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Different members of the alcohol oxidoreductase family can transfer the hydride of NAD(P)H to either the re- or the si-face of the substrate. The enantioselectivity of transfer is very variable, even for a range of substrates reduced by the same enzyme. Exploiting quantitative isotopic (2)H NMR to measure the transfer of (2)H from NAD(P)(2)H to ethanol, a range of enantiomeric excess between 0.38 and 0.98, depending on the origin of the enzyme and the nature of the cofactor, has been determined. Critically, in no case was only (R)-[1-(2)H]ethanol or (S)-[1-(2)H]ethanol obtained. By calculating the relative energies of the active site models for hydride transfer to the re- or si-face of short-chain aldehydes by alcohol dehydrogenase from Saccharomyces cerevisiae and Lactobacillus brevis, it is shown that the differences in the energy of the systems when the substrate is positioned with the alkyl group in one or the other pocket of the active site could play a role in determining stereoselectivity. These experiments help to provide insight into structural features that influence the potential catalytic flexibility of different alcohol dehydrogenase activities.

The nuclear magnetic resonance study of site-specific natural isotope fractionation (SNIF-NMR) produced in the glycolytic conversion of glucose into ethanol and glycerol provides isotopic transfer coefficients, a ij , which relate sites in the products to sites j in the reactants. The isotopic connection between the carbon-bound hydrogens of glycerol and those of glucose and water in fermentation reactions carried out with Saccharomyces cerevisiae has been investigated. The a ij coefficients provide mechanistic information on the genealogy of the glycerol hydrogens, on the relative rates of triose phosphate isomerization and reduction of dihydroxyacetone phosphate into glycerol 3-phosphate, on the stereospecificity of the reduction, on the percentages of intra- and intermolecular hydrogen transfer occurring in the course of the reaction, and on the order of magnitude of the active overall kinetic and thermodynamic isotope effects. Thus a close connection is determined between sites 3 pro-R of glycerol (stereospecific numbering) and H 6 pro-R and to a lesser extent H 2 of glucose and between site 3 pro-S of glycerol and H 6 pro-S and H 1 of glucose. Moreover site 2 of glycerol, which exhibits a strong correlation with water is also partly connected with glucose. Possible changes in the values of the isotopic transfer coefficients, as a function of the composition of the medium or of the environmental conditions, enable mechanistic perturbations such as variations in the percentage of intermolecular transfers to be detected. Analyzed in terms of stereospecificity of the reduction step the isotopic results provide a direct chemical shift assignment of the enantiomeric pairs (1 S , 3 R ) and (1 R , 3 S ) of glycerol triacetate. The influence of added bisulfite, which strongly increases the yield in glycerol is estimated. The isotopic characterization of the bioconversion producing both ethanol and glycerol has been extended to the determination of the carbon isotopic parameters by isotope ratio mass spectrometry. Although it usually occurs as a by-product of the fermentation, glycerol can be considered as a useful complementary probe for characterizing the glycolytic pathway and for inferring various properties of the carbohydrate precursors.

The isotopic parameters of fermentation products provide reliable criteria for characterizing their carbohydrate precursors on the condition that the isotopic coefficients which relate individual sites in the products and in the reactants have strictly reproducible or predictible values. Since fermentation may be performed either in natural media (such as fruit juices) which exhibit variable compositions or in “synthetic” media prepared from appropriate nutrients, it is necessary to estimate the influence of the medium on the carbon and hydrogen isotope ratios. By combining isotope ratio mass spectrometry and nuclear magnetic resonance investigations it has been shown that variations in the reaction rates induced by the medium are not connected with isotopic fractionation effects. At natural concentrations the nutrients present in juices, for instance, ensure a significant acceleration of the bioconversion with respect to pure water without introducing undesirable isotopic effects. The role of different nutrients added in relatively large amounts to synthetic media has been analyzed. It has been shown in particular that a significant decrease in the isotope ratio of the methylenic site of ethanol is due to the combined influence of both phosphates and assimilable nitrogen which are also the key components governing the growth and activity of yeasts. For a given nutrient composition the effect of the medium on the isotopic coefficients which connect the individual sites of ethanol to the starting water can be estimated. The observed behavior can be explained by modifications in the percentages of intra- and intermolecular hydrogen transfers (methyl site) and by variations in the kinetic isotope effects and exchange rates involving the hydrogens originating from water. Carbohydrates from different origins (beet and cane) have been fermented in different “natural” media prepared by eliminating sugars from orange, apple, and grape juices. In spite of very different fermentation rates the isotopic ratios of the products are then nearly independent of the composition of the medium. These results support the use of the ethanol probe for characterizing endogenous or exogenous sugars contained in different fruit juices.

For a given biochemical transformation, such as the fermentation reaction, the redistribution coefficients, which relate the natural site-specific isotope contents in end products to those of their precursors, are a source of mechanistic information. These coefficients characterize the traceability of specific hydrogens in the products (ethanol and water) to their parent hydrogens in the starting materials (glucose and water). In conditions of complete transformation, they also enable intermolecular exchanges with the water medium to be estimated. Thus it is directly confirmed that hydrogens 1, 2, 6, and 6′ of glucose are strongly connected to the methyl site I of ethanol obtained by fermentation by Saccharomyces cerevisiae. However, whereas hydrogens 6 and 6′ are transferred to a great extent, transfer is only partial for hydrogen 2, and it is even less for hydrogen 1. Because the two moieties of glucose corresponding to carbons 1-2-3 and 4-5-6 are scrambled by the aldolase and triose-phosphate isomerase reactions, additional exchange of hydrogens at positions 1 and 2 must have occurred before these steps. The value of the coefficient that relates site 2 of glucose to site I of ethanol in particular can be used to quantify the contribution of intermolecular exchange occurring in the course of the transfer from site 2 of glucose 6-phosphate to site 1 of fructose 6-phosphate mediated by phosphoglucoisomerase. The average hydrogen isotope effects associated with the transfer of hydrogen from the water pool to the methyl or methylene site of ethanol are estimated. In contrast to conventional experiments carried out in strongly deuterium-enriched media where metabolic switching may occur, the NMR investigation of site-specific natural isotope fractionation, which operates at tracer isotopic abundance, faithfully describes the unperturbed metabolic pathways.

In order to apply Site-specific Natural Isotope Fractionation (SNIF) to chemical, biochemical, and environmental studies of d -glucose, there is a need for readily prepared compounds which give 2 H NMR spectra with all or most signals resolved. Changing substituents of C-1 and/or C-6 of α- d -glucopyranose pentaacetate improved the dispersion of deuterium signals, the best results being achieved with 1,2,3,4-tetra- O -acetyl-6-deoxy-6-thiocyanato-α- d -glucopyranose, for which only the 2 H-2 and 2 H-4 signals were not resolved, and with 2,3,4-tri- O -acetyl-6-bromo-6-deoxy-α- d -glucopyranosyl bromide, for which only the 2 H-6a and 2 H-6b signals were not resolved. Periodate oxidation of methyl 4,6- O -benzylidene-α- d -glucopyranoside and 4,6-dichloro-4,6-dideoxy- d -galactose was also examined as a possible source of useful compounds. Products obtained from the benzylideneglucoside gave inadequate resolution and broad deuterium signals. The oxidation of 4,6-dichloro-4,6-dideoxy- d -galactose was not straightforward. The α anomer was oxidised more rapidly than the β anomer. The oxidation product, 2,4-dichloro-2,4-dideoxy-3- O -formyl- d -threose, was too labile to be isolated pure and its hydrolysis to 2,4-dichloro-2,4-dideoxy- d -threose was accompanied by a slow elimination to form 2,4-dichlorobut-2-enal.

Resume La synthese de tBu2MeCCOOH, acide qui se place parmi les structures les plus encombrees a ete realisee. Diverses voies d'acces potentielles a cette molecule ont ete envisagees. Seule la sequence decrite precedemment par Newman via tBu2CO, tBu2MeCOH, tBU2CCH2, tBU2CHCH2OH, tBu2CHCOOH, tBU2CHCOCl, tBu2CCO et tBu2MeCCOH nettement amelioree dans ce travail par un acces direct a tBu2CHCOOH conduit atBU2MeCCOOH avec un maximum d'efficacite. La condensation magnesienne du chlorure d'acide derive de cette structure en presence de chlorure cuivreux permet la preparation de nouvelles cetones particulierement encombrees tBu2MeCCOR. L'alkylation de ces cetones peut conduire a des composes encore plus congestionnes. Les possibilites, les limites et les performances de chaque sequence ont ete definies.