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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 -
Angewandte Chemie (International Ed. in... Nov 2020Radiolabelling is fundamental in drug discovery and development as it is mandatory for preclinical ADME studies and late-stage human clinical trials. Herein, a general,...
Radiolabelling is fundamental in drug discovery and development as it is mandatory for preclinical ADME studies and late-stage human clinical trials. Herein, a general, effective, and easy to implement method for the multiple site incorporation of deuterium and tritium atoms using the commercially available and air-stable iridium precatalyst [Ir(COD)(OMe)] is described. A large scope of pharmaceutically relevant substructures can be labelled using this method including pyridine, pyrazine, indole, carbazole, aniline, oxa-/thia-zoles, thiophene, but also electron-rich phenyl groups. The high functional group tolerance of the reaction is highlighted by the labelling of a wide range of complex pharmaceuticals, containing notably halogen or sulfur atoms and nitrile groups. The multiple site hydrogen isotope incorporation has been explained by the in situ formation of complementary catalytically active species: monometallic iridium complexes and iridium nanoparticles.
Topics: Catalysis; Coordination Complexes; Deuterium; Heterocyclic Compounds; Iridium; Isotope Labeling; Tritium
PubMed: 33463019
DOI: 10.1002/anie.202008519 -
Chirality Feb 2022The study of enantiodiscriminations in relation to various facets of enantiomorphism (chirality/prochirality) and/or molecular symmetry is an exciting area of modern... (Review)
Review
The study of enantiodiscriminations in relation to various facets of enantiomorphism (chirality/prochirality) and/or molecular symmetry is an exciting area of modern organic chemistry and an ongoing challenge for nuclear magnetic resonance (NMR) spectroscopists who have developed many useful analytical approaches to solve stereochemical problems. Among them, the anisotropic NMR using chiral aligning solvents has provided a set of new and original tools by making accessible all intramolecular, order-dependent NMR interactions (anisotropic interactions), such as residual chemical shift anisotropy (RCSA), residual dipolar coupling (RDC), and residual quadrupolar coupling (RQC) for spin I > 1/2, while preserving high spectral resolution. The force of NMR in enantiopure, oriented solvents lies on its ability to orient differently in average on the NMR timescale enantiomers of chiral molecules and enantiotopic elements of prochiral ones, leading distinct NMR spectra or signals to be detected. In this compendium mainly written for all chemists playing with (pro)chirality, we overview various key aspects of NMR in weakly aligning chiral solvents as the lyotropic liquid crystals (LLCs), in particular those developed in France to study (pro)chiral compounds in relation with chemists needs: study of enantiopurity of mixture, stereochemistry, natural isotopic fractionation, as well as molecular conformation and configuration. Key representative examples covering the diversity of enantiomorphism concept, and the main and most recent applications illustrating the analytical potential of this NMR in polypeptide-based chiral liquid crystals (CLCs) are examined. The latest analytical strategy developed to determine in-solution conformational distribution of flexibles solutes using NMR in polypeptide-based aligned solvents is also proposed.
Topics: Deuterium; Liquid Crystals; Magnetic Resonance Spectroscopy; Molecular Conformation; Stereoisomerism
PubMed: 34936130
DOI: 10.1002/chir.23386 -
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 -
Immunological Reviews Sep 2018Deuterium is a non-toxic, stable isotope that can safely be administered to humans and mice to study their cellular turnover rates in vivo. It is incorporated into newly... (Review)
Review
Deuterium is a non-toxic, stable isotope that can safely be administered to humans and mice to study their cellular turnover rates in vivo. It is incorporated into newly synthesized DNA strands during cell division, without interference with the kinetics of cells, and the accumulation and loss of deuterium in the DNA of sorted (sub-)populations of leukocytes can be used to estimate their cellular production rates and lifespans. In the past two decades, this powerful technology has been used to estimate the turnover rates of various types of leukocytes. Although it is the most reliable technique currently available to study leukocyte turnover, there are remarkable differences between the cellular turnover rates estimated by some of these studies. We have recently established that part of this variation is due to (a) difficulties in estimating deuterium availability in some deuterium-labeling studies, and (b) assumptions made by the mathematical models employed to fit the data. Being aware of these two problems, we here aim to approach a consensus on the life expectancies of different types of T cells, B cells, monocytes, and neutrophils in mice and men. We address remaining outstanding problems whenever appropriate and discuss for which immune subpopulations we currently have too little information to draw firm conclusions about their turnover.
Topics: Animals; Cell Division; DNA; Deuterium; Humans; Isotope Labeling; Leukocytes; Longevity; Mice; Models, Theoretical
PubMed: 30129193
DOI: 10.1111/imr.12693 -
Journal of Mass Spectrometry : JMS Feb 2022Stable isotope tracing can be safely used for metabolic studies in animals and humans. The endogenous biosynthesis of lipids (lipogenesis) is a key process throughout...
Stable isotope tracing can be safely used for metabolic studies in animals and humans. The endogenous biosynthesis of lipids (lipogenesis) is a key process throughout the entire life but especially during brain and lung growth. Adequate synthesis of pulmonary surfactant lipids is indispensable for life. With this study, we report the use of deuterium-depleted water (DDW), suitable for human consumption, as metabolic precursor for lipogenesis. We studied 13 adult rabbits for 5 days. Four rabbits drank tap water (TW) and served as controls; in four animals, DDW was substituted to drinking water, whereas five drank deuterium-enriched water (DEW). After 5 days, a blood sample and a bronchoalveolar lavage (BAL) sample were collected. The H/ H (δ H) of BAL palmitic acid (PA) desaturated phosphatidylcholine (DSPC), the major phospholipid of pulmonary surfactant, and of plasma water was determined by high-resolution mass spectrometry. We found that the δ H values of DDW, DEW and TW were -984 ± 2‰, +757 ± 2‰ and -58 ± 1‰, respectively. After 5 days, plasma water values were -467 ± 87‰, +377 ± 56‰ and -53 ± 6‰, and BAL DSPC-PA was -401 ± 27‰, -96 ± 38‰ and -249 ± 9‰ in the DDW, DEW and TW, respectively. With this preliminary study, we demonstrated the feasibility of using DDW to label pulmonary surfactant lipids. This novel approach can be used in animals and in humans, and we speculate that it could be associated with more favourable study compliance than DEW in human studies.
Topics: Animals; Deuterium; Drinking Water; Phosphatidylcholines; Phospholipids; Pulmonary Surfactants; Rabbits
PubMed: 35060656
DOI: 10.1002/jms.4808 -
Analytical Methods : Advancing Methods... Mar 2023Determining the purity of deuterium labelled compounds is important due to the increasing use of these compounds in mass spectrometry (MS) based quantitative analyses...
Determining the purity of deuterium labelled compounds is important due to the increasing use of these compounds in mass spectrometry (MS) based quantitative analyses for targeting metabolic flux, reducing toxicity, confirming reaction mechanisms during synthesis, predicting enzyme mechanisms, and enhancing the efficacy of drugs, in quantitative proteomics, and also as internal standards. In the present study, a strategy using liquid chromatography electrospray ionization high resolution mass spectrometry (LC-ESI-HR-MS) and nuclear magnetic resonance (NMR) spectroscopy was proposed to determine the isotopic enrichment and structural integrity of deuterium labelled compounds. The proposed strategy involves recording full scan MS, extracting and integrating isotopic ions, and calculating the isotopic enrichment of the desired labelled compounds. NMR analysis confirms structural integrity or positions of labelled atoms and can provide insights into the relative percent isotopic purity. This strategy was used to evaluate the isotopic enrichment and structural integrity of in-house synthesized compounds as well as a series of commercially available deuterium labelled compounds. The % isotopic purity for labelled compounds of a benzofuranone derivative (BEN-), tamsulosin- (TAM-), oxybutynin- (OXY-), eplerenone- (EPL-), and propafenone- (PRO-) was calculated and found to be 94.7, 99.5, 98.8, 99.9, and 96.5, respectively. All the samples were run in triplicate and the results were observed to be reproducible.
Topics: Deuterium; Spectrometry, Mass, Electrospray Ionization; Magnetic Resonance Spectroscopy; Chromatography, Liquid; Magnetic Resonance Imaging
PubMed: 36876453
DOI: 10.1039/d2ay01980a -
Journal of the American Chemical Society Apr 2022Deuterated amino acids have been recognized for their utility in drug development, for facilitating nuclear magnetic resonance (NMR) analysis, and as probes for enzyme...
Deuterated amino acids have been recognized for their utility in drug development, for facilitating nuclear magnetic resonance (NMR) analysis, and as probes for enzyme mechanism. Small molecule-based methods for the site-selective synthesis of deuterated amino acids typically involve de novo synthesis of the compound from deuterated precursors. In comparison, enzymatic methods for introducing deuterium offer improved efficiency, operating directly on free amino acids to achieve hydrogen-deuterium (H/D) exchange. However, site selectivity remains a significant challenge for enzyme-mediated deuteration, limiting access to desirable deuteration motifs. Here, we use enzyme-catalyzed deuteration, combined with steady-state kinetic analysis and ultraviolet (UV)-vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of l--Ile. We show that an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze Cα and Cβ H/D exchange of amino acids, while reactions without DsaE lead exclusively to Cα-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for Cα/Cβ-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.
Topics: Amines; Amino Acids; Catalysis; Deuterium; Hydrogen; Kinetics; Proteins
PubMed: 35416652
DOI: 10.1021/jacs.2c00608 -
Anti-cancer Drugs Nov 2023Colorectal cancer is one of the most common types of cancer in the world and the study of the role of nutrients in preventing or inhibiting the growth of this cancer is...
Colorectal cancer is one of the most common types of cancer in the world and the study of the role of nutrients in preventing or inhibiting the growth of this cancer is of interest to scientists. In this article, the synergistic effect of deuterium-depleted water(DDW) and crocin at specific concentrations on HT-29 cells was investigated. In this regard, HT-29 cells were grown in RPMI medium containing DDW, alone and in combination with crocin for 24, 48 and 72 h. Cell viability, cell cycle changes and antioxidant enzymes status were determined by MTT assay, flow cytometry and quantitative luminescence methods, respectively. The results of these analyses proved the cell growth inhibitory effect of deuterium alone and its synergistic effect in combination with crocin. The cell cycle analysis showed an increase in the number of cells in the G0 and G1 phases whereas there was a decrease in the number of cells in the S, G2 and M phases. The activities of superoxide dismutase and catalase enzymes also decreased compared to the control group that is a reason to increase Malonyl dialdehyde factor. The results suggested that a combination of DDW and crocin can open a new strategic approach in the prevention and treatment of colorectal cancer.
Topics: Humans; Deuterium; HT29 Cells; Water; Colorectal Neoplasms
PubMed: 36847076
DOI: 10.1097/CAD.0000000000001512 -
BMC Bioinformatics Dec 2014Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is...
BACKGROUND
Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation.
RESULTS
Here, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com .
CONCLUSION
Isotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis.
Topics: Carbon Isotopes; Deuterium; Deuterium Exchange Measurement; Humans; Hydrogen; Isotope Labeling; Mass Spectrometry; Peptide Fragments; Proteins; Software
PubMed: 25495703
DOI: 10.1186/s12859-014-0403-1