Hydrogen-Deuterium Addition and Exchange in N -Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.

Glutathione (GSH) is an important and ubiquitous thiol compound abundantly present in virtually every living cell. It is a powerful antioxidant critically required to protect cells from oxidative damage and free radical injury. Its quantification in ex vivo analysis remains a major challenge because it spontaneously oxidizes to form glutathione disulfide. N -Ethylmaleimide (NEM) is a well-known Michael acceptor, which reacts rapidly and irreversibly with thiol and prevents disulfide bond formation. Based on thiol conjugation to NEM, recently, the concentration of GSH was determined in human blood using NMR spectroscopy [ Anal. Chem , 2021, 93(44): 14844-14850]. It was found that hydrogen-deuterium addition and exchange occur during the thiol-maleimide reaction as well as NMR analysis, generating a series of poorly explored diastereomers/isotopomers. Here, we establish a general NMR approach to identify the thiosuccinimide diastereomers/isotopomers derived from the thiol-maleimide reaction. The thiol-Michael addition reaction was conducted for GSH and another thiol compound, cysteine, separately, using D 2 O and H 2 O. The conjugates were characterized by 1 H/ 13 C 1D/2D NMR under different solvent, buffer, and pH conditions. The Michael addition combined with the H/D exchange formed twelve unique diastereomers/isotopomers. NMR measurements allowed the distinct assignment of all structures in solutions and quantification of H/D addition and exchange. Interestingly, the deuterium exchange rate was dependent on structure, pH, and buffer. The elucidation of the thiol-maleimide reaction and H/D exchange mechanism can potentially impact areas including metabolomics, small molecule synthesis, and bioconjugation chemistry.