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Nature Communications Aug 2022Site- and enantioselective incorporation of deuterium into organic compounds is of broad interest in organic synthesis, especially within the pharmaceutical industry....
Site- and enantioselective incorporation of deuterium into organic compounds is of broad interest in organic synthesis, especially within the pharmaceutical industry. While catalytic approaches relying on two-electron reaction manifolds have allowed for stereoselective delivery of a formal deuteride (D) or deuteron (D) at benzylic positions, complementary strategies that make use of one-electron deuterium atom transfer and target non-benzylic positions remain elusive. Here we report a photochemical approach for asymmetric radical deuteration by utilizing readily available peptide- or sugar-derived thiols as the catalyst and inexpensive deuterium oxide as the deuterium source. This metal-free platform enables four types of deuterofunctionalization reactions of exocyclic olefins and allows deuteration at non-benzylic positions with high levels of enantioselectivity and deuterium incorporation. Computational studies reveal that attractive non-covalent interactions are responsible for stereocontrol. We anticipate that our findings will open up new avenues for asymmetric deuteration.
Topics: Alkenes; Catalysis; Deuterium; Light
PubMed: 35915119
DOI: 10.1038/s41467-022-32238-8 -
Journal of Labelled Compounds &... 2013Cytochrome P450 (P450) enzymes account for ~75% of the metabolism of drugs. Most of the reactions catalyzed by P450s are mixed-function oxidations, and a C-H bond is... (Review)
Review
Cytochrome P450 (P450) enzymes account for ~75% of the metabolism of drugs. Most of the reactions catalyzed by P450s are mixed-function oxidations, and a C-H bond is (usually) broken. The rate-limiting nature of this step can be analyzed using the kinetic isotope effect (KIE) approach. The most relevant type of KIE is one termed intermolecular non-competitive, indicative of rate-limiting C-H bond breaking. A plot of KIE versus kcat for several P450s showed a correlation coefficient (r(2) ) of 0.62. Deuterium substitution has been considered as a potential means of slowing drug metabolism or redirecting sites of metabolism in some cases, and several general points can be made regarding the potential for application of deuterium in drug design/development based on what is known about P450 KIEs.
Topics: Cytochrome P-450 Enzyme System; Deuterium; Drug Discovery; Humans; Kinetics; Oxidation-Reduction
PubMed: 24285515
DOI: 10.1002/jlcr.3031 -
Biomolecules Jun 2021Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division...
Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including , a well-established model organism in cell cycle studies, are no exception. , a green unicellular alga of the class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in . Synchronous cultures of were cultivated in growth medium containing 70 or 90% DO. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of , contradicting the role of the "sizer" in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.
Topics: Cell Cycle; Cell Division; Chlamydomonas reinhardtii; Deuterium
PubMed: 34207920
DOI: 10.3390/biom11060861 -
The FEBS Journal Oct 2011The study of protein structure and function has evolved to become a leading discipline in the biophysical sciences. Although it is not yet possible to determine 3D... (Review)
Review
The study of protein structure and function has evolved to become a leading discipline in the biophysical sciences. Although it is not yet possible to determine 3D protein structures from MS data alone, multiple MS-based techniques can be combined to obtain structural and functional data that are complementary to classical protein structure information obtained from NMR or X-ray crystallography. Monitoring gas-phase interactions of noncovalent complexes yields information on binding constants, complex stability, and the nature of interactions. Ion mobility MS and chemical crosslinking strategies can be applied to probe the architecture of macromolecular assemblies and protein-ligand complexes. MS analysis of hydrogen-deuterium exchange can be used to determine the localization of secondary structure elements, binding sites and conformational dynamics of proteins in solution. This minireview focuses first on new strategies that combine these techniques to gain insights into protein structure and function. Using one such strategy, we then demonstrate how a novel hydrogen-deuterium exchange microfluidics tool can be used online with an ESI mass spectrometer to monitor regional accessibility in a peptide, as exemplified with amyloid-β peptide 1-40.
Topics: Animals; Deuterium; Deuterium Exchange Measurement; Humans; Hydrogen; Mass Spectrometry; Microfluidics; Models, Molecular; Proteins; Structure-Activity Relationship
PubMed: 21668648
DOI: 10.1111/j.1742-4658.2011.08215.x -
Protein Science : a Publication of the... Aug 1999A database of hydrogen-deuterium exchange results has been compiled for proteins for which there are published rates of out-exchange in the native state, protection... (Review)
Review
A database of hydrogen-deuterium exchange results has been compiled for proteins for which there are published rates of out-exchange in the native state, protection against exchange during folding, and out-exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359-360) is reexamined in a detailed comparison of the specific amide protons (NHs) and the elements of secondary structure on which they are located. For each pulsed exchange or competition experiment, probe NHs are shown explicitly; the large number and broad distribution of probe NHs support the validity of comparing out-exchange with pulsed-exchange/competition experiments. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native state, NHs first protected during folding, and NHs most protected in partially folded species. There is not a one-to-one correspondence of individual NHs. Proteins for which there are published data for native state out-exchange and theta values are also reviewed. The elements of secondary structure containing the slowest exchanging NHs in native proteins tend to contain side chains with high theta values or be connected to a turn/loop with high theta values. A definition for a protein core is proposed, and the implications for protein folding are discussed. Apparently, during folding and in the native state, nonlocal interactions between core sequences are favored more than other possible nonlocal interactions. Other studies of partially folded bovine pancreatic trypsin inhibitor (Barbar E, Barany G, Woodward C, 1995, Biochemistry 34:11423-11434; Barber E, Hare M, Daragan V, Barany G, Woodward C, 1998, Biochemistry 37:7822-7833), suggest that developing cores have site-specific energy barriers between microstates, one disordered, and the other(s) more ordered.
Topics: Animals; Cattle; Deuterium; Hydrogen; Protein Folding; Protein Structure, Secondary
PubMed: 10452602
DOI: 10.1110/ps.8.8.1571 -
Analytical Chemistry Jul 2022During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding...
During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during liquid chromatography (LC)-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD), in which the protein is deuterated at positions and analyzed with the same buffer components, %DO, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here, we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ∼40 min for nonmembrane proteins. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with earlier protocols that either require much more time or require isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed for maximum deuteration in a short time (∼10 min) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy-to-perform protocol should enhance routine preparation of maxD controls.
Topics: Deuterium; Deuterium Exchange Measurement; Hydrogen; Hydrogen Deuterium Exchange-Mass Spectrometry; Mass Spectrometry; Proteins
PubMed: 35796687
DOI: 10.1021/acs.analchem.2c01446 -
Drug Discovery Today Jan 2014The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in... (Review)
Review
The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this review, HDX-MS based workflows designed for protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS in protein therapeutics characterization are also described.
Topics: Animals; Deuterium; Humans; Hydrogen; Mass Spectrometry; Protein Binding; Protein Interaction Maps; Protein Structure, Secondary; Protein Transport
PubMed: 23928097
DOI: 10.1016/j.drudis.2013.07.019 -
Methods in Enzymology 2022Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a valuable technique to investigate the dynamics of protein systems. The approach compares the...
Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a valuable technique to investigate the dynamics of protein systems. The approach compares the deuterium uptake of protein backbone amides under multiple conditions to characterize protein conformation and interaction. HDX-MS is versatile and can be applied to diverse ligands, however, challenges remain when it comes to exploring complexes containing nucleic acids. In this chapter, we present procedures for the optimization and application of HDX-MS to studying RNA-binding proteins and use the RNA helicase Mtr4 as a demonstrative example. We highlight considerations in designing on-exchange, bottom-up, comparative studies on proteins with RNA. Our protocol details preliminary testing and optimization of experimental parameters. Difficulties arising from the inclusion of RNA, such as signal repression and sample carryover, are addressed. We discuss how chromatography parameters can be adjusted depending on the issues presented by the RNA, emphasizing reproducible peptide recovery in the absence and presence of RNA. Methods for visualization of HDX data integrated with statistical analysis are also reviewed with examples. These protocols can be applied to future studies of various RNA-protein complexes.
Topics: Deuterium; Deuterium Exchange Measurement; Hydrogen; Hydrogen Deuterium Exchange-Mass Spectrometry; Mass Spectrometry; Proteins; RNA
PubMed: 35965017
DOI: 10.1016/bs.mie.2022.04.002 -
Amino Acids Sep 2016Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging...
Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl-forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age.
Topics: Asparagine; Deuterium; Oligopeptides
PubMed: 27169868
DOI: 10.1007/s00726-016-2250-z -
Communications Biology Jun 2022Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for...
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for specific peptides. This rate will be altered upon structural perturbation and detecting significant changes to this rate requires a statistical test. To determine rates of incorporation, HDX-MS measurements are frequently made over a time course. However, current statistical testing procedures ignore the correlations in the temporal dimension of the data. Using tools from functional data analysis, we develop a testing procedure that explicitly incorporates a model of hydrogen deuterium exchange. To further improve statistical power, we develop an empirical Bayes version of our method, allowing us to borrow information across peptides and stabilise variance estimates for low sample sizes. Our approach has increased power, reduces false positives and improves interpretation over linear model-based approaches. Due to the improved flexibility of our method, we can apply it to a multi-antibody epitope-mapping experiment where current approaches are inapplicable due insufficient flexibility. Hence, our approach allows HDX-MS to be applied in more experimental scenarios and reduces the burden on experimentalists to produce excessive replicates. Our approach is implemented in the R-package "hdxstats": https://github.com/ococrook/hdxstats .
Topics: Bayes Theorem; Deuterium; Deuterium Exchange Measurement; Hydrogen Deuterium Exchange-Mass Spectrometry; Mass Spectrometry; Peptides
PubMed: 35705679
DOI: 10.1038/s42003-022-03517-3