A catalytic metal-free approach for the H/D exchange in aromatic compounds using D2O as the terminal deuterating reagent has been developed. This metal-free protocol employs a triaryl carbenium as the mediator and showcases a wide applicability in the late-stage deuteration of various natural products and small-molecule drugs. Gram-scale deuteration was successfully demonstrated with β-Estradiol, highlighting the method\'s practicability. Detailed mechanistic insights, supported by DFT calculations, unveiled the essential role of in-situ generated acidic species in this electrophilic aromatic substitution process. This newly developed method offers a sustainable and versatile alternative to traditional metal-catalyzed H/D exchange techniques, addressing challenges such as the use of expensive metals, impurity formation, and the necessity for residual metal removal from the final products.

We present the photoreaction of newly prepared bis(6,6\'-dimethyl-2,2\'-bipyridine)(oxalato)rhodium(III) ([Rh(N^N)2(ox)]+) in CD3OD. Photoirradiation of this complex causes the dissociation of ox, followed by the formation of the unprecedented Rh(III) complex with Rh-H and Rh-C s bonds, [Rh(N^N)(C^N)(H)(CD3OD)]+ (C^N = [6,6\'-dimethyl-2,2\'-bipyridine]-3-yl-κC3,κN1\'). This hydride formation and cyclometallation spontaneously proceed owing to the conflict between the steric hindrance arising from the methyl groups of N^N and the driving force for the structural change due to [Rh(N^N)2]+ formation. Although [Rh(N^N)(C^N)(H)(CD3OD)]+ is initially converted to [Rh(N^N)2]+ by photoirradiation, it is immediately regenerated by the rollover cyclometallation of the [Rh(N^N)2]+ complex. [Rh(N^N)(C^N)(H)(CD3OD)]+ undergoes H/D exchange for the H atoms in the Rh-H bond and at the 3, 3\'-positions of the N^N ligand during the photoirradiation. DFT calculations predict with reasonable certainty the spontaneous structural change of [Rh(N^N)2]+ to [Rh(N^N)(C^N)(H)(CD3OD)]+ and the subsequent photodriven Rh-C bond rupture leading to the formation of [Rh(N^N)2]+ accompanied by H/D exchange reactions.

The increasing demand for deuterium-labeled amino acids and derivatives has heightened interest in direct hydrogen/deuterium exchange reactions of free amino acids. Existing methods, including biocatalysis and metal catalysis, typically require expensive deuterium sources or excessive use of deuterium reagents and often struggle with site selectivity. In contrast, our pioneering binary catalysis system, employing benzaldehyde and Cs2CO3 in the presence of inexpensive D2O with minimal stoichiometric quantities, facilitates efficient hydrogen/deuterium exchange at the α-position of amino acids without the need for protecting groups in the polar aprotic solvent DMSO. The process is highly compatible with most natural and non-natural α-amino acids and derivatives, even those with potentially reactive functionalities. This advancement not only addresses the cost and efficiency concerns of existing methods but also significantly broadens the applicability and precision of deuterium labeling in biochemical research.

Identification of molecules in complex natural matrices relies on matching the fragmentation spectra of ions under investigation and the spectra acquired for the corresponding analytical standards. Currently, there are many databases of experimentally measured tandem mass spectrometry spectra (such as NIST, MzCloud, and Metlin), and considerable progress has been made in the development of software for predicting tandem mass spectrometry fragments in silico using combinatorial, machine learning, and quantum chemistry approaches (such as MetFrag, CFM-ID, and QCxMS). However, the electrospray ionization molecules can be ionized at different sites (protonated or deprotonated), and the fragmentation spectra of such ions are different. Here, we are using the combination of the in-ESI source hydrogen/deuterium exchange reaction and MSn fragmentation for the investigation of the fragmentation pathways for different protomers of organic molecules. It is shown that the distribution of the deuterium in the fragment ions reflects the presence of different protomers. For several molecules, the distribution of deuterium was traced up to the MS5 level of fragmentation revealing many unusual and unexpected effects. For example, we investigated the loss of HF from the ciprofloxacin and norfloxacin ions and observed that for ions protonated at -COOH group, the eliminating hydrogen always comes from -NH group. When ions are protonated at another site, the elimination of hydrogen with a probability of 30% occurs from the -NH group, and with a probability of 70%, it originates from other sites on the molecule. Such effects were not described previously. Quantum chemical simulation was used for the verification of the protonated structures and simulation of the corresponding fragmentation spectra.

The deuteration of organic molecules is considerably important in organic and medicinal chemistry. An electrochemical membrane reactor using proton-conducting graphene oxide (GO) nanosheets was developed to synthesize valuable deuterium-labeled products via an efficient hydrogen-to-deuterium (H/D) exchange under mild conditions at ambient temperature and atmospheric pressure. Deuterons (D+) formed by the anodic oxidation of heavy water (D2O) at the Pt/C anode permeate through the GO membrane to the Pt/C cathode, where organic molecules with functional groups (C≡C and C═O) are deuterated with adsorbed atomic D species. Deuteration occurs in outstanding yields with high levels of D incorporation. We also achieved the electrodeuteration of a drug molecule, ibuprofen, demonstrating the promising feasibility of the GO membrane reactor in the pharmaceutical industry.

Neutron crystallography is a highly effective method for visualizing hydrogen atoms in proteins. In our recent study, we successfully determined the high-resolution (1.2 Å) neutron structure of high-potential iron-sulfur protein, refining the coordinates of some amide protons without any geometric restraints. Interestingly, we observed that amide protons are deviated from the peptide plane due to electrostatic interactions. Moreover, the difference in the position of the amide proton of Cys75 between reduced and oxidized states is possibly attributed to the electron storage capacity of the iron-sulfur cluster. Additionally, we have discussed about the rigidity of the iron-sulfur cluster based on the results of the hydrogen-deuterium exchange. Our research underscores the significance of neutron crystallography in protein structure elucidation, enriching our understanding of protein functions at an atomic resolution.

Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th century. Due to its capability to resolve isotopically labeled species, it can be applied together with stable isotope tracers to reveal the transformation of particular biologically relevant molecules. However, low-resolution techniques, which were used for decades, had limited capabilities for untargeted metabolomics, especially when a large number of compounds are labelled simultaneously. Such untargeted studies may provide new information about metabolism and can be performed with high-resolution mass spectrometry. Here, we demonstrate the capabilities of high-resolution mass spectrometry to obtain insights on the metabolism of a model plant, Lepidium sativum, germinated in D2O and H218O-enriched media. In particular, we demonstrated that in vivo labeling with heavy water helps to identify if a compound is being synthesized at a particular stage of germination or if it originates from seed content, and tandem mass spectrometry allows us to highlight the substructures with incorporated isotope labels. Additionally, we found in vivo labeling useful to distinguish between isomeric compounds with identical fragmentation patterns due to the differences in their formation rates that can be compared by the extent of heavy atom incorporation.

Steroids are tetracyclic aliphatic compounds, and most of them contain carbonyl groups. The disordered homeostasis of steroids is closely related to the occurrence and progression of various diseases. Due to high structural similarity, low concentrations in vivo, poor ionization efficiency, and interference from endogenous substances, it is very challenging to comprehensively and unambiguously identify endogenous steroids in biological matrix. Herein, an integrated strategy was developed for the characterization of endogenous steroids in serum based on chemical derivatization, ultra-performance liquid chromatography quadrupole Exactive mass spectrometry (UPLC-Q-Exactive-MS/MS), hydrogen/deuterium (H/D) exchange, and a quantitative structure-retention relationship (QSRR) model. To enhance the mass spectrometry (MS) response of carbonyl steroids, the ketonic carbonyl group was derivatized by Girard T (GT). Firstly, the fragmentation rules of derivatized carbonyl steroid standards by GT were summarized. Then, carbonyl steroids in serum were derivatized by GT and identified based on the fragmentation rules or by comparing retention time and MS/MS spectra with those of standards. H/D exchange MS was utilized to distinguish derivatized steroid isomers for the first time. Finally, a QSRR model was constructed to predict the retention time of the unknown steroid derivatives. With this strategy, 93 carbonyl steroids were identified from human serum, and 30 of them were determined to be dicarbonyl steroids by the charge number of characteristic ions and the number of exchangeable hrdrogen or comparing with standards. The QSRR model built by the machine learning algorithms has an excellent regression correlation, thus the accurate structures of 14 carbonyl steroids were determined, among which three steroids were reported for the first time in human serum. This study provides a new analytical method for the comprehensive and reliable identification of carbonyl steroids in biological matrix.

A method for the technically easy-to-implement synthesis of deuterium-labeled pyrazolo[1,5-a]pyrimidines and 1,2,4-triazolo[1,5-a]pyrimidines have been developed. The regioselectivity of such transformations has been shown. 1H NMR and mass spectrometric methods have proved the quantitative nature of such transformations and the kinetics of deuterium exchange has been studied. Spectrally, at different temperatures (+30 °C, -10 °C and -15 °C), the kinetics of the process was studied both in CD3OD and in deuterated alkali.

The work is dedicated to further development of our described method for analyzing mass spectra of biomolecules acquired as a result of hydrogen-deuterium exchange reactions (HDXs). The modified method consists of separating HDX distributions via their approximations by a minimum number of components corresponding to independent H/D substitutions and independent charge carrier retentions in different spatial isoforms or conformations of biomolecules with unknown primary structures. In this case, neither the natural isotopic distribution nor the exact number of active sites involved in HDXs and H+ or D+ attachments can be determined in advance. Original H/D electrospray mass spectra of an apamin solution were taken from our previous work. In that work, taking into account the natural isotopic distribution of apamin molecules, three main conformations of apamin ions were found as a result of separating the H/D mass spectra of the apamin solution for the gas flow with the addition of about 10% ND3 molecules. Using the proposed modified method that does not require knowledge of the primary structure of the biomolecules gave similar results with slight deviations of calculated HDX distributions of the apamin ions from those obtained earlier. The maximum difference between mean values of the calculated HDX distributions for ions of the same charge in both cases does not exceed a few percent. In addition, HDX mass spectra of the apamin complex with an adduct of unknown structure were processed. Such analysis gave also three main fractions of ions with relatively large contributions when ND3 was injected into a radio-frequency quadrupole. In the absence of ND3 flow, the results of calculations for apamin and its complex were close to each other too. The formation of the apamin complex most probably in solution was confirmed by performed calculations.