Stereoselective hydrogen isotope exchange on nicotinamide cofactors through flavoenzyme microscopic reversibility

We describe a panel of flavoenzymes with the ability to catalyse stereoselective hydrogen isotope exchange (HIE) between ²H₂O (D₂O) and reduced nicotinamide cofactors, enabling a simple and redox-neutral route to deuterated NAD(P)H isotopomers. In screening the FMN- and FAD-dependent enzymes, which have diverse native functions, we identified catalysts (many of which are commercially available) that selectively yield the full suite of [4-²H]-NAD(P)H stereoisotopomers in a single step. In combining stereo-complementary enzymes, we also identify simple one-pot routes to dideuterated [4-²H₂]-NAD(P)H. The biocatalytic methods provide near-quantitative ²H-incorporation for both NADPH and NADH under mild conditions, using readily available ²H₂O as the isotope source. The observed activity indicates a reversible flavoenzyme hydride-transfer cycle, reliant on hydrogen/deuterium exchange on the transiently reduced flavin cofactors. We provide computational analysis to rationalise the stereochemical outcomes of the screened reactions. Finally, preparative-scale syntheses are described that deliver isolated deuterated cofactors in excellent yield and isotopic purity. We envisage this easily implemented procedure will simplify access to these important biochemical compounds for mechanistic studies, and may open up wider ²H₂O-driven biocatalytic deuteration reactions.