2H‐NMR resolution of the methylenic isotopomers of ethanol applied to the study of stereospecific enzyme‐catalysed exchange

We have shown that site-specific natural isotope fractionation of hydrogen studied by NMR (SNIF-NMR) is an important source of information on the mechanistic and environmental effects which govern the photosynthesis of sugars and their fermentation into ethanol. Three isotope ratios associated with the methyl, methylene, and hydroxyl sites of ethanol are determined in achiral media. In this study we show that complementary information about possible stereospecific mechanisms involving the methylenic hydrogens is also rendered accessible by 2H-NMR enantiomeric resolution. The synthesis of mandelate esters enables exchange between the pro-R site of ethanol and water to be investigated. Simultaneous access to the three site-specific isotope ratios of the ethyl group is obtained at isotopic dilutions close to the natural ones. Mediation of the exchange by the enzymic system alcohol dehydrogenase-alpha-lipoyldehydrogenase and by the yeast Saccharomyces cerevisiae are compared. The progress of the reaction can be followed quantitatively as a function of time and the occurrence of glycolytic metabolism of endogeneous materials by yeast can be substantiated in a one-pot experiment.