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In the 1H NMR spectra (DMSO-d6) of 1,3-disubstituted ureas containing the 4,7,7-trimethyl-3-oxo-4-oxabicyclo[2.2.1]heptan-1-yl substituent, the signals for the CH2—CH2 moiety appear as an ADMP-type spin system consisting of four doublets of doublets of doublets with chemical shifts in the range from 1.54 to 2.80 ppm. The experiment in CD3OD showed the disappearance of the signal at δ 2.80 and a change in the multiplicity of the other signals, which is indicative of the selective deuterium exchange of one of the four protons of the CH2—CH2 group for a deuterium atom. This allows the selective modification of compounds containing the oxacamphanyl moiety. [ABSTRACT FROM AUTHOR]

Funding: This project was supported with funds from the US Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development Program Award number i01 BX003312 (M.K.R.). M.K.R. is a recipient of a VA Research Career Scientist Award (14S-RCS001). Research reported in this publication was also supported by the US National Institutes of Health under award numbers R01AI100569 and R01AI141412 (M.K.R.) and by the US Department of Defense Peer Reviewed Medical Research Program (PR181134) (M.K.R.). This work was also funded by VA Merit Review Award BX004522 to JSD from the US Department of Veterans Affairs Biomedical Laboratory Research and Development. The National Science Foundation provided instrument funding for the BioAnalytical Mass Spectrometry Facility at Portland State University (NSF, MRI 1828573), which was used to generate HRMS analytical measurements. [ABSTRACT FROM AUTHOR]

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for 'forward' Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict 'one way' detoxication enzyme. [ABSTRACT FROM AUTHOR]

Due to significant safety tolerances on maximum levels of visible and sub-visible particles in parenterally dosed drug products like monoclonal antibodies (mAbs), particle formation rates must be determined during development and minimized. Agitation stress, encountered during transportation and manufacturing, increases particle formation rates in a protein and formulation dependent fashion in a phenomenon thought to be partially mediated by mAb adsorption to the continuously regenerating air-water interface that results from agitation. The goal of this study was to explore the structural dynamics of three mAbs with variable sensitivity to agitation to develop a mechanistic understanding of exactly what occurs at the air-water interface that leads to aggregation and particle formation. We observed preferential orientation at the interface and subsequent cooperative unfolding for the molecule which aggregates most extensively under agitation, and also that the magnitude of destabilization appears to scale with particle formation rates. We also show that polysorbate, a widely-used excipient in parenteral formulations to protect against particle formation, eliminates interface-induced destabilization. This study provides direct evidence that local unfolding events resulting from interface exposure precede particle formation and may play a causal role in the process. [ABSTRACT FROM AUTHOR]

Antibody drug conjugates (ADCs) have been at the forefront in cancer therapy due to their target specificity. All the FDA approved ADCs are developed in lyophilized form to minimize instability associated with the linker that connects the cytotoxic drug and the antibody during shipping and storage. We present here solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a tool to analyze protein structure and matrix interactions for formulations of an ADC with and without commonly used excipients. We compared results of the ssHDX-MS with accelerated stability results using size-exclusion chromatography and determined that the former technique was able to successfully identify the destabilizing effects of mannitol and polysorbate 80. In comparison, Fourier-transform infrared spectroscopy results were inconclusive. The agreement between ssHDX-MS and stressed stability studies supports the potential of ssHDX-MS as a method of predicting relative stability of different formulations. [ABSTRACT FROM AUTHOR]

Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix. HX-MS measurements of NIST mAb bound to in-solution and resin forms of ProA revealed regions of the C H 2 and C H 3 domains with increased protection from HX upon ProA binding, consistent with the known ProA binding region. In-solution ProA experiments revealed regions in the F ab with increased HX uptake when the ProA:mAb molar ratio was increased to 2:1, suggesting an allosterically induced increase in backbone flexibility. Such effects were not observed with lower ProA concentration (1:1 molar ratio) or when ProA resin was used, suggesting some kind of change in binding mode. Since all pharmaceutical processes use ProA bound to resin, our results rule out reversible allosteric effects on the NIST mAb during interaction with resin ProA. However, irreversible effects cannot be ruled out since the NIST mAb was previously exposed to ProA during its original purification. [ABSTRACT FROM AUTHOR]

This study aimed at a better understanding of the wood-water interaction, in particular the role of the hydroxyl accessibility during the humidity-dependent change in moisture content. Thin sections (80 µm) of never-dried Norway spruce sapwood that contained early- and latewood were used for the experiments. Sorption isotherm measurements confirmed the humidity-dependent moisture content changes and the effect of the first drying of the wood sections. Changes in hydroxyl accessibility were then determined by deuteration of the sections using deuterium oxide, followed by their re-protonation in water (H2O) vapor at different relative humidity: 15, 55 or 95%. The deuteration and re-protonation of the wood sections were quantified by dry mass changes as well as by changes in the OH and OD stretching vibrations in the Fourier transform infrared spectra. The results showed that the deuterated sections could be almost completely re-protonated in H2O vapor, nearly irrespective of the applied relative humidity. Therefore, changes in hydroxyl accessibility were not the driving force for the humidity-dependent changes in moisture content. However, a slow re-protonation rate at low relative humidity had to be considered. Nonetheless, a small quantity of OD groups persisted the re-protonation in H2O vapor and liquid H2O, which was not related to the drying of the wood. [ABSTRACT FROM AUTHOR]

The volatile thiol compound 3-sulfanylhexan-1-ol (3SH) is a key impact odorant of white wines such as Sauvignon Blanc. 3SH is produced during fermentation by metabolism of non-volatile precursors such as 3-S-gluthathionylhexanal (glut-3SH-al). The biogenesis of 3SH is not fully understood, and the role of glut-3SH-al in this pathway is yet to be elucidated. The aldehyde functional group of glut-3SH-al is known to make this compound more reactive than other precursors to 3SH, and we are reporting for the first time that glut-3SH-al can exist in both keto and enol forms in aqueous solutions. At wine typical pH (~3.5), glut-3SH-al exists predominantly as the enol form. The dominance of the enol form over the keto form has implications in terms of potential consumption/conversion of glut-3SH-al by previously unidentified pathways. Therefore, this work will aid in the further elucidation of the role of glut-3SH-al towards 3SH formation in wine, with significant implications for the study and analysis of analogous compounds.

The objective of the study was to investigate the water vapour sorption behaviour of thermally modified (TM) wood powder, e.g. ground wood prepared from waste streams of TM solid wood, and wood powder that was extracted in pressurised hot water. Solid spruce wood was TM in steam conditions (210°C for 3 h), milled and hot-water-extracted (HWE) at elevated pressure (140°C for 1 h). The results evidence that the hot-water extraction reduced the water sorption and the accessible hydroxyl group concentration by the removal of amorphous carbohydrates. In contrast, the enhanced cross-linking of the cell wall matrix and the annealing of amorphous matrix polymers during thermal modification reduced the sorption behaviour of wood additionally, without further reducing the hydroxyl accessibility. These additional effects of thermal modification were at least partially cancelled by hot-water extraction. The results bring novel insights into the mechanisms that reduce the water vapour sorption of wood by compositional and structural changes induced by heating. [ABSTRACT FROM AUTHOR]

Deamidation is an important degradation pathway for proteins. Estimating deamidation propensities is essential for predicting their long-term stability. However, predicting deamidation rates in folded proteins is challenging because higher-order structure has a significant and unpredictable effect on deamidation. Here, we investigated the correlation between amide hydrogen exchange (HX) and deamidation to assess the potential of using hydrogen exchange–mass spectrometry (HX-MS) to rapidly predict deamidation propensity. Maltose-binding protein and a structurally less stable mutant, W169G, were stored in the dark at pH 7.0 at 23 ± 2°C for 1 year. Deamidation at each asparagine site was measured using liquid chromatography–mass spectrometry after trypsin digestion. Deamidation rates at each deamidation site were determined based on first-order kinetics. HX rates at the deamidation sites were determined before storage using the shortest peptic peptide containing each site using conventional bottom-up HX-MS at pD 7.0 at 25°C. We observed a power law correlation between deamidation half-life and HX half-life for the NG sites with measurable kinetics. For NA sites, slow deamidation was only observed at 2 sites located in rapidly exchanging regions. Our findings demonstrate that HX-MS can be used to reliably and rapidly rank deamidation propensity in folded proteins. [ABSTRACT FROM AUTHOR]

Abstract Earlywood samples of unmodified and acetylated radiata pine were exposed to the brown-rot fungus Rhodonia placenta for 1, 2, 3 and 4 weeks for unmodified samples and 10, 16, 24 and 28 weeks for acetylated samples. Longer incubation periods were used for acetylated samples based on the hypothesis that given enough time under favourable conditions the fungus would eventually degrade the wood. After exposure, samples were weighed and chemically characterized by ATR-FTIR analysis, acetyl content by saponification, and hydroxyl (OH) accessibility by deuterium exchange. Longer incubation times for acetylated samples led to comparable levels of mass loss between unmodified and acetylated wood. Initial brown-rot decay in acetylated wood exhibited a different trend compared to unmodified wood, with an increased OH accessibility and a significant reduction in acetyl content. This was followed by a stable, low OH accessibility and plateau in acetyl content above 10% mass loss in acetylated wood. In unmodified wood, the OH accessibility was nearly constant throughout decay, while the initially low acetyl content decreased linearly with mass loss. ATR-FTIR analysis confirmed the differences in acetyl removal between unmodified and acetylated wood. Wood-water relations before and after brown-rot decay were determined with low-field nuclear magnetic resonance (LFNMR) relaxometry on water saturated samples. For the decayed acetylated wood, the behaviour of the water corresponded well with de-acetylation observed by chemical characterization. The results show that after removal of acetyl groups, degradation of acetylated wood by R. placenta occurred at a similar rate to that of unmodified wood. Highlights • Brown-rot was delayed in acetylated wood but the rate of degradation matched that of unmodified wood once decay started. • Hydroxyl accessibility and acetyl content analysis indicate de-acetylation during initial degradation of acetylated wood. • LFNMR T 2 relaxation times also suggest de-acetylation followed by nanoporosity development in acetylated wood. [ABSTRACT FROM AUTHOR]

Abstract—The deuterium concentration in normal mice and mice inoculated with 4T1 mammary carcinoma has been studied by magnetic resonance imaging (MRI) and 1H and 2H contrast. Mice received deuterium (up to 10%) in drinking water. The reversible effect was observed when mice began to drink ordinary drinking water. The average period that is required to perform hydrogen/deuterium exchange in all parts of the mouse is 2.4 days. Hydrogen/deuterium exchange rates in normal and deuterated mice are not reliably distinguished. The day range for hydrogen/deuterium exchange in biopolymers depends on its localization in the mouse and varies from 8 to 24 days. The nonmonotonic character of the time dependence of the deuterium level was observed after a weekly consumption of drinking water with an increased deuterium content in the tumor. The median survival rate of the inoculated mice that received drinking water with an increased content of deuterium was 4 days longer than that in the control group. [ABSTRACT FROM AUTHOR]

Abstract: A reliable, simple and fast method for quantitative analysis of heavy water (D2O) was developed using deuterium exchange headspace extraction-gas chromatography-mass spectrometry. The method is based on incubating the acetic anhydride with D2O, CH3COOD formation, headspace extraction of CH3COOD (exchange of hydrogen for deuterium) and gas chromatography/mass spectrometry detection. The quantification was performed using single ion monitoring (m/z = 61). The calibration was linear (R2 = 0.9998) in the concentration range of 1 × 103-3 × 105 mg/L). This method can measure D2O concentrations in water from 0.27 g/L. It was applied for determination of D2O in distilled water with accuracy RSD ≤ 6% and precision RSD ≤ 4%. [ABSTRACT FROM AUTHOR]

Acetylated wood (WAc) shows improved properties largely due to the reduced amount of water in its cell wall, but the exact mechanism of water reduction remains unclear. Acetylation reduces hydroxyl (OH) content by acetyl (Ac) substitution but may also limit water access to unmodified OH groups by steric hindrance. In the present work, the accessibility of OH groups in acetylated or propionylated Radiata pine (Pinus radiata D. Don) wood (WAc and WPr) was investigated by deuterium exchange, saponification in sodium hydroxide followed by highperformance liquid chromatography (HPLC) analysis and weight percentage gain determination of the modified samples. Acetylation reduced OH accessibility (OHA) to a greater extent than would be predicted, if OH substitution were the only responsible mechanism for accessibility reduction. The combination of deuterium exchange and saponification results provides strong evidence that steric hindrance plays a key role in reduction of water accessibility to unmodified OH groups in WAc. The supramolecular architecture of WPr samples seems to be modified by the propionylation reaction, which leads to increased OHA at low levels of substitution. This suggests that molecular restructuring within the cell wall exposes new OH groups after propionylation. At higher levels of substitution, however, the WPr exhibited less OHA than expected indicating steric hindrance from the propionyl groups. [ABSTRACT FROM AUTHOR]

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D2O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern. [ABSTRACT FROM AUTHOR]

Conversion of acetyl cedrene ( 2) to its follower ( 3) using acetic anhydride and polyphosphoric acid involves a multi-step cationic molecular rearrangement, which is consistent with deuteriation and 1-C labeling studies of acetyl cedrene. The key step involves cyclopropylcarbinyl cation-cyclopropylcarbinyl cation rearrangement. [ABSTRACT FROM AUTHOR]

The first drying of wood cell walls from the native state has sometimes been described as producing irreversible structural changes which reduce the accessibility to water, a phenomenon often referred to as hornification. This study demonstrates that while changes do seem to take place, these are more complex than what has hitherto been described. The accessibility of wood cell wall hydroxyls to deuteration in the form of liquid water was not found to be affected by drying, since vacuum impregnation with liquid water restores the native cell wall accessibility. Contrary to this, hydroxyl accessibility to deuteration by water vapour was found to decrease to different levels depending on the drying conditions. Vacuum drying at 60 °C for 3 days reduced the accessibility more than drying for 1 day at 103 °C without vacuum. Drying for 3 days at 103 °C increased the hydroxyl accessibility compared to 1 day. Moreover, the decrease in hydroxyl accessibility to deuteration by water vapour induced by the first drying could be at least partially erased by subsequent vacuum impregnation with liquid water, indicating reversibility. For the drying of solid, non-degraded wood cell walls the results challenge the often supposed process of hornification, understood as a permanent decrease in hydroxyl accessibility to water. [ABSTRACT FROM AUTHOR]

The structure of T-2 toxin in the solid-state is limited to X-ray crystallographic studies, which lack sufficient resolution to provide direct evidence for hydrogen-bonding interactions. Furthermore, its solution-structure, despite extensive Nuclear Magnetic Resonance (NMR) studies, has provided little insight into its hydrogen-bonding behavior, thus far. Hydrogen-bonding interactions are often an important part of biological activity. In order to study these interactions, the structure of T-2 toxin was compared in both the solution- and solid-state using NMR Spectroscopy. It was determined that the solution- and solid-state structure differ dramatically, as indicated by differences in their carbon chemical shifts, these observations are further supported by solution proton spectral parameters and exchange behavior. The slow chemical exchange process and cross-relaxation dynamics with water observed between the hydroxyl hydrogen on C-3 and water supports the existence of a preferential hydrogen bonding interaction on the opposite side of the molecule from the epoxide ring, which is known to be essential for trichothecene toxicity. This result implies that these hydrogen-bonding interactions could play an important role in the biological function of T-2 toxin and posits towards a possible interaction for the trichothecene class of toxins and the ribosome. These findings clearly illustrate the importance of utilizing solid-state NMR for the study of biological compounds, and suggest that a more detailed study of this whole class of toxins, namely trichothecenes, should be pursued using this methodology.

Tetraazafulvalene 1 has found broad application in reduction and other related transformations and is conveniently generated from bis-propyl bis-imidazolium salt 4 with a strong base in a non-protic solvent. The proposed mechanism for the formation of 1 involves initial at C(2) to give a mono-carbene 9 followed by reaction at the second azolium centre. Herein, we report the second-order rate constants for deuteroxide-catalysed exchange in aqueous solution of the C(2)-hydrogens of bis-propyl bis-imidazolium di-iodide salt 4 and related monomeric dipropyl imidazolium iodide 10 of kDO = 1.37 × 104 and 1.79 × 102 M−1 s−1, respectively, and used these data to calculate p Ka values of 21.2 and 23.1. The greater C(2)-H acidity of the doubly bridged bis-propyl bis-imidazolium salt 4 relative to 10 may be attributed to the inductive or electrostatic destabilization of the conjugate acid dicationic azolium ion 4 relative to the monocationic carbene 9, which is enhanced by bis-tethering. Formation of tetraazafulvalene 1 was not observed under the aqueous conditions employed highlighting that carbene reprotonation significantly outcompetes under these conditions. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The interaction of water with cellulose stages many unresolved questions. Here 2 H MAS NMR and IR spectra recorded under carefully selected conditions in 1 H- 2 H exchanged, and re-exchanged, cellulose samples are presented. It is shown here, by a quantitative and robust approach, that only two of the three available hydroxyl groups on the surface of cellulose fibrils are exchanging their hydrogen with the surrounding water molecules. This finding is additionally verified and explained by MD simulations which demonstrate that the 1 HO(2) and 1 HO(6) hydroxyl groups of the constituting glucose units act as hydrogen-bond donors to water, while the 1 HO(3) groups behave exclusively as hydrogen-bond acceptors from water and donate hydrogen to their intra-chain neighbors O(5). We conclude that such a behavior makes the latter hydroxyl group unreactive to hydrogen exchange with water. [ABSTRACT FROM AUTHOR]

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for protein structural analysis has been adopted for many purposes, including biopharmaceutical development. One of the benefits of examining amide proton exchange by mass spectrometry is that it can readily resolve different exchange regimes, as evidenced by either binomial or bimodal isotope patterns. By careful analysis of the isotope pattern during exchange, more insight can be obtained on protein behavior in solution. However, one must be sure that any observed bimodal isotope patterns are not artifacts of analysis and are reflective of the true behavior in solution. Sample carryover and certain stationary phases are known as potential sources of bimodal artifacts. Here, we describe an additional undocumented source of deuterium loss resulting in artificial bimodal patterns for certain highly charged peptides. We demonstrate that this phenomenon is predominantly due to gas-phase proton exchange between peptides and bulk solvent within the initial stages of high-transmission conjoined ion guides. Minor adjustments of the ion guide settings, as reported here, eliminate the phenomenon without sacrificing signal intensity. Such gas-phase deuterium loss should be appreciated for all HDX-MS studies using such ion optics, even for routine studies not focused on interpreting bimodal spectra. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Of central importance in chemistry and biology, enolate chemistry is an attractive topic to elaborate on possible contributions of anion-π interactions to catalysis. To demonstrate the existence of such contributions, experimental evidence for the stabilization of not only anions but also anionic intermediates and transition states on π-acidic aromatic surfaces is decisive. To tackle this challenge for enolate chemistry with maximal precision and minimal uncertainty, malonate dilactones are covalently positioned on the π-acidic surface of naphthalenediimides (NDIs). Their presence is directly visible in the upfield shifts of the α-protons in the 1H NMR spectra. The reactivity of these protons on π-acidic surfaces is measured by hydrogen-deuterium (H-D) exchange for 11 different examples, excluding controls. The velocity of H-D exchange increases with π acidity (NDI core substituents: SO2R>SOR>H>OR>OR/NR2>SR>NR2). The H-D exchange kinetics vary with the structure of the enolate (malonates>methylmalonates, dilactones>dithiolactones). Moreover, they depend on the distance to the π surface (bridge length: 11-13 atoms). Most importantly, H-D exchange depends strongly on the chirality of the π surface (chiral sulfoxides as core substituents; the crystal structure of the enantiopure ( R, R, P)-macrocycle is reported). For maximal π acidity, transition-state stabilizations up to −18.8 kJ mol−1 are obtained for H-D exchange. The Brønsted acidity of the enols increases strongly with π acidity of the aromatic surface, the lowest measured p Ka=10.9 calculates to a Δp Ka=−5.5. Corresponding to the deprotonation of arginine residues in neutral water, considered as 'impossible' in biology, the found enolate-π interactions are very important. The strong dependence of enolate stabilization on the unprecedented seven-component π-acidity gradient over almost 1 eV demonstrates quantitatively that such important anion-π activities can be expected only from strong enough π acids. [ABSTRACT FROM AUTHOR]

This article presents a novel method to selectively probe the non-crystalline, hydrated fractions of cellulosic biomass. The method is based on time-resolved infrared spectra analyzed to provide information on spectral and dynamical features of deuterium exchange (OH → OD) in D 2 O atmosphere. We assign deuterium exchange spectral regions (700–3800 cm −1 ) and explore changes due to relative humidity, different cellulosic samples, and infrared polarization. Here, two results are highlighted. First, a wide range of celluloses isolated from plants show remarkable spectral similarities whatever the relative amounts of cellulose and xylan. This result supports an inherent type of hydrated disorder which is mostly insensitive to the molecular identities of the associated polysaccharides. Second, polarized infrared analysis of cotton reveals hydrated cellulose having chains preferentially aligned with those of crystals, while the hydroxyls of hydrated cellulose present much more randomized orientation. Our results provide new insights on molecular and group orientation and on hydrogen bonding in hydrated fractions of cellulosic biomass. [ABSTRACT FROM AUTHOR]

Previous studies of the C(3)-hydrogen/deuterium of the triazolium ion conjugate acids of triazolyl N-heterocyclic carbenes revealed a change of mechanism under acidic conditions with N1-protonation to a dicationic salt. Interestingly, the data suggested an increase in p KaN1 in the presence of a N-pentafluorophenyl substituent relative to other N-aryl substituents with hydrogens or methyl substituents rather than fluorines at the ortho-positions. To probe the presence of an apparent donor effect of a N-pentafluorophenyl substituent, which differs from the more common electron withdrawing effect of this group, we have studied the analogous deuterium of four triazolium salts with heteroatoms or heteroatom substituents in the 2-position and/or 6-position of the N-aryl ring. These include triazolium salts with N-2,4,6-tribromophenyl 11, N-2,6-dichlorophenyl 12, N-2-pyridyl 13 and N-2-pyrimidinyl 14 substituents. The log kex - p D profiles for 11, 12 and 14 were found to show similar trends at lower p Ds as for the previously studied N-pentafluorophenyl triazolium salt, hence supporting the presence an apparent donor effect on p KaN1. Surprisingly, the log kex - p D profile for N-pyridyl salt 13 uniquely showed at lower p Ds. We propose herein that this data is best explained by invoking an general base role for the N-(2-pyridyl) substituent in conjunction with N1-protonation on the triazolium ring. Finally, the second order rate constants for deuteroxide ion catalysed C(3)-H/D exchange ( kDO, M−1 s−1), which could be obtained from data at p Ds >1.5, were used to provide estimates of C(3)-carbon acid p KaC3 values for the four triazolium salts 11, 12, 13, 14. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP+. The neutron data were collected to 2.0-Å resolution using a 3.6-mm3 crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed. [ABSTRACT FROM AUTHOR]

The deuterium exchange rate of the α-methylene of deoxyvasicinone ( 1) and mackinazolinone ( 2) and their seven- ( 3) and eight-membered ( 4) homologs was studied in neutral, acidic, and basic media. It was found that the exchange rate depended on the number of methylenes in the polymethylene ring and the medium. The deuterium exchange rate decreased in the order 2→ 1→ 3→ 4 and was slowest in media close to neutral. The results agreed with those for the reactions of 1- 4 with bromine in acidic medium, condensation with substituted aromatic and heterocyclic aldehydes, and acylation by aromatic acid chlorides. Several trends in the reactivity of the tricyclic quinazolin-4-ones were discovered. [ABSTRACT FROM AUTHOR]

A method for the preparation of multi-gramme quantities of N-methyl- d3- N-nitroso- p-toluenesulfonamide (Diazald- d3) and N-methyl-13 C-N-nitroso- p-toluenesulfonamide (Diazald-13 C) and their conversion to diazomethane- d2 and diazomethane-13 C, respectively, is presented. This approach uses robust and reliable chemistry, and critically, employs readily commercially available and inexpensive methanol as the label source. Several reactions of labelled diazomethane are also reported, including alkene cyclopropanation, phenol methylation and α-diazoketone formation, as well as deuterium scrambling in the preparation of diazomethane- d2 and subsequent methyl esterification of benzoic acid. [ABSTRACT FROM AUTHOR]

Highlights: [•] Concept of sheath-flow chemistry for CE-MS is described and demonstrated. [•] Fundamental effects of capillary diameter investigated using example of hydrogen/deuterium exchange. [•] Antioxidant assay using DPPH with CE-MS developed using these concepts. [Copyright &y& Elsevier]

Background: ADAMTS-13, a plasma metalloprotease, cleaves von Willebrand factor. ADAMTS-13 activity appears to be regulated through allosteric inhibition by its distal C-terminus., Objectives: The objective of this study was to better understand how domain-domain interactions may affect ADAMTS-13 conformations and functions., Methods: We performed deuterium-hydrogen exchange plus mass spectrometry to assess the number and rate of deuterium incorporation into various peptides of full-length ADAMTS-13 and its truncated variants., Results: Under physiological conditions, a bimodal distribution of deuterium incorporation was detected in the peptides from metalloprotease (217-230 and 282-304), cysteine-rich (446-482), and CUB (for complement C1r/C1s, Uegf, Bmp1) domains (1185-1214, 1313-1330, 1341-1347, 1358-1378, and 1393-1407) of full-length recombinant ADAMTS-13, but not of truncated variants. These results suggest that the full-length ADAMTS-13 undergoes conformational changes. On removal of the middle and distal C-terminal domains, the number and rate of deuterium incorporation were increased in the peptides from cysteine-rich (445-467, 467-482, and 495-503) and spacer domains (621-642 and 655-654) but decreased in the peptides from metalloprotease (115-124, 217-230, and 274-281). Moreover, most peptides, except for 217-230 and 1357-1376, exhibited a pD-dependent deuterium incorporation in the full-length ADAMTS-13, but not in the truncated variant (eg, MDTCS or T5C). These results further suggest that the bimodal deuterium incorporation observed in the peptides from the full-length ADAMTS-13 is the result of potential impact from the middle to distal C-terminal domains. Surface plasmon resonance revealed the direct binding interactions between the distal and proximal domains of ADAMTS-13., Conclusion: Our results provide novel insight on how intramolecular interactions may affect conformations of ADAMTS-13, thus regulating its proteolytic functions., (© 2022 The Author(s).)

Highlights: [•] Three kinds of bridging hydroxyls are detected on H-FER. [•] Adsorption of N2 at 100K allows selective measuring of the acidity of the OH groups. [•] CO is not appropriate IR probe for measuring acidity of FER bridging hydroxyls. [ABSTRACT FROM AUTHOR]

The fragmentations of [AA + M] complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag). [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The phase structure of nanocrystalline cellulose (NCC) is studied with solid-state NMR experiments on NCC samples subjected to H/H exchange in order to label regions accessible to water. These novel results show partial but similar exchange in portions of the C spectrum previously assigned to both highly crystalline and disordered/surface cellulose. Exchange in liquid DO occurs rapidly, faster than the time-scale required to dry the samples, while exposure to DO vapor reveals a single time constant of ∼30 h to build up to the liquid exposed exchange level. In both cases ca. 37 % of the expected exchangeable sites are found to contain H. These results, along with those from relaxation experiments requiring multi-component or stretched-exponential fits, together with X-ray diffraction results suggest the NCC particles contain distributions of environments distinguished by their structures and crystalline perfection. The incomplete exchange of the disordered/surface regions suggests that the NCC crystallites contain abundant disordered domains in their interiors that are protected from exchange. Preliminary P/C REDOR experiments on phosphoric-acid hydrolyzed NCC samples provide a C spectrum of accessible surface sites, but are of relatively low signal-to-noise. These experiments suggest a phosphate coverage of 2.6 per 100 anhydroglucose monomers, similar to sulfate coverage measured previously for sulfuric acid hydrolyzed samples. [ABSTRACT FROM AUTHOR]

Irradiation of 2-phenyl-1-naphthol ( 6) in CH3CN/D2O (3:1) leads to very efficient incorporation of deuterium at the ortho-positions of the adjacent phenyl ring (overall Φ=0.73±0.07), along with minor incorporation at the naphthalene positions 5 and 8. These finding are explained by excited state intramolecular proton transfer (ESIPT) from the phenolic OH group to the corresponding carbon atoms, the main pathway giving rise to quinone methide (QM) 7, which has been characterized by LFP ( τ≈20 ns; 460 nm). The ESIPT reaction paths have been explored with the second order approximate coupled cluster (CC2) method. In nonprotic solvents the ESIPT from the naphthol O-H to the ortho-position of the phenyl ring proceeds in a barrierless manner along the 1La energy surface via a conical intersection with the S0 state, delivering 7. In aqueous solvent, clusters with H2O are formed wherein proton transfer (PT) to solvent and a H2O-mediated relay mechanism gives rise to naphtholates and QMs. The results are compared with 2-phenylphenol ( 3) that also undergoes barrierless ESIPT giving a QM via a conical intersection. However, due to an unfavorable conformation in the ground state, the quantum efficiency for ESIPT of 3 is significantly lower ( Φ for D-exchange=0.041). These results show that ESIPT from phenol to carbon need not be an intrinsically inefficient process. [ABSTRACT FROM AUTHOR]

Natural microbial communities are extremely diverse and contain uncharacterized but functionally important small molecules. By coupling a deuterium (D) labeling technique to high mass accuracy untargeted liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) metabolomic analysis, we found that natural acidophilic microbial biofilms dominated by bacteria of the genus Leptospirillum contained unusual lyso phosphatidylethanolamine (PE) lipids in high abundance (more than 10 nmol/mg of dry biomass). The unusual polar head group structure of these lipids is similar to lipids found in phylogenetically unrelated acidophilic chemoautolithotrophs and may be related to the affinity of these lipids for iron and calcium ions. Correlations of lyso phospholipid and proteome abundance patterns suggest a link between the lyso phospholipids and the UBA-type substrain of Leptospirillum group II. By combining untargeted metabolomics with D exchange we demonstrate the ability to identify cryptic but biologically functional small molecules in mixed microbial communities. [ABSTRACT FROM AUTHOR]

Although the use of hydrogen exchange (HX) mass spectrometry (MS) to study proteins and protein conformation is now over 20 years old, the perception lingers that it still has 'issues.' Is this method, in fact, still in the quicksand with many remaining obstacles to overcome? We do not think so. This critical insight addresses the 'issues' and explores several broad questions including, have the limitations of HX MS been surmounted and has HX MS achieved 'indispensable' status in the pantheon of protein structural analysis tools. [ABSTRACT FROM AUTHOR]

The hydrogen-deuterium exchange reaction was performed for hydrogenation of D-1-pentene (4,4,5,5,5) under realistic cobalt Fischer-Tropsch synthesis conditions. In the presence of CO, the added deutero-1-pentene did not show any significant H/D exchange but a step-wise H/D exchange occurs when CO was replaced with N. The inhibition effects of CO and other FT products on H/D exchange of D-1-pentene and a pressure dependency effect on H/D exchange are observed. Graphical Abstract: [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]