6H), 4.11 (s, 6H), 4.17 (s, 6H), 6.78 (s, 2H).</p><p>To a solution of the above perylenequinone product (12 mg, 0.0221 mmol) in THF (2 mL) under an argon atmosphere was added a solution of MgI2 in Et2O (0.07 M, 665 µL, 0.0464 mmol). The dark purple mixture was stirred 10 min (until the mixture turns from purple to black), diluted with EtOAc, washed with saturated aq NH4Cl, and dried (Na2SO4). Concentration yielded a red residue, which was chromatographed (2.5% MeOH/CH2Cl2) to yield product 39 as a red resin (10 mg, 87% (69% for the three steps)): 1H NMR (500 MHz, CDCl3) δ 0.52 (t, J = 7.2 Hz, 6H), 0.88 (m, 2H), 1.25 (m, 2H), 2.76 (m, 2H), 3.33 (m, 2H), 4.09 (s, 6H), 4.21 (s, 6H), 6.59 (s, 2H), 15.82 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 14.1, 24.3, 35.0, 56.3, 61.1, 101.1, 105.7, 116.5, 126.2, 127.5, 140.5, 151.0, 166.5, 174.1, 176.8; IR (film) 3383, 2927, 2858, 1607, 1460, 1274, 1213 cm−1; HRMS (ES) calcd for C30H31O8 (MH+) 519.2019, found 519.2036; See Supporting Information for CD spectrum; CSP HPLC (Chiralpak AD-H, 0.5 mL/min, 95:5 hexanes:i-PrOH) tR (P) = 15.9 min, tR (M) = 17.1 min.</p><!><p>Dedicated to the memory of Ralph Hirschmann.</p><p>Supporting Information Available Additional experimental descriptions, NMR spectra, and crystallographic data. This material is available free of charge via the Internet at http://pubs.acs.org.</p><p>Representative Structures of the Perylenequinone Classes.</p><p>Mold Perylenequinones 2–17.</p><p>Strained Extended Quinone Structures.</p><p>Proposed Racemization Pathway of 69.</p><p>Previous Synthesis of Parent Perylenequinone 1.</p><p>Weiss Attempted Synthesis of Perylenequinone 22 (PPA = polyphosphoric acid).</p><p>Dallacker Synthesis of Perylenequinone 27.</p><p>Conformational Isomers of Hypocrellin B and Scutiaquinone B.</p><p>Proposed Enzymatic Perylenequinone Formation.</p><p>Retrosynthesis of the perylenequinone natural products.</p><p>Perylenequinone Retrosynthetic Analysis.</p><p>Synthesis of Naphthol Substrates.</p><p>Synthesis of C7-Propyl Substrate.</p><p>Synthesis of Bis-ortho-quinone 54 (ORTEP drawing with 30% probability thermal ellipsoids).</p><p>Attempted Synthesis of C7,C7'-Bispropyl Perylenequinone.</p><p>Decarboxylation and Attempted Synthesis of a Perylenequinone.</p><p>Two-step Synthesis of Perylenequinone from Bis-ortho-quinone.</p><p>Isolated Intermediate in the Oxidation to bis-ortho-Quinone 60.</p><p>Proposed Mechanism for Bis-ortho-quinone Formation.</p><p>Attempted C5-Oxidation of a naphthalene model system.</p><p>Regioselection in the PIFA-mediated Hydroxylation.</p><p>PIFA-mediated Hydroxylation.</p><p>Synthesis of Optically Active Perylenequinone 89.</p><p>Palladium-Mediated Decarboxylation and Synthesis of Optically Active 39.</p><p>Enantioselective Total Synthesis of (M)-39.</p><p>Enantioselective Biaryl Coupling with Diaza-cis-decalin (S,S)·51.</p><p>(R,R)·51 catalyst complex was used.</p>

PubMed Author Manuscript

Heteroatom Effects on Quantum Interference in Molecular Junctions: Modulating Antiresonances by Molecular Design

Controlling charge transport through molecular wires by utilizing quantum interference (QI) is a growing topic in single-molecular electronics. In this article, scanning tunneling microscopy-break junction techniques and density functional theory calculations are employed to investigate the single-molecule conductance properties of four molecules that have been specifically designed to test extended curly arrow rules (ECARs) for predicting QI in molecular junctions. Specifically, for two new isomeric 1-phenylpyrrole derivatives, the conductance pathway between the gold electrodes must pass through a nitrogen atom: this novel feature is designed to maximize the influence of the heteroatom on conductance properties and has not been the subject of prior investigations of QI. It is shown, experimentally and computationally, that the presence of a nitrogen atom in the conductance pathway increases the effect of changing the position of the anchoring group on the phenyl ring from para to meta, in comparison with biphenyl analogues. This effect is explained in terms of destructive QI (DQI) for the meta-connected pyrrole and shifted DQI for the para-connected isomer. These results demonstrate modulation of antiresonances by molecular design and verify the validity of ECARs as a simple “pen-and-paper” method for predicting QI behavior. The principles offer new fundamental insights into structure–property relationships in molecular junctions and can now be exploited in a range of different heterocycles for molecular electronic applications, such as switches based on external gating, or in thermoelectric devices.

heteroatom_effects_on_quantum_interference_in_molecular_junctions:_modulating_antiresonances_by_mole

Introduction<!>ECAR-1<!>ECAR-2<!>Methods<!>Molecular Design<!><!>Molecular Design<!>Molecular Conductance Studies<!><!>Molecular Conductance Studies<!>Charge-Transport Simulations<!><!>Charge-Transport Simulations<!>Conclusions<!><!>Author Contributions<!>

<p>Single-molecule conductance values have been determined for a diverse array of molecular wires trapped between metal electrodes since the development of specialized measurement techniques in the late 1990s and early 2000s.1−3 These methods include mechanically controlled break junction4 and scanning tunneling microscopy-break junction (STM-BJ)5 experiments. By combining these techniques with the power of organic synthesis, it has been widely demonstrated that substantial variation in the conductance of molecular wires can be achieved by small structural modifications, such as structural isomerism and/or the presence of heteroatoms.6−13 Particularly, in the case of π-conjugated systems, much of this behavior can be attributed to quantum interference (QI) effects,14−16 which are readily visualized in transmission functions derived from charge-transport simulations.2,17−20</p><p>The transmission function T(E) of a molecular junction is a plot of the probability of electrons with energy E passing from one electrode to the other through the molecule and is proportional to molecular conductance. E is usually considered relative to the system's Fermi energy, EF. Calculated transmission functions from first-principles simulations reliably show qualitative agreement with experimental conductance studies.2 Quantitative agreement is more challenging due to difficulties such as the accurate determination of EF using density functional theory (DFT).17,21 Sharp resonances coincident with the energies of molecular orbitals (e.g. the highest occupied and lowest unoccupied molecular orbitals, HOMO and LUMO, respectively) are key features of a typical transmission function. In low-bias conductance studies, EF usually lies near the center of the HOMO–LUMO gap. Furthermore, the low-bias QI behavior of a molecular junction relates to the characteristics of the transmission function in the HOMO–LUMO gap. QI can be constructive (CQI) or destructive (DQI). Where CQI occurs, a smooth, featureless transmission curve is usually seen between the HOMO and LUMO resonances. A characteristic feature of DQI is a sharp antiresonance in the transmission curve where T(E) approaches zero.</p><p>This work considers two subcategories of DQI, based on the energy at which an antiresonance appears in the transmission function of a molecular junction. DQI refers to cases where an antiresonance occurs close to EF, and significantly reduced low-bias conductance would be expected relative to a similar system without an antiresonance. Shifted DQI (SDQI) refers to systems where an antiresonance occurs in the transmission function but does not lie close to EF and so the conductance of the junction remains high in the low-bias regime.19,20 Where SDQI occurs, an antiresonance can even be shifted beyond the HOMO–LUMO gap,20 meaning that SDQI is not always readily distinguishable from CQI.</p><p>In addition to computationally demanding charge-transport simulations, many simpler methods exist to predict and rationalize the QI behavior of molecular wires. Some methods are based on structural considerations alone, such as "curly arrow" rules (CARs)22−24 and graphical or topological methods.15,25,26 Other methods require a mathematical or computational input, such as orbital symmetry approaches,27,28 QI maps,29 and magic ratio rules.18,30−33 These more straightforward methods necessarily have limitations to their scope compared to charge-transport simulations. They generally work well for bipartite hydrocarbon lattices but can be less accurate for molecular wires that incorporate more elaborate structural features, such as (i) deviation from a framework of fused six-membered rings; (ii) the inclusion of heteroatoms, either as substituents or within the lattice; or (iii) cross-conjugation.</p><p>Two of the present authors recently presented an extension to predictive CARs for QI behavior [extended curly arrow rules (ECARs)].24 This was in part inspired by work from another two of the present authors which showed that simple CARs as widely applied in molecular electronics22 "broke down" when applied to cross-conjugated anthraquinone derivatives.8 ECARs are a "pen-and-paper" method that can predict whether a given molecule will exhibit CQI, DQI, or SDQI. ECARs account for previously reported QI behavior of molecular wires containing heteroatoms, nonbipartite structures, and cross-conjugation.24 However, ECARs cannot predict the relative conductance of wires with respect to one another. Despite this, the conductance of structurally similar materials would usually be expected to follow the trend CQI ≥ SDQI > DQI. The rules24 are as follows:</p><!><p>Identify the two anchoring units of a molecular wire and replace one with a donor group (D) and the other with an acceptor group (A). If the D lone pair can be delocalized onto A using curly arrows, CQI is expected; if not, DQI is expected.</p><!><p>If DQI is expected, identify any electron-withdrawing groups (EWGs) or electron-donating groups (EDGs) present in the molecular wire. If EWGs are present, replace each anchor with D. If a lone pair from each D can be independently delocalized to the same EWG, SDQI is expected. If EDGs are present, replace each contact with A. If a lone pair (or negative charge) from the same EDG can be independently delocalized to each A, SDQI is expected. Otherwise, DQI is expected around EF.</p><p>To further test the validity of ECARs, we have designed and synthesized new heteroatom-containing molecular wires that differ structurally from those considered in the development of the rules. Specifically, the novel feature of these molecules is that when they are held between gold electrodes, the conductance pathway through these molecules must pass through a nitrogen atom. In contrast, a pathway comprising only carbon atoms existed in all of the examples used in the conception of ECARs.24 To our knowledge, this is the first study of QI effects in organic molecules where an all-carbon conductance pathway is not available between the anchoring groups. The predictions made by ECARs for these new wires have been tested experimentally using the STM-BJ technique and investigated computationally by calculating transmission functions using a simple tight-binding method and DFT-based material-specific charge-transport simulations.</p><!><p>Full details of the synthesis and characterization of molecules 1–4 are given in the Supporting Information. In brief, the 1-phenylpyrrole derivatives 1 and 2 were prepared from 3-bromo-1-(triisopropylsilyl)pyrrole. The thiomethyl anchor was first installed through lithiation followed by treatment with dimethyl disulfide.34 The TIPS protecting group was then removed before forming the aryl–aryl C–N bond via Ullmann coupling35 with the appropriate bromothioanisole. The biphenyl species 3 and 4 were prepared based on a reported synthesis of 4(36) using a Suzuki cross-coupling reaction between 3-(methylthio)phenylboronic acid and the appropriate bromothioanisole.</p><p>Molecular conductance measurements were performed using the lab-built STM-BJ technique, which has been reported in previous publications.5,37 In brief, molecular junctions were repeatedly formed by driving the gold tip in and out of contact with a gold substrate. Conductance was measured as a function of the gold tip-substrate displacement, which is mainly controlled by a piezo stack during the repeated formation of junctions (see Supporting Information for more details). All experiments were carried out in a solution of the target molecules (0.1 mM) in mesitylene under ambient conditions with a 0.1 V bias voltage. Logarithmically binned one-dimensional (1D) conductance histograms and two-dimensional (2D) conductance-displacement histograms were plotted by compiling at least 2000 molecular conductance-displacement traces. Statistical analysis was performed using the methods we reported previously.37</p><p>The molecular conductance behavior of molecules 1–4 was investigated computationally using DFT combined with quantum transport calculations.21 From the optimized geometry of each molecule in the gas phase and between two gold electrodes, we obtained a ground-state Hamiltonian from the Siesta38 implementation of DFT and combined it with the Gollum21,39 transport code to obtain a transmission coefficient T(E) for electrons with energy E passing from one electrode to the other (see Computational Methods in the Supporting Information for further details). The low-bias electrical conductance was then calculated from the Landauer formula G = G0T(EF), where G0 is the conductance quantum and EF is the Fermi energy of the electrodes. The room temperature electrical conductance was obtained from the thermal averaging of T(E) (see Computational Methods in the Supporting Information).</p><!><p>We set out to design molecules that could be used to test the validity of ECARs via investigation of their conductance behavior, both computationally and in break junction studies. To test the breadth of applicability of ECARs, we targeted molecules with a clear structural difference to those used in prior studies of QI. The isomeric 1-phenylpyrrole (i.e. N-phenylpyrrole) derivatives 1 and 2 (Figure 1a) contain a nitrogen atom that lies directly in the conductance pathway of the molecules, with no alternative through-bond route between the anchoring groups by which the nitrogen can be avoided. This is in contrast to the species studied previously to which ECARs were applied24 and should maximize the influence of the heteroatom on conductance properties. Past studies of QI effects in molecular junctions have only considered molecular backbones where a heteroatom-containing pathway exists in parallel to an all-carbon pathway10−13 or organometallic systems.40,41 Studies of molecular junctions where the conductance pathway must pass through one or more heteroatoms in the molecular backbone have been reported,35,42 for example, using oligophenyleneimines.43 However, to our knowledge, QI effects have not been investigated in such systems.</p><!><p>(a) Structures of the studied 1-phenylpyrrole (1 and 2) and biphenyl (3 and 4) wires; (b) application of ECAR-1 to the four wires—note that the choice of which anchor is replaced with D and which with A has no impact on the result of ECAR-1 and that it is not possible to delocalize a D lone pair onto the pyrrole nitrogen as no vacant orbitals are available; (c) application of ECAR-2 to wires 1 and 2, for which the nitrogen lone pair can be used as an EDG. Different colored curly arrows represent different delocalization pathways indicated by correspondingly colored resonance arrows.</p><!><p>Each 1-phenylpyrrole isomer has a thiomethyl anchoring group in the pyrrole 3-position and a second thiomethyl anchor on the benzene ring, either para (1) or meta (2) to the pyrrole ring. As shown in Figure 1b, when applying ECAR-1, it is not possible to delocalize electrons from a D group at either anchoring position to an A group at the other for either isomer. However, the nitrogen lone pair can be used as an EDG for ECAR-2 (Figure 1c). For 1, it is possible to independently delocalize the nitrogen lone pair to an A group in either anchoring position, so ECARs predict SDQI. For 2, it is only possible to delocalize the nitrogen lone pair to an A group in the pyrrole-anchoring position (the anchoring group on the benzene ring is meta to the EDG, so delocalization is not possible); therefore, DQI is expected.</p><p>For comparison, the analogous biphenyl derivatives 3 and 4 (Figure 1a), in which the pyrrole ring is replaced by a benzene ring with the anchor in the meta-position, were investigated. Similar to 1 and 2, this means that when applying ECAR-1, it is not possible to delocalize electrons from a D group at either anchoring position to an A group at the other position for either 3 or 4 (Figure 1b). As no EDGs or EWGs are present in 3 or 4, ECAR-2 is not applicable and DQI is expected for both systems. As the four biaryl systems 1–4 form relatively short molecular wires, it was expected that their molecular conductance would be sufficiently high to measure experimentally despite the expected occurrence of DQI in three of the systems. The thiomethyl anchoring groups were selected for their proven and effective anchoring properties44−47 and good compatibility with the synthetic route.</p><p>It was anticipated that direct comparison between the 1-phenylpyrrole (1 and 2) and biphenyl (3 and 4) species could be complicated by differences in the torsional angle (θ) between the connected rings. The angle θ was not expected to vary significantly within each isomer pair, as the steric environment around the aryl–aryl bond remains the same. We, therefore, reasoned that any influence of θ would be overshadowed by comparing the relative effect of changing the position of the second anchoring group (i.e. that on the right of the structures in Figure 1a) from para to meta for the two isomeric pairs. If the prediction of ECARs is correct and 1 shows SDQI (and therefore higher low-bias conductance), while the other three species show DQI, then, the following relationship between molecular conductances GX (where X is the molecule number) should hold around EF:This means that a larger decrease in conductance is expected for the 1-phenylpyrrole backbone than the biphenyl backbone as the second anchor is changed from the para- to meta-position. In practice, the DFT-minimized conformations of 1–4 (see below, and Figure S13 and Table S1 in the Supporting Information) showed that θ was similar for all four species in the gas phase. In the DFT-minimized molecular junction conformations, θ differed by 6° in the 1-phenylpyrrole isomer pair and 8° in the biphenyl pair. We emphasize that these values relate only to the energy-minimized junction conformation. Experimental conductance measurements sample a broad range of conformations, where θ is likely to vary similarly for a given isomer pair. As the barrier to rotation is low at room temperature, we, therefore, anticipated that the proposed conductance relationship would remain valid.</p><!><p>The STM-BJ technique5,37 was used to investigate the molecular conductance of the four molecules (see Methods and the Supporting Information). 1D conductance histograms are shown in Figure 2a, with 2D histograms in Figures 2b and S11 in the Supporting Information and example conductance traces in Figure 2c. The most probable molecular conductances for the four molecules follow the trend 1 (10–3.05G0) > 3 (10–3.50G0) > 2 (10–3.85G0) > 4 (10–4.05G0). The broader conductance histograms observed for 2 and 4 (and to a lesser extent 3) in comparison to 1 can be related to a wider range of possible junction configurations.45 A small shoulder is visible in the 1D histogram of 4 (Figure 2a). This minor feature may be caused by Au-π, rather than Au–S electronic coupling of a meta-anchored phenyl ring.48 However, previous examples of such behavior were observed only when the other anchor was para-connected, and a similar feature is not observed for 2 or 3. The small peaks visible between 10–1 and 100G0 in Figure 2a are attributed to the conductance of single molecules of mesitylene, which was used as the solvent in the measurements.49</p><!><p>(a) Logarithmically binned conductance histograms of molecules 1–4; (b) 2D conductance-displacement histogram of molecule 1 under 0.1 V bias voltage (2D histograms of molecules 2–4 are shown in Figure S11 in the Supporting Information), inset: length distribution; (c) representative conductance traces measured for molecules 1–4 (trace colors match those used in panel a).</p><!><p>The hypothesized relationship between the molecular conductances based on ECARs holds: the ratio between the most probable conductances of molecules 1 and 2 is 100.80 (ca. 6.3), whereas that between 3 and 4 is only 100.55 (ca. 3.5). Changing the position of the second anchoring group from para to meta has nearly double the effect for the 1-phenylpyrrole species 1 and 2 than it has for the biphenyl wires 3 and 4. The higher conductance of 1 relative to 3 (and 2 relative to 4) indicates that the 1-substituted 3-(methylthio)pyrrole unit affords improved conductance relative to a meta-linked benzene ring. Indeed, the conductance of 1 is comparable to that of the para-linked biphenyl species 4,4′-bis(methylsulfide)biphenyl,375, which was determined to be 10–3.10G0 under the same experimental conditions used for 1–4 (Figure S10).</p><p>A similar trend is observed if the relative conductances of molecules 2, 3, and 4 are compared. Each has a meta-anchored benzene bound to a second aromatic system, respectively, 3-(methylthio)pyrrole (via the pyrrole 1-position), para-(methylthio)benzene, or meta-(methylthio)benzene. As molecular conductance increases in the sequence 4 < 2 < 3, 1-linked 3-(methylthio)pyrrole can be considered an intermediate between para- and meta-(methylthio)benzene. This trend in relative conductance is notably compatible with the QI behavior that ECARs predict for wires comprising only a meta-benzene (DQI), 1,3-difunctionalised pyrrole (SDQI), or para-benzene (CQI).</p><!><p>Figure 3a,b shows the calculated conductance for molecules 1–4 between gold electrodes based on DFT material-specific Hamiltonians. The conductance of 1 is higher than 2 for a wide range of EF around the DFT Fermi energy (EF = 0 eV), and the conductance of 3 is higher than that of 4 around EF = 0 eV. This is in qualitative agreement with the experimentally determined conductance values (Figure 2), as is the trend in molecular conductance at EF = 0 eV (see Table S2 in the Supporting Information), which decreases in the sequence 1 > 3 > 2 > 4. (The possibility that the relative conductance was significantly influenced by different anchoring geometries50 of different isomers was ruled out as described in the Supporting Information and Figure S15). Furthermore, Figure 3a,b shows that for EF < ca. 0.25 eV, G1 > G2 and G3 is similar to or < G4. This agrees with the ECARs-predicted relationship between the four molecular conductances. Taking the values at EF = 0 eV as an example, the ratio of the conductances of 1 and 2 is 100.57 (ca. 3.7) and that between 3 and 4 is 100.24 (ca. 1.7). Similar to the trend observed in the STM-BJ data, the effect of switching the second anchoring group from para to meta for the 1-phenylpyrrole species is around twice as large as for the biphenyl wires. However, the DQI-mediated antiresonance feature near EF that was predicted using ECARs is not clearly visible in the transmission function of 2, 3, or 4. We attribute this to the effect of σ-orbitals on transport.51 Molecules 1–4 are short, and therefore the contribution to transport from σ-orbitals is higher than that from π-orbitals at energies around the antiresonance feature, causing it to be masked.</p><!><p>Calculated electron transport through molecules 1–4 between gold electrodes using DFT material-specific Hamiltonians (a,b) and a tight-binding model (c,d). For the tight-binding model, site energies are 0 for all atoms except the nitrogen atom, which has a site energy of −0.5. All couplings between connected sites are −1. Expansion of the indicated region of panel c (e), showing T(E) for 1 and 2 around E = −1 eV, and the energy level coincident with this energy range. Tight-binding molecular orbitals for the energy level around E = −1 eV, showing that the LDOS is zero at both connection points of 1 (f) but non-zero at one connection point of 2 (g), resulting in the respective absence or presence of a Fano resonance around this energy in panel (e).</p><!><p>To illustrate this effect, we note that transmission functions calculated using a simple tight-binding method with a single π-orbital per atom (Figure 3c,d) show clear antiresonance features in the HOMO–LUMO gap for 2, 3, and 4 but not for 1. Note that additional sharp features can be seen outside the HOMO–LUMO gap, at E ≈ ± 1 eV for 2–4 and E ≈ −0.6 and +1.6 eV for 1 and 2. The origin of these features was investigated by calculating the tight-binding energy levels and corresponding wavefunctions for the 1-phenylpyrrole molecular core of 1 and 2 (Figure S14). A transmission resonance is usually expected close to the energy levels of a molecule in a junction. As exemplified in Figure 3e, although an energy level exists at E = −1 eV for the molecular core (indicated by the orange line in Figure 3e), no associated resonance is observed for 1 in the tight-binding transmission function, whereas for 2, a very narrow resonance can be seen. This can be understood by examination of the wavefunctions of the molecular core (Figures 3f,g and S14 in the Supporting Information). The width of a transmission resonance is proportional to the sum of the density of states [local density of state (LDOS), i.e., modulus squared of wavefunctions] at the connection points to electrodes.21 When both LDOSs are zero, a resonance with zero width (a "vanishing resonance") is expected, as seen for 1 in Figure 3f. In contrast, when only one LDOS is non-zero, as seen for 2 in Figure 3g, a resonance or Fano resonance is expected. A Fano resonance is normally due to a localized state (e.g. that shown in Figure 3f,g) that interacts weakly with continuum states. The feature observed for 2 close to E = −1 eV is, therefore, a Fano resonance (a resonance attached to an antiresonance), but the amplitude of the resonance is small because of a large asymmetry in the self-energies due to the coupling to the left and right electrodes.</p><p>To further demonstrate that the absence of antiresonance features in the DFT calculations is due to conduction through the σ-orbitals,51 the electrical conductance of extended analogues of molecules 1 and 2 (molecules 6 and 7 in Figure S12 in the Supporting Information) was calculated. Phenylacetylene units were added between the molecular core and the anchoring groups to lengthen the conductance pathway and weaken the effect of σ-channels on total transport. The resulting calculations (see Figure S12 of the Supporting Information) show that the antiresonance feature predicted by ECARs is present for 7 (i.e., it is no longer masked by σ-orbital contributions), whereas no antiresonance feature is observed in the HOMO–LUMO gap for 6. The observed QI behavior agrees with the predictions of ECARs and the simple tight-binding study.</p><!><p>Molecular wires 1 and 2, based on 1-phenylpyrrole, were designed and synthesized to test recently reported ECARs for predicting QI behavior.24 By comparison with analogous biphenyl wires 3 and 4, it was shown using STM-BJ studies that the presence of an unavoidable nitrogen atom in the through-bond conductance pathway increases the effect of changing the position of the anchoring group on the phenyl ring from para (1) to meta (2). This agrees with ECARs which predict DQI near EF for 2–4 (i.e. a low low-bias conductance) and SDQI for 1 (i.e. a higher low-bias conductance due to a shifted antiresonance). The experimental results are supported by charge-transport simulations of the measured molecules and extended analogues. This work verifies the validity of ECARs as a "pen-and-paper" method for predicting QI behavior and will, therefore, have an impact on the design criteria of new molecular wires.</p><p>Despite the absence of an alternating pathway of single and double bonds between the anchoring units, the conductance of 1 is comparable to that of linearly conjugated 4,4′-bis(methylsulfide)biphenyl, 5. 1,3-Disubstituted pyrroles therefore represent a prototype of heteroaromatic units that can be used to add SDQI behavior to a molecular wire without significantly reducing low-bias conductance. These results offer new fundamental understanding of structure–property relationships in molecular junctions which can now be exploited in a range of molecular electronic applications such as switches based on external gating or in thermoelectric devices.</p><!><p>Synthetic details and molecular characterization; general experimental and computational methods; 1H NMR spectra; 13C NMR spectra; synthetic procedures; UV-visible spectra; single-molecule conductance experiments; comparison of logarithmically binned conductance histograms; 2D conductance-displacement histograms; illustration of molecules; DFT-optimized configurations; torsion angle (θ) between aromatic rings; calculated molecular conductances; molecular orbitals; tight-binding orbitals and transmissions; and transmission coefficients (PDF)</p><p>jp1c04242_si_001.pdf</p><!><p>L.J.O. conceptualized the study and designed, synthesized, and characterized the molecules, under the supervision of M.R.B. The molecular conductance studies were conducted by W.X. under the supervision of W.H. Theory and modeling were provided by S.S. and H.S., and A.D. performed the calculations. L.J.O. led the preparation of the manuscript with contributions from all authors. Funding for the work was acquired by M.R.B., W.H., H.S., and S.S. All authors have given approval to the final version of the manuscript.</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Transgene expression within the spinal cord of hTH-eGFP rats

The enzyme tyrosine hydroxylase (TH) is broadly expressed in catecholaminergic neurons. In the spinal cord, four components contain TH, including A11 diencephalospinal dopaminergic (DA-ergic) pathways, intraspinal DA-related neurons, supraspinal noradrenergic projections, and afferents of TH-expressing sensory neurons. A human TH-enhanced green fluorescent protein (hTH-eGFP) transgenic rat was recently developed to tag TH+ profiles in the nervous system for visualization. Using immunostaining, we found that only A11 pathways express GFP whereas the other 3 components do not in the spinal cord. Thus, this may suggest a genetic difference among these TH+ elements even though they produce the same protein.

transgene_expression_within_the_spinal_cord_of_hth-egfp_rats

Introduction<!>Materials and methods<!>Results<!>Discussion<!>Conclusions

<p>Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine (DA) and adrenaline/noradrenaline synthesis in the central and peripheral nervous system. The enzyme catalyzes tyrosine to levodopa and the latter is further converted to DA by dopamine decarboxylase (DDC). If dopamine-β-hydroxylase (DBH) is present, it transforms DA to norepinephrine. Accordingly, noradrenergic neurons are TH+/DBH+ while DA-ergic neurons are featured with TH+/DBH−. In the mammalian spinal cord, TH+ elements consist of neurons and projections originating from 4 different regions of the nervous system, such as intraspinal DA-related neurons in the lumbosacral spinal cord (Hou et al., 2016), diencephalospinal pathways from the A11 cell group, supraspinal adrenergic/noradrenergic projections from nuclei (A5-7) in the brainstem (Sharples et al., 2014), and afferent fibers of TH-expressing sensory neurons in dorsal root ganglia (DRGs) throughout spinal levels (Brumovsky, 2016).</p><p>The diencephalic A11 neurons are the primary source of DA that emerges in the spinal cord. They project to the entire spinal levels, mainly the superficial dorsal horn and the intermediolateral cell column (IML). The structural characteristics suggest that these neurons are highly related to sensory and autonomic function. Indeed, dysregulation of the A11 pathway leads to painful syndromes, such as migraines and restless leg syndrome (Charbit et al., 2010; Clemens et al., 2006). In parallel, 3 nuclei in the brain, including A5, A6 (locus coeruleus) and A7, project and transport noradrenaline to the spinal cord. A5 neurons mostly project to thoracic sympathetic nuclei, and A6 projections accumulate in the dorsal horn while A7 fibers are dense in the ventral horn (Bruinstroop et al., 2012). This distribution indicates they are involved in autonomic, sensory, and motor functions, respectively. In the 1980s, a group of TH+/DBH− neurons were found in the rat lumbosacral spinal cord. Our recent studies showed these neurons undergo plasticity and contribute to a low level of DA sustained in the lower spinal cord after spinal cord injury (SCI) interrupts DA transportation from A11 descending pathways (Hou et al., 2016). It is assumed that the spinal TH+ population serves as a compensatory DA source when supraspinal DA provision is insufficient. In addition, TH+/DBH− neurons are also found in DRGs at entire spinal levels, which belong to the peripheral nervous system. Although these TH+ DRG neurons sense input from skin or visceral organs (Brumovsky, 2016), their functional details are never determined.</p><p>Recently, a transgenic rat line was generated by infusing a human TH promoter to upstream of an enhanced green fluorescent protein (eGFP) reporter gene. Theoretically, all cells that express TH should be tagged with GFP. The founder of the rat line has shown that the major DA-ergic neurons in the brain express GFP (Iacovitti et al., 2014). As it appears to be an ideal tool for genetic analysis and electrophysiologic study of TH+ neurons, we initially attempted to take advantage of this rat line to differentiate GFP-tagged TH+ neurons in the spinal cord and DRGs of naïve and SCI rats. Unexpectedly, a heterogeneous expression pattern of GFP transgene in the spinal cord was detected. This report provides important information about the transgenic line for researchers who will consider utilizing it in the future research.</p><!><p>Adult hTH-eGFP transgenic rats were purchased from Taconic Biosciences (NTac:SD-Tg (TH-eGFP) 24Xen) and then bred in-house. A total of 8 rats were used, including 3 naïve and 3 SCI female adults (weighing 250-300 g) and 2 postnatal day 10 (P10) pups. The guidelines of the National Institutes of Health and the Institutional Animal Care and Use Committee at Drexel University on animal care and housing were followed to minimize the number of animals used and potential suffering.</p><p>House-bred transgenic pups were identified by PCR to amplify a 264 bp product. The following primers were used: CAGCACGACTTCTTCAAGTCC and GATCTTGAAGTTCACCTTGATGC (Iacovitti et al., 2014). Subsequent electrophoresis was applied to visualize the presence of the eGFP gene (Fig. 1A). We previously observed that the number of TH+ neurons in the lower spinal cord was increased after SCI. To investigate whether the newly emerged cells express GFP after SCI, we performed a complete spinal cord transection at the 10th thoracic (T10) level (n = 3), as previously reported (Hou et al., 2016). Briefly, rats were anesthetized with 4% isoflurane in oxygen and kept anesthetic in 2% isoflurane during the surgery. Rats were placed on the heating pad with a supine position, a skin incision about 2 cm was made in the midline of the back. The paraspinal muscle was removed by scissors to facilitate structural recognition of the T8-10 spine. Then, a laminectomy was performed at T8 vertebrae to expose the dorsal part of the T10 spinal cord segment. The spinal cord was completely transected using a No. 11 blade and the lesion was confirmed visually at the time of surgery and after dissection. Once the bleeding stopped, the muscle was sutured and the skin was closed by wound clips. Animals were subcutaneously administered with cefazolin (10 mg/kg, West-ward), 3 ml Lactated Ringer's solution (VEDCO), and buprenorphine hydrochloride (0.1 mg/kg, Par). Bladder care was manually performed at least three times per day until sacrifice. Animals survived for an additional 3-4 weeks. In addition, 3 naïve adults and 2 P10 pups were also sacrificed. P10 rats were anesthetized by euthasol. A sharp 25G needle was inserted into the left ventricle. We first infused 100 ml 0.1 M phosphate-buffered saline (PBS) by a peristaltic pump, then switched the solution to 100 ml 4% paraformaldehyde (PFA) in PBS. After perfusion, the lumbosacral spinal cord was collected. Adult rats were euthanized by euthasol followed by transcardial perfusion with 200 ml 0.1 M PBS, followed by 200 ml 4% PFA solution. The brain, lumbosacral spinal cord, lumbar DRGs (L1. L2, L5, L6) were dissected. All tissues were post-fixed in 4% PFA overnight and cryoprotected in 30% sucrose in PBS for at least 2 days at 4°C. Tissue samples were embedded in optimum cutting temperature (OCT) compound (Sakura Finetek). Longitudinal spinal cord sections (35 μm) and coronal brain sections (35 μm) were obtained as six series using a cryostat (Microm Laborgerate) and kept in order in 24 well plates at 4°C. DRGs were serially cryosectioned in the longitudinal plane at 20 μm and mounted on slides for subsequent histological analysis.</p><p>For immunostaining, free-floating sections of brain (210 μm apart), lumbosacral spinal cord (210 μm apart) or mounted DRGs (120 μm apart) were washed 3 times in tris-buffered saline (TBS) and then incubated with primary antibodies in blocking buffer (TBS containing 5% donkey serum and 0.5% Triton X-100). Primary antibodies against TH (rabbit, 1:1000, Millipore) and GFP (chicken, 1:1000, Aves Labs) were used to observe the co-expression. DBH antibody (mouse, 1:500, Millipore) was used to identify noradrenergic profiles. Calcitonin gene-related peptide (CGRP, rabbit, 1:1000, Peninsula) and parvalbumin (PV, rabbit, 1:2,000, Abcam) were markers for peptidergic and proprioceptive DRG neurons, respectively. Double immunostaining for GFP/CGRP or GFP/PV was conducted to characterize GFP+ sensory neurons. Triple immunostaining for GFP/TH/DBH was used to detect supraspinal noradrenergic projections. Sections were incubated in primary antibody solution overnight at 4°C and then rinsed 3 times in TBS. Subsequently, sections were moved to secondary antibody solution containing Alexa-488, 594, or 647 conjugated donkey secondary antibodies (1:500, Invitrogen) in blocking buffer for 3 hours at room temperature. After washed, tissue sections were mounted on slides, air-dried, and coverslipped with mounting medium (SouthernBiotech). All slides were examined immediately and preserved in −20°C freezer. Imaging acquisition was processed on a Leica DM5500B microscope connect to a digital camera (Hamamatsu C11440-42U), and optimization of minimum and maximum display value was performed with SlideBook software (Intelligent Imaging Innovations). The far-red channel was pseudo-colored to blue. Adobe Photoshop 7.0 was used only to adjust contrast and construct image combinations.</p><!><p>In the adult intact spinal cord, immunohistochemical analysis revealed many TH+ but DBH− cells in the superficial dorsal horn, parasympathetic nuclei, and dorsal gray commissure (DGC) around the central canal at L6-S3 spinal levels, consistent with previous reports (Hou et al., 2016). However, none of these TH+ cells expressed transgene GFP (Fig. 1B-D). In both the white and gray matter, numerous supraspinal noradrenergic projections were double-labeled with TH and DBH, which were not co-labeled by GFP. Meanwhile, many TH and GFP double-labeled but DBH− neuronal fibers distributed in the entire spinal segments, mainly within the dorsal and medial parts of the cord (Fig. 1E). In addition, there were axon terminals expressing GFP only but not TH that was considered to originate from ectopic expression of the transgene. No GFP and DBH double-labeled cells or axons were detected in the spinal cord (Fig. 1F).</p><p>Based on the preliminary results, intraspinal TH+ population may change dynamically during different life stages or conditions, such as postnatal, adult, and after SCI (unpublished data). To determine the potential alteration of GFP transgene expression, immunostaining was employed in the spinal cord tissue collected from P10 rats as well as adult rats with SCI. Notably, TH+ neurons in the lower spinal cord did not express GFP in these two groups. In the spinal cord of P10 rats, dense TH+ fibers without GFP co-labeling were seen (Fig. 1G, H), and some axon extensions were only positive for GFP. In the spinal cord of adult rats with SCI, TH+ neurons in the parasympathetic nuclei (Fig. 1I, J) and GFP+ axons in the dorsal spinal cord (Fig.1K) were identified. No double-labeled profiles were detected.</p><p>Previous studies reported that DA-ergic but not noradrenergic neurons in the brain express GFP in this transgenic rat line (Iacovitti et al., 2014). In the intact spinal cord, we observed TH+/GFP+ but not TH+/GFP+/DBH+ axon extensions which supports the findings above. To confirm their origination, the brain sections were immunostained to recognize TH+ nuclei projecting to the spinal cord. The results showed that TH+ neurons in the A11 cell group expressed GFP while A6 (locus coeruleus) noradrenergic neurons did not (Fig. 2A-D). Interestingly, very few noradrenergic neurons in the A5 region express GFP (Fig. 2C, E). This validates that the major descending TH+/GFP+ neuronal pathways stem from the A11 DA-ergic neurons. Additionally, some TH− cells in the parabrachial nuclei adjacent to the A6 were labeled with GFP which could be caused by ectopic transgene expression (arrow, Fig. 2C).</p><p>It has been reported that plenty of TH+ sensory neurons are harbored in DRGs throughout the spinal cord level (Brumovsky, 2016). Immunostaining revealed single TH or GFP labeled sensory neurons in lumbar DRGs. However, these two categories did not have overlap (Fig. 2F). Further, GFP+ DRG neurons did not express CGRP and PV, suggesting that they are not nociceptive or proprioceptive neurons (Fig. 2G, H).</p><!><p>In the spinal cord of this hTH-eGFP transgenic rat line, the reporter is mainly expressed in the A11 diencephalospinal DA-ergic pathways, whereas other TH+ components do not produce GFP, such as intraspinal TH+ neurons, supraspinal noradrenergic projections, and afferent fibers of TH+ DRG neurons. The results are in compliance with previous studies in the brain that described DA-ergic but not noradrenergic neurons expressing GFP.</p><p>Among TH+ populations projecting to the spinal cord, GFP was exclusively expressed in the A11 cell group. After SCI interrupts all descending pathways, some GFP+/TH− terminals were still seen in the dorsal spinal cord. In line with detected GFP+/TH− neurons in DRGs, these GFP+ terminals should be the central projections of those DRG neurons. Accordingly, GFP+ axons in the intact spinal cord are comprised of descending A11 projections and afferent fibers of GFP+ DRG neurons. In research of supraspinal pathways, the utilization of neuronal tracing techniques to identify A11 projections often has problems of specificity since tracers injected into the nuclei are easy to diffuse into adjacent areas and label neurons there. It is therefore plausible that the transgenic rat provides a useful tool to visualize the descending A11 DA-ergic projections in the spinal cord by double immunostaining for TH and GFP.</p><p>Why is the expression of GFP transgene different in these distinct groups of TH+ neurons? The founder of the rat line attributed the failure of GFP expression in noradrenergic neurons to the instability of the reporter. If this is the case, it may also be true in intraspinal and sensory TH+ neurons. It is unclear whether TH has different genetic regulation between different categories of neurons because this factor could influence genetic recombination. Another possible reason is that some essential genetic elements may be missing in the hTH promotor plasmid so that only a certain type of TH+ cells, e.g., DA-ergic ones, can recognize for translation (Iacovitti et al., 2014). It should be mentioned that similar transgene expression pattern was shown in the brain when researchers infused the same plasmid into mice single cell embryos (Kessler et al., 2003), indicating no species difference. This made us wonder whether there are discrepancies of TH regulation between human and murine. Indeed, it has been found that humans and mice share merely 46.4% homology in TH promotor sequence, and the degree is even less between humans and rats that is about 30% (Jin et al., 2006). Among these three species, it was only identified 5 conserved regions in this 11kb human TH promotor, which even display functional differences. For example, Nurr1 is a transcription factor that is crucial for TH expression. A recent study unveiled that TH regulation does not depend on Nurr1 in humans but does in murine (Jin et al., 2006). Thus, the human TH promotor is genuinely different from that in rats and mice and this may explain the tissue-specific expression pattern of transgene.</p><p>Approximately 1% sensory neurons in lumbar DRGs are TH+ in rats (Price et al., 1983) and 15% in mice (Li et al., 2011). In the transgenic rat, DRG TH+ neurons were not tagged by GFP. Unexpectedly, we found a certain population of only GFP+ neurons. Two possibilities may explain this ectopic expression. Firstly, DRG neurons may have different repressive gene regulators from those in the brain since they belong to the peripheral nervous system. The binding sites for repressors may exceed the range of this 11kb hTH promotor. Another possibility is that high copies of transgene titrate out repressors in DRG neurons so that the transgene escapes from its specific regulators (Kessler et al., 2003). As a result, ectopic GFP expression occurs in a distinct sensory neuron population. These neurons do not express nociceptive marker CGRP or proprioceptive marker PV, and their identity remains to be determined.</p><!><p>A heterogeneous transgene expression pattern was revealed in the spinal cord of TH-eGFP rats. The reporter was mainly expressed in the A11 DA-ergic pathways whereas other TH+ components within the spinal cord were not tagged by GFP, such as intraspinal TH+ neurons, supraspinal noradrenergic projections, and afferent fibers of TH+ DRG neurons. This may suggest different genetic regulations among these TH+ elements even though they express the same product. Thus, it is pivotal to acknowledge this important information when researchers consider utilizing the transgenic line for spinal cord experiments.</p>

PubMed Author Manuscript

Controlled sequential assembly of metal-organic polyhedra into colloidal gels with high chemical complexity

Assembling many chemical components into a material in a controlled manner is one of the biggest challenges in chemistry. Particularly porous materials with multivariate character within their scaffolds are expected to demonstrate synergistic properties. In this study, we show a synthetic strategy to construct porous networks with multiple chemical components. By taking advantage of the hierarchical nature of a colloidal system based on metal-organic polyhedra (MOPs), we developed a two-step assembly process to form colloidal networks; assembling of MOPs with the organic linker to the formation of MOP network as a colloidal particle, followed 2 by further connecting colloids by additional crosslinkers, leading to colloidal networks. This synthetic process allows not only for the use of different organic linkers for connecting MOPs and colloidal particles, respectively, but for assembling different colloidal particles formed by various MOPs. The proof-of-concept of this tuneable multivariate colloidal gel system offers an alternative to developing functional porous soft materials with multifunction.

controlled_sequential_assembly_of_metal-organic_polyhedra_into_colloidal_gels_with_high_chemical_com

Introduction<!>Controlled synthesis of coordination polymer particle (CPP)<!>Investigation of colloidal gel formation from CPPs<!>Multivariate colloidal assembly<!>OHRhMOP<!>Conclusion<!>Supporting Information

<p>Increasing the chemical complexity by assembling different functional components is key for creating complex architectures with not only material properties beyond a single function but also tailorable characters. [1][2][3] Such complex systems have attracted great interests particularly for crystalline and ordered porous solids such as metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), in which more than two functional moieties (e.g., ligands with different functional groups or various metal clusters) can be integrated within the same structure. [4][5][6] The chemical complexity, together with the decorated pore surface of these multivariate porous frameworks, showed significant benefits in developing functional materials for catalysis [7][8][9][10] , sensing [11] and separation applications [12,13] . Yet, the lack of predictable arrangement of mixed compositions in the structure makes it difficult to control the positioning of the specific molecular components in the framework scaffold. [14,15] The multivariate character of these materials is not limited to a single motif at the molecular level but can be integrated at longer length scales in order for the chemical complexity to emerge, for instance, via epitaxial growth of two distinct MOF [16,17] or the control of the size and shape of MOF particles resulting to different layout of mesostructures (colloidal MOFs) and material behaviors (e.g., photonic or sensing property). [18][19][20] Nevertheless, in the first case, it remains challenging to increase the chemical complexity by integrating various types of material due to the requirement for matching the lattice parameters at the crystal interface, while for the second one, the mesostructures of these types of assembled colloidal MOFs are usually not stable due to weak interactions, and it is still challenging to assemble several distinct nanocrystals into a single mesostructure.</p><p>To tackle these issues, one can consider using a colloidal gel system, in which chemically different mesoscale colloidal particles can be interconnected [21] through electrostatic interaction or chemical bonding [22][23][24][25] to form multicomponent colloidal networks. Interestingly, depending on the interaction between particles, the assembled structures of two chemically distinct colloids can either (i) be self-sorted into a network and form double interpenetrated colloidal networks, or (ii) lead to the preferential interaction between two different colloids (for instance, electrostatic interaction) yielding to the formation of a mixed colloidal network. [26,27] In recent years, a number of MOF particle-based gels have been reported, proving the possibility of creating new types of multicomponent colloidal gels with versatile functionalities. [28][29][30][31] Metal-organic polyhedra (MOPs) with discrete cage structures present a good alternative to MOFs due to their processability in solution and lability. When used as secondary building blocks, MOPs can be assembled with additional linkers into colloidal particles or colloidal gel networks. [32][33][34][35][36][37][38] However, all of these systems have been assembled from a single MOP component. Therefore, integration of more than two distinct components during the self-assembly process remains a major challenge due to the lack of methodology.</p><p>Here, we show a coordination chemistry approach to form multicomponent hierarchical structures from interconnected MOPs. Our methodology takes advantage of available coordination sites exposed on the surface of the MOPs, where first a ditopic N-donor ligand leads to the formation of MOP networks as colloidal particles, which can be further crosslinked into a colloidal gel network by the addition of a secondary N-donor ligand. The transfer of MOPs to complex colloidal gel networks using this two-steps assembly strategy will demonstrate the feasibility to install selected functional moieties at the scales of the colloidal particles or colloidal gel networks (Figure 1). This will allow the precise engineering of the supramolecular structure and advance the multi-functionalities in a controlled manner.</p><!><p>In this study, we chose the stable rhodium-based MOP, [Rh2(bdc)2]12 (HRhMOP), assembled from 12 dirhodium acetate paddlewheels and 24 benzene-1,3-dicarboxylate (bdc) with cuboctahedral geometry for the formation of colloidal particles. [38] By taking advantage of the reactivity of the external axial site of the rhodium paddlewheel, we previously demonstrated the polymerization of MOPs with bisimidazole derivatives such as 1,4-bis[(1H-imidazol-1yl)methyl]benzene (bix) as linkers. On the one hand, the gradual addition of the ditopic Ndonor ligand bix to the MOP solution resulted in the formation of colloidal spheres (named as coordination polymer particles, CPPs). On the other hand, the generation of kinetically-trapped MOP as RhMOP(bix)12 by adding an excess amount of bix to the RhMOP solution produced a colloidal gel network. Note that the latter strategy leads to the spontaneous formation of a colloidal gel network once the MOP and bix are mixed, which does not allow for further control of the assembly process, including its termination at the stage of colloidal formation. In order to achieve a multicomponent colloidal structure, one needs to use colloidal particles as initial building blocks. Here, we first optimized the reaction condition for the CPP formation based on HRhMOP with different types of ditopic N-donor ligands and various stoichiometric ratios.</p><p>Then, we optimized the condition to connect these CPP with another bisimidazole crosslinker to form the colloidal gel network. Finally, two chemically distinct but isostructural CPPs (one with HRhMOP and the other with BrRhMOP) were mixed to yield multicomponent colloidal gel networks (BrRhMOP, [Rh2(5-Br-bdc)2]12, 5-Br-bdc = 5-bromobenzene-1,3dicarboxylate).</p><p>A series of ditopic N-donor bisimidazole ligands, bix, 4,4'-bis(1H-imidazol-1ylmethyl)biphenyl (bibph), and 1,12-di(1H-imidazol-1-yl)dodecane (bidod) were selected as bridging linkers to sequentially connect MOPs or CPPs. In order to process HRhMOP in solution, the cage was solubilized according to a reported coordination solubilizer method, which requires the coordination of monodentate N-dodecylimidazole (diz) to all of the 12 axial sites of Rh paddlewheels exposed on the surface of the molecule to yield soluble HRhMOP(diz)12. [37] Formation of CPPs was then investigated via sequential addition of 0.5 molar equivalents per step of three distinct bisimidazole linkers, bix, bibph, or bidod, into the DMF solution of HRhMOP(diz)12. [37] Replacement of diz by bisimidazole linkers (L) initiates the linkage between HRhMOP(diz)x(L)y cages and leads to a MOP network in the form of CPPs.</p><p>The use of bix as a linker provided dispersible CPPs, while the bibph and bidod generated undesired agglomerates of CPPs, which make it difficult to be further processed (Figure S5). Therefore, a series of CPPs made with bix as linkers were selected for further investigation.</p><p>In the MOP network linked by bix, the HRhMOP acts as a node linked up, in principle, to 12 other MOPs. The effective connectivity of MOP can be estimated from the chemical composition of the CPP determined by proton nuclear magnetic resonance measurement ( 1 H NMR, Figure S8) and represented by the branch functionality, f, a parameter that indicates the number of rhodium sites per MOP that are used to connect with neighboring MOPs (see Supplementary Information). [37,42,43] In order to correlate the added bix quantity with the branch functionality, the corresponding CPPs were synthesized with different molar equivalents of bix linker (3, 4, 6 and 12 molar equivalents) added. The calculated f increases up to 8 by the addition of 6 molar equivalents of bix but decreases by the addition of an excess amount of bix, which is most likely due to the coordination of bix as a mono-dentate fashion (Table 1). As further addition of bridging ligands is required to allow connections between CPPs to form a colloidal gel network, the obtained CPPs should have enough accessible coordinative sites at the exohedral HRhMOP. Therefore, the CPPs formed with 3 molar equivalent addition of bix and having a relatively low f value were selected for further studies. The size of CPP is expected to affect the macroporosity and connectivity of the corresponding colloidal gel networks. As reported previously, the CPP formation process can be tuned by the molar equivalent of bix added at each step, in which the first addition would generate nuclei and the following addition would contribute to particle growth. [36] Hence, the effects of different concentrations of HRhMOP(diz)12 and stepwise amount of bix added on the resulting CPP size were investigated by dynamic light scattering (DLS) titration experiments (Figure 2). When the addition of bix changed from 0.5 molar equivalents to 3.0 molar equivalents for each step, the size of final CPPs was decreased from 98 ± 14 nm to 55 ± 10 nm as also confirmed by scanning electron microscopy (SEM) images (Figure 3). This size reduction is attributed to the larger numbers of nuclei formed, yielding to smaller CPPs. In addition, the increase in the concentration of HRhMOP(diz)12 from 0.93 mM to 1.86 mM raises the collision probability of particles, leading to a larger size of CPP (189 ± 30 nm). In contrast, lowering the concentration to 0.46 mM led to a smaller CPP particle size (62 ± 10 nm).</p><!><p>In order to demonstrate the possibility of forming colloidal gels with CPPs, two types of CPPs with different sizes were produced by different addition rates of bix (CPP-large: 98 ± 14 nm and CPP-small: 55 ± 10 nm) were selected for further investigations. We firstly attempted the gelation with various concentrations of CPP-large (4 mg/mL, 8.5 mg/mL, 17 mg /mL, and 42 mg/mL in DMF) by adding 12 molar equivalents bix and reacting at 80 ºC for 2 h. Note that the molar equivalence of added bix here was calculated as a relative molar ratio to HRhMOP in the CPP, which was estimated from the weight and 1 H NMR spectra. Only the high concentration of CPP-large (42 mg/mL) formed corresponding gel, but the CPP with lower concentrations gave suspensions (Figure S10).</p><p>To gain more insight into the gelation mechanism, the gelation process of 42 mg/mL CPP-large and CPP-small, after the addition of 12 molar equivalents of bix, were monitored by time-resolved dynamic light scattering (TRDLS) measurements at 50 ºC. [36][37]44] The timeaveraged scattering light intensity ⟨𝐼⟩𝑇 was found to be almost constant at the early stage of the reaction before fluctuating at 38 min for CPP-large (Figure 4a). This random fluctuation is a clear indication of the sol to gel transition due to the loss of ergodicity during the percolation of colloidal particles. Insight into the gelation mechanism can be extrapolated from the timeevolution of the intensity correlation functions (ICFs), g (2) (τ)-1, which is a function measuring the similarity between scattering light signal at different delay times (Figure 4b). Indeed, ICFs were fitted by a single stretched exponential function and give access to the following parameters:</p><p>where σI 2 is the initial amplitude of the ICF, τf is the relaxation time related to the diffusion rate of particles, and β is the stretched exponent that reflects the distribution of τf . τf is further related to the correlation length ξ, which reflects the evolution of the density of the network during the reaction time in the solution phase, through Equation 2:</p><p>where q, kB, T, η are the scattering vector, the Boltzmann constant, the absolute temperature, and the viscosity of media, respectively (see the Supplementary Information for the derivation of Equation 2). In the first 10 min of the reaction, τf and the corresponding correlation length ξ were almost constant, indicating that the CPPs were individually diffusing and not aggregating with each other (Figures 4c and S13h). This was also proven by the values of β close to unity (≈ 0.9), which reflects the relatively narrow size distribution of the CPP as observed by the SEM measurements. After this induction period, τf increased while β decreased toward the gelation point. This simultaneous change of τf and β indicates that the average particle size and size distribution are no anymore uniform, which is most likely explained by the stochastic aggregation of colloidal particles and the formation of a gel network. This agrees with the drastic increase of the correlation length ξ, (Figure S13h). The TRDLS experiment on CPPsmall also showed a similar trend to the CPP-large as shown in Figure S13. Differences in gelation time as a function of temperatures were also observed by TRDLS measurements. At 50 ºC, the sol-gel transition for CPP-small occurs at 107 min after the addition of bix. In contrast, the gel was formed in 19 min for the same system heated at 80 ºC (Figure S12). This is most likely caused by the increase in the diffusion rate of particles and the kinetics of ligand exchange reaction accompanying the colloidal aggregation when increasing the temperature. In addition, the gelation time is dependent on the CPP size as seen from the gelation thresholds of 107 min and 38 min measured for CPP-small and CPP-large, respectively at 50 ºC (Figure S13). This can be explained by the difference of van der Waals interaction between colloidal particles, which is estimated to be proportional to the particle diameter from Hamaker's theory. [45] These examples illustrate the possibility of controlling gelation time by changing reaction temperature and CPP size, while retaining the chemical composition of the resulting colloidal gel network. Moreover, the reduction of the number of bix added from 12 to 6 eq. led to an increase in the gelation time (Figure S14), which is most likely attributed to the reduced coordinative interactions between CPPs that hindered the gelation process. For the purpose of accumulating more data points during the gelation process, the gelation reaction at 50 ºC is used for the following investigation. To further confirm that the CPP gelation is induced by the second addition bix, a control experiment was performed using TRDLS to follow the reaction of CPP-large without the addition of bix (Figure S14). CPPlarge suspension showed almost constant τf and β during the 6 h of heating at 50 ºC. This indicates that CPP is stable itself in DMF and gelation is indeed driven by crosslinking of CPPs mediated by bix molecules.</p><p>This successful assembly via two-step addition of bix to form CPP-based gel envisaged incorporating different crosslinkers (bibph and bidod) in a second step. However, only the addition of bidod to more than 42 mg/mL CPP-large was able to form a gel under the synthetic condition of 80 ºC for 2 h. In contrast, the addition of bibph gave a suspension after the 2 h reaction at 80 ºC (Figure S15). We attributed this difference in gelation to the variation in crosslinking reactivity of the linkers as: bidod > bix > bibph, which was proven by the difference of gelation time observed by TRDLS (Figure S16). Therefore, to investigate the multivariate colloidal gel, the system with CPP-large as cores and bidod as secondary linkers was selected and its gelation condition was optimized to obtain more mechanically durable colloidal gel. It is expected that the strength of the gels is predominantly affected by the addition of bidod due to the cross-linkage, interaction between CPPs, and the chemical environment. To clarify the effect of crosslinker amount on the strength of the gels, a series of CPP-large/bidod gels with addition of 3, 6, 9, 12 molar equivalents bidod have been synthesized (Figure S18) from which the compositions were determined by 1 H NMR spectroscopy (Figure S19). The average number of diz, bix, and bidod per HRhMOP was estimated using the average integration of selected representative signals (Figure 5a). Interestingly, the relative number of bix molecules to HRhMOP remained almost the same compared to the original CPP itself regardless of the quantity of bidod added. In contrast, the relative number of diz linearly decreased and that of bidod linearly increased as a function of the amount of bidod added. This clearly indicates that bix and HRhMOP molecules are forming a stable coordination network inside CPPs and newly added bidod can only replace diz molecules in CPP. Unlike the CPP formation, branch functionality f is no longer suitable to reflect the connectivity between CPPs since bidod can crosslink MOPs within the same CPP as well as with the neighboring CPPs.</p><p>Therefore, the relation between the quantity of bidod addition and the stiffness of the gels was further investigated by rheology measurements (Figures 5b and S20). The CPP-large/bidod gels were prepared in situ in the rheometer by heating the mixture of CPP-large suspension and 6 or 12 equivalents bidod at 80 ºC for 20 min, followed by determination of the storage (G') and loss (G'') modulus at 25 ºC. The G' was always higher than G'' and they were almost constant independently of the shearing frequency and strain, which demonstrate their linear elastic property. It was found that the G' and G'' of CPP-large/12 eq. bidod gel are greater than those of the CPP-large/6 eq. bidod gel, which agrees with the increased number of bidod present in the gel network. This result further confirms that the addition of bidod enhances the crosslinking between CPPs and forms a more robust gel. Furthermore, the successful formation of gels from CPP-small with either bix or bidod was confirmed (Figures 6 and S13), which illustrates the possibility of altering the network structures.</p><!><p>In order to demonstrate the versatility of material composition, isostructural MOPs, with a substitution at 5 th position of benzene-1,3-dicarboxylate with hydrophilic functionality (-OH), To demonstrate the possibility to create a multivariate colloidal gel network formed from the mixing of CPP-Br with 'pristine' CPP based on HRhMOP, the presence of Br component in the colloidal aerogel network was mapped via energy dispersive X-ray analysis (EDX) in transmission electron microscope (TEM) due to its electron-rich property (Figure 7).</p><!><p>The EDX image in Figure 7a shows a homogeneous distribution of Br (green) and Rh (red) atoms in the system, which is also confirmed by a 1:1 ratio of Br and Rh from the elemental composition analysis (Figure S22c). Then the possibility to increase the complexity of this</p><!><p>The two-step hierarchical construction strategy developed here has allowed us to fine-tune the properties of the colloidal gel network via the control of the CPP size, gel composition (CPP core and its connectivity of gel network) and crosslinking degree as demonstrated by the combination of TRDLS, 1 H NMR and rheology techniques. In addition, the possibility to i) have control over the colloidal size without altering the core components, ii) modify the mesoscale connectivity with different crosslinkers and iii) install functional moieties via alteration of RhMOPs and CPPs open up a new alternative to develop multivariate colloidal gel via hierarchical assembly to form multi-functional materials for potential applications such as sensing and separation.</p><!><p>Supporting Information is available from the Wiley Online Library or from the author.</p>

ChemRxiv

Cd-driven surface reconstruction and photodynamics in gold nanoclusters

With atomically precise gold nanoclusters acting as a starting unit, substituting one or more gold atoms of the nanocluster with other metals has become an effective strategy to create metal synergy for improving catalytic performances and other properties. However, so far detailed insight into how to design the goldbased nanoclusters to optimize the synergy is still lacking, as atomic-level exchange between the surfacegold (or core-gold) and the incoming heteroatoms is quite challenging without changing other parts. Here we report a Cd-driven reconstruction of Au 44 (DMBT) 28 (DMBT ¼ 3,5-dimethylbenzenethiol), in which four Au 2 (DMBT) 3 staples are precisely replaced by two Au 5 Cd 2 (DMBT) 12 staples to form Au 38 Cd 4 (DMBT) 30 with the face-centered cubic inner core retained. With the dual modifications of the surface and electronic structure, the Au 38 Cd 4 (DMBT) 30 nanocluster exhibits distinct excitonic behaviors and superior photocatalytic performances compared to the parent Au 44 (DMBT) 28 nanocluster.

cd-driven_surface_reconstruction_and_photodynamics_in_gold_nanoclusters

Introduction<!>Results and discussion<!>Conclusions<!>Conflicts of interest

<p>Metal synergy is of paramount importance as the rationale to modulate the intrinsic properties of metal nanoparticles. 1,2 However, the precise synergistic interaction in an intermetallic nanoparticle has so far been elusive, due to the challenges in determining the atomic-level arrangement of the metal heteroatoms in the nanoparticle. Atomically precise metal nanoparticles (oen called nanoclusters) lead to unprecedented opportunities in signalling clear directions to exploit the cooperativity between the two metal elements within a single nanocluster. 3 Thiolate-protected gold nanoclusters, Au n (SR) m , where n is the number of gold atoms and m is the number of thiolate ligands, SR, have gained momentum over the past few years as an exciting area and have opened up new horizons in precise tailoring of the composition and structure to control the physicochemical properties. [4][5][6][7][8][9][10][11][12] The Au n (SR) m nanoclusters are typically congured with an inner gold core (or kernel) and various surface motifs, in which the motifs containing both gold and thiolate resemble staples. Both the gold core and the surface motifs can contribute to the physicochemical properties such as the optical and electronic properties, as well as catalysis. [13][14][15][16][17][18] It has been recognized that substituting one or more gold atoms in either the core or the motifs with other metals can tune the overall performances of the parent nanoclusters. [19][20][21][22][23][24][25][26] Therefore, it has become possible to access the previously inaccessible metal synergy in the bimetallic nanoclusters with atomic-precision.</p><p>Among the gold-based bimetal nanoclusters, cadmiumcontaining bimetal clusters provide synergistic strategies to adjust the electronic structures and further modulate the physicochemical properties in the clusters, since Cd has one more valence electron than Au. 21,26,27 Cd introduction usually causes surface reconstruction of gold nanoclusters. For example, Au 19 Cd 2 (SR) 16 was obtained through the substitution of two neighboring surface Au atoms with one Cd with the cuboctahedral Au 13 unchanged. 26 Au 19 Cd 3 (SR) 18 was formed by retaining the icosahedral Au 13 core but only changing the surface of Au 25 (SR) 18 . 27 However, the surface reconstruction strategy remains challenging and no examples of bimetal clusters formed without breaking the face-centered cubic (fcc) core of the parent gold clusters have been documented, which might thus impede gaining a higher understanding of how to tailor the surface structure of gold-based nanoclusters and accordingly optimize their synergy.</p><p>Herein, we report our success in synthesizing a Au 38 Cd 4 (-DMBT) 30</p><!><p>X-ray crystallography analysis shows that the parent Au 44 (-DMBT) 28 nanocluster is composed of an Au 26 kernel, six Au 2 (SR) 3 and two Au(SR) 2 staples (Fig. 1a, c and Table S1 †). The formula of Au 44 (DMBT) 28 is further conrmed by electrospray ionization mass spectroscopy (ESI-MS, Fig. S1a †). The structural framework of Au 44 (DMBT) 28 is identical to that of the reported Au 44 (TBBT) 28 (TBBT ¼ 4-tert-butylbenzenethiol) (Fig. S2 †), 28 both of which can be assembled into the layered structures (Fig. S3-S5 †). Notably, a signicant difference is observed in the layer's interior, where all the molecules of Au 44 (TBBT) 28 in the layer (marked with the same color, Fig. S3 †) are packed along the same direction, while Au 44 (DMBT) 28 molecules are arranged in different directions (Fig. S5 †). Such a difference may be ascribed to the different steric hindrance between TBBT and DMBT. The UV-vis-NIR spectra of the two Au 44 (SR) 28 nanoclusters show only small deviations. As shown in Fig. S6, † the prominent peak at 380 nm for Au 44 (TBBT) 28 is slightly red-shied to 388 nm for Au 44 (DMBT) 28 , and the broad peaks at 650 and 725 nm become apparent when TBBT is replaced by DMBT.</p><p>With Au 44 (DMBT) 28 as a starting unit, a Cd-doped nanocluster was further synthesized via an ion-exchange strategy. From ESI-MS data (Fig. S1b †), the prominent peak at 6025.43 m/ z with a +2 charge is assigned to Au 38 Cd 4 (DMBT) 30 (theoretical value: 6025.48 m/z), which is further conrmed by the excellent match between experimental and calculated isotopic patterns (inset of Fig. S1b †). Single crystallography analysis reveals that Au 38 Cd 4 (DMBT) 30 contains a 26-Au-atom kernel, two Au 5 Cd 2 (-SR) 12 staples, two Au(SR) 2 staples and two bridging SR ligands, as shown in Fig. 1b, d, and Table S2. † Note that the retained kernel of Au 38 Cd 4 (DMBT) 30 experiences a slight distortion from "slender" to "stocky" in comparison with that of the parent Au 44 (DMBT) 28 (Fig. 1e-h). Further analysis shows that the Au 26 kernel in Au 38 Cd 4 (DMBT) 30 can be viewed as the assembly of tetrahedral Au 4 units in a double-helical mode, as well as that in Au 44 (DMBT) 28 (Fig. 2). Furthermore, the two nanoclusters have almost identical distances between neighboring Au 4 units, which is clearly manifested in the similar Au-Au bond lengths according to the different positions of the Au atoms (Fig. S7 †). Therefore, Au 38 Cd 4 (DMBT) 30 can be viewed as the gentle surface reconstruction without breaking the double-helical Au 26 kernel based on the parent Au 44 (DMBT) 28 . In addition, Au 38 Cd 4 (-DMBT) 30 is also patterned along different directions in the layer structure (Fig. S8 †).</p><p>To gain an in-depth insight into the Cd-induced surface reconstruction mechanism, density functional theory (DFT) calculations were performed. Starting from the Au 44 (SR) 28 cluster, as presented in Fig. S9, † To investigate the electronic structure changes induced by Cd-atom surface modication, the optical adsorption spectra of the Au 44 (DMBT) 28 and Au 38 Cd 4 (DMBT) 30 nanoclusters were measured. The absorption peaks of Au 38 Cd 4 (DMBT) 30 are mainly centered at 400, 465, 550 and 678 nm (Fig. 3b), which differ from those observed in the parent nanocluster (387, 452, 635 and 725 nm; Fig. 3a). These optical features can be well reproduced by theoretical calculations (Fig. 3a, b and S10 †). The Kohn-Sham (KS) molecular orbital (MO) energy levels and atomic orbital components in each KS MO of Au 44 (SR) 28 and Au 38 Cd 4 (SR) 30 suggest that the absorption peaks mainly involve the Au(sp) / Au(sp) transitions (Fig. 3c and d). In particular, for Au 44 (SR) 28 , the rst absorption peak centered at 734 nm originates from the highest occupied molecular orbital / the lowest unoccupied molecular orbital (HOMO / LUMO) transition, while for Au 38 Cd 4 (SR) 30 , the rst absorption peak centered at 695 nm originates from the HOMO / LUMO, HOMO / LUMO+1, HOMO / LUMO+4, HOMOÀ1 / LUMO, HOMOÀ1 / LUMO+1, and HOMOÀ1 / LUMO+5 transitions. The more complex orbital transitions in Au 38 Cd 4 (SR) 30 than in Au 44 (SR) 28 can be attributed to the dopant Cd. This behaviour can also be observed for other absorption peaks.</p><p>Moreover, femtosecond and nanosecond carrier dynamics of the two nanoclusters were measured via time-resolved transient absorption (TA) spectroscopy to decipher their potential energyrelated applications. The femtosecond-resolved TA spectra of the Au 44 (DMBT) 28 and Au 38 Cd 4 (DMBT) 30 nanoclusters are provided in Fig. 4a and b. Similarly, both Au 44 (DMBT) 28 and Au 38 Cd 4 (DMBT) 30 nanoclusters showed broad excited state absorption (ESA) signals overlapped with ground state bleaching (GSB) peaks near 675 nm. We selectively extracted the TA spectra at different delay times, combined with the dynamic traces probed at 515 and 675 nm to study the transient evolution and the relaxation dynamics (Fig. 4c-f). A 0.6 ps process at the early stage, which is attributed to the ultrafast internal conversion from higher excited states to lower excited states, 29 was observed in the two nanoclusters (Fig. S11 and Table S3 †). It is worth noting that the major divergence between the two nanocluster systems emerged aer a delay of 2 ps. For Au 44 (-DMBT) 28 , the TA spectra remained nearly unchanged aer 2 ps (Fig. 4c), which is consistent with the at decay kinetic traces shown in Fig. 4e. A 19 ps process obtained by exponential tting was ascribed to the structural relaxation caused by conformational changes aer pumping. [29][30][31] For Au 38 Cd 4 (DMBT) 30 , interestingly, an obvious spectral transformation was observed and the lifetime of this component was determined to be 57 ps (Table S3 †), which differs from the 19 ps structural relaxation observed in Au 44 (DMBT) 28 and might be related to the charge transfer states between the ligand and the metal core of Au 38 -Cd 4 (DMBT) 30 , [32][33][34][35] The distinguishable electronic and optical properties of the two nanoclusters would apparently impact their catalytic properties. Thus, visible light-driven degradation of methyl orange was selected to explore the photocatalysis of the two nanoclusters. From Fig. 5a and b, within 50 min, methyl orange can be completely degraded on the Au 38 Cd 4 (DMBT) 30 catalyst under visible light illumination, while on the Au 44 (DMBT) 28 catalyst it was completed in 70 min. The plots of methyl orange degradation on the catalysts versus reaction time further indicate the better photocatalytic performance of the Au 38 Cd 4 (-DMBT) 30 catalyst (Fig. 5c). Electrochemical impedance spectroscopy was performed to investigate the interfacial transfer of electrons. In Fig. 5d, the semicircular diameter of Au 38 Cd 4 (DMBT) 30 was smaller than that of Au 44 (DMBT) 28 , which implies faster electron-transfer in the Au 38 Cd 4 (DMBT) 30 system. The photocatalysis difference in the two cluster catalysts is suggested to arise from their different equilibria established between the carrier recombination and the electron transfer inuenced by metal synergy.</p><!><p>In summary, we have developed a Cd-driven surface reconstruction strategy for synthesizing a new Au 38 Cd 4 (DMBT) 30 bimetallic nanocluster with the fcc Au 26 core retained from the parent Au 44 (DMBT) 28 nanocluster. The two nanoclusters that exhibit elegant patterns of Au 4 tetrahedra show distinct differences in the electronic structures, optical properties, and photocatalytic performances. Beyond the Cd-mediated surface reconstruction case, we anticipate that this heteroatom-doping mechanism will nd applications in using gold and other metals in a series of challenging gold-based nanocluster formations and tuning of their intrinsic properties.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Purification of indium by solvent extraction with undiluted ionic liquids

A sustainable solvent extraction process for purification of indium has been developed from a chloride aqueous feed solution using the ionic liquids Cyphos® IL 101 and Aliquat® 336. The high affinity of indium(III) for the ionic liquid phase gave extraction percentages above 95% over the HCl concentration range from 0.5 to 12 M. Attention was paid to the loading capacity of the ionic liquid phase and the kinetics of the extraction process. An extraction mechanism was proposed based on the relationship between the viscosity of the ionic liquid phase and the loading with indium(III) ions. Even for loadings as high as 100 g L −1 , equilibrium was reached within 10 min. Due to the very high distribution ratio for indium(III), stripping of indium(III) from the ionic liquid phase was very difficult with water or acid solutions. However, indium could conveniently be recovered as In(OH) 3 by precipitation stripping with a NaOH solution. Precipitation stripping has the advantage that no ionic liquid components are lost to the aqueous phase and that the ionic liquid is regenerated for direct re-use. The extraction of some metal ions that are commonly found as impurities in industrial indium process solutions, i.e. cadmium(II), copper(II), iron(III), manganese(II), nickel(II), tin(IV) and zinc(II), has been investigated. The distribution ratios for the different metal ions show that indium(III) can be purified efficiently by a combination of extraction, scrubbing and stripping stages. This new ionic liquid process avoids the use of volatile organic solvents.

purification_of_indium_by_solvent_extraction_with_undiluted_ionic_liquids

Introduction<!>Chemicals<!>Instrumentation and analysis methods<!>Extraction experiments<!>Mono-element system<!>Multi-element system<!>Distribution ratio and separation factor<!>Mono-element system<!>Multi-element system<!>Conclusions

<p>Indium is a scarce metal with a 0.1 ppm abundance in the earth's crust comparable to that of silver. 1,2 It is labeled as a critical raw material by the European Commission, due to its high supply risk. 3,4 This risk is due to two supply and demand factors, namely, the increasing demand for applications in photovoltaics, flat screen TVs, laptops and mobile phones as well as the Chinese production monopoly. In 2008, China had a 58% share in the primary global indium production. 5 The two factors are further compounded by low substitutability and low recycling rates, resulting in higher prices and the need to raise production. 3,4 Indium is derived as a by-product from ores and is most commonly found in association with zinc ores such as sphalerite, although it also occurs in lead, copper, iron and tin ores. [1][2][3] Indium is accumulated in low concentrations in residues formed during the processing of these ores. Therefore, indium is often regarded as an impurity that increases the production costs. 5 Furthermore, indium recycling is mainly limited to production scrap. To keep up with the rising demand for high-tech applications, the development of industrial processes for the successful recovery of indium from various primary and secondary sources is crucial. Possible sources of indium are by-products of zinc refining, flue dusts, slags and metallic intermediates, e-waste and impure indium (<99%). 1,2 Hydrometallurgical separation methods are very suitable for extracting indium from these sources. They compromise an essential part of extractive metallurgy utilized for treating complex and low-grade materials. Typically, hydrometallurgical solutions are generated by leaching the metals present in concentrates with strong acids or bases. Metals are already separated partially or completely in the leaching step, if one or more of the metals does not solubilize. Further concentration or separation generally takes place via solvent extraction or ion exchange. Once the metals have been separated, pure metals or metallic compounds can be produced by, for example, precipitation, cementation or electrolysis.</p><p>Solvent extraction (SX) has been widely used as a process for separation, purification and recovery of metals, due to its simplicity of equipment and operation. Solvent extraction is based on selective and efficient transfer of the desired metal species from one phase to another, usually from an aqueous to an organic phase. 6,7 The aqueous phase, in which the metals are present, is brought into contact with a water-immiscible organic phase, consisting of one or more extractants, a diluent and sometimes a modifier. The separation is based on differences in the solubility of the metal ions in both the organic and aqueous phases due to the variations in the strength of the chemical interaction between the metal ion and the extractant. The efficiency of the extraction process depends on several parameters such as the pH, the temperature, the concentration of the metal ions in the aqueous feed and the concentration of the extractant in the organic phase.</p><p>The traditional solvent extraction process for indium makes use of water-immiscible organic solvents, many of which are flammable, volatile or toxic. 8 Ionic liquids are good alternatives as an extraction medium for the development of sustainable separation processes. [9][10][11] Ionic liquids are solvents consisting completely of ions, mostly an organic cation and an inorganic anion. 12,13 The physicochemical properties of ionic liquids can be tuned adequately for a given application. 14 Therefore, it is not surprising that water-immiscible ionic liquids have already been investigated as extractants for metals from aqueous solutions. 11,[15][16][17][18][19][20][21] These ionic liquids often contain fluorinated anions, such as the hexafluorophosphate (PF 6 − ) or the bis(trifluoromethylsulfonyl)imide (Tf 2 N − ) anion. 22,23 Besides their high prices, in some cases, these type of ionic liquids pose a severe risk due to hydrolysis and formation of hydrofluoric acid. 24 Therefore it is better from an environmental and economical point of view to use ionic liquids with long alkyl chains instead of fluorinated anions. 22 Several non-fluorinated hydrophobic ionic liquids have already been used for the extraction of metal ions but generally not in a pure state. These ionic liquids such as Aliquat® 336 [25][26][27] and tri(hexyl)tetradecylphosphonium chloride (Cyphos® IL 101) [27][28][29][30][31][32] are usually diluted in molecular solvents prior to use. Diluents such as toluene, kerosene or chloroform are added to decrease the viscosity of the organic phase leading to an increase in mass transfer and faster kinetics. However, recently in some cases, the problem of viscosity was overcome by saturating the ionic liquid with water, by working at slightly elevated temperatures and/or by using intermediate metal feed concentrations. [33][34][35][36] Major advantages of ionic liquids for application in solvent extraction processes, in comparison with traditionally used water-immiscible organic solvents, are their negligible vapor pressure and low flammability. [12][13][14] Due to their low volatility, they can be considered as more environmental-friendly and safer alternatives to organic solvents. While the use of ionic liquids for metal extraction does offer many advantages, there are some disadvantages as well. First of all, not all types of ionic liquids can be used for extraction. Water-immiscible ionic liquids are required for solvent extraction. Secondly, extraction often takes place through an ion-exchange mechanism which leads to losses of ionic liquid components to the aqueous phase. [37][38][39][40] Ionic liquids are also quite expensive, so that their use for conventional solvent extraction processes cannot be justified solely from an economical point of view. Furthermore, their generally high viscosity often leads to slow mass transfer so that long contact times are required to reach equilibrium. 33,34 In this paper, an efficient solvent extraction process for the purification of indium from a chloride medium was developed based on the use of quaternary phosphonium and ammonium ionic liquids. The feasibility of the quaternary phosphonium ionic liquid trihexyl(tetradecyl)phosphonium chloride (Cyphos® IL 101) and the quaternary ammonium ionic liquid Aliquat® 336 to serve as a possible undiluted organic phase/ extractant for the extraction of indium is investigated. Special attention was paid to the difficult stripping of indium from the ionic liquid phase after extraction. Is it shown that precipitation stripping of In(OH) 3 with NaOH is a convenient sustainable method for recovery of indium and for regeneration of the ionic liquid.</p><!><p>Trihexyl(tetradecyl)phosphonium chloride (Cyphos® IL 101, purity >97%) was purchased from Cytec Industries Inc. (Niagara Falls, Ontario, Canada). Aliquat® 336 (mixture of quaternary ammonium chlorides, with 88.2-90.6% quaternary ammonium content), InCl 3 •4H 2 O (97%), MnCl 2 (98%) and phosphor standard (1000 ppm) were obtained from Fluka (Sigma-Aldrich, Diegem, Belgium). CdCl 2 (99%), CuCl 2 (99%), NiCl 2 (98%), PbCl 2 (99%), SnCl 4 (99.99%), Na 2 EDTA•2H 2 O (99+ %), Na 2 SO 4 (99%, extra pure) and ethanol (99.8+%, absolute) were purchased from Acros Organics (Thermo Fisher Scientific, Geel, Belgium). As 2 O 3 (99.5%) was obtained from Alfa Aesar (Karlsruhe, Germany), FeCl 3 (98.5%) from Carl Roth (Karlsruhe, Germany) and ZnCl 2 (98-100.5%), CaCl 2 •2H 2 O (99.5+%) and lanthanum standard (1000 ppm) from Chem-Lab (Zedelgem, Belgium). NaOH pellets (>99% AnalaR Norma-pur®) were purchased from VWR (Leuven, Belgium). The silicone solution in isopropanol was obtained from SERVA Electrophoresis GmbH (Heidelberg, Germany) and the rhodium standard (1000 ppm) from Merck (Overijse, Belgium). Hydrochloric acid solutions were prepared from HCl (37%, Acros Organics) and Milli-Q® water. All chemicals were used as received, without further purification.</p><!><p>After each extraction, the mixtures were centrifuged with a Heraeus Megafuge 1.0 centrifuge. Metal concentrations were determined using a bench top total reflection X-ray fluorescence spectrometer (TXRF; Bruker S2 Picofox) for simple matrices (mono-element system) or inductively coupled plasma optical emission spectrometer (ICP-OES; Agilent, type E730) with an axial plasma configuration for the more complex matrices (multi-element system) due to the spectral overlap and matrix effects that were encountered with TXRF.</p><p>For analysis of the aqueous phase by TXRF, part of the aqueous phase was mixed with a lanthanum internal standard and Milli-Q® water until a total volume of 1 mL was obtained. The quartz glass sample carriers were first treated with 20 μL of a silicone solution in isopropanol, dried for 5 min in a hot air oven at 60 °C, followed by the addition of 5 μL of the sample and drying for 30 min at the same temperature. The metal concentrations in the aqueous phase were measured for 1000 s. For the organic phase, the lanthanum internal standard was added to a small amount of the organic phase (27 mg) and was further diluted with absolute ethanol until 1 mL. The sample carrier pre-treatment, the drying procedure and the measuring time were performed in the same way for the organic phase as described for the aqueous phase but the sampling volume was reduced to 2 µL. For analysis of the aqueous phase by ICP-OES, a calibration curve was prepared with multi-element solutions over a concentration range of 0-10 mg L −1 with a quality control of 2 mg L −1 and scandium as an internal standard. The calibration solutions and the samples were diluted in 10 vol% HCl. The spectra were measured with a power of 1.4 kW, an argon flow of 15 L min −1 and an auxiliary argon flow of 1.5 L min −1 . For the organic phase, the sample was first digested with a mixture of H 2 SO 4 and HNO 3 in a quartz beaker on a heating plate. H 2 SO 4 and HNO 3 were evaporated (∼400 °C) and the residue was dissolved in 10 vol% HCl before measurement. The calibration and measuring procedure have been performed in the same way for the organic phase as described for the aqueous phase.</p><p>The viscosity of the organic phase was measured using a falling-ball type viscometer (Anton Paar, Lovis 2000 ME), densities were determined using a density meter with an oscillating U-tube sensor (Anton Paar, DMA 4500 M) and pH measurements were performed with an S220 SevenCompact™ pH/Ion meter (Mettler-Toledo) and a Slimtrode (Hamilton) electrode. A Mettler-Toledo DL39 coulometric Karl Fischer titrator was used with Hydranal® AG reagent to determine the water content of the ionic liquid. 31 P NMR spectra have been recorded on a Bruker® Avance™ II 600 MHz Spectrometer, operating at 242.94 MHz. The samples for 31 P NMR measurements were dissolved in toluene-d 8 and measured with respect to a H 3 PO 4 external reference. A delay time (d1) of 60 s was applied in the NMR pulse sequence to avoid saturation effects in the 31 P NMR spectra. All NMR spectra were analyzed with the TOPSPIN software package.</p><p>The loss of Cyphos® IL 101 to the aqueous phase was determined by ICP-OES. All measurements were carried out using an ICP-OES spectrometer, model Optima 8300 DV (Perkin Elmer) equipped with axially viewed plasma. 31 P was analyzed at 177.434 nm. The internal standard was measured at 343.489 nm (Rh). Standard solutions of 31 P (3, 1, 0.5, 0.1, 0.05 ppm) were prepared in 5 vol% HCl. 1 ppm of Rh was added as an internal standard. 5 vol% HCl was used as a blank solution for calibration. All samples were diluted 10 times with a 5 vol% HCl solution and 1 ppm of Rh was added as an internal standard. All aqueous solutions were prepared with Milli-Q® water. A reagent blank solution of 5 vol% HCl containing 1 ppm of Rh as an internal standard was used for correcting the Ca-free samples. The Ca-containing samples were corrected using a reagent blank having the same Ca concentration as the samples and containing 1 ppm of Rh as an internal standard. The loss of Aliquat® 336 to the aqueous phase was determined using TOC measurements. The total organic carbon content (TOC) was analyzed with a HiPerTOC TOC analyser (Thermo Scientific) using the non-purgeable organic carbon (NPOC) method. The total inorganic carbon (TIC; dissolved CO 2 , HCO 3 − and CO 3 2− ) of the sample (30 mL) was converted to CO 2 by addition of 400 µL of a 10 vol% H 3 PO 4 solution. The formed CO 2 gas was removed from the solution by flushing with O 2 gas. Subsequently, 1 mL of the sample was transported to the UV reactor together with 1.5 mL of an oxidizing solution (120 g L −1 Na 2 S 2 O 8 ) under O 2 atmosphere (99.999%) with an O 2 flow rate of 250 mL min −1 . The UV reactor converted the remaining carbon to CO 2 . Next, the formed gas was dried by a Peltier cooler and separated from the liquid components by a gas-liquid separator. Afterwards, the dried CO 2 was carried to a non-dispersive infrared detector, where CO 2 was measured. A calibration curve was prepared to relate the detector signal to the CO 2 concentration and hence to the corresponding total carbon concentration of the sample.</p><!><p>Extraction experiments were performed using two phases: metal chloride salts in an HCl acidified water phase and undiluted ionic liquid comprising the organic phase. The IL was presaturated with Milli-Q® water and hydrochloric acid in a volume ratio of 4.5 : 1, having the same chloride concentration as the aqueous phase before extraction, at 60 °C to prevent co-extraction of Milli-Q® water and hydrochloric acid. Therefore, the ratio of the volume of the aqueous and the ionic liquid phase remains constant. Extractions were performed by intensive stirring at 500 rpm for 60 min at 60 °C with a magnetic stirring bar. Hydrochloric acid was used as the chloride source. After the extraction, separation of the phases was assisted by centrifugation for 15 min at 3500 rpm.</p><!><p>Distribution ratio studies. The distribution ratios of indium(III) at different HCl concentrations were determined using equal volumes of ionic liquid and an acidified water phase (5 mL) containing 5 g L −1 of indium(III). Further experiments studied the distribution ratios of indium(III) as a function of feed concentration at the optimal hydrochloric acid concentration of 0.5 M and using feed solution concentrations ranging from 5 to 120 g L −1 . The viscosity of the organic phase was determined for the various feed solution concentrations.</p><p>Kinetics. Reaction kinetics were studied by shaking equal volumes of ionic liquid and an acidified water phase (5 mL) containing 5 to 100 g L −1 of indium(III) for different times ranging from 1 to 180 min.</p><p>Stripping. First, extractions were performed from a feed solution containing 5 g L −1 indium(III) at the optimal hydrochloric acid concentration of 0.5 M. Afterwards, the loaded ionic liquid phase was stripped with a water phase (5 mL) containing different stripping agents: Milli-Q® water, ethylenediaminetetraacetic acid disodium salt dihydrate (Na 2 EDTA•2H 2 O) and sodium hydroxide. All stripping experiments were executed using equal volumes of ionic liquid and a water phase at 60 °C, except for the stripping with sodium hydroxide which was performed at room temperature. After stripping, separation of the phases was assisted by centrifugation for 15 min at 3500 rpm.</p><p>Recycling of the ionic liquid for reuse. Extractions were performed from a feed solution containing 5 g L −1 indium(III) at the optimal HCl concentration of 0.5 M. Afterwards, the loaded ionic liquid phase was stripped with an aqueous NaOH solution. The loaded Cyphos® IL 101 and Aliquat® 336 were stripped by 4.5 and 4 equivalents of NaOH, respectively. All extraction and stripping experiments were executed with equal volumes of ionic liquid and water phase. After extraction and stripping the ionic liquid was equilibrated by a 0.5 M HCl solution in a volume ratio 1 : 5. The equilibrated ionic liquid was reused in a second extraction step using a feed solution containing 5 g L −1 indium(III) at the optimal HCl concentration of 0.5 M.</p><p>Loss of ionic liquid to the aqueous phase. First, Cyphos® IL 101 was washed 4 times with a 0.05 M NaCl solution in a 1 : 25 volume ratio at room temperature until the water soluble phosphor-containing impurities present in the ionic liquid were removed. The P content of the aqueous phase after washing remained constant. A 0.05 M NaCl solution was used as a washing solution instead of Milli-Q® water due to emulsion formation with Milli-Q® water. Subsequently, extractions were performed using equal volumes of ionic liquid and aqueous phase (5 mL) at 60 °C. The solubility of the ionic liquid in the aqueous phase was determined at different HCl (0.5 M, 6 M and 12 M), indium(III) (5 g L −1 , 40 g L −1 and 120 g L −1 ) and CaCl 2 (0.25 M, 3 M and 6 M) concentrations in the aqueous phase. The loss of Cyphos® IL 101 to the aqueous phase was determined by ICP-OES. Aliquat® 336 was first saturated with a 0.05 M NaCl solution in a 1 : 1 volume ratio at room temperature. Afterwards, extractions were performed using equal volumes of ionic liquid and aqueous phase (250 mL) at 60 °C. The solubility of the ionic liquid in the aqueous phase was determined at different HCl (0.5 M, 6 M and 12 M) and CaCl 2 (0.25 M, 3 M and 6 M) concentrations in the aqueous phase. The loss of Aliquat® 336 to the aqueous phase was determined by TOC measurements. Aliquat® 336 is considered to be a mixture quaternary ammonium chloride with a 2 : 1 molar ratio of octyl groups to decyl groups. A molar weight of 432 g mol −1 was used instead of 404 g mol −1 which is valid for pure trioctylmethylammonium chloride. Based on the molar ratio, the following composition of quaternary ammonium chloride was assumed (not including water and other impurities) according to random statistical distribution: trioctylmethylammonium chloride (33 wt%), dioctyldecylmethylammonium chloride (28 wt%), octyldidecylmethylammonium chloride (22 wt%) and tridecylmethylammonium chloride (17 wt%). 41 These assumptions were made to calculate the solubility of Aliquat® 336 in the aqueous phase based on TOC analyses results.</p><!><p>Distribution ratio studies. The distribution ratio of cadmium(II), copper(II), indium(III), iron(III), manganese(II), nickel(II), tin(IV) and zinc(II) at different HCl concentrations was studied using equal volumes of ionic liquid and an acidified water phase (10 mL) containing 5 g L −1 of each metal added as chloride salts. Additionally, 1 g L −1 of lead(II) as a chloride salt and arsenic(III) as an oxide were added to the water phase. These elements are commonly found in primary and secondary sources of indium. Extraction experiments of the multi-element system were carried out only once.</p><!><p>The distribution ratio D of a metal M is defined as</p><p>where [M] IL is the metal concentration in the ionic liquid and [M] aq is the metal concentration in the aqueous phase after extraction. For metals which are strongly extracted to the ionic liquid phase (%E ≥ 50%) only the remaining metal concentration in the aqueous phase was measured after extraction and eqn (1) can be rewritten as</p><p>where [M] 0 is the initial metal concentration in the aqueous phase before extraction. The metal concentration of the ionic liquid phase was measured for metals which are poorly extracted (%E < 50%), and eqn (1) becomes</p><p>The percentage extraction (%E) is defined as the amount of metal extracted to the ionic liquid phase over the initial amount of metal present in the aqueous phase:</p><p>The separation efficiency between two metals can be described by the separation factor α, which is defined as the ratio of the respective distribution ratios of two extractable solutes measured under the same conditions:</p><p>where D M1 and D M2 are the distribution ratios D of metal M 1 and M 2 , respectively. By definition, the value of the separation factor is always greater than unity.</p><p>Metals are removed after extraction from the organic phase by a stripping agent. The percentage stripping (%S) in the stripping phase can be defined as the amount of metal stripped from the organic phase to the total amount of metal in the organic phase before stripping:</p><p>where [M] IL,0 is the metal concentration in the organic phase after extraction or before stripping.</p><!><p>Distribution ratio studies. In the first series of experiments, the distribution ratios and the extraction percentages of indium(III) between the ionic liquid and aqueous phase were determined as a function of hydrochloric acid concentration (Fig. 1, S1, † and Table 1). Fig. 1 shows bell-shaped curves; i.e., the distribution ratios increase as a function of HCl concentration until a value from where they begin to decrease. Bellshaped curves of the distribution ratio versus the HCl content have also been observed by other authors for extraction of Cu(II), Co(II), Fe(III), Ga(III), In(III), Mn(II) and Zn(II) by quaternary ammonium salts. [42][43][44][45] The average distribution ratio of indium(III) between Cyphos® IL 101 and hydrochloric acid aqueous solution, increases with increasing chloride concentration, with a maximum D In = 4966 ± 262, at a HCl concentration of approximately 5 M. The same trend but with lower values was observed for the distribution ratio for the HCl-Aliquat® 336 system, where a maximum distribution ratio of D In = 340 ± 28 was found at the same 5 M HCl concentration. In both cases, the initial indium(III) concentration in the aqueous phase was 5 g L −1 .</p><p>The bell-shaped curve of the distribution ratio is tentatively attributed to a change of the indium speciation in the aqueous and/or in the ionic liquid phase in combination with HCl extraction. Narita et al. 46 already described that the indium speciation in the aqueous phase can change as a function of HCl concentration. Co-extraction of HCl by phosphonium and ammonium ionic liquids has also been previously observed by Komasawa et al., 47 Sato et al. 48 and Wellens et al. 49 The distribution ratios of indium(III) for the HCl-Aliquat® 336 system were significantly lower, minimum by a factor of 5, than those for the HCl-Cyphos® IL 101 system over the whole HCl concentration range (Fig. 1). This can be explained by the higher water uptake of Aliquat® 336 (21.27 wt%) compared to Cyphos® IL 101 (13.65 wt%). The hydrophobicity of ionic liquids with a common anion is dependent on the total number of carbon atoms in the alkyl chains attached to the corresponding central atom. Shorter alkyl chains attached to the ammonium cation core lead to a lower hydrophobicity of Aliquat® 336 compared to Cyphos® IL 101, and therefore to a higher mutual miscibility with water.</p><p>Furthermore, the charge delocalization at the ammonium cations makes the central part of these cations more charged, and thus overall more polar, than the corresponding phosphonium cations. 50,51 Very high values for the distribution ratio were observed over the entire HCl concentration range for the HCl-Cyphos® IL 101 system indicating a strong affinity of indium(III) for the ionic liquid phase. The distribution ratio observed for the quaternary ammonium ionic liquid over the entire HCl concentration range are not exceptionally high. High distribution ratios are not desirable for solvent extraction since they make stripping in many cases very difficult. Following studies regarding the kinetics, maximal loading, the extraction mechanism and the stripping of the mono-element systems are executed at a low HCl concentration of 0.5 M where the distribution ratio is high enough to ensure almost complete extraction of indium(III), but not too high so that stripping difficulties are avoided.</p><p>Kinetics. For industrial applications, it is more interesting to expand the indium(III) concentration range of the feed solu- tion. The distribution ratios of indium(III) as a function of feed concentration were studied at the optimal HCl concentration of 0.5 M and with feed solution concentrations ranging from 5 to 120 g L −1 for both Cyphos® IL 101 and Aliquat® 336 (Fig. 2). A stirring time of 1 h was used to ensure that equilibrium was reached. Due to the strong affinity between the ionic liquid and indium(III), the organic phase can be loaded with high amounts of indium(III), while it is still possible to obtain a high percentage extraction (Fig. S2 †). Phase inversion was observed for Cyphos® IL 101 and an initial indium(III) concentration of 100 g L −1 and higher and for Aliquat® 336 and an initial indium(III) concentration of 120 g L −1 . At high loading of the ionic liquid phase, its density becomes larger than the density of the aqueous phase so that a phase inversion occurs. The density at 25 °C of the 100 g L −1 indium(III) loaded Cyphos® IL 101 and the 120 g L −1 indium(III) loaded Aliquat® 336 was 1.0242 ± 0.0023 g mL −1 and 1.0303 ± 0.0021 g mL −1 , respectively The density of the corresponding aqueous phases was 1.0078 ± 0.0003 g mL −1 for the Cyphos® IL 101 system and 1.0303 ± 0.0021 g mL −1 for the Aliquat® 336 system at 25 °C. The avoidance of phase inversion is essential to industrial extraction processes, since the design of the contacting equipment is based on preferred direction of solute transfer, giving optimum mass transfer rates. Phase inversion, can cause considerable operating problems, especially in mixer-settlers, in which the change in properties of both the continuous phase and the drop size can lead to a delay in the settling process. Next, the influence of the extraction time was evaluated from 1 min to 60 min at the optimal HCl concentration of 0.5 M, with feed solution concentrations ranging from 5 to 100 g L −1 (Fig. 3 and S3 †). The results show that equilibrium is reached after 10 min, regardless of the indium(III) concentration. However, longer stirring times to reach the equilibrium were expected at higher indium(III) concentrations due to an increased viscosity of the ionic liquid phase at high indium(III) loadings. This increase in viscosity as a function of indium(III) concentration in the aqueous feed solution is shown in Fig. 4. The influence of the viscosity on the reaction time is thus most probably negligible due to the high affinity of indium for the ionic liquid phase. The viscosity of the ionic liquid phase increased only slightly when it was loaded with higher indium(III) concentrations (Fig. 4). This indicates that the chloroindate(III) anion formed during extraction has the same charge as the chloride anion in the pure ionic liquid, i.e. that the [InCl 4 ] − anion is formed instead of higher charged species such as [InCl 5 ] 2− or [InCl 6 ] 3− . The presence of species with a higher charge are known to lead to a sharp increase of the viscosity at higher loadings. 33,34,46,52 The same speciation was also observed in the organic phase for the extraction of indium(III) using long chain alkylamines and quaternary ammonium chlorides from chloride media. 43,44,46,53,54 The proposed extraction mechanism is therefore:</p><p>Indium(III) is probably extracted as a neutral InCl 3 or the single negatively charged [InCl 4 ] − complex. The more negatively charged chloroindate(III) complexes, [InCl 5 ] 2− and [InCl 6 ] 3− , are more strongly hydrated, thus more difficult to dehydrate and to convert to extractable anionic species.</p><p>Stripping. The ease of recovery of the metal from the ionic liquid phase and regeneration of the ionic liquid are as important as obtaining a high extraction efficiency. 7 Stripping of the ionic liquid phase after extraction was tested using several stripping agents. Cyphos® IL 101 and Aliquat® 336 are basic extractants (anion exchangers). The driving force for extraction is the presence of chloride anions. Stripping of metals is efficient if the distribution ratio is low. This can be achieved by decreasing the chloride concentration by addition of water. However, the stripping with water did not result in an efficient stripping; 6.1 ± 0.7% indium was stripped from the loaded Cyphos® IL 101 and 29 ± 3% from the loaded Aliquat® 336 phase. Furthermore, the stripping of the loaded Cyphos® IL 101 ionic liquid gave also rise to a difficult disengagement (formation of an emulsion). A small amount of Na 2 SO 4 (±15 mg) was added as a salting-out agent to 5 mL of the aqueous phase. Although the stripping of the loaded ionic liquids with water resulted in a low percentage stripping, changing the temperature or the volume ratio could increase the stripping efficiency. By increasing the volume ratio of the aqueous to the organic phase from 1 : 1 to 3.5 : 1, stripping percentages of 19% and 78% were obtained for Cyphos® IL 101 and Aliquat® 336, respectively. In contrast to stripping with a 1 : 1 volume ratio, a white In(OH) 3 precipitate was found in the aqueous phase after stripping. Ethylenediaminetetraacetic acid (EDTA) forms a very stable, highly water-soluble chelate complex with indium(III) (log β = 24.90). 55,56 Therefore, it was tested to strip indium(III) from the ionic liquid with an aqueous solution of Na 2 EDTA. Although reasonable percentages of stripping were acquired, 52 ± 1% for loaded Cyphos® IL 101 and 67 ± 1% for the loaded Aliquat® 336, the use of EDTA in the industry is not favored due to its low biodegradability and difficulties associated with recovery of EDTA in a continuous solvent extraction process due to its high level of complexing capacity with respect to heavy metals. [57][58][59][60] If wanted, higher percentages stripping can probably be obtained by adjusting the pH with an alkaline solution, neutralizing the HCl present in the ionic liquid phase and removing the remaining protons from Na 2 EDTA. Care must be taken to avoid ionic liquid decomposition in alkaline environment [61][62][63][64] and anion exchange with EDTA. 34 Aside from the complexing agent EDTA, stripping was also tested with sodium hydroxide as a precipitation agent. Precipi-tation stripping from a metal-loaded ionic liquid phase has already been reported for the rare earths using oxalic acid as the stripping agent. [65][66][67] Indium is directly stripped from the organic phase with sodium hydroxide forming an insoluble hydroxide. The precipitation stripping reaction can be represented as:</p><p>As the pH increases, the equilibrium of the hydrolysis reaction is shifted to the right and more indium is precipitated from the aqueous phase. According to Pourbaix, the solubility of In(OH) 3 is minimal at pH 6.79. 68 The loaded ionic liquid phase was stripped with an aqueous phase containing different equivalents of sodium hydroxide (Fig. 5). Using sodium hydroxide as a stripping agent, it was possible to obtain a percentage stripping close to 100%. The 0.5 M HCl-Aliquat® 336 system was easier to strip compared to the 0.5 M HCl-Cyphos® IL 101 system due to the lower indium distribution ratio obtained during extraction. 4 equivalents of NaOH were needed to achieve a percentage stripping over 99% for the 0.5 M HCl-Aliquat® 336 system. The equilibrium pH under these conditions was 4.35. However, at this point not all of the indium was precipitated yet as In(OH) 3 . Part of it is dissolved in the aqueous phase. 4.75 equivalents of NaOH were needed to fully precipitate all indium. A percentage stripping over 99% was reached for the 0.5 M HCl-Cyphos® IL 101 system if 4.5 equivalents of NaOH were used. Unlike the previous stripping experiments with water and Na 2 EDTA, the stripping was performed at room temperature instead of 60 °C, because quaternary phosphonium and ammonium ionic liquids tend to decompose in alkaline conditions, especially at elevated temperatures. Thus, in this context, quaternary phos- phonium ionic liquids can decompose to yield a tertiary phosphine oxide and alkane under alkaline conditions. [61][62][63]</p><p>Quaternary ammonium ionic liquids can easily undergo Hofmann elimination or β-elimination to yield a tertiary amine and an alkene in the presence of a base (Fig. 6). 61,62,64 Trihexyl(tetradecyl)phosphonium chloride stability was investigated by comparing the integration of the degradation products 31 P resonance signal, i.e. phosphine oxide, formed after mixing for 1 h with an aqueous NaOH solution to the integration of the trihexyl(tetradecyl)phosphonium cation 31 P resonance signal at 32.9 ppm. The resonance signal of phosphine oxide was situated at 45.8 ppm. 31 P NMR spectra were measured for the ionic liquid before and after stripping since commercial Cyphos® IL 101 already contains small amounts of phosphine oxide impurities. Peak integration ratios were then compared and it was concluded that no decomposition occurs because no change of the ratio of the integration of the phosphine peak over the integration of the phosphonium cation peak took place when stripping with NaOH at room temperature.</p><p>Recycling of the ionic liquid for reuse. An important aspect of green chemistry is the recyclability and reusability of the ionic liquid. The recyclability was shown by reusing the ionic liquid in a second extraction step after stripping. The results of each individual step are given in Table 2. The results indicate that the ionic liquid can be recycled for reuse in liquid/liquid extraction of indium(III) without any loss of activity.</p><p>Loss of the ionic liquid to the aqueous phase. Ionic liquids are often considered as green solvents primarily due to their negligible vapor pressure and low flammability. However, the avoidance of loss to the aqueous phase and the recyclability of the ionic liquid are equally as important in establishing a sustainable process. The loss of Cyphos® IL 101 and Aliquat® 336 to the aqueous phase was determined. First, the phosphor con-taining water soluble impurities present in Cyphos® IL 101 were removed by four consecutive washing steps with a 0.05 M NaCl solution using a 1 : 25 volume ratio at room temperature (Fig. S4 †). If not removed these impurities will insinuate a higher solubility of Cyphos® IL 101 in the aqueous phase. Subsequently, the ionic liquid was brought into contact with several aqueous solutions at 60 °C: HCl (0.5 M, 6 M and 12 M), indium(III) (5 g L −1 , 40 g L −1 and 120 g L −1 ) and CaCl 2 (0.25 M, 3 M and 6 M). The solubility of Cyphos® IL 101 in the different aqueous phases is presented in Table 3. The purity of Aliquat® 336 (88.2-90.6%) is low in comparison with Cyphos® IL 101 (>97%). Therefore, no prior washing steps were executed to remove possible water soluble organic impurities. For result comparison reasons, Aliquat® 336 was saturated with a 0.05 M NaCl solution in a 1 : 1 volume ratio at room temperature. Afterwards, the solubility of Aliquat® 336 was determined in several HCl (0.5 M, 6 M and 12 M) and CaCl 2 (0.25 M, 3 M and 6 M) solutions after contacting the ionic liquid with the aqueous solutions at 60 °C. The solubility results are displayed in Table 3. The solubility of Aliquat® 336 in aqueous phase is higher than the solubility of Cyphos® IL 101 due to it higher mutual miscibility with water. As the salt concentration in the aqueous phase increases, the solubility of the ionic liquid in the aqueous phase decreases due to the salting-out effect. In general, the solubility of the ionic liquids in the aqueous phase is very limited thereby having a low associated economical and environmental impact.</p><!><p>Distribution ratio studies. A useful purification process needs to have a reasonable selectivity for the targeted metal. All the experiments described above have been carried out on mono-element solutions but industrial solutions are generally complex mixtures of several elements. In a second series of experiments, the separation of indium(III) from arsenic(III), cadmium(II), copper(II), iron(III), lead(II), manganese(II), nickel(II), tin(IV) and zinc(II) was investigated. More elements are added to the feed solution compared to previous experiments, to a more industrial relevant elemental composition: 5 g L −1 of each of the other elements with the exception of arsenic(III) and lead(II) present in a concentration of 1 g L −1 . A concentration of 1 g L −1 for arsenic(III) and lead(II) was chosen due to the limited solubility of arsenic(III) oxide and lead(II) chloride in the aqueous phase. 69,70 Fig. 7 and Fig. 8 show the distribution ratios between the ionic liquid phase and aqueous phase of the elements as a function of HCl concentration for the HCl-Cyphos® IL 101 and HCl-Aliquat® 336 systems. Percentage extraction as a function of HCl concentration for both systems is given in Fig. S5 and S6. † Many elements display a bell-shaped distribution ratio curve. Indium(III) has a maximum distribution ratio at 5 M in both systems in agree-ment with the previous experiments. For copper(II) and manganese(II), the maximum distribution ratio was found at a HCl concentration of 4 M and 6 M, respectively for the HCl-Cyphos® IL 101 system and at 5 M and 7 M, respectively for the HCl-Aliquat® 336 system. For nickel(II) and arsenic(III), the maximum distribution ratio was found at higher HCl concentration of 8 M and 10 M, respectively, independent of the extraction system. Moreover, the distribution ratios of nickel(II) were significantly lower than those of the other elements over the whole HCl concentration range, indicating a low affinity of nickel(II) for the ionic liquid phase. For lead(II) the behavior was distinctly different from that of the other elements. Here the distribution ratios were high at low HCl concentrations and decreased with increasing HCl concentration. It is very likely that only the right side of the bell-shaped curve is represented. The maximum value for the distribution ratios of cadmium(II), iron(III), tin(IV) and zinc(II) could not be determined with ICP-OES as the analysis method due to the lower detection limit of 1 mg L −1 . However, from the partial shape of the distribution ratio curves of cadmium(II), iron(III) and zinc(II), it can be concluded that also a bell-shaped path is followed. The maximum distribution ratio and corresponding HCl concentration for each of the elements are given in Table 4. Advantage can be taken of the low affinity of arsenic(III), manganese(II) and nickel(II) for the ionic liquid phase at lower HCl concentrations to separate these ions from cadmium(II), iron(III), lead(II), tin(IV), zinc(II) and most importantly from indium(III). Due to the lower distribution ratios obtained for most of the elements, that allows an easier stripping of indium(III) from the ionic liquid phase after extraction, Aliquat® 336 is the preferred undiluted extractant.</p><p>In the following paragraphs, information is given on the capabilities of the 0.5 M HCl-Aliquat® 336 system for purification of indium(III), and possible process steps are suggested. The low distribution ratios of arsenic(III), manganese(II) and nickel(II) in combination with high separation factors between these elements and indium(III), imply an easy separation (Table 5). The contaminating ions will remain largely in the aqueous phase during extraction. A high separation factor was also obtained for the In(III)/Cu(II) couple, but due to the slightly elevated distribution ratio of copper(II) in comparison with arsenic(III), manganese(II) and nickel(II), extraction of copper(II) cannot be avoided completely (Table 5). However, reasonably low distribution ratio enables the scrubbing of copper(II) from the ionic liquid phase together with traces of arsenic(III), manganese(II) and nickel(II) extracted. A solution with a low HCl concentration has to be used for scrubbing to avoid loss of indium(III) to the aqueous phase. After extraction and scrubbing, cadmium(II), iron(III), lead(II), tin(IV), zinc(II) will still be present in the ionic liquid phase together with indium(III). The high separation factors for the couples In(III)/ Cd(II), In(III)/Sn(IV) and In(III)/Zn(II) suggest an easy separation (Table 5). Although they all possess a high affinity for the ionic liquid phase (D In = 30.7, D Cd , D Sn , D Zn > 5.00 × 10 3 ), indium(III) will be easier to strip due to its distribution ratio being 160 times smaller. It was demonstrated previously that indium can be removed from the loaded Aliquat® 336 phase by stripping with water or NaOH. Both methods can be satisfactory, but stripping with water will require larger volume ratios of the aqueous to the organic phase. Also selectivity will play a vital role in choosing a stripping method. Selective stripping of indium(III) from iron(III) and lead(II) will be difficult due to the small separation factors, α Fe(III)/In(III) and α Pb(II)/In(III) (Table 5).</p><p>Hydrolysis curves have to be constructed for the stripping of the ionic liquid phase with NaOH to get a better understanding about the hydrolysis behavior of the various metals, elaborating a more selective stripping. After stripping, the ionic liquid phase has to be scrubbed so that it can be reused. Scrubbing of the ionic liquid phase with water will have little effect due to the high distribution ratios of cadmium(II), tin(IV) and zinc(II). Scrubbing with a NaOH solution will in most cases be the best option.</p><!><p>It is shown that the commercial ionic liquids Cyphos® IL 101 and Aliquat® 336 are very efficient for extraction of indium(III) from chloride feed solutions. The ionic liquids are used in an undiluted form so that volatile molecular organic solvents can be avoided. The extraction process is selective for indium(III), over many other metal ions that are commonly found as impurities in process solutions of indium refineries. Due to the very high distribution ratios, co-extracted impurities can easily be scrubbed from the ionic liquid phase without affecting the extracted indium(III) ions. Indium could be recovered in the form of In(OH) 3 by precipitation stripping with a NaOH solution. This stripping step also regenerates the ionic liquid. This work indicates that precipitation stripping with NaOH is a very convenient methods for recovering metal ions that show high affinities for the organic phase, such as indium(III) for undiluted chloride ionic liquids. The method is environmentally friendly because NaCl is the sole waste product and the ionic liquids can be reused. system and TOC analyses were executed in the analytical laboratory of Umicore Group Research & Development. a According to IUPAC, by convention the ratio of the respective distribution ratios has to be chosen so that α > 1. 71</p>

Royal Society of Chemistry (RSC)

Chalcogen bonds: Hierarchical ab initio benchmark and density functional theory performance study

AbstractWe have performed a hierarchical ab initio benchmark and DFT performance study of D2Ch•••A− chalcogen bonds (Ch = S, Se; D, A = F, Cl). The ab initio benchmark study is based on a series of ZORA‐relativistic quantum chemical methods [HF, MP2, CCSD, CCSD(T)], and all‐electron relativistically contracted variants of Karlsruhe basis sets (ZORA‐def2‐SVP, ZORA‐def2‐TZVPP, ZORA‐def2‐QZVPP) with and without diffuse functions. The highest‐level ZORA‐CCSD(T)/ma‐ZORA‐def2‐QZVPP counterpoise‐corrected complexation energies (ΔE CPC) are converged within 1.1–3.4 kcal mol−1 and 1.5–3.1 kcal mol−1 with respect to the method and basis set, respectively. Next, we used the ZORA‐CCSD(T)/ma‐ZORA‐def2‐QZVPP (ΔE CPC) as reference data for analyzing the performance of 13 different ZORA‐relativistic DFT approaches in combination with the Slater‐type QZ4P basis set. We find that the three‐best performing functionals are M06‐2X, B3LYP, and M06, with mean absolute errors (MAE) of 4.1, 4.2, and 4.3 kcal mol−1, respectively. The MAE for BLYP‐D3(BJ) and PBE amount to 8.5 and 9.3 kcal mol−1, respectively.

chalcogen_bonds:_hierarchical_ab_initio_benchmark_and_density_functional_theory_performance_study

<!>INTRODUCTION<!><!>INTRODUCTION<!>Ab initio geometries and energies<!><!>DFT geometries and energies<!>Ab initio geometries<!><!>Ab initio geometries<!>Ab initio Chalcogen bond energies<!><!>Ab initio Chalcogen bond energies<!><!>Ab initio Chalcogen bond energies<!>Performance of density functional approximations<!><!>Performance of density functional approximations<!>CONCLUSIONS<!>

<p>de Azevedo Santos L , Ramalho TC , Hamlin TA , Bickelhaupt FM . Chalcogen bonds: Hierarchical ab initio benchmark and density functional theory performance study. J Comput Chem. 2021;42:688–698. 10.1002/jcc.26489 33543482</p><p>Funding information Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: CNPq; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Grant/Award Number: CAPES; Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Grant/Award Number: FAPEMIG; Netherlands Organization for Scientific Research, Grant/Award Number: NWO</p><!><p>Chalcogen bonding has emerged as a key noncovalent interaction with several applications including supramolecular chemistry, 1 biochemistry, 2 and catalysis. 3 The chalcogen‐bond (ChB) is defined as the net‐attractive noncovalent interaction, in a D2Ch•••A complex, between a chalcogen‐bond donor D2Ch, a Lewis‐acid, and a chalcogen‐bond acceptor A− (or A), a Lewis‐base, in which Ch stands for a chalcogen atom, i.e., an atom of group 16 (Scheme 1). 4a The "σ‐hole interaction" between a positive region on the electrostatic potential surface on the chalcogen atom and a negatively charged density on the ChB acceptor is usually invoked to characterize the ChB. 4 Despite this, recent studies have shown that the strength of the ChB is, instead, correlated to the electron‐accepting capacity of the σ*‐type LUMO of the chalcogen molecule. 5 The debate over the origin and fundamental bonding mechanism of the ChB continues to stimulate much interest in the literature.</p><!><p>Chalcogen‐bonded D2Ch•••A− model complexes (Ch = S, Se; D, A = F, Cl)</p><!><p>Density functional theory (DFT) based Kohn‐Sham molecular orbital analysis has been paramount for our understanding of bonding mechanisms and the nature of chemical phenomena. 6 Selection of the appropriate density functional approximation to investigate chalcogen bonding is critical to ensure trust‐worthy results, but unfortunately this is not entirely straightforward, as the question of which approximate functional works best is highly dependent on the property and system of interest.</p><p>The first purpose of this work is to provide a detailed benchmark study of high‐level relativistic ab initio methods and focus on the investigation of ChB, using the D2Ch molecules as chalcogen‐bond donors and the halides A− as chalcogen‐bond acceptors (see Scheme 1). Our model complexes systematically varies the substituent (D), the chalcogen atom (Ch), the acceptor (A−), and is the perfect archetype for strongly bound chalcogen systems studied experimentally. 2 , 3 This is done by computing the D2Ch•••A− complexation energies ΔE for the first time in a procedure involving both a hierarchical series of ab initio methods [HF, MP2, CCSD, and CCSD(T)] 7 in combination with a hierarchical series of Gaussian‐type basis sets of increasing flexibility, polarization (up to g functions), and diffuseness, thereby eclipsing the two other benchmarks based on a single‐shot CCSD(T) approach. 7i,j Interestingly, the predictions of ΔE by both benchmarks for the same systems can differ by up to 10 kcal mol−1. The basis set superposition error (BSSE) has been accounted for through the counterpoise correction (CPC) of Boys and Bernardi. 8</p><p>The second purpose of this work is to evaluate the performance of 13 different density functionals in combination with ADF's Slater‐type QZ4P basis set (vide infra) for predicting the ChB energy ΔE against our best ab initio benchmark. Thus, we perform an extensive analysis to highlight the importance of diffuse and polarization functions in the basis set, the role of the BSSE, and the necessity of Coulomb correlation as well as the extent to which the approach has converged with respect to the level of correlation treatment and basis set quality. Our analyses identify the B3LYP and M06‐2X functionals, along with the M06 DFT approach as appropriate and computationally efficient alternatives to expensive high‐level ab initio computations of chalcogen‐bonded complexes.</p><!><p>All ab initio calculations were carried out using ORCA. 9 The atomic orbitals were described by the all‐electron scalar relativistically contracted variants of Gaussian‐type def2‐XVP(P) (X = S, TZ, QZ) basis sets with polarization functions (up to g functions) in the series BS1 to BS3 (see Table 1). 10 The series BS1+ to BS3+ result from BS1 to BS3 after adding extra s and p minimally augmented (ma) diffuse functions (see Table 1). 10c For each of the six basis sets (BS#), the equilibrium geometry was computed using coupled‐cluster singles and doubles with perturbative triples, i.e., at CCSD(T)/BS#. 11 Then, for each BS# and corresponding CCSD(T)/BS# geometry, energies were evaluated along the following hierarchical series of quantum chemical methods: Hartree‐Fock theory (HF/BS#), second‐order Møller‐Plesset perturbation theory (MP2/BS#), 12 coupled‐cluster with single and double excitations (CCSD/BS#) 13 and CCSD(T)/BS#. 11 The scalar relativistic effects were accounted for using the scalar zeroth‐order regular approximation (ZORA). 14 Inclusion of relativistic effects are necessary for heavier chalcogen‐bonded systems and without ZORA, our counterpoise‐corrected complexation energies ΔE CPC are significantly under‐bound. For example, for Cl2Se•••Cl− the ΔE CPC is −31.2 kcal mol−1 at CCSD(T)/BS3+ and −34.3 kcal mol−1 at ZORA‐CCSD(T)/BS3+. For the lighter chalcogen systems, such as F2S•••F−, this effect is smaller and ΔE CPC is −45.1 kcal mol−1 at CCSD(T)/BS3+ and −45.2 kcal mol−1 at ZORA‐CCSD(T)/BS3+.</p><!><p>Number of relativistically contracted basis functions for ZORA‐def2‐ basis sets without (BS) and with (BS+) diffuse functions for F, S, Cl, and Se elements.</p><!><p>All DFT calculations were carried out using the Amsterdam Density Functional (ADF) program. 15 The equilibrium geometries and energies of chalcogen‐bonded complexes were computed at different DFT levels using (i) the GGA based functionals: PBE, 16 BP86, 17 and BLYP 17 , 18 ; (ii) the hybrid functionals: B3LYP 19 and BHANDH (50% HF exchange, 50% LDA exchange, and 100% LYP correlation 18 ); (iii) the meta‐GGA based functionals: SSB‐D 20 and M06‐L 21 ; (iv) the meta‐hybrid functionals: M06, 21 M06‐2X, 21 and M06‐HF. 21 The long range dispersion corrections were included into the B3LYP, BLYP, and SSB‐D functionals with Grimme's empirical D3 correction using the Becke‐Johnson (BJ) damping function. 22 Energies and geometries were computed for each of the various DFT approaches with the QZ4P basis set. 23 This is a large, uncontracted and relativistically optimized, all‐electron (i.e., no frozen core approximation) basis set of Slater‐type orbitals (STOs), which is of quadruple‐ζ quality for all atoms and has been augmented with the following sets of polarization and diffuse functions: two 3d and two 4f on fluorine, three 3d and two 4f on sulfur and chlorine, two 4d and three 4f on selenium. The molecular density was fitted by the systematically improvable Zlm fitting scheme. Scalar relativistic effects were accounted for using the zeroth‐order regular approximation (ZORA). 14</p><!><p>First, we examine the equilibrium geometries of D2Ch•••A− complexes (Ch = S, Se; D, A = F, Cl) which were fully optimized at the ZORA‐CCSD(T) level along with a hierarchic series of Gaussian‐type basis sets both with and without diffuse functions (see Table 1; for optimized Cartesian coordinates see Tables S10, S11 in the Supporting Information). The isolated halide and C 2v symmetric D2Ch neutral fragment form the stable T‐shaped, chalcogen‐bonded complexes D2Ch•••A− which are of C 2v (D = A) or C s symmetry (D ≠ A) (see Figure 1). All species have been verified through a vibrational analysis to represent equilibrium structures (no imaginary frequencies). Thus, we have a set of geometries that have been optimized at the same relativistic ab initio level along with each basis set considered in this work, without any structural or symmetry constraint (for complete structural details, see Tables S2 and S3 of the Supporting Information).</p><!><p>Geometries (in Å and degrees) and point group symmetries of D2Ch•••A− complexes computed at ZORA‐CCSD(T)/BS3+.</p><!><p>The chalcogen bond distance in the D2Ch•••A− complexes become longer as the chalcogen atom (Ch) varies from S to Se and as the accepting halide (A−) varies from F− to Cl−, and shorter as the substituent D varies from F to Cl (see Figure 1). Furthermore, the Θ1 and Θ2 angles (see Scheme 1) are slightly smaller than 90° for D = F and slightly larger than 90° for D = Cl. The key structural parameters (chalcogen bond distance and angles) converge faster as a function of basis‐set flexibility and polarization if diffuse functions are included in the basis set. For example, chalcogen bond lengths converge within 0.004–0.015 Å along the BS1 to BS3 series and within 0.000–0.010 Å along the BS1+ to BS3+ series (see Tables S2 and S3 in the Supporting Information). Interestingly, the differences in bond distances and angles of the D2Ch•••A− complexes between using quadruple‐ζ basis sets basis sets with (BS3+) or without diffuse functions (BS3) are small, only ca. 0.001 Å and 0.1°. In the following, all ZORA‐CCSD(T) calculated geometries are used in the series of high‐level ab initio calculations that constitute our benchmark study of chalcogen bonds (ChB) complexation energies.</p><!><p>Here, we report the first systematic investigation of the complexation energies, with (ΔE CPC) and without (ΔE) counterpoise corrections, as a function of a hierarchical series of ab initio methods and basis sets. The results of our ab initio computations are collected in Tables 2, 3, 4, 5 (ΔE CPC, ΔE, and BSSE; for thermodynamic values see Tables S8 and S9 in the Supporting Information) and graphically displayed in Figures 2, 3, 4, 5 (ΔE CPC and BSSE). In general, we find that the same trends in chalcogen‐bond strengths emerge at all levels of theory, that is, chalcogen bonds become stronger as the chalcogen Ch varies from S to Se, the halide A− varies from Cl− to F−, and the substituents D from F to Cl (see Figure 2). Our best reference data, obtained using counterpoise‐corrected ZORA‐CCSD(T)/BS3+ energies, show that the D2Ch•••A− chalcogen‐bond strength increases along F2S•••F− to F2Se•••F− from −45.2 to −56.4 kcal mol−1 and along F2Se•••Cl− to F2Se•••F− from −31.6 to −56.4 kcal mol−1. On the other hand, along F2S•••Cl− to Cl2S•••Cl−, the chalcogen‐bond strength only marginally strengthens from −20.8 to −22.8 kcal mol−1. For smaller basis sets in combination with ZORA‐CCSD(T), this minor difference in stability along the variation on the substituent D becomes even smaller and, for BS1+ basis sets, the selenium bonds D2Se•••F− become marginally stronger for D = F. Our best level ZORA‐CCSD(T)/BS3+ has converged within 1.5–3.1 kcal mol−1 in respect to the basis set series and, in combination with the BS3+ basis set, ΔE CPC have converged within 1.1–3.4 kcal mol−1 along the series of ab initio methods.</p><!><p>Complexation energies (in kcal mol−1) of D2S•••A− chalcogen‐bonded complexes with (ΔE CPC) and without (ΔE) counterpoise correctionsa</p><p>Note: aComputed at ZORA‐Method/BS#//ZORA‐CCSD(T)/BS#.</p><p>Complexation energies (in kcal mol−1) of D2Se•••A− chalcogen‐bonded complexes with (ΔE CPC) and without (ΔE) counterpoise corrections.a</p><p>Note: aComputed at ZORA‐Method/BS#//ZORA‐CCSD(T)/BS#.</p><p>Basis set superposition error (BSSE, in kcal mol−1) of D2S•••A− chalcogen‐bonded complexes.a</p><p>Note: aComputed at ZORA‐Method/BS#//ZORA‐CCSD(T)/BS#.</p><p>Basis set superposition error (BSSE, in kcal mol−1) of D2Se•••A− chalcogen‐bonded complexes.a</p><p>Note: aComputed at ZORA‐Method/BS#//ZORA‐CCSD(T)/BS#.</p><p>Trends in D2Ch•••A− chalcogen‐bond strength relative to the most stable Cl2Se•••F− complex along (a) ZORA‐CCSD(T)/BS# and (b) ZORA‐method/BS3+. Sulfur complexes in full lines and selenium complexes in dashed lines</p><p>Counterpoise‐corrected ZORA‐CCSD(T) complexation energies (∆E CPC) for D2Ch•••A− chalcogen‐bonded complexes along (a) BS1 to BS3 and (b) BS1+ to BS3+ basis sets</p><p>Basis set superposition error (BSSE) calculated at ZORA‐CCSD(T) level for D2Ch•••A− chalcogen‐bonded complexes along (a) BS1 to BS3 and (b) BS1+ to BS3+ basis sets</p><p>Counterpoise‐corrected ZORA‐CCSD(T) complexation energies (ΔE CPC) for D2Ch•••A− chalcogen‐bonded complexes along the ab initio method in combination with (a) BS3 and (b) BS3+</p><!><p>Despite the trend in D2Ch•••A− chalcogen‐bond strength being qualitatively the same at all levels of ab initio theory in our double hierarchical series (in QM method and in basis set), major variations of up to ca. 20 kcal mol−1 in absolute values are observed between the various levels (see Tables 2 and 3). For example, with Cl2S•••F− the ΔE CPC varies from −60.0 to −49.6 kcal mol−1 at both ZORA‐HF/BS1 and ZORA‐CCSD(T)/BS3+ levels, respectively. The high accuracy of our best level ZORA‐CCSD(T)/BS3+ can be attributed to four main factors: i) inclusion of additional s and p diffuse functions to accurately describe anions, as one would expect; ii) use of a highly flexible basis set with diffuse functions to minimize BSSE; iii) introduction of Coulomb correlation; and iv) inclusion of polarization functions especially for highly correlated methods.</p><p>We first examine ΔE CPC as a function of the basis set. In general, a strengthening of the D2Ch•••A− chalcogen bond occurs as the flexibility of the basis set is increased, and ΔE CPC is only converged at larger basis sets (see Figure 3). An exception to this trend is observed for ChB ΔE CPC values computed with the small basis set BS1, which lacks diffuse functions. For example, the ΔE CPC for Cl2Se•••F− that is already −62.0 kcal mol−1 at ZORA‐CCSD(T)/BS1 slightly weakens to −58.4 kcal mol−1 at ZORA‐CCSD(T)/BS3 (see Figure 3(A)), whereas the ΔE CPC is −50.5 kcal mol−1 at ZORA‐CCSD(T)/BS1+ and strengthens to −56.7 kcal mol−1 at ZORA‐CCSD(T)/BS3+ (see Figure 3(B)). This is caused by the breathing orbitals of the anionic halide fragments going from diffuse in the isolated anion to more compact upon forming the ChB complex, which leads to charge delocalization over the molecular system. 24 , 25 In the absence of diffuse functions, the complexation energy is overestimated due to the artificially high energy of the anion because the charge density cannot breath, i.e., expand, in order to relieve electron–electron repulsion in the negatively charged species. This explains the possibly misleading conclusion that the ΔE CPC converges faster along the BS1 to BS3 series compared to the BS1+ to BS3+ series and, therefore, the use of the basis set series without diffuse functions would be more appropriate. Later on, we illustrate that this is only a consequence of these complexation energies being 'corrected' by the BSSE.</p><p>The BSSE becomes significantly smaller with the addition of diffuse functions and decreases from 1.2–3.9 kcal mol−1 at ZORA‐CCSD(T)/BS3 to 0.9–1.8 kcal mol−1 at ZORA‐CCSD(T)/BS3+ (see Tables 4, 5, 6). However, the BSSE is large, in particular, for highly correlated methods and smaller basis sets without diffuse functions, that is, at the ZORA‐CCSD(T)/BS1 level (see Figure 4). As a result, the ZORA‐CCSD(T) ΔE CPC are better for the BS1+ to BS3+ series but become similar to the series without diffuse functions as the BSSE simultaneously decreases as the basis sets size increases. Both basis sets series, indeed, converge to a similar value independently of the number of diffuse functions, but this result is fortuitous due to the BSSE correction that damps any fluctuations along the BS1 to BS3 series. In fact, the uncorrected ZORA‐CCSD(T) complexation energies ΔE converges significantly faster along the BS1+ to BS3+ series (within 0.3–1.5 kcal mol−1) compared to the BS1 to BS3 series (within 1.9–3.5 kcal mol−1) (see Tables 2 and 3). This is, again, due to the poor description of the anionic reactants by basis sets without diffuse functions. This effect is particularly apparent at HF where Coulomb correlation is absent, mainly for systems involving the compact atom F−. 24a For example, the ΔE for Cl2Se•••F− that is −86.7 kcal mol−1 at ZORA‐HF/BS1 significantly weakens to −57.0 kcal mol−1 at ZORA‐HF/BS1+, whereas, for Cl2Se•••Cl−, the ΔE is −38.8 kcal mol−1 at ZORA‐HF/BS1 and weakens to −25.9 kcal mol−1 at ZORA‐HF/BS1+ (see Table 3).</p><!><p>ZORA‐DFT/BS complexation energies (in kcal mol−1) of representative D2Ch•••A− chalcogen‐bonded complexes.a</p><p>Note: aΔE CPC computed at ZORA‐CCSD(T)/BS3+.</p><!><p>Lastly, inclusion of Coulomb correlation is critical to achieve accurate chalcogen‐bond energies. At HF, the D2Ch•••A− complexes are weakly bound and enter into stronger chalcogen bonds as Coulomb correlation is introduced (see Figure 5). For example, from HF to CCSD(T), the ΔE CPC for F2S•••F− strengthens from −38.1 to −46.6 kcal mol−1 for BS3 and from −37.4 to −45.2 kcal mol−1 for BS3+ (see Table 2). We also note that the stabilization of ΔE CPC due to the increasing of basis set size is more pronounced for high correlated methods. For example, from BS1+ to BS3+, the ΔE CPC for F2Se•••F− slightly varies from −51.8 to −51.4 kcal mol−1 at HF level and strengthens from −51.9 to −56.4 kcal mol−1 at CCSD(T) level (see Tables 2 and 3). This is due to the well‐known fact that correlated ab initio methods strongly depend on the extent of polarization functions to generate configurations through which the wavefunction can describe the correlation hole. 7c On the other hand, at the HF level without Coulomb correlation, there is much less sensitivity of ΔE CPC towards increasing the flexibility and polarization functions of the basis set. Taken altogether, our benchmark approach, based on hierarchical series, reveals that our best estimates are converged with regards to correlation and basis set within 1.1–3.4 kcal mol−1 and 1.5–3.1 kcal mol−1, respectively, and provides the most accurate benchmark to date, surpassing the recently published benchmark based on a single‐shot CCSD(T) approach. 7i In the next section, we discuss the ability of DFT to describe Coulomb correlation compared to our ZORA‐CCSD(T)/BS3+ benchmark.</p><!><p>Finally, we have computed the complexation energies ΔE for various GGAs, meta‐GGAs, hybrid, and meta‐hybrid functionals in combination with the all‐electron QZ4P basis set and ZORA for relativistic effects on optimized geometries at the same level. The performance of the density functionals is discussed by comparing the resulting ΔE with our best ab initio ZORA‐CCSD(T)/BS3+ level. These results are graphically illustrated by the bar diagrams in Figure 6 (mean absolute error, mean error, and largest deviation) and collected in Tables S4 and S5 (complexation energies, mean absolute error, mean error, and largest deviation, see Supporting Information).</p><!><p>Mean absolute error (MAE, red), mean error (ME, black), and largest deviation (LD, blue) of the ZORA‐DFT/QZ4P functionals relative to the ZORA‐CCSD(T)/BS3+ (a) Ch•••A− bond lengths, (b) bond angles Θ2, and (c) D2Ch•••A− counterpoise‐corrected complexation energies</p><!><p>The ΔE computed at the DFT levels follow the same trends as those at ZORA‐CCSD(T)/BS3+, that is, chalcogen bonds D2Ch•••A− become stronger as the chalcogen Ch varies from S to Se, the halide A− varies from Cl− to F− and the substituents D from F to Cl. SSB‐D and SSB‐D3(BJ) are exceptions, whereby the ChB becomes more stabilizing when D varies from Cl to F (see Table S4 in the Supporting Information). The main trends in bond lengths and angles are also in line with the ab initio methods where the D2Ch•••A− chalcogen bond becomes longer as Ch varies from S to Se and as A− varies from F− to Cl− and shorter as D varies from F to Cl (see Tables S6 and S7; for optimized Cartesian coordinates see Tables S12‐S27 in the Supporting Information). In general, we find that the density functionals give longer chalcogen bonds and bigger bond angles Θ2 (Scheme 1) compared to our best level ZORA‐CCSD(T)/BS3+ geometries (see Figure 6). The best overall agreement with our best ab initio level geometries is with the meta‐hybrid M06, M06‐HF, M06‐2X functionals (MAE of 0.006–0.017 Å for bond lengths and MAE of 0.7–1.5 degrees for bond angles). The GGAs BLYP and BLYP‐D3(BJ) perform the worst and have the largest MAEs up to 0.063 Å and 7.2 degrees.</p><p>The mean absolute error (MAE), mean error (ME), and largest deviation (LD) for the 13 density functionals are computed relative to ZORA‐CCSD(T)/BS3+. Three main observations emerge: (i) M06‐2X, B3LYP, and M06 perform the best; (ii) BHANDH, BLYP‐D3(BJ), and BP86 perform the worst; and (iii) all 13 density functionals overestimate the ΔE compared to ZORA‐CCSD(T)/BS3+. The best overall agreement with the ab initio benchmark is with the meta‐hybrid functionals, M06‐2X and M06 (MAE of 4.1–4.3 kcal mol−1 and LD of 6.6–6.8 kcal mol−1) and by the popular B3LYP hybrid functional (MAE 4.2 kcal mol−1 and LD of 6.4 kcal mol−1) (see Figure 6(c)). GGAs perform the worst and have the largest MAEs up to 9.3 kcal mol−1. BLYP is the best GGA with a MAE of 6.9 kcal mol−1 and LD of 8.6 kcal mol−1. Addition of an explicit dispersion correction (D3) and damping function (BJ) for the BLYP and B3LYP functionals results in less accurate ΔE values and increases the MAE to 8.5 and 5.7 kcal mol−1, respectively.</p><p>The ME is negative, and its absolute value is equal to the MAE for all density functionals, that is, the stabilization of the D2Ch•••A− chalcogen‐bonded complexes is overestimated by all functionals in this study. Nevertheless, our best performing density functionals together with the Slater‐type QZ4P basis set have the same trends in chemical stability and geometry as our ZORA‐CCSD(T)/BS3+ benchmark, with relatively small deviations from the ab initio ΔE CPC. For larger chalcogen‐bonded systems, the smaller Slater‐type TZ2P basis set may be used, which also provides satisfactory results in comparison with our best ab initio level. For our three‐best density functionals, B3LYP, M06‐2X, and M06, the ΔE is ca. 2 kcal mol−1 more over‐binding for TZ2P than for QZ4P (see Table 6), that is, the overestimation on the stability of chalcogen‐bonded systems increases. This results in larger errors relative to our best estimate and the B3LYP, M06‐2X, and M06 density functionals in combination with TZ2P basis set turn out to have similar accuracy as the ZORA‐BLYP/QZ4P. Thus, we identify not only B3LYP and M06‐2X, 7i but also M06, in combination with the all‐electron QZ4P basis set, to be reasonable approaches for computing the complexation energies of chalcogen bonds without relying on expensive ab initio methods.</p><!><p>We have computed a ZORA‐CCSD(T)/BS3+ benchmark for the archetypal chalcogen‐bonded model complexes D2Ch•••A− (Ch = S, Se; D, A = F, Cl) that derives from a hierarchical series of relativistic ab initio methods and basis sets. The counterpoise‐corrected ZORA‐CCSD(T)/ma‐ZORA‐def2‐QZVPP level is converged within 1.5–3.1 kcal mol−1 and 1.1–3.4 kcal mol−1 with respect to the basis set size and ab initio method, respectively. Our benchmark data show that chalcogen bonds (ChB) in D2Ch•••A− become stronger for the heavier chalcogen Ch, the lighter halide A−, and for the less electronegative halogen substituent D.</p><p>Basis sets including diffuse functions are required for the calculation of accurate complexation energies for the chalcogen‐bonded complexes D2Ch•••A− involving anions. Addition of diffuse functions yields smaller BSSE and faster convergence with respect to the basis set size and ab initio method. However, as the BSSE simultaneously decreases as the flexibility of the basis set size increases, the uncorrected and counterpoise‐corrected complexation energies become similar for larger basis sets, with or without diffuse functions. Coulomb correlation is also crucial, and, for highly correlated methods, addition of polarization functions is necessary to accurately describe the correlation hole.</p><p>The performance of 13 relativistic (ZORA) density functionals for describing the complexation energies of ChB was evaluated. Best agreement with our hierarchical ab initio benchmark is achieved by hybrid and meta‐hybrid DFT functions, which overestimate the bond strength with mean absolute errors up to 4.3 kcal mol−1. Neither GGA nor meta‐GGA DFT approaches can achieve this accuracy. The BLYP functional, which is the best performing GGA approach, overestimates complexation energies by 6.9 kcal mol−1. Taken altogether, M06‐2X and M06 and B3LYP in combination with the all‐electron QZ4P basis are accurate, efficient, and non‐expensive methods for the routine investigation of chalcogen bonds.</p><!><p>Table S1 Number of relativistically contracted basis functions for ZORA‐def2‐ basis sets without (BS) and with (BS+) diffuse functions for F, S, Cl and Se elements.</p><p>Table S2. Ab initio bond lengths and angles (in Å and degrees) of D2S∙∙∙A− chalcogen‐bonded complexes.</p><p>Table S3. Ab initio bond lengths and angles (in Å and degrees) of D2Se∙∙∙A− chalcogen‐bonded complexes.</p><p>Table S4. Complexation energies (in kcal mol−1) of D2Ch∙∙∙A− chalcogen‐bonded complexes.</p><p>Table S5. The mean error (ME), mean absolute error (MAE), and largest deviation (LD) of ZORA‐DFT/QZ4P approaches relative to the geometries (in Å and degrees) and counterpoise corrected complexation energies (in kcal mol−1) of D2Ch∙∙∙A− complexes computed at ZORA‐CCSD(T)/BS3 + .</p><p>Table S6. Representative DFT bond lengths and angles (in Å and degrees) of D2S∙∙∙A− chalcogen‐bonded complexes.</p><p>Table S7. Representative DFT bond lengths and angles (in Å and degrees) of D2Se∙∙∙A− chalcogen‐bonded complexes.</p><p>Table S8. Thermodynamic values (in kcal mol−1 at 298 K) associated with formation of D2S∙∙∙A− chalcogen‐bonded complexes for representative methods.</p><p>Table S9. Thermodynamic values (in kcal mol−1 at 298 K) associated with formation of D2Se∙∙∙A− chalcogen‐bonded complexes for representative methods.</p><p>Table S10. Cartesian coordinates, electronic energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐CCSD(T) with ZORA‐def2 basis sets in the gas phase using ORCA.</p><p>Table S11. Cartesian coordinates, electronic energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐CCSD(T) with ma‐ZORA‐def2 basis sets in the gas phase using ORCA.</p><p>Table S12. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐B3LYP/QZ4P in the gas phase using ADF.</p><p>Table S13. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐B3LYP‐D3(BJ)/QZ4P in the gas phase using ADF.</p><p>Table S14. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐BHANDH/QZ4P in the gas phase using ADF.</p><p>Table S15. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐BLYP/QZ4P in the gas phase using ADF.</p><p>Table S16. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐BLYP‐D3(BJ)/QZ4P in the gas phase using ADF.</p><p>Table S17. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐BP86/QZ4P in the gas phase using ADF.</p><p>Table S18. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐M06/QZ4P in the gas phase using ADF.</p><p>Table S19. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐M06‐HF/QZ4P in the gas phase using ADF.</p><p>Table S20. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐M06‐L/QZ4P in the gas phase using ADF.</p><p>Table S21. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐M06‐2X/QZ4P in the gas phase using ADF.</p><p>Table S22 Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐PBE/QZ4P in the gas phase using ADF.</p><p>Table S23. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐SSB‐D/QZ4P in the gas phase using ADF.</p><p>Table S24. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐SSB‐D3(BJ)/QZ4P in the gas phase using ADF.</p><p>Table S25. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐B3LYP/TZ2P in the gas phase using ADF.</p><p>Table S26. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐M06/TZ2P in the gas phase using ADF.</p><p>Table S27. Cartesian coordinates, bonding energies, H, TS, and G (in a.u. at 298 K) for all stationary points computed at ZORA‐M06‐2X/TZ2P in the gas phase using ADF.</p><p>Click here for additional data file.</p>

PubMed Open Access

Sugar recognition by CscB and LacY

The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H+ symporter sub-family of the Major Facilitator Superfamily, and both catalyze sugar/H+ symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C3-OH of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of the CscB is directed towards the fructofuranosyl moiety of substrate, while the specificity of LacY is directed towards the galactopyranosyl moiety.

sugar_recognition_by_cscb_and_lacy

<!>Materials<!>Expression Analysis<!>Transport Assays<!>MIANS labeling<!>Sucrose or fructose transport by CscB<!>Lactulose transport by CscB or LacY<!>DISCUSSION

<p>Bacterial sugar transporters homologous to the lactose permease of Escherichia coli (LacY) belong to the oligosaccharide/H+ symporter (OHS) sub-family of the Major Facilitator Superfamily (MFS) (1), and LacY is the best characterized member. Like LacY, other members of the OHS are believed to catalyze the coupled translocation of a sugar and an H+ (sugar/H+ symport). These proteins are also likely to have a structure similar to that of LacY (2, 3) with twelve mostly irregular transmembrane α-helices that transverse the membrane in a zigzag fashion connected by hydrophilic loops with both N- and C-termini on the cytoplasmic face and a large water-filled cavity facing the cytoplasm (4–6).</p><p>The second most well studied symporter in the OHS is the sucrose permease of E. coli (CscB), encoded by the cscB gene, which transports sucrose, but not lactose or other galactopyranosides (7, 8). Another OHS symporter, the melibiose permease of Enterobacter cloacae (MelY), which has high sequence similarity with LacY, does not transport methyl-1-thio-β-d-galactopyranoside, a good substrate for LacY (9), while both proteins recognize melibiose and lactose as substrates. In any case, CscB exhibits 28% sequence identity with LacY and an overall homology of 51% (2, 10). Most of the irreplaceable residues in LacY with respect to activity are conserved in CscB, and site-directed mutagenesis confirms their importance (3, 11, 12). Moreover, homology threading of CscB with the LacY crystal structure as a template, as well as functional studies of site-directed mutants in CscB, predicts similar organization of the sugar- and H+-binding sites (2, 3). Glu126 (helix IV), Arg144 (helix V) and Trp151 (helix V) in LacY, which are directly involved in sugar binding, are homologous with Asp129, Arg147 and Tyr154 in CscB. Although Glu270 is one helix turn closer to the cytoplasmic side of CscB than the homologous residue Glu269 (helix VIII) in LacY, the functional role appears to be the same (3). In addition, the spatial organization of the residues involved in H+ translocation in LacY is almost identical in CscB, as judged from homology modeling (2, 3).</p><p>There are many bacterial genes encoding proteins with significant similarities to LacY and CscB although their expression and transport specificities have yet to be studied (2). LacY specifically recognizes d-galactose and the d-galactopyranosyl moiety of its disaccharide substrates, but has no affinity for d-glucopyranosides or d-glucose (13–16). Therefore, it was assumed that the specificity of CscB would be directed at the d-glucopyranosyl moiety of sucrose. To test this notion, we identified several sugars that might hypothetically be transported by CscB and found that CscB catalyzes transport of not only sucrose but also lactulose and fructose (Fig. 1). Moreover, d-glucopyranoside has no detectable affinity for CscB. Thus, the specificity of CscB is directed toward the fructofuranosyl ring of sucrose and not the glucopyranosyl moiety. In addition, the C3-OH group on the fructofuranosyl ring appears to be important for recognition. Finally, lactulose is an excellent substrate for LacY, as predicted from its specificity for the galactopyranosyl moiety of its multiple substrates.</p><!><p>Fructose, glucose, sucrose, lactulose, turanose, and palatinose were of the highest available grade and purchased from Sigma-Aldrich. Octyl-α-d-galactopyranoside was from Carbosynth Limited (UK). [U-14C]sucrose was purchased from Perkin Elmer (Boston, MA), D-[U-14C]fructose from Moravek Biochemicals (Brea, CA), and [galactose-6-3H]lactulose from American Radiolabeled Chemicals, Inc (St. Louis, MO). DNA plasmid purification kits and Penta-His antibody-horseradish peroxidase (HRP) conjugate was obtained from QIAGEN (Valencia, CA). The Supersignal West Pico Chemiluminescent substrate kit was from Pierce Inc (Rockford, IL).</p><!><p>Plasmids pSP72/CscB and pT7-5/LacY constructs were engineered to encode the appropriate permease with a C-terminal 6-His-tag to enable identification of protein expression by Western blot analysis. Both permeases CscB and LacY were expressed to similar levels in the membrane of E. coli as detected by using penta-His HRP conjugated antibody and Supersignal West Pico Chemiluminescent substrate.</p><!><p>E. coli T184 [lacI+O+Z−Y−(A), rspL, met−, thr−, recA, hsdM, hsdR/F' lacIqOZD118(Y+A+)] was transformed with the appropriate expression vector and grown aerobically overnight at 37 °C in Luria-Bertani culture medium containing 100 μg/ml of ampicillin. A ten-fold dilution of the culture was grown for 2 h before induction with 1mM IPTG. Following induction, growth was continued for further 2 h, after which the cells were harvested by centrifugation, washed with 100 mM potassium phosphate (KPi; pH 7.0)/10mM MgSO4 and adjusted to an absorbance at 420 nm (A420) of 20 (approximately 1.4 mg/ml of protein) for transport measurements. Transport of a given radiolabeled sugar was assayed at room temperature in the absence or presence of given unlabeled sugars by rapid filtration as described (12, 17). Transport was initiated by addition of 2 μl radiolabeled sugar to 50 μl aliquots of cells containing 70 μg of total protein and stopped by dilution followed by rapid filtration.</p><p>Rates of transport at various substrate concentrations were measured by mixing 50 μl aliquots of cells with 50 μl of radiolabeled sugars and stopped after 1 min incubation at room temperature. Total level of radioactivity was maintained constant in samples with different sugar concentrations. The rates of transport were estimated after correction for sugar uptake by cells carrying vector without inserted transporter. Data were analyzed by using the Michaelis-Menten equation (18) and Sigmaplot 10 (Systat software).</p><!><p>Apparent affinity of purified LacY for galactosidic sugars was measured by substrate protection of Cys148 against alkylation by 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) as the effect of sugar concentration on the initial rate of MIANS labeling as described (19, 20). Fluorescence change was monitored at room temperature using SLM-Aminco 8100 spectrofluorimeter (Urbana, IL) modified by OLIS, Inc. (Bogart, GA) with excitation and emission wavelengths of 330 nm and 415 nm, respectively. Data fitting was carried out by using SigmaPlot 10 (Systat Software Inc., Richmond, CA).</p><!><p>E. coli overexpressing CscB catalyze transport of either sucrose or fructose, while cells transformed with vector devoid of cscB exhibit essentially no transport of either sugar (Fig. 2 A&C). Sucrose transport by cells expressing CscB increases at a rapid rate for ~5 min and reaches a steady-state level of ~140 nmol/mg of protein within ~20 min (Fig. 2A). In contrast, the same cells catalyze fructose transport at a relatively low rate to a steady-state level of ~20 nmol/mg (Fig. 2C). Rates of transport as a function of sucrose or fructose concentration follow a hyperbolic relationship with a Km for sucrose of 6.7 mM and a Vmax of 130 nmol/min·mg protein (Fig. 2B); fructose transport exhibits a Km of 36 mM and a Vmax of 60 nmol/min·mg protein (Fig. 2D). Transport of neither sucrose nor fructose by cells overexpressing LacY is observed (Fig. 2 A&C, filled triangles).</p><p>Sucrose transport by CscB is not inhibited to any degree whatsoever by glucose since kinetic parameters of transport remain unchanged even in the presence of 30 mM glucose (Fig. 3). Moreover, sugars devoid of a fructose moiety, which include galactose, lactose, melibiose, mannose, rhamnose or ribose, have no effect on sucrose transport (data not shown). The results indicate that the specificity of CscB is directed toward the fructofuranosyl ring of substrate. In order to further evaluate the importance of the fructose moiety, different fructofuranosides were tested for their ability to inhibit the sucrose transport by CscB (Fig. 4). The sugars tested include turanose (3-O-α-d-glucopyranosyl-D-fructose), lactulose (4-O-β-D-galactopyranosyl-D-fructose), and palatinose (6-O-α-D-glucopyranosyl-D-fructose). While 10 mM palatinose or lactulose inhibit sucrose transport, albeit relatively weakly, turanose has no effect on the transport, thereby providing suggestive evidence that the C3-OH group on the fructofuranosyl ring may be important for recognition by CscB. Detailed analysis reveals that fructose is a competitive inhibitor of sucrose transport with a Ki of 30 – 50 mM (Fig. 4 B), which is in the same range as the Km for fructose transport (Fig. 2 D).</p><!><p>E. coli overexpressing CscB catalyze transport of lactulose (Fig. 5). Accumulation of this disaccharide occurs at a relatively slow rate and it does not reach steady state even by 60 min at 20 mM lactulose. In contrast, E. coli overexpressing LacY catalyze transport of lactulose at a rapid rate to a steady-state level of ~85 nmol/mg protein in approximately 3 min (Fig. 6A). Kinetic parameters for lactulose transport by LacY estimated from concentration dependence of initial rate (Fig. 6B) are: Km = 0.24 mM and a Vmax = 49 nmol/min·mg protein. These values are similar to those obtained for transport of lactose by LacY (21).</p><p>As shown previously by substrate protection against alkylation of Cys148 in LacY, galactose or lactose exhibit apparent affinities of 50 or 9 mM, respectively (15), while β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) or 4-nitrophenyl-α-D-galactopyranoside (NPG), exhibit apparent affinities of 0.85 or 0.03 mM, respectively (20). By using the same method, the apparent affinity of LacY for lactulose (Kdapp) is estimated ~8 mM (Table 1), a value approximately the same as that observed for lactose.</p><!><p>Although there is a clear similarity in the structural organization of key residues involved in sugar binding in LacY and CscB (2, 3), as shown here, there is an unexpected and surprising difference in substrate recognition between the two symporters. Thus, CscB catalyzes transport of sucrose, fructose or even lactulose, but exhibits no recognition of glucopyranosides, glucose in particular, as evidenced by the inability of glucose to inhibit sucrose transport. Taken together, the results lead to the conclusion that the specificity of CscB is directed toward the fructofuranosyl moiety of sucrose. In contrast, extensive studies demonstrate that the specificity of LacY is directed toward the galactospyranosyl moiety of substrate, and the C4-OH plays the predominant role by far in recognition and binding (22, 23). The monosaccharide galactose is the most specific substrate for LacY, although it binds with very low affinity (13, 14, 23), while galactopyranosides in the α configuration with anomeric substitutions, particularly those that are hydrophobic, exhibit increased affinity with little or no effect on specificity (16, 23).</p><p>Asp129 and Arg147 in CscB, which are positioned similarly to Glu126 and Arg144 in LacY respectively, probably also play a direct role in sugar recognition and binding, and Tyr154 in CscB, which is homologous to Trp151 in LacY, likely stacks hydrophobically with the fructofuranosyl ring of sucrose (2, 3). Glu270 in CscB, although positioned one helix turn closer to the cytoplasmic side of CscB than Glu269 in LacY, is essential and is probably also important for substrate recognition and binding. Although replacement of Ser151 (homologous to Cys148 in LacY) with Cys causes CscB to become highly sensitive to N-ethylmaleimide in a manner similar to that of LacY, substrate affords no protection whatsoever against inactivation of transport or alkylation with CscB (12). Thus, the overall architecture of the substrate binding sites appears to be conserved in CscB and LacY, but there are important differences in detailed interactions, and the fructofuranosidyl moiety of sucrose likely occupies a position homologous to that of the galactopyranosyl moiety of lactose in LacY (Fig. 7).</p><p>Inhibition studies with several fructofuranosides (Fig 4) provide some evidence that the C3-OH of the fructose moiety in sucrose is important for binding. In lactulose and palatinose, the galactose moiety is attached to the C4 and C6 atoms of the fructose moiety, respectively. These fructofuranosides partially inhibit sucrose transport by CscB, suggesting that the C4-OH and C6-OH groups are not important for binding. On the other hand, in turanose, the galactose moiety is attached to the C3 atom of fructose moiety, and turanose does not inhibit sucrose transport, thereby suggesting that the C3-OH is likely an important player in the interaction of the fructose moiety with CscB.</p><p>Transport of lactulose is catalyzed by CscB, but at a low rate. In complex with sugar binding proteins lactulose is in an extended, planar conformation with respect to the galactose and fructose moieties, which are in the β-configuration (24). In contrast, sucrose is in a bent conformation with the glucose and fructose rings positioned at approximately a right angle (PDB ID 1AF6, 1PT2, 1IW0). Therefore, in order for the C3-OH group of the fructose moiety in a disaccharide to be maximally accessible in the sugar-binding site of CscB, the anomeric ring may need to be attached to fructose moiety in such a manner that the C3-OH is readily accessible. With lactulose, the galactose ring may sterically interfere with the accessibility of the C3-OH of the fructose moiety, making it a relatively poor substrate for CscB. Therefore, it is clear that high-resolution crystal structures with bound substrates are needed to identify critical contacts of side chains with ligands that determine binding specificity.</p><p>In contrast to CscB, lactulose is an excellent substrate for LacY. In lactulose, the galactose moiety is bonded to fructose by a β-1,4 glycosidic bond, making the OH groups at each position on the galactose moiety as accessible as in lactose. Moreover, both protein-bound lactose (PDB ID 1DLL, 1ULC) and lactulose are in extended conformations. Although the C4-OH is the major determinant for recognition and binding by LacY, each OH group makes a contribution to binding affinity (16). Thus, it is not surprising that lactulose is a good substrate for LacY.</p>

PubMed Author Manuscript

Coal-based 3D hierarchical porous carbon aerogels for high performance and super-long life supercapacitors

Coal-based 3D hierarchical porous carbon aerogels (3D HPCAs) has been successfully fabricated from a freeze-drying method and with subsequent of calcination process, using coal oxide as carbon precursors, and PVA as both cross-linking agent and sacrifice template. The 3D HPCAs, using as electrode materials for supercapacitors, display outstanding electrochemical performance. The optimal sample (HPCAs-0.4-800) presents a high specific capacitance of 260 F g −1 at 1 A g −1 , and exhibits considerable rate capability with the retention of 81% at 10 A g −1 . Notably, HPCAs-0.4-800 shows an excellent cycling stability with 105% of the capacitance retention after 50000 cycles at 10 A g −1 , attributing to its unique hierarchical porosity, high surface area up to 1303 m 2 g −1 , and improved conductivity. This work offers a promising route to synthesize coal-based porous carbon aerogels electrode materials for supercapacitors. Supercapacitors (SCs), also called ultracapacitors or electrochemical capacitors, have caused a large amount of interest owing to excellent electrochemical stability, fast charge/discharge, high power density and environmental friendly [1][2][3][4][5] . Supercapacitors store electrical charge on high-surface-area conductive materials, so its performance mainly relies on the electrode materials. Outstanding electrode materials should possess ion approachable high surface areas for high specific capacitance and fasted electron transfer for excellent rate capacity 6,7 . So it is very crucial for supercapacitors with high performance to prepare electrode materials with proper architecture structure, suitable pore size distribution and high specific surface area (SSA) 8 . Among the numerous electrode materials of supercapacitors, carbon materials have attracted more attention because of their unique physical and chemical properties 9,10 . Carbon aerogels (CAs), as one of carbon materials, show outstanding characteristics, such as low density, developed porosity, and multi-branched network structure [11][12][13] . These structural features can afford the quick transfer channel for ion migration and more active sites, which can lead to the excellent electric double layer performance in supercapacitors. To improve the specific surface area and porosity, most of the CAs are prepared by using pore-forming agents, such as strong bases [14][15][16] , hard templates [17][18][19] , soluble salts 20,21 , soft templates and so on [22][23][24] . Among of them, the soft templates can be directly decomposed during the carbonization process instead of etching procedure using harmful and toxic or corrosive chemicals. Therefore, it has been attracting extensive attention to prepare CAs using soft template for the application of supercapacitors.Currently, the researches of CAs are mainly focused on precursors, such as resorcinol-formaldehyde 25 , polymers 26 , nanotubes 27,28 , graphene 29,30 , and natural precursors such as cellulose and glucose 31,32 . In our previous works, we have fabricated some functional materials on coal of traditional fossil, such as porous spheres 33 , fibers 34 , bamboo-like carbon nanotubes (CNTs) 35 , graphene quantum dots (GQDs) 36 and hierarchical porous carbon 37 . All of them demonstrate that the coal can be used to fabricate functional carbon materials. However, so far, coal-based porous carbon aerogels have been few reported and the preparation processes were very complicated and the yield was low in a few studies. Therefore, it is still a great challenge to design simple and productive approaches for the controllable synthesis coal-based porous carbon aerogels.

coal-based_3d_hierarchical_porous_carbon_aerogels_for_high_performance_and_super-long_life_supercapa

<!>Results and discussion<!>Conclusion<!>Experiment Section<!>Electrochemical characterization.<!>S

<p>In our work, we developed an efficient method to construct coal-based 3D HPCAs by carbonization of freezing-dried PVA/coal-based hydrogels, in which coal oxide serves as the carbon source and PVA serves as the sacrificial template and cross-linking agent, respectively. The amount of mesoporous and micropores of the 3D HPCAs can be controlled by tuning the mass ratio of coal oxide and PVA. The performance of the obtained 3D HPCAs are evaluated as the electrode materials of supercapacitors. The optimal sample displays an excellent electrochemical performance. It exhibits a specific capacitance up to 260 F g −1 in the three-electrode system at 1 A g −1 , and a high rate performance of 187 F g −1 at 20 A g −1 , as well as a remarkable cycling stability (105% of capacitance retention after 50000 cycles). More importantly, the specific capacitance measured was 201.1 F g −1 at the current density 1 A g −1 in an assembled symmetrical cell system, and good cycling stability with 108% over 10000 cycles at 4 A g −1 . The excellent electrochemical performance may be attributed to the characteristic of 3D cross-linked structure with SSA up to 1303 m 2 g −1 , hierarchical porous structure and appropriate ratio of micropore volume to total volume of 65.6%. The materials with hierarchical porous structure can be used as potential electrode materials for energy conversion and storage, and this work provides a green way for high-value utilization of coal in energy storage.</p><!><p>The synthesis procedure of the 3D HPCAs is illustrated in Fig. 1. Firstly, PVA/coal-based hydrogels were prepared by using PVA as a crosslinking agent of coal oxide fragment, and then the 3D network porous structures were formed through a freeze-drying and with subsequent of calcination. To understand the role of PVA and coal oxide in 3D HPCAs, thermogravimetric (TG) analyses of PVA and coal oxide were studied (Fig. S1). The mass loss of PVA is 96.6% of initial weight from 260 °C to 490 °C, and 98.8% when heated to 800 °C, while that of coal oxide is about 62.6% when heated up to 800 °C under an argon atmosphere. The results demonstrate that coal oxide is the primary carbon source in the 3D HPCAs, while PVA is the cross-linking agent for formation of hydrogels and the sacrifice template to fabricate 3D network porous structures.</p><p>This result is also directly proved by the optical and SEM images of the pure oxidized coal-800, prepared by the same proceduce only without addition of PVA. As shown in Fig. S2a,b, the pure oxidized coal-800 shows powder shape but non-aerogel on the macro level, and block shape on the micro level, with dense surface and no obvious macropores and mesopores. The morphology of fabricated 3D HPCAs were directly observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Fig. 2). The SEM images show that the pore structure of 3D HPCAs changed obviously as the increase of PVA content. Consistent well with SEM images, the TEM images of HPCAs-0.4-800 display a richer porous structure and more even pore size distribution obviously. The results demonstrate that an appropriate amount of PVA is crucial for the formation of the pore structure of 3D HPCAs. Coal oxide will stack seriously when crosslinking dose of PVA is excessive, on the contrary, too little sacrificial dose of PVA is not conducive for the formation of holes during the carbonization process. In addition, the influence of calcination temperature on the pore structure of 3D HPCAs was also studied. As shown in Fig. S3, the pore size of HPCAs-0.4-700 and HPCAs-0.4-900 increase due to the change of carbonized temperatures. The broken mesopores can be observed in the HPCAs-0.4-900 due to the skeleton collapse at high carbonization temperature.</p><p>The effect of different components and calcination temperatures on the structure of the HPCAs was investigated by X-ray powder diffraction (XRD) (Fig. 3). XRD patterns of 3D HPCAs all display two weak peaks located at 24° and 43°, associated with diffraction of the (002) and (101) planes carbon. Compare with the peaks of coal oxide (Fig. S4), the peaks around 23° of the HPCAs shift up to a relatively high angle, indicating that the degree of graphitization is reduced during the process of carbonization [39][40][41] . In addition, the HPCAs-0.4-800 displays the greatest peak intensity at 23° among of all HPCAs, indicating that the HPCAs-0.4-800 has the highest degree of graphitization associated with conductivity. Raman spectra are shown in Fig. 3b and S3, two obvious peaks around 1350 and 1590 cm −1 are attributed to D and G bands. The D peak is attributed to the sp 3 defects of disordered ones in the hexagonal graphitic layers and sp 2 carbon with O-containing groups and H-sites, as well as domain boundary. The G peak reflects the vibration of sp 2 -bonded carbon atoms [42][43][44] . The I D /I G values of HPCAs-0.4-700, HPCAs-0.2-800, HPCAs-0.4-800, HPCAs-0.8-800, HPCAs-0.4-900 and coal oxide are 0.99, 0.97, 0.87, 0.90, 0.97 and 0.81 respectively. Compared HPCAs with coal oxide (Fig. S4), the HPCAs show higher values of I D /I G , because the surface carbon atoms of HPCAs were carried off during the activation operation, thus, leaving the free bond at the surface and forming a disordered carbon structure 1,45,46 . In addition, the HPCAs-0.4-800 has www.nature.com/scientificreports www.nature.com/scientificreports/ the lowest I D /I G value in the samples of different calcination temperatures, which likely resulted from the reduction reaction at high temperature, on the other hand, the defects of materials increase as the structure collapse at a too high carbonization temperature (900 °C). And among the samples of HPCAs-0.2-800, HPCAs-0.4-800, and HPCAs-0.8-800, HPCAs-0.4-800 also exhibits the highest graphitization degree, which acts as a key role in improving the conductivity. This is because excessive PVA decomposition at high temperatures causes the pore structure to collapse, thus forming an amorphous carbon. The above results prove that appropriate carbonization temperature and PVA content are crucial to the pore structure and the degree of graphitization, which finally reflected in its electrochemical performance.</p><p>The XPS spectra were carried out to evaluate the surface atomic composition of HPCAs-0.4-800. Fig. S5 shows the XPS spectra of samples. The survey spectrum confirmed the existence of C, O, and N elements in the sample of HPCAs-0.4-800 (Fig. S5a). The results are consistent with the FT-IR spectra data analysis reported in our previous work 38 . The N and O in samples are mainly come from the raw coal and nitric acid and sulfuric acid used in the oxidation process. The high-resolution spectrum of C 1s (Fig. S5b) can be divided into three peaks at 288.6, 286.2 and 284.5 eV, which are assigned to C=O, C-O and C-C, respectively 47,48 . The O 1s spectrum (Fig. S5c) consists of four peaks located at 530.9, 531.5, 532.4 and 533.5 eV, which corresponding to the carbonyl groups (C=O), bridge-bonded oxygen (C-O-C), ester groups (O-C=O) and chemisorbed oxygen or water (COOH carboxylic groups or water), respectively. The high resolution spectrum of N 1s (Fig. S5d) shows three peaks with binding energy values of 398.1, 400.5 and 404.3 eV for the pyridinic-N, pyrrolic-N and oxidized-N, respectively 49,50 . The elemental contents (atomic%) of the HPCAs-0.4-800 obtained from XPS data are presented in Table S1. HPCAs-0.4-800 has high contents of oxygen and nitrogen, with a ratio of 7.27% and 1.6%, respectively, which can ameliorate the wettability between the electrode material surface and electrolytes, further facilitate the immersion of electrolyte into the interior of the electrode materials, and ultimately reflect in high capacitance performance. Besides, the pyridinic-N and pyrrolic-N can introduce the faradaic pseudocapacitance in aqueous electrolytes and thus also enhance electrochemistry capacitance 51 .</p><p>The electric double layer capacitor (EDLC) is a surface regulated phenomenon, so a larger surface area is crucial for acquiring a high capacitance 52 . The N 2 adsorption-desorption isotherms and the pore distribution of samples with the different mass ratios of coal oxide/PVA are presented in the Fig. 4a-f. The isotherms of the 3D HPCAs show classical type-IV curves with an H4 type hysteresis loop in the relative pressure region between 0.45 and 1.0, suggesting that the existence of a silt-shaped pore structure. Compared with the pure oxidized coal-800 (Fig. S2c), the volume of adsorbed N 2 increase steeply at relatively low pressure, which stands for the existence of a large number of micropores, and the remarkable hysteresis loop between N 2 adsorption and desorption branch manifests the existence of mesopores. It is well known that micropores and mesopores are in favor of the improvement of charge storage and ion transport, respectively 53 . DFT pore-size-distribution curves show that the HPCAs have broad micropores size distribution (0.65-2 nm) and a narrow mesopores size distribution (2-10 nm). The sizes of micropores are close to the size of hydrolyzed K + ions (0.331 nm), which are beneficial for their capacitive performance 54 . The data of S BET and pore distribution of 3D HPCAs are shown in Table 1. S BET of HPCAs-0.2-800, HPCAs-0.4-800, HPCAs-0.8-800 are 1018, 1303, and 847 m 2 g −1 , respectively. Distinctly, the contribution of micropores to S BET of HPCAs-0.4-800 (66%) was greatest among all samples (44% to HPCAs-0.2-800 and 55% to HPCAs-0.8-800). In general, the large specific surface area of micropores can result in a high capacity 6,55 . The N 2 adsorption-desorption isotherms and the pore distribution of samples with different calcination temperatures (HPCAs-0.4-700 and HPCAs-0.4-900) are shown in Fig. S6. They show similar types of type-IV curves with HPCAs-0.4-800. The effects of the calcination temperature on distribution of hierarchical porous have also been studied, as shown in Table S2. The S BET of HPCAs was 971, 1303, and 900 m 2 g −1 at 700, 800, and 900 °C, respectively. The contribution of micropores to S BET of HPCAs-0.4-800 (66%) was also larger than that of HPCAs-0.4-700 (54%) and HPCAs-0.4-900 (46%). This is because the high temperature promotes the decomposition of the large amounts of coal oxide and PVA, leading to achieve abundant pores, but too high a temperature (900 °C) will cause the stacking of carbon layers and/or generation of isolated pores, reflecting a smaller S BET and the amount of micropores. Such result is related to their electrochemical performances.</p><p>The electrochemical performances of 3D HPCAs were evaluated through CV curves at the scan rate of 50 mV s −1 and GCD curves at the current density of 1 A g −1 (Fig. 5a,b). All 3D HPCAs electrode materials display similar rectangles, suggesting that the energy storage type of the 3D HPCAs are the electric double layer capacitor (EDLC). The values of specific capacitance are 232, 260, 218, 210 and 184 F g −1 at the current density of 1 A g −1 for HPCAs-0.2-800, HPCAs-0.4-800, HPCAs-0.8-800, HPCAs-0.4-700 and HPCAs-0.4-900, respectively, which is all are superior to that of pure oxidized coal-800 (35.7 F g −1 Fig. S2e). The 3D HPCAs-0.4-800 exhibits the highest specific capacitance among all the electrode materials due to advantages of the large accessible surface area, more available mesoporous channels and advisable proportion of micropore volume to total volume. As shown in the Fig. 5c,d, specific capacitances of HPCAs-0.4-800 obtained from the discharge curve are 267, 260, 242, 224 and 210 F g −1 at the current density of 0.5, 1, 2, 5 and 10 A g −1 . On account of the insufficient surface contact and hindrance of ions diffusing into the internal pores, the capacitance decreases as the current density increased 56 . The calculated specific capacitances of the HPCAs at different current densities are presented in Fig. 5e. The 3D HPCAs-0.4-800 electrode material displays the specific capacitance of 260 F g −1 at 1 A g −1 , which is higher than HPCAs-0.2-800, HPCAs-0.8-800, and the commercial activated carbon (Kuraray YF-50, 87 F g −1 ) (Fig. S7). Such result is probably ascribed to that sufficient micropores and mesopores channel to be used. The specific capacitance of HPCAs-0.4-800 is 210 F g −1 at 10 A g −1 , which is about 80.7% of the capacitance retention of 260 F g −1 at 1 A g −1 . However, when the current density increases from 1 A g −1 to 10 A g −1 , HPCAs-0.2-800 and HPCAs-0.8-800 have the only 74.8% and 78.9% capacitance retention, respectively. HPCAs-0.4-800 manifests a considerably better rate performance than HPCAs-0.2-800 and HPCAs-0.8-800 samples, because of HPCAs-0.4-800 have uniformed and well-interconnected hierarchical porous structure. On the other hands, compared to the samples of HPCAs-0.4-700 and HPCAs-0.4-900, HPCAs-0.4-800 has the highest SSA and the suitable ratio of micropore volume to total volume of 65.6%, which is good for charge storage. Therefore, it has the highest specific capacitance.</p><p>Electrochemical impedance spectroscopy (EIS) were measured to compare the electrochemical kinetics of the samples. Nyquist plots of HPCAs electrode materials consist of a vertical line and a semicircle at the low Table 1. BET Specific surface area and porous structure of HPCAs-0.2-800, HPCAs-0.4-800 and 800. a BET surface area. b The total pore volume at P/P o = 0.99, c The mesopore volume calculated using the BJH method based on the Kelvin equation. d Micropore surface area calculated using the V-t plot method. e Average pore size (4V t /S BET ). (2020) 10:7022 | https://doi.org/10.1038/s41598-020-64020-5</p><p>www.nature.com/scientificreports www.nature.com/scientificreports/ frequency and the high frequency area, respectively (Fig. 5f, and S8c). The intercept at the real axis of high frequency of all electrodes are nearly the same, indicating their similar ohmic resistance (R s ) of 0.5 Ω. A smaller semicircle at the high frequency, reflects a relatively lower charger transfer resistance (R ct ) 57 . The R ct values of HPCAs are 3.1, 2.5, 1.8, 1.5, and 1.1 Ω for HPCAs-0.4-900, HPCAs-0.4-700, HPCAs-0.2-800, HPCAs-0.4-800 and HPCAs-0.8-800, respectively. The HPCAs-0.4-800 has the smallest R ct among three samples of different carbonization temperature. The result is due to that HPCAs-0.4-800 has the highest degree of graphitization and comparatively abundant pore structure. Besides, the R ct decreased with the increase of the PVA content in samples, which is likely attributed to more available mesoporous channels and more N content coming from PVA. On the other hand, all samples show nearly perpendicular to the imaginary axis in the low frequency region, which indicated that the electrolyte ions had the best diffusion ability in electrode structure. And the straight line demonstrates the ideal EDLC behavior of electrode materials 58,59 .</p><p>From the above results, the HPCAs-0.4-800 exhibits the excellent electrochemical property. The specific capacitance performance of HPCAs-0.4-800 is superior to some previously reported porous carbon materials (Table S3). As mentioned above, the excellent capacitive performance of HPCAs-0.4-800 can be ascribed to the following aspects: (i) the carbon yields of coal oxide and PVA are different, which leads to the formation of The stable cycling life is an important factor for the practice application of supercapacitor electrode materials. Fig. 6 shows that the specific capacitance of the HPCAs-0.4-800 still reaches 230 F g −1 at the high density of 10 A g −1 , and superior capacitance is maintained up to 105% after 50000 cycles. What impressed us most is that the capacitance presents an increasing trend during the repeating process of cycling at the high current density of 10 A g −1 . On the basis of the pore distribution, this phenomenon has to do with the porous structure of the electrode materials. To be specific, in the beginning stages, only large pores and mesopores are infiltrated by electrolyte, the micropore structure is not fully utilized due to the thin film on the electrode of supercapacitors 60 . However, the K + hydrated ions can gradually penetrate into the micropores and participate in the establishment of electric double layers 61 . The electrode material of 3D HPCAs-0.4-800 tends to a stable capacitance value due to the full utilization of pores as the cycles increasing. Therefore, the sample has a considerable reversibility and satisfactory cycle stability during the repeated charge-discharge process.</p><p>Simultaneously, the electrochemical performance of the HPCAs-0.4-800 in symmetric cell system was also investigated. Fig. 7a shows that the HPCAs-0.4-800 has rectangular CV curves at different scan rates, indicating distinct capacitive behavior and fine reversibility. Fig. 7b shows the galvanostatic charge-discharge (GCD) curves at different current densities from 1 to 20 A g −1 in the potential range from 0 to 1 V. The HPCAs-0.4-800 shows an excellent specific capacitance of 201.1 F g −1 at 1 A g −1 and 160.0 F g −1 at 20 A g −1 , respectively. And the electrode has a good rate capability with about 80% capactive retention at 20 A g −1 . As shown in Fig. 7c, the HPCAs-0.4-800-based device has a pretty high energy density (7.2 Wh kg −1 at 500 W kg −1 ). Fig. 7d manifests the cycling stability of the HPCAs-0.4-800//HPCAs-0.4-800 cell. The specific capacitance retention up to 108% after 10,000 cycles due to the fully wetting of sufficient pores during the long time charged/discharged process, exhibiting its outstanding stable cycling. To sum up, the excellent electrochemical performances of HPCAs-0.4-800, such as high specific capacitance, and good cycling stability can be attributed to the 3D hierarchical porous and the appropriate microspores and mesopores size distribution as well as improved conductivity.</p><!><p>In summary, we design a low cost and facile strategy to obtain coal-based 3D HPCAs by carbonization of the freeze dried PVA/coal-based hydrogels. The structure and electrical performances of 3D HPCAs are adjusted and optimized by changing the content of PVA and carbonization temperature. Among of all samples, the HPCAs-0.4-800, as an electrode material of supercapacitors, exhibits excellent specific capacitances of 260 F g −1 and 201.1 F g −1 at 1 A g −1 in 6.0 M KOH electrolyte for the three-and two-electrode systems, respectively. It also displays an excellent cycling stability of 105% capacitance retention after 50000 cycles. This work provides a promising route to construct coal-based 3D HPCAs as highly efficient electrodes materials for supercapacitors.</p><!><p>Materials. Coal was obtained from Heishan, Xinjiang, China. The similar analysis of coal has been reported by our group 38 . Polyvinyl alcohol (PVA) (molecular weight is 44.05 MW) purchased from Sigma-Aldrich. H 2 SO 4 (98%), HNO 3 (63%), KOH were analytical grade.</p><p>The preparation of 3D HPCA. Coal oxide was firstly obtained by treating raw coal using a mixed acid (VHNO 3 /VH 2 SO 4 = 1:3) as previously reported by our group 38 . Then PVA/coal-based hydrogels were synthesized as following processes. Firstly, coal oxide (0.8 g), PVA (0.4 g) were dissolved in 10.0 mL deionized water and the pH of the solution was neutralized with the KOH, and then stirred at 80 °C continuously for 12 h. Secondly, the product was frozen in refrigerator (−70 °C) for 24 h and freeze-dried in vacuum for 24 h to obtain the xerogels. Finally, the as prepared xerogel was calcined at 800 °C for 2 h with a heating rate of 5 °C min −1 under flowing N 2 atmosphere for formation of 3D coal-based HPCAs, the sample was called HPCAs-0.4-800. Similarly, HPCAs-0.2-800 and HPCAs-0.8-800 were prepared by the nearly same methods with the only different mass ratios of coal Structural characterization. SEM and TEM images were recorded on field emission scanning electron microscopy (FESEM Hitachi SU-4800) and transmission electron microscopy (TEM, JEM-2100F), respectively. Thermogravimetric analysis (TGA) was tested by using a NETZSCH STA449F3-QMS403C instrument under N 2. XRD measurements were carried out on an X-ray diffractometer (XRD, Bruker D8, using filtered Cu Kα radiation). X-ray photoelectron spectroscopy (XPS) spectra and the Raman spectrum were recorded with a Thermo ESCALAB 250 instrument (Al Ka X-ray source) and a Bruker Senterra spectrometer (532 nm) Raman spectrometer, respectively. SSA and pore size distribution of coal-based 3D HPCAs were determined on Autosorb-IQ, Quantachrome by BET method.</p><!><p>The electrochemical experiments of coal-based 3D HPCAs were tested in the three-electrode system in 6.0 M KOH electrolyte at room temperature, in which the 3D HPCAs use as working electrode, Ag/AgCl and Pt foil (1 cm 2 ) as the reference electrode and counter electrode, respectively. The working electrodes were prepared by mixing 3D HPCAs, carbon black and [Poly (trafluoroethylene)] at a mass ratio of 8:1:1 in absolute ethyl alcohol (the active mass is about 2.0 mg), and then the mixture was pasted on the nickel form, and dried at 60 °C for 12 h. The electrochemical performances of these electrodes were carried out using the CHI 660D electrochemical workstation (Chenhua, China). The current density of 10 A g −1 was applied in cyclic GCD measurements for over 50,000 cycles (the potential is −1.0 -−0.1 V). The specific capacitance (C S ) of the 3D HPCAs electrode materials were calculated via Eq. (1).</p><!><p>Where I, ∆t, ∆V, m is current, discharging time, potential window and the mass of the active material, respectively.</p><p>In two-electrode system, CV and GCD curves were measured using the product in 6.0 M KOH as the electrode (the total mass of the active on two working electrodes is 4.0 mg). The specific capacitance for the single electrode (C sp ) was obtained via Eq. (2). Where E, P are the energy density (Wh kg −1 ), power density (W kg −1 ), respectively.</p>

Scientific Reports - Nature

Facile reduction of graphene oxide suspensions and films using glass wafers

This paper reports a facile and green method for conversion of graphene oxide (GO) into graphene by low-temperature heating (80 °C) in the presence of a glass wafer. Compared to conventional GO chemical reduction methods, the presented approach is easy-scalable, operationally simple, and based on the use of a non-toxic recyclable deoxygenation agent. The efficiency of the proposed method is further expanded by the fact that it can be applied for reducing both GO suspensions and large-scale thin films formed on various substrates prior to the reduction process. The quality of the obtained reduced graphene oxide (rGO) strongly depends on the type of the used glass wafer, and, particularly, magnesium silicate glass can provide rGO with the C/O ratio of 7.4 and conductivity of up to 33000 S*cm −1 . Based on the data obtained, we have suggested a mechanism of the observed reduction process in terms of the hydrolysis of the glass wafer with subsequent interaction of the leached alkali and alkali earth cations and silicate anions with graphene oxide, resulting in elimination of the oxygencontaining groups from the latter one. The proposed approach can be efficiently used for low-cost bulkquantity production of graphene and graphene-based materials for a wide field of applications.

facile_reduction_of_graphene_oxide_suspensions_and_films_using_glass_wafers

<!>Results<!>Component Defects C=C<!>Conductivity measurements.<!>Discussion<!>Methods<!>Reduction of GO.

<p>Graphene continues to inspire interest in various fields of science due to its outstanding physical and chemical properties [1][2][3] , even though intensive studies devoted to this unique nanocarbon material 4,5 have been carried out during the last ten years. It seems to have a wide field of applications in different technologies, including fabrication of transparent electrodes, supercapasitors and nanoelectronic devices, gas sensing and catalysis, biochemistry and microbiology [6][7][8][9] .</p><p>However, the preparation of graphene in large scales remains to be a challenging task. Several strategies have been developed to overcome this challenge, such as epitaxial growth of graphene on silicon carbide (SiC) 10 , growth of graphene on the surface of transition metals by chemical vapor deposition (CVD) 11 , and reduction of graphene oxide (GO) 12 . Among these methods, the reduction proved to be an effective approach to produce graphene with an optimal quality at relatively low cost 13,14 .</p><p>Numerous approaches are applied to achieve conversion of graphene oxide to graphene, for instance, high-temperature annealing in a reducing enviroment 15 , ultraviolet 16 and laser 17 irradiation of GO films, electrochemical 18 and chemical 19 treatment of graphene oxide suspensions and films. Compared to other techniques, the chemical reduction of GO offers great ease of large-scale production of rGO in various forms such as suspensions, rGO paper or thin films on various substrates.</p><p>Various chemicals, namely, hydrazine monohydrate 20 , dimetilhydrazine 21 , hydroquinone 22 or sodium borohydride 23 are typically employed in chemical reduction of GO. However, these reducing agents are highly toxic, unrecyclable, highly unstable, or generate hazardous by-products. Moreover, strong acidic or alkaline conditions are commonly required in these reduction processes thus limiting their applicability for preparing graphene-containing polymer composites 24 when the reduction process must be carried out simultaneously with introducing graphene into the polymer matrix.</p><p>On the other hand, the use of mild reducing agents, such as green molecules presented by different types of sugars (glucose or sucrose) 25 or L-ascorbic acid 26 , results in a drastic increase in time required for the reduction of GO, and usually takes place in hot solutions. Furthermore, effective reduction of GO by these chemicals commonly requires addition of ammonia that limits the subsequent use of the obtained rGO suspensions. As such, the development of new chemical methods able to provide rapid and efficient reduction of graphene oxide under mild experimental conditions with the use of a reusable deoxygenation agent is of a high interest nowadays.</p><p>In this paper we report a new facile method to convert GO to graphene under low-temperature heating by using sodium, alkali-barium and magnesium silicate glass wafers as reducing agents. The method can be applied for both GO aqueous suspensions and large-scale GO films formed on various substrates prior to reducing. A possible model of the GO reduction using different glass wafers is proposed. Overall, we demonstrate that reduction of GO via low-temperature heating in the presence of glass wafers appears to be a green, efficient and easily scalable method that is based on the use of recyclable non-toxic reducing agent and can be effectively employed for low-cost bulk-quantity production of graphene and graphene-based derivatives.</p><!><p>Optical images and UV-Vis spectra. Figure 1a presents photographs of the initial GO aqueous suspension and GO suspensions (GO) after heating them at 80 °C in the presence of sodium silicate glass (rGO_S-gl), alkali-barium silicate glass (rGO_AB-gl) and magnesium silicate glass (rGO_Mg-gl) wafers. The suspension color change from yellow to black is an obvious visible characteristic of the successful conversion of GO into graphene 27 . The removal of the hydrophilic functional groups is further evidenced by the aggregation of rGO sheets as a result of π-π stacking interactions. The rate of the aggregation rises from the rGO_S-gl to rGO_Mg-gl sample. This fact suggests more complete elimination of oxygen-containing functional groups and larger areas of the graphene network in the latter one.</p><p>Figure 1b shows the UV-Vis spectra of GO and rGO samples. The initial GO exhibits two distinctive features, the main absorption peak at 230 nm due to π-π* transitions of C=C bonds and a broadband absorption shoulder centered at 300 nm. The latter one is commonly attributed to n-π* transitions of C=O bonds of carbonyl and carboxyl groups 28 . However, it also can arise from optical transitions between π and π* states in the nanometer-size sp 2 clusters remained in the structure of GO after its oxidation 29 . Upon the reduction, the 230 nm absorption peak progressively shifts towards higher wavelengths, and overall absorption in the range up to near-infrared (NIR) region rises significantly due to the restoration of sp 2 -conjugated graphene network. As seen, the UV-Vis spectra of rGO_Mg-gl and rGO_AB-gl are almost similar with the peak of the π-π* transition lying at 265-268 nm, which is a characteristic feature of the high-degree GO reduction 30 . At the same time, the position of the main absorption peak (at 247 nm) and highly non-linear character of the absorption in the visible and NIR regions in the rGO_S-gl spectrum signifies incomplete elimination of the oxygen functionalities in the case of using sodium silicate glass as a reducing agent.</p><p>FTIR and XPS spectra. Figure 2a presents the initial GO IR spectrum that exhibits a number of characteristic absorption bands related to the oxygen functionalities and interlayer water 31,32 . Particularly, the broadband absorption feature at 3000-3700 cm −1 originating from the set of overlapping bands of O-H stretching in water molecules, hydroxyls and carboxyls is observed. Additionally, distinguishable bands at 1720 cm −1 , 1620 cm −1 , 1415 cm −1 , 1225 cm −1 and 1040 cm −1 are presented. These absorption lines correspond to the stretching and bending vibrations of the carbonyl/carboxyl groups, water molecules, basal-plane hydroxyls, epoxides and edge-located hydroxyls, respectively. The distinguishable features at 980 cm −1 and 1280 cm −1 are related to the presence of the five-membered ring lactols 32 and ethers 33 .</p><p>The emergence of the prominent absorption band at 1580 cm −1 that corresponds to C=C vibrations within the recuperated conjugated aromatic structure and vanishing of the absorption bands at 3000-3700 cm −1 indicates successful deoxygenation of the rGOs. However, the IR spectrum of the rGO-Sgl sample still exhibits noticeable absorption bands of epoxides (1225 cm −1 ), edge-located hydroxyls (1040 cm −1 ) and carboxyls/carbonyls (1720 cm −1 ). This suggests retention of some amount these functionalities after the reduction. At the same time, the IR spectra for both rGO_AB-gl and rGO_Mg-gl samples demonstrate nearly complete elimination of all oxygen-containing functionalities. The only absorption feature that can be distinguished is related to edge-located hydroxyl groups (phenols). The preservation of these groups is due to their high resistivity to elimination 34 .</p><p>Figure 2b-d show the survey, high-resolution C 1 s and high-resolution O 1 s core level XPS spectra of the samples, respectively. The presence of prominent peaks of Na 1 s and Na KLL in the rGO_S-gl survey spectrum (Fig. 2b) demonstrates that a certain amount of Na (~4.9 at.%,) retains in the structure of this sample. The observed preservation of sodium in such concentrations can be understood in terms of substitution of the hydrogen ion in the residual hydroxyl and carboxyl groups with a sodium cation. At the same time, the analysis of the survey spectra of rGO_AB-gl and rGO_Mg-gl indicates that concentration of residual alkaline-earth metals in these samples is considerably low, appearing to be less than 0.8 at % and 0.3 at%, respectively.</p><p>In the C1s XPS spectra (Fig. 2c), six distinct peaks can be discerned. The peak at 283.9 eV is attributed to carbons that are the nearest neighbors of graphene vacancy defects 35 (peak C-V). The peaks 284.6 eV and 284.9 eV are related to sp 2 -bonded carbons of perfect graphene lattice (peak C=C) and to carbon atoms being partially sp 3 -hybridized due to strong graphene network distortion caused by attachment of oxygen-containing groups (peak C-C), respectively 36 . Note that the C=C peak is asymmetric due to the natural asymmetry inherent for C1s XPS spectra of highly sp2-conjugated graphene-like structure observed in highly reduced GO films 37,38 . Other three peaks located at 286.7 eV, 288.2 eV and 288.9 eV correspond to hydroxyl and epoxide groups (C-OH and C-O-C), carbonyl groups (C=O) and carboxyl groups (COOH), respectively 36,39 . Three main O1s components (Fig. 2d) positioned at binding energies of 531.0, 532.5 and 533.6 eV are assigned, respectively, to the C=O bonds, C-O bonds within the basal plane groups (C-OH and C-O-C) and C-O bonds within phenols (C-OH(ph)) and carboxyls (O=(C-OH)) 34 . Table 1 represents the results obtained by quantitative analysis of the deconvoluted C1s XPS spectra. High content of the oxygen-containing functional groups and low calculated C/O ratio give a hint that the initial GO is highly oxidized. After the reduction, the intensities of the C 1s peaks related to the oxidized groups decrease significantly, which is accompanied by a significant rise in the C/O ratios determined to be 4.22, 5.3 and 7.41 for rGO_S-gl, rGO_AB-gl and rGO_Mg-gl, respectively. These values are very close to those of rGO prepared by chemical reduction using common reducing agents, namely, hydrazine, benzylamine, various alcohols and sodium borohydride 19,40 . Beside the difference in the C/O ratio, the rGOs obtained using different glass wafers also exhibit distinct compositions of residual functionalities. The rGO_S-gl sample is characterized by the presence of a high number of carbonyl groups, which is indicated by the prominent 288.2-eV peak in the C 1 s spectrum and domination of the C=O component in the O 1 s spectrum. This suggests that reduction of GO by using sodium silicate glass lead to formation of new carbonyls. At the same time, according to the quantitative analysis of the deconvoluted C1s XPS the content of carbonyls in rGO_AB-gl is nearly the same as in GO, suggesting absence of their elimination during the treatment. This is also indicated by the relatively high intensity of the C=O peak in the rGO_AB-gl O 1 s spectrum, which significantly differs from the rGO_Mg-gl one. The observed difference in the O 1s spectra of these samples is also related to the higher concentration of retained alkaline earth carbonates formed during the reduction. This is evidenced by a higher content of the retained Ca, Ba, and Mg in rGO_AB-gl sample in comparison to the amount of the residual Mg in rGO_Mg-gl as can be seen from the survey XPS spectra.</p><!><p>XRD patterns, Raman spectra and TEM images. Figure 3a shows the diffraction pattern of the initial GO that exhibits a narrow peak at 2θ = 11.3° that corresponds to diffraction reflection from the (00.2) planes with the basal spacing (d basal ) of 7.8 Å. This value is consistent with the published data 41 . Additionally, the less intense broadened peaks at 2θ = 43° and 2θ = 77.7° characteristic of the (10) and (11) reflections can be observed. These peaks arise from the 2D diffraction from the planar carbon network of GO flakes. The intensity ratio between the crystalline reflections of type (00.l) and lattice reflections of type (hk) indicates that the GO flakes have a lamellar structure, and their lateral size is larger than several micrometers 42 . Further analysis of the SEM images of arrays of GO flakes on the silicon wafer and laser diffraction measurements of the GO aqueous suspension (Figure S1) confirm this assumption, demonstrating that GO flakes have lateral size of up to 100 μm which complies with the highest values reported previously 43 .</p><p>After the GO reduction, the (00.2) XRD-pattern diffraction peak has shifted to higher angles due to the decrease in the rGO interlayer spacing, which proves elimination of the oxygen-containing groups. However, this peak positions are different for different types of the glass wafers. The interlayer spacing has been determined to be 4.1 Å for rGO_S-gl, 3.9 Å for rGO_AB-gl, and 4.5 Å for rGO-Mg-gl. These values are noticeably lower than the GO interlayer distance, which confirms elimination of interlayer water and oxygen-containing groups. On the other hand, these values are larger than both the graphite interlayer spacing of 3.4 Å and that of 3.7 Å published for the reduced graphene oxide 44 . This is due to retention of metal ions and carbonates formed during the reduction process, which cause an increase in the interlayer distance.</p><p>The diffraction patterns of the rGO_AB-gl and rGO_Mg-gl samples also contain distinguishable asymmetric (10.l) and (11.l) reflections which are superposition of reflections of the (hk.l) and (hk) types. The shapes and positions of these diffraction features coincide with those in microcrystalline graphite 45 and suggest that an average lateral size of coherent scattering regions (CSR) corresponding to the defect-free regions in rGO_AB-gl and rGO_Mg-gl is 200 nm.</p><p>Figure 3b shows Raman spectrum of the initial GO with a broad G peak around 1595 cm −1 related to in-plane stretching of the graphene lattice and D peak around 1349 cm −1 caused by the lattice disorder, e.g., edges of the sp 2 clusters and boundaries of the flakes 46,47 . After reduction, the frequency of the D band and G band in Raman spectra of all rGOs is equal to that of GO. The intensity ratio of these bands (I D /I G ) is commonly used to evaluate the stacking order and defect density in the obtained graphene samples 12 . Upon the reduction, the I D /I G ratio does not significantly change and remains within the range of 1.1, although commonly applied chemical reduction procedures lead to the significant rise of the I D /I G ratio 39,48,49 . This evidences that the used reduction procedure does not cause considerable structural disorder if alkali-barium silicate glass or magnesium silicate glass are applied. The absence of the observable decrease in I D /I G ratio is related to the high number of layers in the studied films that are about 500 nm in thick.</p><p>The absence of a high number of defects, e.g., nanosized holes and rips, which commonly arise due to removal of oxide groups 33,47 in the obtained rGO_AB-gl and rGO_Mg-gl samples, is also shown by the obtained TEM images (Fig. 3e,f). The initial GO exhibits a continuous defect-free structure with the absence within GO flakes of any rips or holes with lateral size of more than tens of nanometers. The sharpness of the obtained diffraction spots and ratio between their intensities collectively demonstrate the monolayer character of the GO flakes. After the reduction with the alkali-barium silicate glass and magnesium silicate glass, no nanosized defects are observed in the structure of the rGO platelets (Fig. 3e,f). Moreover, a set of distinguishable hexagonal diffraction patterns rotated relative to each other can be observed in the case of rGO_Mg-gl (Fig. 3f (Inset)). This indicates that the obtained rGO_Mg-gl consists of the lamellar platelets combined in stacks of several layers having well-preserved crystalline structure with the long-range order of minimum several tens of nanometers. Note that this estimation coincides well with the aforementioned CSR area evaluated based on the X-ray diffraction data. In turn, the electron microdiffraction pattern of rGO_AB-gl (Fig. 3e (Inset)) is more ring-shaped (still having the six-fold graphene symmetry). This may be caused by high density of nanowrinkles arising due to the aforementioned retention of metal carbonates on the surface of rGO platelets.</p><p>The crumpled structure of the rGO platelet is also observed for the rGO_S-gl sample where sodium-containing species have been retained on the layer surfaces. However, in opposite to rGO_AB-gl and rGO_Mg-gl samples, rGO_S-gl exhibits quite defective structure. TEM image of this sample (Fig. 3d) demonstrates that large number of holes 5-10 nm in lateral size distributed within the structure of the layer. The low structural quality of the rGO_S-gl sample is also evidenced by low intensities of (10.l) and (11.l) reflections in the XRD pattern, although the I D /I G ratio for rGO_S-gl is comparable to that for rGO_AB-gl. This discrepancy may be related to the sufficiently large distances between the formed holes and aforementioned large lateral size of the GO platelets, since the overall length of boundaries strongly affecting the intensity of the D band is considerably small in this case.</p><!><p>The difference in the efficiency of the GO reduction using different types of glass wafers is further evidenced by the conductivity measurements. The values of sheet resistance and corresponding conductivity values are summarized in Supplementary Table S1. The rGO_Mg-gl sample exhibits the highest conductivity of 33000 S*cm −1 , whereas the conductivities of rGO_AB-gl and rGO_S-gl have been determined to be 10500 S*cm −1 and 117 S*cm −1 , respectively. The values obtained for rGO_Mg-gl and rGO_AB-gl are comparable to those of rGO reduced by using borohydrides 49 , metal-acid solutions in the mild conditions 19 , and high-temperature annealing 50 . At the same time, rGO prepared using sodium silicate glass exhibits quite low conductivity due to highly defective nature of the rGO platelets.</p><p>Reduction of the prior-formed GO films. One of the main issues that limits the use of the liquid-media chemical reduction method for preparing graphene for its further use is restacking of the suspension rGO platelets into graphite-like multilayer aggregates 48,51 . To solve this problem, the liquid-phase reduction of GO is carried out in strong basic or acidic solutions where the electrostatic repulsion of the remained functional groups prevents restacking 52 . Another approach is to modify rGO with various surfactants 53 .</p><p>However, the use of reducing agents that efficiently convert GO to rGO under mild conditions gives the opportunity to straightforwardly reduce not GO suspensions but GO films formed on various substrates prior to the reduction process. To analyze whether the method under consideration is applicable for effective reduction of GO films without their disruption, an additional series of experiments was performed. Particularly, GO films on quartz and silicon substrates were placed in aqueous media containing magnesium silicate glass wafer with its subsequent heating at 80 °C during 5 hours. Further characterization of the obtained sample by the means of UV-Vis and FTIR spectroscopy (Supplementary Figure S2), as well as elemental analysis (Supplementary Figure S3), indicates that the treated GO films were successfully converted to rGO with a high degree of reduction. This assumption is further confirmed by the conductivity measurements demonstrating that conductivity of the obtained reduced graphene oxide film is about 30000 S*cm −1 .</p><p>Importantly, the applied reduction procedure also does not result in peeling of the GO film (Supplementary Figure S4) and formation of any observable defects, e.g., rips and tears, as is indicated by the obtained SEM images (Fig. 4). Note that bright areas observed in the SEM images of the rGO film originate from the oxidation of the silicon wafer surface (Figure S5). Thus, the considered reduction procedure can be applied to reduce not only the GO suspension but also GO films formed on the surface of various substrates that are widely used in graphene-based optoelectronic devices.</p><p>Recyclability of the applied reducing agent. The efficiency of using glass wafers as a reducing agent in converting GO into rGO further improved by their recyclability and simplicity of use. While conventional reducing agents are usually completely consumed during the reduction process, the glass wafer can be simply withdrawn from the aqueous media, washed by deionized water, and used again. The obtained UV-Vis and FTIR spectra (Fig. 5) demonstrate that a single glass wafer may be reused 5 times, providing effective reduction of GO. After the 5th cycle the reduction efficiency begins drastically decrease, and after the 7th cycle no significant elimination of the oxygen functionalities occurs. This is indicated by the shape of the corresponding UV-Vis spectrum (Fig. 5a, magenta curve) and retention of the distinguishable peaks at 1225 cm −1 , 1365 cm −1 and 1720 cm −1 corresponding to basal-plane and edge-located oxygen-containing groups in the FTIR spectrum (Fig. 5b). Nevertheless, glass wafers are highly recyclable as GO-reducing agents and are much easier to be reused than other deoxygenating agents.</p><!><p>The observed conversion of GO into rGO both by low-temperature heating in the presence of a glass wafer can be explained by the following mechanism. In heating a GO suspension with a glass wafer immersed into it to 60-80 °C, the wafer begins dissolving due to acid-catalyzed bimolecular displacement reactions [54][55][56] . This process results in leaching of the alkali and alkaline-earth cations (Na + , Mg 2+ , Ba 2+ , Ca 2+ ) along with metasilicate (SiO3 2− ) and ortosilicate (SiO4 4− ) anions from the glass surfaces into the suspension [57][58][59] . In the process, the pH value rises from 3.4 to 8-8.5. Beside the formation of alkali and alkaline-earth silicates, the leached silicate anions may also interact with the oxygen-containing functional groups of GO. In this case intermediates are formed, composed by the metasilicate or ortosilicate anion that is attached to the GO layer by the oxygen-bridge bond originated from rearrangement of chemical bonds in epoxides (Fig. 6a), hydroxyls (Fig. 6b) or carboxyls (Fig. 6c). The obtained spectra demonstrate that glass wafers as a reducing agent can be reused up to five times without significant loss in the effectiveness.</p><p>The assembled intermediates can be further eliminated by two possible ways depending on the chemical composition of the used glass wafer. If the alkali silicate glass wafer is used then further redistribution of the electron density in the intermediate may lead to the subsequent removal of the silicate anion. This results in the cleavage of carbon bond in the graphene network with formation of the carbonyl group (Fig. 6d,e). This way for eliminating oxygen functionalities from GO in the presence of alkali silicate glass is well supported by the observed rise in the carbonyl group concentrations and perforation of the carbon network in the rGO_S-gl sample.</p><p>In turn, the use of glass wafer that contains alkaline-earth metals, such as Mg or Ba, provides an alternative way of the intermediate transformation. In this case, an alkali-earth cation existing in the suspension interacts with the formed intermediate, resulting in their removal from GO with formation of the alkaline-earth metasilicate and alkali hydroxydes (Fig. 6f,g), while the graphene network remains intact. Furthermore, the presence of the alkaline earth cation also provides elimination of the carboxyl and carbonyl groups. In the case of carboxyls, the intermediate formed from the carboxyl groups and ortosilicate anion is removed and the carbonate along with phenol group are formed (Fig. 6h). Note that the supposed increase in the number of phenols groups after the reduction clearly manifests itself in arising of the 533.6-eV component in the O 1 s XPS spectra (Fig. 2d) of the rGO_AB-gl and rGO_Mg-gl samples. Elimination of carbonyls does not require silicate anions and is based on hydration of carbonyl group 60 with formation of two adjusting hydroxyl groups. These groups further interact with the alkaline earth cation, resulting in their elimination and formation of alkaline earth hydroxide. Magnesium exhibit the lowest value of heat of hydroxide formation in the set of Mg, Ca and Ba 61 . This results in more effective removal of carbonyls using magnesium silicate glass then by using alkali-barium silicate glass, as is indicated by XPS data (Table 1). Moreover, Mg cation diffuse out more easily then Ba or Ca cation due to the lower ionic radius of the former one 62 . As a net result, magnesium silicate glass provide more effective reduction of GO.</p><p>Taking into account the data of the relative concentration of oxygen-containing groups in the initial GO and rGOs we further estimated the number of alkali-, alkali earth-cations and silicate anions consumed for reduction of the studied suspensions. Details of the calculations can be found in Supplementary materials and the obtained results are presented in Supplementary Tables S3 and S4. As seen, for the 200 µl of GO suspension 0.003 wt% in concentration the number of alkali and alkaline earth cations and silicate anions for all the used glass wafers lies within the range of 0.16 µmol, 0.085 µmol and 0.08 µmol, respectively. These values consist with the published data on the concentration of the anions and cations leached during the glass dissolving with the comparable mass and surface area 55,57 . The number of cations and anions required for conversion of 1 mg of GO into graphene was additionally calculated and the obtained values can be found in Supplementary Table S5.</p><p>To validate the proposed mechanism, a series of control experiments was carried out. In these experiments GO was heated under otherwise identical conditions in the presence of either a quartz wafer (rGO_Quartz), or magnesium sulphate (rGO_MgSO4), or sodium hydroxide (rGO_NaOH), or sodium silicate (rGO_Sil), or a combination of sodium silicate and magnesium sulphate (rGO_Sil-Mg). The degree of reduction and chemical composition determined for the resulting rGOs is indicative of the role of each component (silicate anions, alkali and alkaline-silicate cations) in reducing GO with glass wafers. The low-temperature (80 °C) heating of the GO aqueous suspension in the presence of the quartz wafer does not result in the deoxygenation of graphene oxide, as shown by UV-Vis, FTIR and XPS spectra (Figure S6). The treatment of GO with magnesium sulphate results in some elimination of the oxygen containing groups (Figure S7), but the reduction degree (C/O = 1.47) is drastically low and is not comparable to the values for the rGOs obtained using glass wafers. These results collectively indicate that introduction of both silicate anions and metal cations into the medium as a result of glass dissolving is of paramount importance for the GO reduction.</p><p>The primary role of the silicate anions in the observed reduction process was further verified by analyzing the additional rGO_NaOH, rGO_Sil and rGO_Sil-Mg samples. Figure 7 demonstrates C1s and O1s XPS spectra of the obtained rGO samples and results of quantitative analysis of these XPS spectra presented in Table S2. Although strong alkaline solution has been reported to deoxygenate exfoliated GO sheets at the temperatures above 55 °C36,39 , the rGO obtained in alkaline solution with pH~8 has the C/O ratio of 2.9 which is significantly lower than those even in the rGO_S-gl samples. On the other hand, the reduction degree of rGO_Sil (C/O = 4.99) is very close to that in rGO_S-gl (C/O = 4.2), and an increase in concentration of carbonyl groups is observed upon the reduction with both sodium silicate glass and sodium silicate powder. Moreover, the formation of nanoscale holes in the rGO_Sil sample is also demonstrated by the obtained TEM image (Figure S8). In turn, the rGO samples obtained either in the presence of glass wafers containing alkaline-earth oxides or by using sodium silicate mixed with magnesium sulphate exhibit almost equal C/O ratios and compositions of the residual groups. Thus, the obtained experimental results confirm the assessment that the studied reduction process originates from the presence of silicate anions. At the same time, the structural parameters and chemical composition of the obtained rGOs are determined by the type of the metal cations, presented in the suspension.</p><p>In summary, we for the first time have demonstrated that GO can be easily reduced by low-temperature heating in the presence of various glass wafers, namely, sodium silicate, magnesium silicate and alkaline-barium silicate glass wafers. The discussed method can be used to reduce both GO suspensions and GO films formed on various substrates without any considerable effect on the film morphology in the latter case. The additional studies have also confirmed recyclability of the glass wafers used as reducing agents, i.e., the possibility to efficiently reduce GO five times with a single glass wafer. The mechanism of the observed reduction process has been studied as well, revealing that the GO to rGO conversion by using glass wafers occurs due to cooperative interaction of the leached silicate anions and metal cations with oxygen-containing function groups of GO. The advantages of the proposed reduction method, i.e., its simplicity, low reaction temperature, recyclability and non-toxicity of the reducing agent, and the absence of strong acids and bases, make it attractive for the large-scale production of graphene and graphene-based materials for various applications, e.g., fabrication of composite fillers, graphene-based inks, and graphene coatings for optoelectronic devices.</p><!><p>Formation of GO suspensions and films. Graphene oxide was synthesized by the Hummers method 63 . In brief, graphite powder (4 g) was oxidized by using concentrated H 2 SO 4 , KMnO 4 , NaNO 3 , and H 2 O 2 solutions. The resulting mixture was centrifuged (3500 rpm for 1 hour), and the supernatant was decanted away. The material remaining after this was additionally centrifuged (1500 rpm for 10 min) to obtain aqueous GO suspension as a supernatant. In the process of synthesis, sonication was excluded to prevent damaging of graphene oxide flakes and obtain suspensions with the utmost size of GO flakes (with lateral size of up to 100 μm).</p><p>To prepare GO films for the subsequent reduction, 200 μL of GO aqueous suspension 0.003 wt % in concentration was drop-casted on silicon and quartz wafers and dried overnight at room temperature.</p><!><p>Three types of glass wafers with different chemical compositions were used as possible reducing agents for GO deoxygenation: sodium silicate glass containing only sodium oxide, magnesium silicate glass containing only sodium oxide and magnesium oxide, and alkali-barium silicate glass, as one of the most common glass types, containing various alkali and alkaline earth oxides. The chemical compositions of the used glass wafers and their price are presented in Table S6.</p><p>The reduction of GO aqueous suspensions was performed as follows: a piece of sodium silicate glass (10 × 6 mm wafer, 0.135 g), alkali-barium glass (16 × 7 mm wafer, 0.53 g) or magnesium silicate glass (14 × 8 mm wafer, 0.45 g) was immersed into GO aqueous suspension (40 mL) 0.01 wt% in concentration with subsequent stirring of the suspension at 80 °C for 5 hours in a fluoroplastic flask. The obtained rGO suspensions were copiously washed by centrifuging (centrifuge Sigma 3-30KS) at 26,200 rpm (60.600 g) and rinsing the obtained sediment with de-ionized water. The described purification procedure was repeated five times. The obtained rGO samples were denoted as rGO_S-gl (reduced by sodium silicate glass), rGO_AB-gl (reduced by alkali-barium silicate glass) and rGO_Mg-gl (reduced by magnesium silicate glass). The quantity of graphene, obtained from the 0.5 g piece of glass wafer (Sodium, Alkali Barium or Magnesium Silicate) with actual size of 15 × 7.5 × 1.0 mm was determined to be about 50 mg. Magnesium silicate glass wafer was applied for the reduction of graphene oxide suspension up to 7 times with successful conversion of GO into rGO during 5 cycles. As a result, the maximum quantity of the produced graphene from a single glass wafer in the applied conditions was determined to be about 0.25 g.</p><p>To analyze applicability of the studied method for reducing GO films on substrates, the GO films on quartz or silicon substrates were put into a fluoroplastic flask filled with de-ionized water (40 mL); after that, a piece of magnesium silicate glass was added, and the flask was heated at 80 °C for 5 hours. After the reduction, substrates with the rGO film were carefully withdrawn from the solution, washed several times with de-ionized water, and dried overnight at room temperature.</p><p>For better understanding of the processes that lie behind the observed deoxygenation of GO, a series of control experiments was carried out. Namely, GO aqueous suspensions were heated at 80 °C during 5 hours in the presence of the quartz wafer, after adding 0.01 mol. of magnesium sulphate powder (obtained from Acros Organics Company), or 150 µL of NaOH solution (0.1 M, obtained from Acros Organics Company), or 0.7 mmol. of sodium silicate powder (obtained from Acros Organics Company), or 0.8 mmol. of sodium silicate together with 0.8 mmol. of magnesium sulphate. The obtained samples were washed according to the aforementioned procedure.</p><p>To provide the correct alignment and deconvolution of the XPS spectra of GO and whole series of the studied rGOs, an additional rGO sample denoted as rGO_HT was prepared by annealing the GO film at 600 °C during 2 hours.</p><p>Characterization of the obtained rGO samples. The pH values of the solutions were determined with a Fisher Scientific Accumet Basic AB15 pH meter. The UV-vis absorption spectra of the GO and rGO samples were collected with a Shimadzu-2450 spectrophotometer. Fourier transform infrared spectroscopy was performed on the Infralum-08 FTIR spectrometer equipped with the attenuation of total reflectance attachment. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a Thermo Fisher ESCALAB 250Xi XPS system with a monochromatic Al Kα X-ray source (1486.6 eV). The spectra were calibrated with respect to the Au 4f7/2 line (84.0 eV). A surface charging revealed for low-conducting GO (and some rGOs) was taken into account by the aligning their XPS spectra with respect to the C1s line position (284.6 eV) for a well-conductive rGO-HT sample (see Supplementary Figure S9). The quantification and curve fitting of the obtained XPS spectra were performed by using standard CasaXPS software.</p><p>The X-ray diffraction (XRD) analysis was carried out using a Bruker Smart Apex Duo installation with a CuKα source and Apex 2D detector. The sample for diffraction measurements was fixed with nitrocellulose lacquer at the end of a cactus needle. Diffraction patterns were measured at various angles between the normal to the detector surface and the X-ray direction, the 2D data being subsequently recalculated to the 2θ configuration. The obtained diffraction patterns were analyzed using the DIFFRAC.EVA (Bruker Cor.) software based on the data from Powder Diffraction File ICCD PDF-2 release [JCPDS-International Centre for Diffraction Data (http:// www.icdd.com)].</p><p>Raman spectra were obtained on a Horiba Jobin-Yvon LabRam HR800 installation equipped with a Laser Quantum Torus 532-nm laser 50 mW in output power. The exciting light was additionally attenuated with a filter having optical density of 1 and focused with a 20x objective lens into a spot approximately 30 μm in diameter. The power reaching the sample after passing the lightpath and objective was 0.11 mW.</p><p>Size distribution of GO and rGO flakes in aqueous solution was determined by laser diffraction measurements using Mastersizer 2000. Transmission electron microscopy (TEM) images were acquired with a Jeol JEM-2100F microscope (accelerating voltage 200 kV, point-to-point resolution 0.19 nm). Samples for TEM were prepared by deposition of aqueous GO and rGO suspensions 7•10−4 wt % in concentration onto conventional lacey carbon films. SEM images were collected with a JSM-7001F, Jeol microscope. Monolayer films for SEM imaging were prepared by the Langmuir−Blodgett method according to the procedures published elsewhere 64 . Surface morphology and thickness of the rGO films were analyzed with a Veeco Dimension 3100 atomic force microscope operating in the tapping mode by using RTESP probes.</p><p>Electrical conductivity measurements of the GO and rGO samples were performed on the base of two-electrode system. The GO and rGO films were deposited by the drop-casting method onto the surface of quartz substrates with two comb Au electrodes 80 nm thick separated by 500 µm. The electrode comb consisted of 8 pairs of the electrode bars (Figure S10).</p>

Scientific Reports - Nature

Effects of GPI-anchored TNAP on the dynamic structure of model membranes

Tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role during skeletal mineralization, and TNAP deficiency leads to the soft bone disease hypophosphatasia. TNAP is anchored to the external surface of the plasma membranes by means of a GPI (glycosylphosphatidylinositol) anchor. Membrane-anchored and solubilized TNAP displays different kinetic properties against physiological substrates, indicating that membrane anchoring influences the enzyme function. Here, we used Electron Spin Resonance (ESR) measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPI-anchored TNAP with model membranes. The goal was to systematically analyze the ESR data in terms of line shape changes and of alterations in parameters such as rotational diffusion rates and order parameters obtained from non-linear least-squares simulations of the ESR spectra of probes incorporated into DPPC liposomes and proteoliposomes. Overall, the presence of TNAP increased the dynamics and decreased the ordering in the three distinct regions probed by the spin labeled lipids DOPTC (headgroup), and 5- and 16-PCSL (acyl chains). The largest change was observed for 16-PCSL, thus suggesting that GPI-anchored TNAP can give rise to long reaching modifications that could influence membrane processes halfway through the bilayer.

effects_of_gpi-anchored_tnap_on_the_dynamic_structure_of_model_membranes

Introduction<!>Materials<!>Cell culture and preparation of membrane fractions rich in TNAP<!>Solubilization and partial purification of GPI-anchored TNAP with polyoxyethylene-9-lauryl ether (polidocanol)<!>Liposome preparation and incorporation of GPI-anchored TNAP into liposomes<!>Electron spin resonance (ESR) measurements<!>Results<!>Discussion

<p>Alkaline phosphatases belong to a multigene family encoded in humans by 4 distinct gene loci: ALPP, ALPP2 and ALPI genes encode the placental, germ cell and intestinal isozymes, while ALPL encodes the tissue-nonspecific alkaline phosphatase isozyme, also known as liver–bone–kidney type.1 In vivo, TNAP plays a crucial role during the process of endochondral ossification restricting the concentration of inorganic pyrophosphate (PPi), a potent mineralization inhibitor.2–4 Hypomorphic mutations in ALPL lead to hypophosphatasia, an inherited error of metabolism characterized by soft bones in children and/or adults (rickets or osteomalacia) due to accumulation of PPi in the extracellular matrix.5 In vitro, TNAP is a nonspecific phosphomonohydrolase (E.C.3.1.3.1) that is able to hydrolyze phosphate monoesters (ATP, ADP, AMP, p-nitrophenylphosphate, glucose-6-phosphate, glucose-1-phosphate, and glyceraldehyde-3-phosphate), PPi, and phosphate diesters (bis-p-nitrophenylphosphate and cyclic AMP), as well catalyze transphosphorylation reactions.6–19</p><p>Regardless of their tissue of origin, alkaline phosphatases are homodimeric enzymes and each catalytic site has three metal ions (two zinc ions and one magnesium ion) required for the enzymatic activity.1,20–22 Studies regarding the involvement of TNAP in the calcification process have suggested that the enzyme can be found either in a soluble form or associated with membranes.18,23–26 TNAP is associated with the membrane through a glycosylphosphatidylinositol (GPI) anchor. The anchor structure provides lateral mobility in the membrane and allows for TNAP release by the action of phospholipases.1,13,18</p><p>Our research group has demonstrated that the catalytic properties of TNAP vary depending on the microenvironment where the enzyme is located. Thus, different forms of the enzyme (membrane-bound, detergent-solubilized or phospholipase-treated) show different specificities for various substrates, suggesting that the enzyme's kinetic properties are significantly affected by the presence of the GPI anchor and/or other membrane components.27–32 However, little is known about the effects of the GPI anchor on the dynamic properties of the membrane's acyl chains. To address this issue we carried out an ESR study using spin labeled phospholipids that allow monitoring the headgroup region (DOPTC) and two different positions along the lipid acyl chain (n-PCSL). Spectral simulations of the ESR spectra measured before and after TNAP addition to a membrane mimetic were used to assess the profiles of ordering and molecular mobility of the membrane in the presence of the GPI-anchored protein.</p><!><p>Mammalian CHO-K1 cells were purchased from Rio de Janeiro Cell Bank (Rio de Janeiro, RJ). Spin labels DOPTC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and n-PCSL (1-acyl-2-[n-(4,4-dimethyloxazolidine-N-oxyl)stearoyl]-sn-glycero-3-phosphocholine) with n = 5 and 16 were purchased from Avanti Polar Lipids. Synthetic phosphatidylcholine DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was from Avanti Polar Lipids (Alabaster, AL). The chemical structures of the labeled lipids can be found elsewhere.33 Organic solvents and other chemicals were from Sigma-Aldrich.</p><!><p>Cells were prepared and cultured according to Simão et al.28 Membrane-bound recombinant human TNAP was obtained from transfected CHO-K1 cells as described by Simão et al.34</p><!><p>Membrane-bound TNAP (0.2 mg mL−1) was solubilized with 1% polidocanol (w/v, 10 mg mL−1 final concentration) for 1 h, with constant stirring, at 25 °C. After centrifugation at 100 000 × g for 1 h at 4 °C, the detergent-free solubilized enzyme was obtained using 200 mg of Calbiosorb resin and 1 mL of polidocanol-solubilized enzyme (~0.03 mg of protein per mL) as previously described.35 All protein concentrations were estimated in the presence of 2% (w/v, 20 mg mL−1) SDS.36 Bovine serum albumin was used as a standard.</p><!><p>Dipalmitoyl phosphatidylcholine (DPPC) liposomes were prepared from a 10 mg mL−1 chloroform stock solution of lipids. Spin-labeled phosphatidylcholines were incorporated at a concentration of 0.5 mol% of total lipids by drying down the chloroform lipid solutions under a stream of nitrogen, followed by further drying in a SpeedVac Concentrator system (Thermo Scientific) overnight. The dried lipid film was resuspended in 50 mM Tris-HCl buffer, pH 7.5, containing 2 mM MgCl2, and the mixture was incubated at 50 °C for 1 h, with vigorous stirring using a vortex at 10 min intervals. The mixture was passed through an extrusion system (Liposofast, Sigma) using a polycarbonate membrane of 100 nm, and the suspension of relatively homogeneous unilamellar vesicles was stored at 4 °C.</p><p>DPPC-proteoliposomes containing TNAP were prepared by mixing and incubating 1mL of liposomes and 10 mL of detergent-free TNAP in 50 mM Tris-HCl buffer, pH 7.5, containing 2 mM MgCl2 for 1 h, at 25 °C. The mixture was then centrifuged at 100 000 × g for 1 h, at 4 °C. The pellet was resuspended in 0.5 mL of the same buffer. TNAP activities in the supernatant and in the resuspended pellet were assayed and used to calculate the percentage of protein incorporation. p-Nitrophenylphosphate (pNPP) activity for TNAP was assayed discontinuously, at 37 °C, in a spectrophotometer by following the release of the p-nitrophenolate ion as described before.19,34,37</p><p>The enzymatic release of TNAP from proteoliposomes was performed as described before by Pizauro et al.13,27,29,34,35 The proteoliposomes were incubated in 50 mmol L−1 Tris-HCl buffer, pH 7.5, with specific phosphatidylinositol phospholipase C (0.1 U PIPLC from B. thuringiensis) for 1 h under constant rotary shaking, at 37 °C. The incubation mixture was centrifuged at 100 000 × g for 1 h, at 4 °C.</p><!><p>Continuous wave ESR spectroscopy was carried out at room temperature (22 ± 1 °C) on a Jeol JES-FA200 spectrometer operating at the X-band (9.2 GHz). Solutions containing TNAP incorporated into the spin-labeled DPPC liposomes were drawn into capillary tubes for ESR experiments. All ESR spectra were recorded with the following experimental parameters: field range of 100 G, microwave frequency of 9.2 GHz, modulation frequency of 100 kHz, modulation amplitude of 100 kHz, and microwave power of 10 mW. The ESR spectra were processed utilizing OriginPro8 software.</p><p>Spectral simulations of the ESR spectra were performed using the NLSL program developed by Freed et al.,38 and available for download at http://www.acert.cornell.edu/index_files/acert_resources.php. The simulations yielded the average rotational diffusion rate R1 and the coefficients c20 and/or c22 of an orienting potential experienced by the nitroxide moiety. Order parameters, S0 and/or S2, can be calculated from those coefficients as described elsewhere.38 Other parameters used as input in the simulations, such as magnetic tensor (g- and hyperfine) components, were obtained from previously published data.38 The fitting procedure followed similar strategies as those described in Basso et al.39 To avoid local minima and to obtain error estimates for each varied parameter, the fits were initiated from different sets of seed values. Once minimization of the parameters was concluded, their final values were compared and their average along with the respective standard deviation was used as final results.40 The procedure led to percent error estimates of R1 (2%) and order parameter (5%).</p><!><p>Several reports suggest that the lipid membrane composition can modulate the TNAP phosphomonohydrolase activity.27–30,32 In addition, evidence indicates that TNAP is found in special regions of the membrane called lipid rafts and microdomains rich in cholesterol and sphingomyelin.1,41–45 However, the docking mechanisms of most GPI-anchored proteins, such as TNAP, and the possible effects of this anchoring mechanism on the enzymatic activity are still unclear.</p><p>Sharom et al.46 used Förster resonance energy transfer (FRET) to examine the anchoring of PLAP and concluded that the GPI-anchored protein is close to the membrane bilayer, and the calculated distance was about 10 Å from the membrane. Here, we carried out ESR spectroscopy of labeled phospholipids to examine the anchoring mechanism of TNAP. The line shape of the ESR spectrum is very sensitive to the spin label ordering and dynamics in membrane bilayers.47–51 Moreover, spectral simulations, based on the routines developed by Freed et al.,38 allow for a detailed quantification of both ordering and dynamics.</p><p>The headgroup region of the membrane models was probed by the DOPTC spin label, and the corresponding ESR spectra of DOPTC incorporated into liposomes of pure DPPC or into proteoliposomes harboring TNAP are shown in Fig. 1. A qualitative analysis of these DOPTC ESR spectra indicates that the three typical lines observed in the nitroxide spectrum become narrower in the presence of TNAP, suggesting that the spin probes are undergoing a somewhat faster motion. To clearly quantify this finding we performed NLSL simulations of both spectra. The best fits to the experimental data are seen as red lines in Fig. 1. From the fits, an R1 value of 0.11 × 109 s−1, which corresponds to a correlation time of 3.30 ns, and an order parameter of ca. 0.37 were found. In the presence of TNAP, R1 is increased to 0.16 × 109 s−1 (τc of 2.23 ns) and ordering is reduced to 0.30. Hence, when TNAP is in the membrane mimetic, ordering and dynamics are changed such that the spin probes are in a more fluid and less ordered environment at the headgroup region. This result suggests that the enzyme itself does not touch the membrane surface, thus allowing for the headgroup to experience a less hindered motion that leads to slightly higher dynamics and less ordering.</p><p>Besides probing the headgroup of the phospholipids with DOPTC, we also checked for changes in the lipid carbon acyl chains of the membrane caused by the presence of GPI-anchored TNAP. To do so we used two additional spin probes: one with a nitroxide radical at carbon 5 (close to the water/lipid interface) and the other with the probe at carbon 16 (at the end of the acyl chain).</p><p>The environment probed by 5-PCSL is closer to the membrane surface, where the nitroxide radicals are subjected to a natural, more restricted motion due to the higher degree of packing of the bilayer.52 This more restricted type of motion is clearly reflected in the low-field line of the 5-PCSL ESR spectrum (Fig. 2) in the absence of the enzyme. The lines are much broader and the lineshape much "distorted" than those observed for DOPTC. The simulation of the 5-PCSL spectrum in the control experiment yielded an R1 value of 0.083 × 109 s−1 (τc of 2.00 ns), in agreement with our qualitative observation of slower dynamics probed by 5-PCSL when compared to DOPTC, and an order parameter of 0.32. A lineshape change is observed in the presence of GPI-anchored TNAP. The simulation also shows such an alteration with the R1 value increasing slightly to 0.091 × 109 s−1 (τc of 1.83 ns) and the order parameter decreasing to 0.24.</p><p>Finally, the spin-labeled phospholipid 16-PCSL was used to probe TNAP-induced alterations further down the acyl chain. 16-PCSL is located in the middle of the bilayer and thus gives rise to ESR spectra associated with much higher mobility than the other probes used in this work. This is clearly seen in its spectra (Fig. 3) that present sharp and intense lines, especially the low- and mid-field resonances, whose intensity ratio is closer to 1 (this ratio can be used as a measure of label mobility). To quantify the order and dynamics of the labels in the absence and in the presence of TNAP, NLSL simulations of the spectra in Fig. 3 were again performed. The best fit to the experimental spectrum in the absence of the enzyme was achieved with an R1 value of 0.66 × 109 s−1 and a negligible order parameter of 0.06. It can be readily seen that this is the fastest regime of motion observed amongst the different regions probed by the set of labels chosen in this paper. In the presence of GPI-anchored TNAP, the ESR spectrum shows differences in its line shape that qualitatively indicate an increase in dynamics (low- and midfield line intensities become very similar). The simulation of the spectrum led to R1 of 2.40 × 109 s−1 and again a negligible order parameter of 0.04.</p><!><p>ESR spectroscopy of spin labeled lipids has proved to be one of the most valuable spectroscopic methods for studying protein interactions with biological membranes. This is due to the sensitivity of spin labeled lipids undergoing ESR to dynamics in the ns timescale.53 In this sense the study of GPI-anchored TNAP performed here using ESR spectroscopy allowed us to obtain valuable insights into the effects caused by the presence of TNAP on the dynamic organization of lipids in the membrane mimetic system. Previous studies, such as those reported by Ciancaglini's group27,29,32 and Roux's group,44,45,54 have dealt with the same problem. However, here we can dissect the effects of TNAP presence as a function of the depth within the bilayer due to the localization of the spin labeled moiety in a very specific region of the bilayer, especially for DPPC membranes in the gel phase. Furthermore, Murphy and Messersmith55 have shown that DPPC liposomes are an adequate initial choice of model of matrix vesicles involved in biomineralization.</p><p>The ESR spectra of all probes used in DPPC-containing models revealed that the enzyme increased the membrane dynamics and decreased the bilayer order (Fig. 4). The quantitative results obtained from the NLSL simulations of the ESR spectra are expressed in terms of the rotational diffusion rate (R1), interpreted as the fluidity of the membrane, and the order parameter. From these values, we can see that the presence of the GPI-anchored TNAP led to an intermediate increase of R1 at the polar headgroup (from 0.11 × 109 s−1 to 0.16 × 109 s−1) and in the region closer to the bilayer surface (from 0.083 × 109 s−1 to 0.091 × 109 s−1), whereas the order parameters reported a decrease in both cases. This means that the lipids experience a higher degree of freedom to move around when TNAP is present, which induces a smaller orienting potential and higher fluidity29 (Fig. 4). In particular, at the headgroup, the increase in R1 and the decrease of the order parameter indicate that the TNAP structure itself does not lie on the surface of the membrane as also observed for PLAP by Sharom et al.46 and for another GPI-anchored alkaline phosphatase by Ronzon et al.54</p><p>The largest change observed in our ESR results comes from the dramatic increase of the R1 values for the 16-PCSL probes. These probes are located half way through the bilayer and are naturally in a more fluid and less ordered environment when compared to the probes that report on other regions of the bilayer as one can see from the R1 values obtained from pure DPPC liposomes. In our case, 16-PCSL is not subjected to an orienting potential, which gives rise to close to zero values of the order parameter that were not affected much by the presence of the GPI-anchored enzyme (it changes from 0.06 to 0.04). However, R1 values showed a four-fold increase when TNAP is in the proteoliposomes, changing from 0.66 × 109 s−1 to 2.40 × 109 s−1. This result indicates that the GPI-anchored TNAP is able to affect the bilayer organization deep down to the end of the lipid acyl chain, inducing higher fluidity (Fig. 4).</p><p>Ronzon et al.56 showed that GPI-anchored alkaline phosphatase was able to disorder the hydrocarbon chains of DPPS monolayers in a much more pronounced way when compared to DPPC monolayers. Here, using ordered DPPC bilayers, we showed that TNAP is capable of perturbing the whole extension of the acyl chains with a great effect right in the middle of the bilayer. Any process that needs some sort of long extent change within the bilayer can then be affected by the presence of TNAP and its GPI-anchor. For example, GPI-anchored TNAP has the ability to spontaneously insert into the lipid bilayer, and several reports have suggested that this protein associates preferentially with cholesterol- and sphingolipid-rich regions called raft domains.29,32,37,57 It has been proposed that acyl and alkyl chain lengths of GPI-anchors in proteins could determine raft association.58 Thus, the length and the order of aliphatic chains in both the fluid and ordered phases are expected to affect the GPI-anchored protein–domain interactions, and the fluidity changes induced by TNAP could be related to the recruitment and the association of other raft related proteins like annexins. Moreover, since TNAP has been shown to preferentially partition in lipid ordered domains, our results could be extended to reveal the effect of TNAP presence on the dynamic structure of ordered bilayers such as DPPC in the gel phase liposomes used in this study.</p><p>In order to check whether the observed changes were due to the presence of the whole GPI-anchored TNAP structure or whether they could also be induced by the GPI motif only, the TNAP-containing proteoliposomes were treated with PIPLC from B. thuringiensis, which specifically cleaves GPI anchors, allowing a selective release of the TNAP protein chain into the solution.13,29,59 This sample was then submitted to ESR experiments using DOPTC and 5-PCSL probes. The corresponding spectra are shown in Fig. 5 and one can see that there were much smaller changes in this case. About 70% of TNAP activity was lost after the treatment (data not shown), indicating that TNAP is not completely removed from the proteoliposomes and the smaller changes observed in Fig. 5 could be attributed to the presence of GPI-anchored TNAP that remained in the sample even after treatment. This result highlights the importance of having the whole protein structure along with the GPI motif in order to promote in full the alterations described above.</p><p>Our data underscore the importance of obtaining direct structural information on this physiologically relevant GPI-anchored enzyme in a lipid bilayer environment. We conclude that TNAP is probably close to the membrane surface and that this proximity can be related to the modulation of catalytic activity by the lipid composition as previously reported.27–30,32,52 Further studies are necessary to fully understand the implications of the GPI-anchoring mechanism on the TNAP structure and function and on membrane protein organization in matrix vesicles.</p>

PubMed Author Manuscript

Selective N1/N4 1,4-Cycloaddition of 1,2,4,5-Tetrazines Enabled by Solvent Hydrogen Bonding

An unprecedented 1,4-cycloaddition (vs 3,6-cycloaddition) of 1,2,4,5-tetrazines is described with preformed or in situ generated aryl-conjugated enamines promoted by the solvent hydrogen bonding of hexafluoroisopropanol (HFIP) that is conducted under mild reaction conditions (0.1 M HFIP, 25 \xc2\xb0C, 12 h). The reaction constitutes a formal [4+2] cycloaddition across the two nitrogen atoms (N1/N4) of the 1,2,4,5-tetrazine followed by a formal retro [4+2] cycloaddition loss of a nitrile and aromatization to generate a 1,2,4-triazine derivative. The factors that impact the remarkable change in the reaction mode, optimization of reaction parameters, the scope and simplification of its implementation through in situ enamine generation from aldehydes and ketones, the reaction scope for 3,6-bis(thiomethyl)-1,2,4,5-tetrazine, a survey of participating 1,2,4,5-tetrazines, and key mechanistic insights into this reaction are detailed. Given its simplicity and breath, the study establishes a novel method for the simple and efficient one-step synthesis of 1,2,4-triazines under mild conditions from readily accessible starting materials. Whereas alternative protic solvents (e.g., MeOH vs HFIP) provide products of the conventional 3,6-cycoladdition, the enhanced hydrogen bonding capability of HFIP uniquely results in promotion of the unprecedented formal 1,4-cycloaddition. As such, the studies represent an example of not just an enhancement in the rate or efficiency of a heterocyclic azadiene cycloaddition by hydrogen bonding catalysis, but also the first to alter the mode (N1/N4 vs C3/C6) of cycloaddition.

selective_n1/n4_1,4-cycloaddition_of_1,2,4,5-tetrazines_enabled_by_solvent_hydrogen_bonding

INTRODUCTION<!>Reaction Discovery, Key Parameters, and Optimization.<!>Substrate Scope.<!>Mechanistic Insights.<!>Application Potential.<!>Conclusions.

<p>The inverse electron demand Diels–Alder reaction of electron-deficient heterocyclic azadienes is an effective method for the synthesis of highly functionalized heterocycles widely used in organic synthesis,1 medicinal chemistry, and chemical biology.2 Previously, we have reported systematic explorations and applications of the cycloaddition reactions of 1,2,4,5-tetrazines,3 1,2,4-triazines,4 1,3,5-triazines,5 1,3,4-oxadiazoles,6 1,2-diazines,3b,7 1,2,3-triazines,8 and most recently a 1,2,3,5-tetrazine.9 Among all heterocyclic azadienes, the readily available 1,2,4,5-tetrazines are the most widely used due to their superb cycloaddition reactivity with an unusually broad range of dienophiles (Figure 1A).1,2 In the >60 years since its first disclosure and among the now countless examples, a single cycloaddition mode is observed that occurs across the two carbon atoms (3,6-cycloaddition) independent of the 1,2,4,5-tetrazine substitution pattern or nature of the dienophile.10 To the best of our knowledge, no example of the alternative [4+2] cycloaddition across two nitrogen atoms (1,4-cycloaddition) of a 1,2,4,5-tetrazine has been disclosed. Best defined and articulated by Houk in computational studies,11 the remarkable rate of cycloaddition, the preferential 3,6-cycloaddition mode, and lack of 1,4-cycloaddition can be attributed to orbital interactions and differential distortion energies enroute to the transition state along with the energetically preferential formation of two C–C versus two C–N bonds, and the subsequent release of N2 rather than a nitrile.</p><p>Until recently and although examined for decades, no general approach to catalysis of the inverse electron demand Diels–Alder reactions of heterocyclic azadienes had been described.12,13 Typically, additives such as Lewis acids lead to non-productive consumption of the electron-rich dienophiles without productive activation of the electron-deficient heterocyclic azadienes. We found that heterocyclic azadienes can be activated for cycloaddition by H-bonding14 with the non-nucleophilic solvents hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE).11 The H-bonding was established in mechanistic 1H NMR studies, the catalysis was found to be unique to HFIP and TFE versus other protic solvents due to the reduced basicity of such heterocyclic azadienes, and our conclusions were verified in subsequent computational studies by Houk.15 Since its discovery, we have continued to investigate the scope of solvent H-bonding assisted inverse electron demand Diels-Alder cycloadditions.13 In these studies, which have focused on defining the rate accelerations, improved conversions, regioselectivity enhancements or alterations, and expansion of the productive reactive diene/dienophile pairs, we discovered an unprecedented formal 1,4-cycloaddition of 3,6-bis(methylthio)-1,2,4,5-tetrazine (1a) with the enamine 4a, 1-styrylpyrrolidine. When HFIP was used as the solvent at room temperature open to air, the reaction provided the 1,2,4-triazine 3a in 75% yield, the structure of which was established with a single-crystal X-ray structure determination (Figure 1C).16 Given the unprecedented nature of the reaction coupled with the importance of the 1,2,4-triazine core in drugs and biologically active molecules (Figure 2),17 we examined and herein detail features that impact this change in the cycloaddition mode, optimization of the reaction parameters, the scope and further simplification of the reaction as it relates to the 1,2,4,5-tetrazine and preformed or in situ generated enamine, and mechanistic insights into this remarkable reaction. Given its breath, it establishes a new simple method for the efficient one-step synthesis of 1,2,4-triazines under mild reaction conditions from easily accessible starting materials.</p><!><p>In order to better understand the role of each factor, especially the solvent, a series of experiments was conducted (Figure 3). An increase in the reaction temperature to 60 °C (entry 2, vs 25 °C), an increase in the reaction time to 22 h (entry 3, vs 13 h), and an increase in the reaction concentration (entry 4) had little impact on the yield of product. Importantly, only the conventional 3,6-cycloaddition product 5a, without elimination of pyrrolidine, was generated when the reaction was conducted in non-fluorinated solvents, including CHCl3 (entry 5) and methanol (entry 6). The reaction in methanol was found to generate the aromatic pyridazine product 5a' (66%) with elimination of pyrrolidine when it was further warmed to 50 °C for 24 h (eq 1). The use of HFIP as solvent was found to be essential for the altered 1,4-cycloaddition, although use of mixed solvent of CHCl3–HFIP (1:1) led to the same product 3a, albeit in lower yield (entry 7). Finally, addition of strong acid, TFA (1 equiv) and conducting the reaction in CHCl3 resulted in no cycloaddition products, likely due to protonation of the enamine 4a (entry 8). The unique behavior of HFIP arises from its ability to H-bond the 1,2,4,5-tetrazine thereby activating it for reaction, and yet not consume either the starting 1,2,4,5-tetrazine because of the attenuated nucleophilic character of HFIP or the conjugated enamine through protonation because of its weakly acidic nature (pKa 9.3). (1)</p><p>A more refined solvent survey for the reaction of 1a with enamine 4b in a series of perfluoroalcohols was conducted. Despite the variations in yields, the ratio of formal 1,4-cycloaddition versus the conventional 3,6-cycloaddition that provides 5b and pKa of the perfluoroalcohols, which is a measure of their H-bonding capability,18 were found to correlate exceptionally well (Figure 4). In fact, a clean switch from exclusive 3,6-cycloaddition to exclusive formal 1,4-cycloaddition was observed as the pKa of the solvent decreased from 15.5 (MeOH) to 9.3 (HFIP). As such, the results highlight the unique behavior of HFIP and indicate that the extent of the H-bonding interaction between the tetrazine and solvent is the feature controlling the 1,4- versus 3,6-cycloaddition selectivity. Experimentally, we observed that the HFIP alcohol proton exhibits a pronounced downfield chemical shift upon titration with 3,6-bis(thiomethyl)-1,2,4,5-tetrazine (1a, Δ0.53 ppm, 0–2 equiv) consistent with this H-bonding interaction with 1a (Supporting Information Figure S1). It is possible that the selectivity is altered due to change in the LUMO molecular orbital distribution that is induced by solvent H-bonding, leading to a conjugated nitrogen now more susceptible to nucleophilic attack than carbon. Simple AM1 computation of the LUMO energy of free and protonated 1a (−1.93 eV vs −6.17 eV) and sum of squared coefficients as they relate to tetrazine carbons (C3, C6) and nitrogens (N1, N2, N4, N5) (free 1a: 0.62 for C and 0.26 for N; protonated 1a: 0.43 for C and 0.39 for N, see Supporting Information Figure S2 for details) supports both the enhanced reactivity (rel ELUMO) and a shift from C to N for attack of a nucleophile.</p><p>Based on precedent that we first introduced4b and with recognition that a preformed enamine may not always be readily available, easily prepared and stored, or stable in open air, we examined whether the enamine could be generated in situ from the corresponding aldehyde and amine. To our delight, replacement of enamine 4a (3 equiv) with phenylacetaldehyde (2a, 3 equiv) and pyrrolidine (1 equiv) provided 3a in an improved yield (88%) under otherwise identical conditions (0.1 M HFIP, 25 °C, 13 h, open flask) (Scheme 1). A screen of alternative secondary amines revealed that the in situ generated pyrrolidine enamine provided the highest conversion to 3a of those examined (Scheme 1). No reaction was observed either when a tertiary amine such as Et3N was used or in the absence of an added secondary amine and both the 1,2,4,5-tetrazine 1a and aldehyde 2a were recovered unchanged. Use of 0.5 equiv pyrrolidine (with 1 equiv of 1a/3 equiv of 2a) as above provided similar results (79% yield) indicating productive turnover, although use of ≤0.25 equiv pyrrolidine resulted in lower yields even with extended reaction times (Supporting Information Figure S4). This sub-stoichiometric use of pyrrolidine with 2b improved with the faster reaction of in situ generated 4b, where good conversion was observed even with 0.25 equiv and dropped off only at 0.1 equiv of pyrrolidine (Supporting Information Figure S4). The limited pyrrolidine turnover is possibly due to acid-promoted self-condensation of the in situ generated enamine (Supporting Information Figure S5).</p><!><p>The carbonyl substrate scope for this transformation was explored (Scheme 2). With the 1,2,4,5-tetrazine 1a as the diene, 2-aryl acetaldehyde substrates bearing either electron-rich (3b, 3f, 3h, 3i, 3k, 3l) or electron-deficient (3c-e, 3g, 3j, 3m-o) arenes are well tolerated, all participating in the reaction effectively, although electron-deficient arenes were found to display a lower reactivity. As a result of the benign reaction conditions, a wide range of functional groups are expected to be well tolerated, including those illustrated herein, consisting of methoxy (3b), halides (3c-e, 3j), phenyl (3f), trifluoromethyl (3g), ester (3m), and nitrile (3o) substituents. Arenes with ortho-substitution (3i, 3k, 3n) that might suffer steric issues also provide the 1,2,4-triazines in satisfactory yields. Heterocyclic as well the all-carbon arenes are also compatible, including thiophene (3h), indole (3k), and quinoline (3n). Nonconjugated enamines (e.g., 1-pyrrolidinocyclopentene) did not react with 1a under current reaction conditions likely due to their protonation by HFIP (for unreactive substrates, see Supporting Information Figure S6). Although the ketone 1-phenylacetone was unreactive, conjugated cyclic ketones (see below) and two related substrates containing 5-membered heterocycles, 1-(thiophen-2-yl)propan-2-one (2p) and 1-(furan-2-yl)propan-2-one (2q), were found to be suitable substrates under the current reaction conditions, providing the 1,2,4-triazines 3p (79%) and 3q (26%), respectively. This differentiated behavior can be attributed to a lower steric barrier (Me/O repulsion vs Me/C–H repulsion) to achieving a conjugated coplanar enamine conformation, increasing the enamine stability toward nonproductive HFIP protonation and self-condensation (Figure 5). This conclusion was further supported by computation (AM1, Gaussian 09) of the C1-C2-C3-C4 dihedral angle of 1-phenylacetone (62.8°) and C1-C2-C3-O dihedral angle of 2p (10.0°).</p><p>Significantly, conjugated cyclic ketones of which 2-tetralones and 2-indanones are prototypical members participate effectively in the reaction under the current reaction conditions (Scheme 3). In addition to the parent 2-tetralone (3r) and 2-indalone (3s), derivatives bearing methoxy (3t) and bromine (3u) substituents were also suitable substrates for this reaction. Cyclic ketones that form conjugated enamines with fused heterocycles, such as pyrrole (3v), indole (3w), pyridine (3x, 3y), and thiophene (3z) all provided the 1,2,4-triazine products in good to excellent yield. The structures of 3r and 3v were confirmed by single-crystal X-ray structure determinations.16</p><p>The substrate scope as it relates to symmetrical 1,2,4,5-tetrazines was also briefly explored (Scheme 4). This was not done with the intent of comprehensively defining the scope and was conducted without alteration of the reaction parameters or optimization of the conditions (temp, time) including choice of perfluoroalcohol. Rather, it was done to establish whether the 1,4-addition extends beyond 1a and to define the range of productive substituents, and the results portend a broad tetrazine scope. 3,6-Bis(benzylthio)-1,2,4,5-tetrazine (1a') displayed a reactivity similar to that of 1a toward the in situ generated pyrrolidine enamine of phenacetaldehyde, providing the corresponding 1,2,4-triazine 6a in excellent yield (78%). Notably, the nonvolatile benzylthiocyanate was observed and characterized by NMR (76%) as a released product in this reaction. As such and while the reaction is unlikely to represent a true cycloaddition across the tetrazine N1/N4, the products are the same as those expected of such a reaction. More remarkable and without an effort at optimization, 1,2,4,5-tetrazines that are less reactive or unreactive in traditional cycloaddition reactions also provided the corresponding 1,2,4-triazines, including 3,6-dimethoxy (6b), diamino (6c), dimethyl (6d), and diphenyl (6e) 1,2,4,5-tetrazines, although in more modest conversions under the present reaction conditions. Unsubstituted 1,2,4,5-tetrazine (s-tetrazine) and dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate were found to undergo the conventional 3,6-cycloaddition without observation of the formal 1,4-cycloaddition product under current conditions (Supporting Information Figure S7). A series of unsymmetrical 1,2,4,5-tetrazines were also examined as is shown below, some of which displayed a remarkable regioselectivity for the 1,4-cycloaddition. Representative of this, the reaction of 4-methyl-1-thiomethyl-1,2,4,5-tetrazine (7d) with the in situ generated pyrrolidine enamine of phenylacetaldehyde under the standard reaction conditions (HFIP, 25 C̊, 12 h) provided exclusively the 1,2,4-triazine 3a (71%) with retention of the thiomethyl group and loss of acetonitrile.</p><p>In addition to the symmetrical 1,2,4,5-tetrazine substrates presented in Scheme 4, a larger representative series of unsymmetrical 1,2,4,5-tetrazines (7a-f) were prepared and examined, with one site substituted with a methylthio group and the remaining site bearing an electron-withdrawing (7a, S(O)Me), electron-donating (7b and 7c, OMe and NHAc), neutral (7d, Me), conjugated (7e, Ph), or no substituent (7f, H). They were allowed to react directly with enamine 4a in HFIP (0.1 M, 25 °C) to further probe factors impacting the mode of cycloaddition, its regioselectivity, and plausible mechanisms (Figure 6). The results revealed that only two, tetrazine 7b (R = OMe, entry 3, 1:1) and 7e (R = Ph, entry 6, 5:1), led to two 1,2,4-triazine products that bear each substituent (3a:6) in reactions that display the 1,4 mode of cycloaddition but represent two regioselectivities for the formal 1,4-cycloaddition. Like the symmetrical tetrazine 1a, the remaining tetrazines provided the single 1,2,4-triazine 3a in a regioselective 1,4-cycloaddition. In addition, the reactions of the electron-deficient tetrazine 7a (R = S(O)Me, entry 2, 2:1) and the unsubstituted tetrazine 7f (R = H, entry 7, 1:3) exhibited competitive 1,4-cycloaddition and 3,6-cycloaddition. Aside from the observation that an electron-withdrawing substituent increases competitive 3,6-cycloaddition as does removal of a substituent, 3a is exclusively (7a,c,d,f) or predominately (7e) formed in all cases with exception of 7b, bearing a methoxy substituent.</p><!><p>The unique 1,4-cycloaddition was examined in greater detail to gain insights into the potential reaction mechanism. Given that no prior example of cycloaddition across two nitrogen atoms (N1/N4) of 1,2,4,5-tetrazines has been described, it is unlikely that a concerted [4+2] cycloaddition followed by retro [4+2] nitrile extrusion is operative in this transformation. Moreover, analysis of the reaction indicates that not only is a concerted cycloaddition unlikely, but even a stepwise addition-cyclization prior to nitrile loss is not able to account for formation of a single 1,2,4-triazine product rather than a mixture of two isomeric products (Figure 7). Further reinforcing this analysis is the observation that room temperature retro [4+2] nitrile extrusion from an initial bicyclic intermediate is unlikely since related isolated bicyclic intermediates obtained from the cycloadditions of 2,4,6-tris(methylthio)-1,3,5-triazine require elevated reaction temperatures, extended reaction times, and often additional acid catalysis for nitrile extrusion.19</p><p>First and foremost, to determine whether a fragmented tetrazine, potentially releasing an acyclic azadiene, might be responsible for the observed reaction, we examined the fate of 1,2,4,5-tetrazine 1a in HFIP alone (25 C̊, 12 h), in the presence of pyrrolidine (1 equiv), and in the presence of phenylacetaldehyde (2a, 1 equiv) (eq 2). Under all conditions, tetrazine 1a was recovered quantitatively without change, verifying that 1a, and not a more reactive fragment, is the compound undergoing reaction. (2)</p><p>We then undertook the detection, trap, or isolation of a reaction intermediate to help clarify the origin of the altered cycloaddition mode and the overall reaction process. After extensive attempts on intermediate capture, compound 3a' was isolated in 36% yield when 1a and enamine 4a were allowed to react in a MeOH/HFIP (1:1) mixed solvent system for 2 h (Figure 8A). The structure of 3a' was unambiguously established with a single crystal X-ray structure determination.16 Compound 3a' converts to product 3a in 78% yield when subjected to the standard reaction conditions (HFIP, 25 °C, 13 h; Figure 8B). A crossover experiment where 3a' was mixed with the enamine 4b under the standard reaction conditions afforded 3a as the sole product without observation of 3b, indicating that at least one step in the generation of 3a' from starting materials is not reversible (Figure 8C). Notable in this structure is a connectivity that indicates enamine attack on a tetrazine nitrogen (N1) as well as the necessary cleavage of a N-N bond (N1/N2) for 1,2,4-triazine formation prior to methylthiocyanate loss, and that it is the alkylated, not distal, N-N bond (N4/N5) that is cleaved.</p><p>The reaction between 1a (0.1 M, HFIP-d2) and enamine 4a (2 equiv, phenacetaldehyde pyrrolidine enamine) was also monitored by 1H NMR (Supporting Information Figure S8) as was that with 4b (4-methoxyphenacetaldehye pyrrolidine enamine). For 4a, the consumption of 1a and accumulation of product 3a was directly observed as the reaction proceeded, monitoring the emerging diagnostic C6-H signal of 3a and a discernable chemical shift in the SMe signal of 3a versus 1a. In addition, a diagnostic signal for 3a' (aminal CH, δ 5.48 in HFIP-d2) was observed to rise as the reaction progressed. No other prominent signals attributable to a thiomethyl group of other potential intermediates were observed to accumulate over the course of the reaction. Notably, enamine 4a was not observed at any stage of the reaction. Instead, its deuterated iminium ion (4a-iminiun) was observed, suggesting that the enamine is in rapid equilibration with the protonated iminium ion and the former rapidly reacts. With the concentration of all major components able to be quantified by 1H NMR (Supporting Information Table S1), a reaction time course profile was plotted and is depicted in Figure 9. As the reaction proceeds and as the concentration of starting 1a and 4a-iminiun decrease, the concentration of 3a' increases until it reached the maximum concentration of 0.015 M, from where it slowly decreases. The appearance of product 3a increases faster than the appearance of 3a' for the first 200 min (2.2–2.7 fold), at which point nearly all 1a is consumed, followed by a slower increase in product that now nearly matches the rate of disappearance of 3a'. The decrease in the 4a-iminiun was found to be faster than 1a, likely attributable to non-productive competitive consumption of 4a-iminium by acid-promoted enamine self-condensation. A more refined treatment of the data, plotting the relative concentration of 3a and 3a' over time for the first 20 min, revealed that the 3a':3a ratio (0.45, 1:2.2) was time-independent. This conclusively establishes that generation of 3a and 3a' are formed in parallel reactions derived from a mutual intermediate (Supporting Information Figure S9). This is consistent with 3a' serving as an off route reversibly-generated compound rather than being a primary reaction intermediate.</p><p>Further consistent with this, the reaction profile of the reaction of 1a with enamine 4b (4methoxyphenacetaldehyde pyrrolidine enamine) was much more straightforward (Figure 9). The analogous intermediate 3b' was not observed in detectable amounts, indicating that it is either not generated in significant amounts under the conditions of the reaction or is much more rapidly converted to product precluding detection. To address this question, we identified conditions that allowed the isolation of 3b' (Figure 10) and measured the rate at which it and 3a' convert to the triazine products under the reaction conditions (25 °C, HFIP). Both 3a' and 3b' converted to the triazine products at slow and near equivalent rates (apparent first order k(obs) = 2.0 × 10−3 min−1 (3a') and 4.5 × 10−3 min−1 (3b')), requiring ca. 25.0 h (3a', t1/2 = 5.8 h) and 11.1 h (3b', t1/2 = 2.6 h) to run to completion (reach 95% yield). Thus, the lack of detection of 3b' in the reaction of 1a with 4b is not due to a much more rapid conversion to 3b. Rather, it can be attributed to 3b' not being generated in detectable amounts under the conditions of the faster direct reaction of 1a with 4b to provide 3b. Together, the combined observations indicate that compound 3a' is and 3b' would be reversibly generated off route compounds that ultimately also convert to the triazine products but do not appear to be on the direct pathway to the products. Finally, the extent of consumption of 1a nearly matches the accumulation of product 3b, indicating that essentially no non-productive consumption of tetrazine 1a occurs in the first 160 min.</p><p>With these key observations in hand, a plausible pathway from 1a and 4a to 3a and 3a' is summarized in Figure 11. H-Bonding activation of 3,6-dimethylthio-1,2,4,5-tetrazine and irreversible enamine nucleophilic attack at N1, either para (N4, shown) or ortho (N2) to the site of H-bonding (interconvertible by tautomerization), generates intermediate a or its 2H tautomer. Regeneration of the enamine from the iminium ion, and 6π electrocyclic rearrangement with required cleavage of the N1/N2 bond leads to c,21 followed by facile cis to trans azo-isomerization provides d. Intermediate d may undergo a 6π electrocyclic cyclization (C8-N5 bonding) to generate f or a reversible 5-endo-trig cyclization22 (C7-N5 bonding) followed by reversible 5-exo-trig ring closure to generate compound 3a' (red arrow). Final aromatization of f with loss of pyrrolidine and MeSCN through a 6-membered transition state, of which the latter two steps both may benefit from an intramolecular H-bond, provides the products of the reaction (3a and MeSCN). Direct generation of f from 3a' via e through a migrative ring expansion (blue arrows) is also possible. In this mechanism, 3a' is also eventually converted to 3a although the possibility of direct transformation of 3a' to 3a without passing through e cannot be ruled out. A related alternative route to intermediate c involves an initial addition to provide a and its stepwise cyclization to the [2 + 2] adduct23 followed by a strain induced 6π electrocyclic rearrangement with cleavage of the N1/N2 bond, followed by aminal cleavage and conversion to c (Figure 10). In addition to accounting for the generation of single 1,2,4-triazine product (3-methylthio-5-phenyl-1,2,4-triazine but no 3-methylthio-6-phenyl-1,2,4-triazine, Figure 5), key elements of the mechanism are a solvent H-bonding activation of the 1,2,4,5-tetrazine for enamine nucleophilic addition to N1 and a 6π electrocyclic rearrangement for the needed cleavage of the N1/N2 bond, representing a pathway to 3a where overall stepwise 1,4-cycloaddition occurs prior to elimination of thiomethylcyanate (MeSCN).</p><!><p>The unprecedented 1,4-cycoladdition has provided rapid access to a suite of analogs of 3a, which itself has been used both to access functionalized 1,2,4-triazines and as a key intermediate in the synthesis of biologically active compounds (Figure 12). Thus, the thiomethyl group in the product 1,2,4-triazines 3 can be used as an effective functional group for further diversification of the product 1,2,4-triazine.24,25 This serves to complement the use of alternatively substituted 1,2,4,5-tetrazines (e.g., 6c). An example that we would like to especially highlight is the use of 3a as a precursor for a second heterocyclic azadiene Diels–Alder reaction,26 setting the foundation for sequential cycloaddition strategies for rapidly accessing a diverse set of highly substituted or fused pyridines that are challenging to prepare by other means.1b Similarly, representative examples of the direct use of 3a in the preparation of biologically active compounds can be highlighted with the preparation of inosine monophosphate dehydrogenase (IMPDH) inhibitors27 and adenosine A2A antagonists,28 for which the work herein provides a simple synthesis of a suite of 3a analogs.</p><!><p>An unprecedented 1,4-cycloaddition of 1,2,4,5-tetrazines with enamines has been discovered. The reaction is conducted under mild reaction conditions (0.1 M HFIP, 25 °C, 12 h) with a broad scope of in situ generated acyclic and cyclic aryl-conjugated enamines to provide 5-aryl-1,2,4-triazines in good yields. Factors that impact this unprecedented change in the reaction mode (N1/N4 vs C3/C6 cycloaddition), optimization of the reaction conditions, the substrate scope of the reaction partners, simplification of its implementation with preformed, in situ formed, or sub stoichiometrically-generated enamines, and mechanistic insights into this remarkable cycloaddition are detailed. Given its breath, it establishes a new method for the simple and efficient one-step synthesis of 1,2,4-triazines under mild conditions from readily accessible starting materials. Mechanistic studies revealed several important features of the reaction and highlight the unique behavior of HFIP, where the strength or extent of its H-bonding interaction with the tetrazines is responsible for the alteration of the cycloaddition mode from the typical 3,6-cycloaddition to 1,4-cycloaddition. As such, the studies represent the first example of not just an enhancement in the rate and efficiency of a heterocyclic azadiene cycloaddition by H-bonding catalysis, but an alteration in the mode of cycloaddition as well. Key elements of a plausible stepwise addition-cyclization mechanism that accounts for the generation of a single 1,2,4-triazine product (3-methylthio-5-phenyl-1,2,4-triazine and no 3-methylthio-6-phenyl-1,2,4-triazine) and other observations to date, are a solvent H-bonding activation of the 1,2,4,5-tetrazine that promotes enamine nucleophilic addition to N1 and a 6π electrocyclic rearrangement for the needed cleavage of the N1/N2 bond, providing a pathway to the products where overall 1,4-cycloaddition occurs prior to elimination of a nitrile. Continued efforts that examine and further expand solvent hydrogen bonding catalysis of other heterocyclic and acyclic azadienes are ongoing and will be reported in due course.</p>

PubMed Author Manuscript

High-throughput discovery of Hf promotion on the formation of hcp Co and Fischer-Tropsch activity

A proxy-based high-throughput experimental approach was used to explore the stability and activity of Co-based Fischer-Tropsch Synthesis catalysts with different promoters on a variety of supports. The protocol is based on XRD estimation of the active phase polymorph, particle size and ratio of crystalline phases of Co to support. Sequential sample libraries enabled exploration of four Co loadings with five different promoters on six support materials. Catalysts stable to aging in syngas, i.e. displaying minimal change of particle size or active phase concentration, were evaluated under industrial conditions. This procedure identified SiC as a support that confers catalyst stability and that a combination of Ru and Hf promotes the formation of hcp Co. Unsupported bulk samples of Co with appropriate amounts of Ru and Hf revealed that the formation of hcp Co is independent of the support. The hcp Co-containing catalyst afforded the highest catalytic activity and C5+ selectivity amongst the samples tested in this study, confirming the effectiveness of the proxy-based high-throughput method.

high-throughput_discovery_of_hf_promotion_on_the_formation_of_hcp_co_and_fischer-tropsch_activity

Introduction<!>Sample preparation<!>Scale up of samples<!>Unsupported samples<!>Characterization<!>Fischer -Tropsch Synthesis<!>Results of the first library<!>Fischer-Tropsch Synthesis -First test<!>Results of the second library<!>Scale up of samples<!>Temperature programmed reduction<!>Fischer-Tropsch Synthesis -Second test<!>Unsupported Samples<!>Fischer-Tropsch Synthesis -Third test<!>Conclusions

<p>The Fischer-Tropsch Synthesis (FTS) is an important process for the production of long chain hydrocarbons from syngas (a mixture of CO and H2) using catalysts with Co, Fe or Ru as active metals. The development of active and stable catalysts is a key factor for the implementation of FTS in the production of fuels and chemicals [1,2]. The activity of FTS catalysts is attributed to different factors such as the particle size, active metal phase, the metal surface area, and the interactions with the support material [3][4][5][6][7][8][9]. The presence of different Co polymorphs (fcc and hcp) for example plays an important role in the activity of the catalysts and has been subject to many studies recently, showing that the hcp Co phase is more active than the fcc Co phase in FTS [3,4,[10][11][12]. The deactivation of the catalysts can occur through different mechanisms such as sintering, oxidation, formation of metal-support compounds, carbon deposition and poisoning [6,9,13,14]. Realistic investigations addressing the activity and stability of FTS catalysts require extended testing times and diverse catalyst sets to delineate the effect of the different factors.</p><p>The formation and stabilization of the hcp Co phase for FTS catalysts has been thoroughly investigated [3, 4, 10-12, 15, 16]. The hcp Co phase is destabilized with increasing temperature because of its higher surface energy compared to fcc Co [17]. Thus, above a temperature of approximately 400 °C fcc Co is the main phase observed [18]. De la Pena et al. [19] reported the formation of Co hcp particles by reducing Co3O4 nanoparticles in a H2 and CO gas mixture. The authors observed the formation of a graphitic layer encapsulating the hcp Co particles, which stabilizes the hcp phase up to a temperature of 700 °C by decreasing the surface energy of those particles. To achieve the formation of the hcp phase on a support material, Gnanamani et al. [4] treat supported Co samples under CO flow and moderate temperatures (230 °C) to form Co2C. The reduction of the Co2C at 230 °C under H2 leads to the formation of the hcp Co, which is attributed to structural similarity between these phases as both crystallize in the hexagonal system. No investigation has facilitated the formation of the Co hcp phase by adding promotors and reducing the samples under H2 at temperatures as high as 500 °C.</p><p>High throughput (HT) experimentation is used to accelerate the screening of large catalyst sets, it has been successfully applied in the development of heterogeneous catalysts [20][21][22], and can be applied for the identification of stable FTS catalysts, by parallelizing the preparation, ageing and characterization of the samples. While HT testing of the catalytic properties of the materials demands access to specialised equipment [23], the use of a proxy can accelerate the identification of suitable, stable and active materials without having to test all of the elements of a library. In a previous paper [7] we reported the development of a proxy-based method for the accelerated discovery of stable FTS catalysts. The workflow (Supplementary Note 1) developed includes the parallelized preparation, reduction, characterization of the samples augmented by an aging procedure under FTS similar conditions (H2:CO = 3; 230 o C, 1 bar) for 100 h. The core of the proxy is the assessment of the stability and activity of the samples by estimating the change in particle size and the amount of metallic Co with the aging procedure. This is carried out by comparing the Co peak width and area (calculated relative to the support peak area). The last step is to run high throughput TGA on selected samples to estimate the degree of reduction. The use of this method identified the composition of a series of highly stable, high surface area supported Co catalysts promoted by Mg and Ru, and the optimum preparation protocol (i.e. order of addition, calcination steps).</p><p>The proxy protocol described above is applied here to investigate the effect of different support materials and promotors on the stability of Co based catalysts. The iterative workflow was used to first screen six different support materials (active carbon, Al2O3, SiC, SiO2, TiO2, ZrO2) and five promotors (Ru, Re, Mo, Mn, La). This set of experiments identified SiC as a suitable support material and the screening of further promotors (Ru, La, Ce, Pr, Nd, Hf) demonstrated the positive effects of a combination of Co, Ru, Hf on SiC, which lead to the formation of hcp Co. Catalytic experiments under FTS conditions revealed higher C5+ selectivity for a catalyst promoted with Ru and Hf. A more comprehensive study of the effects of Ru and Hf on the formation and stabilization of the hcp Co without support materials, shows that Ru and Hf alone stabilize this phase, but the effect increases significantly, when both are present, stabilizing the hcp Co phase to a temperature up to 500 o C and 12 h.</p><!><p>Catalysts were synthesized by incipient wetness impregnation of the support materials. The support materials were weighed by a Quantos automated balance (Mettler Toledo XPE206) (250 mg) into 24 vials. An Eppendorf ep Motion 5075 was used for the liquid deposition onto the support materials; a plate holding an array of 4 x 6 vials containing the weighed support materials, allowed a variation of four different Co loadings and six different promotors on each support material this gives a total of 24 samples with different composition on each of the six support materials (Table S1), i.e., a total of 144 samples.</p><p>The impregnation of the support materials is a multistep procedure. In a typical synthesis of a material with 20 wt.-% of Co, 0.212 ml of a 4.0 M Co(NO3)2•6H2O solution were added to the 24 vials each containing 250 mg of SiC using the Eppendorf ep Motion 5075, followed by the addition of 0.047 ml of water. After mixing the materials with a spatula, the vials are placed on a shaker for 1 h to obtain a homogenous distribution of the solution on the support material. The samples were dried at 100 °C for 16 h. The impregnation of each promoter (Ru, Re, Mo, Mn, and La in the first library and Ru, La, Ce, Pr, Nd, and Hf in the second library) is performed with the respective solution and water amounts. The exact amount of promoter precursors and solutions used in each impregnation are provided in the Supplementary Information file (Supplementary Note 2). The addition of promoter solution is followed by a drying step at 100 °C for 16 h and in samples with two promotors the same drying step is performed between the additions. Finally the samples are calcined at 300 °C under air for 6 h for the decomposition of the nitrates. The following step is the reduction of the samples under pure H2 flow (50 ml/min) at 500 °C for 12 h in ceramic well plates (128x43x6 mm, Figure S2). The ceramic well plate can hold 48 different samples and three of the plates can be reduced each time, making a total of 144 samples per run. Before taking the catalysts out of the furnace they are passivated with 1 % O2 in N2 (100 ml/min) at room temperature. The ceramic well plates are directly placed in a high throughput XRD (HT XRD, Panalytical X-pert Pro diffractometer with an XYZ stage) for the first characterization of the samples.</p><p>The aging procedure of the samples was performed using the ceramic well plates under conditions approximating low temperature FTS with the flow of syngas (H2:CO = 2:1, 90 ml/min) at 230 °C and ambient pressure for 100 h. After the aging procedure the samples were passivated with 1 % O2 in N2 (100 ml/min) at room temperature and transferred to the HT XRD for characterization.</p><!><p>Samples selected for scale up were prepared manually in an identical manner to the procedure described above. In a typical synthesis, a sample with 20 wt.-% Co, 0.1 wt.-% Ru, and 5 wt.-% Hf supported on SiC (CoRuHf SiC) was prepared as follows: 1.697 ml of the 4.0 M Co(NO3)2•6H2O solution was added to 0.463 ml water and deposited on 2 g of SiC in a porcelain dish (80 mm diameter) placed on a shaker. The sample was dried at 100 °C for 16 h, prior to the next impregnation. The next impregnation with the 0.1 M Ru(NO)(NO3)3 solution is performed in an identical manner, but with the respective solution (0.197 ml) and water (2.083 ml) amounts, followed by a drying step at 100 °C for 16 h. Finally, the impregnation with 0.47 M HfCl2O is performed with the respective solution (1.192 ml) and water (0.974 ml) amounts, followed by a last drying step at 100 °C for 16 h and a posterior heat treatment at 300 °C under air for 6 h for the decomposition of the nitrates. The second step is the reduction of the samples, which was performed in a tube furnace under pure H2 flow (50 ml/min) at 500 °C for 12 h in ceramic boats (80 mm). Before taking the catalysts out of the furnace they are passivated at room temperature with 1 % O2 in N2 (100 ml/min).</p><!><p>Unsupported samples were prepared by mixing quantities of the solutions to obtain the desired atomic ratios. In a typical synthesis of a sample with Ru:Co = 0.003 and Hf:Co = 0.08 atomic ratios 1.073 ml of the 4 M Co(NO3)2•6H2O solution, 0.127 ml of the 0.1 M Ru(NO)(NO3)3 solution and 0.722 ml of the 0.47 M HfCl2O solution were mixed together. After mixing the solutions, the drying, calcination and reduction steps are identical as for the supported samples described above.</p><!><p>High-throughput X-ray diffraction, XRD, was performed on a Panalytical X-pert Pro diffractometer with an XYZ stage using Co Kα radiation between 38°-55° 2θ, with a 0.039° step size and 2 s/step. The particle size of the Co metal phase was calculated from the line broadening of the most intense fcc Co peak (111) using the Scherrer equation in X'Pert HighScore Plus software using a Si standard to determine the instrumental line width. Relative crystallinity was determined from the Co (111) to support XRD peak area ratio.</p><p>XRD of bulk samples was performed with a Panalytical X-pert Pro in Bragg-Brentano geometry laboratory X-ray diffractometer with Co Ka1 radiation = 1.78901 Å. Synchrotron XRD data was recorded on beamline I11 (λ = 0.825972 Å) at Diamond Light Source, UK.</p><p>Temperature programmed reduction (TPR) was measured using a Quantachrome ChemBET 3000 unit with a TPD; ca. 50 mg of the calcined sample were loaded into a quartz cell and heated up to 100 °C for 30 min under He (100 ml/min) to remove moisture and other adsorbed species from the samples. After cooling down to room temperature the sample was heated to 750 °C at 5 °C min −1 under a flow of 5% H2 in N2 (100 cm 3 min −1 ) to perform the analysis.</p><!><p>The activity tests in the FTS were performed at Drochaid Research Services Ltd in St. Andrews. The high throughput catalyst testing was done using a custom-built 32-tube fixed bed reactor test plant, designed by Integrated Lab Solutions and built by Premex. One unit, consisting of two heating blocks with eight reactors each, was used for the tests. The reactors (L = 300 mm; ID = 2.4 mm; OD = 6.5 mm) were accommodated in a heating furnace with an isothermal zone of 100 mm. The catalyst bed length is of 7 cm with in the isothermal zone. SiC was used as an inert diluent material. The flow of the gases CO (CP grade, BOC), H2 (CP grade, BOC) and Ar (CP grade, BOC) as an internal standard was controlled by mass flow controller and the pressure by a digital back pressure regulator. The concentration of reaction educts and products was measured using online GC techniques (Agilent Refinery Gas Analyser with a FID channel for the quantification of light hydrocarbons (DB-1 column (2m x 0.32mm x 5μm) and HP-AL/S column (25m x 0.32mm x 8 μm)) and two TCD channels; TCD1 for the permanent gases CH4, CO2, Ar, N2, and CO (HayeSep Q column (6 ft., 80/100 mesh) and a Mol Sieve 5A column (6 ft., 60/80 mesh)) and TCD2 for H2 (HayeSep 3 ft.,80/100 mesh and Molsieve 8 ft., 60/80 mesh). The activity of the catalysts is expressed with cobalt time yield, which was calculated based on molar consumption per gram cobalt per second.</p><!><p>The HT exploration of FTS catalysts is based on the prior assessment of stability of the active phase, metallic Co, on treatment with syngas as proxy for their activity. The applied workflow involves the parallel synthesis, syngas treatment and XRD characterisation of sample libraries. These are prepared by automated incipient wetness impregnation and reduced concurrently by H2 at 500 o C. HT-XRD analysis is employed to determine the phase of Co and its particle size (Supplementary Note 3) before and after the aging of samples under a syngas mixture, H2:CO = 2:1, at 230 o C for 100 h. Large changes in the particle size of Co or in its overall crystallinity, estimated by the peak area ratio of Co to support, are taken as indicators of the lack of stability and predictors of low catalytic activity for FTS.</p><p>The effects of the support material, the Co loading and the addition of promotors were investigated in the first library, which consisted of 144 samples. Support materials Al2O3 [3,13,[24][25][26][27][28][29][30][31][32], TiO2 [8,13,30,[33][34][35][36][37][38][39] ZrO2 [30,34,35], SiO2 [4,13,30,34,[40][41][42][43][44][45][46][47] active carbon (AC) [29,32,48,49], and SiC [50,51] were used. Despite the reactivity of metal oxides, particularly Al2O3, with Co they were selected as supports for their porosity and thermal stability. Non-oxide supports do not interfere with Co, which can be reduced at relatively low temperatures. Ru, Re, Mo, Mn and La were used as promotors. Ru and Re are known to increase the reducibility of cobalt on support materials [8,13,27,28,30,33,35] and increase the resistance to deactivation. Furthermore, Ru is known to be the most active element in FTS. The transition metals Mo and Mn were selected too, since they have been reported to increase the chaingrowth probability factor to larger hydrocarbons in the product stream [32,37,42,46,47,52,53] and finally the rare earth metal La, which has been reported to increase the dispersion of Co on the support [30,31,39,44,46,48,54]. Based on a first assessment of the HT XRD patterns, samples showing no metallic Co peaks after the reduction or after the aging procedure were immediately ruled out. This is the case for the samples supported on ZrO2 and samples with only 5 wt% Co loading on every other support. The results of crystallite size analysis for aged samples are plotted versus the crystallite size before aging (Figure 1a). The y=x line is used to display the increase, points above the line, or the decrease, points below the line, of the crystallite size after the syngas aging process. Samples prepared on SiO2, AC and TiO2 show an increase in the crystallite size after aging, up to a doubling of the particle size after the aging procedure observed for SiO2, AC and TiO2, while the samples supported on SiC show an increase to a lesser extent, from 27 to 36 nm. For samples supported on Al2O3 a decrease of the particle size from 33 to 16 nm, in the most extreme case, was observed. Figure 1b shows the peak area ratio between Co and support, for the three crystalline supports (Al2O3, TiO2 and SiC), before and after the aging procedure. TiO2 and Al2O3 show a decrease of the peak area ratio after the aging procedure (up to 70 % Co peak area ratio loss on samples supported on TiO2 and up to 60 % Co peak area ratio loss on samples supported on Al2O3), while the area ratio of samples on SiC show the smallest change (between 8 and 23 % Co peak area ratio increase). The effect of the different promotors can clearly be observed for the samples supported on SiC (Figure 1c and d). The samples that show the smallest change in the Co peak area ratio are those promoted with Ru (from 0.63 to 0.75), La (from 0.65 to 0.83) and Mo (from 0.70 to 0.77).</p><!><p>Since the samples prepared on SiC show the smallest change of Co crystallite size and peak area ratio after the aging procedure, they were identified as hits from the first library. The samples with a Co loading of 15 wt%; promoted with Ru and Re, at 0.1 wt% loading, and Mn, Mo or La, 5 wt% loading were scaled up and tested in FTS under industrial conditions. These samples were named after the active component and the promoter, e.g. the sample CoRu had 15 wt% Co and 0.</p><!><p>The proxy screening on the first library proved the suitability of SiC as a support material and the positive effect of Ru and La on the stability of Co after the aging procedure. Furthermore, the catalytic test demonstrated the positive effects of the simultaneous promotion of La and Ru enhancing the CO conversion rate and the C5+ selectivity. A second library focussed on the effect of the content of Ru and the inclusion of rare earth elements (La, Ce, Pr, Nd), and Hf as promotors for Co-based materials supported on SiC.</p><p>While the effect of rare earths on Co-based catalysts has been thoroughly investigated [46,47,54,55], little is known about the effect of the promotion of Hf [36] and its effect on the phase formation and stabilization of Co particles. Hf was chosen, because it is in the same group as Ti and Zr, whose oxides are known promotors and support materials for FT catalysts and because it is in the same period as the rare earth metals. Many patents mention the use of Hf as a promoter for Co based FTS catalysts, but only one patent [36] could be found, where experimental data showing the positive effect of Hf promotion of Co-based catalysts is presented.</p><p>The effect of rare earths (La, Ce, Pr, Nd) or Hf addition at different loading levels was investigated for samples loaded with Co and Co and Ru, as shown in Table 3. The results obtained after submitting the samples to the workflow described above can be seen in Figure 3, where peak area ratio of Co after the aging procedure is plotted over the peak area ratio before the aging procedure. Each colour represents a different promoter, the shape of the symbols represents different levels of each promoter and the open symbols represent samples without Ru. Samples with Ru and 2.5 or 5 wt% Hf, along with samples promoted with Ru and Ce and samples with Ru and Pr, show a good stability under aging conditions. Samples promoted without Ru prove to be stable under the aging conditions but show lower Co peak area ratios than samples promoted with Ru. The samples promoted with Ru and Hf are particularly striking, whilst the sample with 5 wt% Hf and 0.1 wt% Ru stabilizes the Co peak area as shown in Figure 3, it also shows a further peak on the XRD patterns, which corresponds to the hcp Co phase (Figure S3). The formation of the hcp Co phase is also observed for the rest of the samples promoted with Ru and different levels of Hf.</p><!><p>The samples with 5 wt% loading of promotors displayed the highest stability in the proxy screening of the second library and they were selected for scale up, characterisation and FTS testing. All samples have fixed loadings of Co (20 wt%), Ru (0.1 wt%), and either Ce, Pr, La or Hf (5 wt%) or no promoter.</p><p>A sixth sample with standard Co loading (20 wt%) alone supported on SiC was made for comparison purposes. The samples are named according to the active component and the promotors in a similar fashion to the first set of samples.</p><p>The synchrotron PXRD patterns of the six scaled up samples and of the pure SiC are presented in</p><!><p>In order to understand the effect of each promoter on the reduction behaviour of Co, the samples were submitted to a TPR analysis under 5 % H2 in N2 (Figure 5). The sample containing only 20 wt% Co shows two peaks, the first one at a lower temperature (300-375 °C) is associated with the reduction of the Co(III) species to Co(II), and the second one, at a higher temperature (400-500 °C) with the reduction of the Co(II) species to Co(0) [30]. The addition of Ru to the samples enhances the reducibility of the samples lowering the temperature of both reduction steps. The addition of both Ru and rare earth metals as promotors leads to an increase of the reduction temperature for both steps, compared to those promoted by Ru alone, as illustrated by the TPR for the samples promoted with Ru and La or Pr. The sample promoted with mixture of Ru and Ce shows similar reduction behaviour in relation to the sample promoted only with Ru. Again it is the sample promoted with Ru and Hf that exhibits the most differentiated reduction behaviour. No reduction is observed up to 330 °C and the peaks for both reduction steps merge together in a single peak between 330-460 °C.</p><!><p>The scaled-up materials were tested under industrial FTS conditions in a parallel reactor system at 20 bar and three different temperatures (Figure 6a). At 210 °C the materials CoRu, CoRuLa, CoRuCe and CoRuPr show similar Co time yield and only the material CoRuHf shows higher performance. At 220 °C all the materials display an increase in their activity with CoRuHf being the most active and CoRuPr showing higher yield than the rest of the samples. At 230 °C all the materials show different CO conversion rates in the FTS with the following activity order CoRuHf> CoRuPr> CoRuLa> CoRuCe> CoRu. The material promoted with Ru and Hf not only shows the highest yield at all temperatures, it also displays the highest selectivity values towards C5+ components as Table 4 shows. (11.84 %). Methane, being a raw material for the production of syngas, and CO2, are undesired by-products in FTS [56].</p><p>A comparison between the fresh and used samples is shown on Figure 6b. Compared with the fresh catalysts the XRD characterization of the spent catalysts shows no significant loss of the Co phases for any of the catalysts. The CoRuHf shows the presence of the hcp Co phase in the fresh and in the spent material. Both fcc and hcp Co phases are stable under the testing conditions, and the deactivation observed at 230 °C during the catalytic test is not caused by the formation of Co oxides. The higher activity and C5+ selectivity values of the Hf containing material compared to the rest of the catalysts is consistent with the formation of the hcp Co phase. The effect of the different Co phases on the activity of the catalysts has been studied previously [3-5, 11, 12, 25, 43] and it was demonstrated hcp phase is more active the fcc one.</p><!><p>The formation of the hcp Co phase supported on SiC and promoted with Hf and Ru motivated us to further investigate the effect of Hf and Ru on formation of hcp Co. To eliminate the effect from the support material a set of samples with different Hf:Co and Ru:Co atomic ratios were prepared and reduced at temperatures between 300 and 500 o C for different times. Samples showing a reduction time of 0 h were held at the reduction temperature for 5 min before cooling down at the natural rate of the furnace. The PXRD characterization results of the samples with three different Hf:Co ratios are provided in Figure 7. The sample with Hf:Co = 0 is reduced completely at 300 °C and shows the formation of the hcp Co phase. An increase in the reduction temperature to 500 °C leads to the formation of the fcc Co phase, which is clearly observed by the appearance of the (200) peak at 2θ = 60.34°. Hcp Co is further converted to the fcc phase with longer reduction times, 2 and 12 h, at 500 °C. The phase composition of each sample has been obtained by Rietveld refinement of the PXRD patterns (Supplementary Note 4).</p><p>The final sample of this series, reduction for 12 h at 500 °C, contains hcp Co phase, 48 wt% and fcc Co, 52 wt% (Table S2). The addition of Hf to the samples shifts the temperature window of Co reduction and hcp to fcc conversion, as the PXRD patterns of the samples with Hf:Co = 0.04 and Hf:Co = 0.08 show (Figures 7b and c). The sample with Hf:Co = 0.04 reduced at 300 °C show mainly the peaks of CoO and some hcp Co (8 wt%). At 400 °C only the metallic phases of Co can be observed and hcp Co remains the dominant phase, 80 wt%, even after reduction at 500 °C for 12 h. Higher Hf content, Hf:Co = 0.08, increases further reduction and phase transformation temperatures. At 300 o C only the oxide phases CoO and Co3O4 are the present and their reduction has been completed at 500 °C. After reducing this sample at 500 °C for 12 h the most of Co is present as the hcp phase, 78 wt%, while very broad peaks corresponding to monoclinic HfO2 have been appeared.</p><!><p>The promoting effect of Hf on the stability of hcp Co and FTS activity that was observed on the samples of second library motivated the further investigation its effect on catalytic activity when combined with two Ru contents, 0.01 wt% and 0.1 wt%. Five samples were prepared with fixed loading of Co (20 wt%), Ru loading 0.01 and 0.1 wt% with and without Hf (5 wt%). PXRD measurements (Figure S7) confirmed the presence of both Co phases in samples containing Hf while the samples without Hf display mainly fcc Co and only traces of hcp Co. The materials were tested in FTS in four different temperatures (Figure 9) and the catalytic activity of the samples containing Hf at each temperature is higher than the noncontaining Hf counterparts by factor of ~ 1.6. Moreover, the sample 20Co/0.01Ru/5Hf display slightly higher Co time yield than the sample 20Co/0.1Ru at 230 o C and 240 o C. This result demonstrates that promotion with Hf at 5 wt% and the subsequent formation of hcp Co compensate the loss of activity caused by the decrease of Ru content by an order of magnitude.</p><!><p>Application of the described high throughput protocol enabled the efficient screening of different support materials and promotors for the Co based FTS catalysts. The protocol allowed us to identify SiC as a suitable support for active and stable catalysts. Furthermore the promotors Ru and Hf were found to not only increase the stability of the catalysts, but also to enhance the formation of the hcp Co phase, known to be more active in FTS than the fcc Co phase.</p><p>Samples containing Co, Ru and Hf were scaled up alongside other samples promoted with Ru and rare earth elements and tested in the FTS under industrial conditions (230 °C, 20 bar). Here the material containing Co, Ru and Hf showed higher CO conversion than other catalysts tested and also exhibited the highest selectivity towards desirable C5+ components. The higher conversion and selectivity of this materials is attributed to the enhanced formation of the hcp Co phase.</p><p>Further investigation of the impact of Hf in the formation of the different cobalt phases demonstrates that Hf shifts the reduction of CoO to higher temperatures and stabilizes the hcp phase at higher temperatures (500 °C) and also during longer reduction times. The addition of Ru to the samples facilitates the reduction of CoO, such that all samples, even those with higher amounts of Hf (up to 0.08 molar ratio) are reduced at temperatures of 300 °C. The simultaneous addition of Ru and Hf to the samples inhibits the transformation of the hcp to the fcc phase, to a greater extent than is achieved by the addition of Hf alone.</p>

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