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Vitiligo is a skin depigmentation disorder. GATA3 expression is downregulated in vitiligo patients, and its role and regulatory mechanism in vitiligo are unclear. GATA3 and HMGB1 levels were detected by qRT-PCR in peripheral blood cells of vitiligo patients and healthy controls, as well as H

Objectives Osteoarthritis (OA) is a common degenerative joint disease with the pathological features of the reduced cartilage cellularity. Celastrol, a compound from Tripterygium wilfordii, exerted therapeutic effects on arthritis, but the potential mechanism remains unclear. Methods Tunicamycin was used to establish a model of OA in vitro, and ACLT surgery model in rats was applied to verify the mechanism. Chondrocytes were isolated from the knee articular cartilage of rabbit. MTT and flow cytometry assay were used to detect cell viability and apoptosis rate. Haematoxylin–eosin staining was used to assess for the histopathological changes. The activity and expression of apoptosis-related factors and ERs (endoplasmic reticulum stress)-related factors were detected by ELISA, WB, PCR and IHC, respectively. Key findings Celastrol exhibited significant enhancement on cell viability and reduced the rate of apoptosis in Tm-exposed chondrocytes. Celastrol reduced enzyme activity and protein expression of caspase-3, caspase-6 and caspase-9, decreased Bip, Atf6, Chop and Xbp-1 expression both at protein and mRNA levels. Celastrol showed a more significant effect on cell apoptosis rate and mRNA expression in the combination with 4-PBA. Conclusions This study reveals that celastrol may prevent OA by inhibiting the ERs-mediated apoptosis. All these might supply beneficial hints for celastrol on OA treatment.

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 origin of hydrogen in fatty acids produced in vivo by chicken was studied by using isotope tracing monitored by 2 H NMR. Chickens were fed with deuterium-labeled water, deuterium-labeled glucose/fructose and trideuterated acetate. The site-specific isotope distribution of the fatty acids produced in vivo by the chickens was analyzed after transformation into methyl esters. A connection was observed between the hydrogen atoms of the feeds and water and those of the different molecular positions of the fatty acids produced by chicken. Water hydrogen makes an important contribution to the formation of the lipids. The hydrogen of the enzyme-bound acetyl or malonyl moiety in acyl-CoA may be transferred from the C-1, C-2 or C-6 of glucose or fructose and indirectly from water. The hydrogen introduced by the cofactor NADPH may be abstracted from C-1 and C-3 of glucose, through its regeneration in the pentose phosphate metabolism pathway, and contains hydrogen atoms of water. The exogenous acetate enters into the in vivo fatty acid biosynthesis pathway in an efficient way and can make an important contribution to the biosynthesis of the lipids.

In asymmetric reduction of carbonyl compounds mediated by microorganisms, the cofactors that transfer hydride should be regenerated by using a recycling system. In most cases, this recycling system consists of carbohydrate molecules, especially glucose or sucrose. Other molecules such as ethanol and acetate have been used as electron donors too. The reduction can even be conducted without added electron donors. To improve biocatalytic synthesis, it is important to understand the cofactor recycling mechanism. In this work, the hydride-transfer mechanism in cofactor regeneration, which takes place in bioreduction mediated by yeast, was studied by means of an isotope tracing technique. The results show that, when glucose was used, the NADH involved in the glycolysis was consumed directly in the formation of ethanol and was not used in the bioreduction. Hence, the regeneration of cofactors in the reduction is not coupled with glycolysis. Nevertheless, glucose is an efficient electron donor that transfers hydride through the hexose monophosphate (HMP) pathway in which the main hydrogen source is C-1 and C-3 hydrogen of glucose. Ethanol is not a good electron donor, since, when it was used, only a small quantity of hydrogen was transferred from this molecule, and the main hydrogen source was water. Therefore, the ethanol oxidation pathway may not be efficient. In the absence of added auxiliary substrates, the yeast cells may use electron donors stored in its cellules. However, in this case we observed that the main hydrogen source for cofactor recycling was water, while only very few hydrogen atoms were from unexchangeable sites. This is similar to the case in which ethanol is used, and is in contradiction with the HMP pathway if stored glucose was the electron donor. The question that remains to be investigated is "what is the efficient electron donor recycling mechanism in the yeast cellules?"

In glucose fermentation, the hydrogen source of products such as ethanol and glycerol is the medium and the sugar. The site-specific natural isotope ratios of the products, (D/H)(i), and that of the medium and sugar, (D/H)(k), may be related by a matrix, A, of redistribution coefficients, a(ik), that characterizes the specific genealogies of the hydrogen atoms. (D/H)(i) = [A](D/H)(k), where (D/H)(i) and (D/H)(k) are the column vectors of the isotope ratios of sites i and k that can be measured by (2)H NMR. The complete redistribution matrix was determined in a set of isotope labeling experiments. Thus, we obtained a mathematical model representing the hydrogen isotope affiliation during alcoholic fermentation. It not only provides information about the biochemical reaction mechanism but also can be used to estimate the isotopic data of the products, based on those of the substrate and the medium. The results prove, in a quantitative way, that the metabolites contain isotopic information about the precursor in a biotransformation and can be used to identify its origin. The method established for the study of the hydrogen-transfer mechanism can be applied to other chemical and biochemical reactions.

During the biosynthesis of natural products, the intra-molecular distribution of isotopes is introduced as a result of different isotope effects associated with the reactions involved. Due to the sensitivity of certain enzymes to the presence of a heavy isotope, the isotope selection effects related to some transformations can be high, especially for hydrogen. The effect of a series of isotope effects specific to each enzyme-catalysed step are additive during a biosynthetic pathway, leading to fractionation of the isotopes between the starting substrate and the final product. As the individual reactions are acting on different positions in the substrate, the net effect is a non-statistical distribution of isotope within the final product. Quantitative 2H NMR spectroscopy can be used to measure the distribution of 2H at natural abundance in natural products. In the first example, the fermentation of glucose is examined. Glucose can act as a primary carbon source for a wide range of fermentation products, produced by a variety of pathways. In many cases, competing pathways are active simultaneously. The relative fluxes are influenced by both environmental and genetic parameters. Quantitative 2H NMR spectroscopy is being used to obtain mechanistic and regulatory information about isotopic fractionation from glucose during such fermentations. Quantitative 2H NMR spectroscopy can also be used to examine the fractionation in 2H that occurs in long-chain fatty acids during chain elongation and oxygenation. It has been found that the (2H/1H) ratio shows an alternating pattern along the length of the chain and that the residual hydrogen atoms at the sites of desaturation are asymmetrically impoverished. The extent to which the non-statistical distribution of isotopes can be related to the mechanism of enzymes involved in the biosynthetic pathway via kinetic isotopic effects will be discussed.

A method for the determination of kinetic isotope effects (KIEs) in organic reactions using natural abundance deuterium NMR is proposed. The method consists of the determination of the site-specific H/D isotopic ratios of the starting reactant and the product in a reaction run under pseudo-first-order kinetic conditions. Using simple and easy experimental procedures, primary and/or multiple secondary KIEs can be measured simultaneously with high precision. The application of this approach to different types of reaction is described. The primary and α-secondary KIEs for C—H(D) bond breaking of —CDH— can be measured separately. The uncertainty in the determination of small secondary KIEs can reach less than 1%. The results show that some remote secondary KIEs are not negligible. The advantages and limitations of the method are discussed. One of its merits is that the method can even be used in complex situations when there are branch reactions and when the reaction is reversible. The performance depends essentially on the signal separation in the NMR spectra. Copyright © 2001 John Wiley & Sons, Ltd.

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.

Quantitative determinations of natural-abundance carbon isotope ratios by nuclear magnetic resonance (SNIF-NMR) have been optimized by appropriate selection of the experimental conditions and by signal analysis based on a dedicated algorithm. To check the consistency of the isotopic values obtained by NMR and mass spectrometry (IRMS) the same glycerol samples have been investigated by both techniques. To have access to site-specific isotope ratios by IRMS, the products have been degraded and transformed into two derivatives, one of which contains carbons 1 and 3 and the other carbon 2 of glycerol. The sensitivity of the isotopic parameters determined by IRMS to fractionation effects possibly occurring in the course of the chemical transformations has been investigated, and the repeatability and reproducibility of both analytical chains have been estimated. The good agreement observed between the two series of isotopic results supports the reliability of the two different approaches. SNIF-NMR is therefore a very attractive tool for routine determination, in a single nondestructive experiment, of the carbon isotope distribution in glycerol, and the method can be applied to other compounds. Using this method, the isotopic distributions have been compared for glycerol samples, obtained from plant or animal oils, extracted from fermented media, or prepared by chemical synthesis. Typical behaviors are characterized.

The site-specific natural isotope fractionation of carbon and hydrogen has been investigated by SNIF-NMR for glycerol samples, extracted from plant lipids, obtained in the fermentation of sugars, or from commercial sources. In the first two cases, several plant species from either C3 or C4 metabolic origin have been considered. Large deviations with respect to a statistical distribution of deuterium in the glycerol skeleton are observed and the dispersion ranges of the isotopic parameters are much larger than the precision of the measure. The NMR determination of the site-specific carbon isotopic parameters (positions 2 and 1,3 of glycerol) provides an easy method for distinguishing natural and synthetic samples. More generally, the discriminating potential of the carbon and hydrogen isotopic fingerprint of glycerol has been estimated on a statistical basis in terms of the nature of the raw material (plant oil or sugar fermentation) and, in each case, in terms of C3 or C4 metabolism of the precursor. Glyc...

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.

The thermokinetic fractionation coefficients of 2 H and 18 O in water of orange and grape juices were determined in experiments modeling the low-temperature evaporation processes carried out by agroindustries to concentrate fruit juices. These results can be used to refine the isotopic methods developed to control the adulteration of orange juices by sucrose and the concentration of musts to enrich wines. From the determination of the Brix value and the δ 2 H and δ 18 O isotope ratios of the remaining water of an orange concentrate, it is possible to infer the initial values of δ 2 H and δ 18 O in the pure juice. Knowledge of these parameters enables the detection level of sugar addition in the concentrate to be lowered. Conversely, by modeling the isotope fractionation processes that take place during must concentration, it is possible to use the δ 2 H and δ 18 O isotope ratios of wine water to improve the detection of wine enrichment by both sucrose addition and must concentration.

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.

Several series of wines were prepared in standard conditions from well defined varieties (Cabernet-Sauvignon, Carignan, Chasselas, Chardonay, Grenache Noir, Riesling, Sauvignon, Syrah et Ugni Blanc) harvested in 1991 in three different regions of production in France (Alsace, Bordeaux and Languedoc). In order to study the influence of the year of production as well, the same varieties grown in Anjou were considered for three different periods of time : 1983, 1984 and 1988. The stable isotope composition of these wines was determined by 2H-NMR spectroscopy ((D/H)I and (D/H)II isotope ratios of ethanol) and by Mass Spectrometry for water (2H and 18O) and ethanol (13C). The variations observed for the wines of the different varieties are explained in terms of the climatic conditions (temperature, precipitation and insolation) which governed the regions of production during the vine vegetation cycles considered. It is shown that similar behaviour is observed for the 2H and 18O contents of the water of musts and wines but they differ from the Craig relationship existing in meteorological waters. The early vine varieties cultivated in the different regions considered give wines with a higher concentration in the heavy isotopes than the later varieties as far as the ethanol (D/H)I parameter is concerned.

Stable isotope analysis of grape juices and fermented products involving 2H-NMR spectroscopy and isotope ratio mass spectroscopy was carried out in combination with elemental determinations performed by atomic absorption spectroscopy using flame and thermal ionisation. One hundred and sixty five grape samples were carefully collected in well-defined vineyards of France situated in Alsace, Burgundy, Beaujolais and the Loire Valley. Moreover, the samples from Burgundy were picked from four typical Appellations (Cǒtes de Nuits, Cǒtes de Beaune, Cǒtes Chalonnaises and Mǎconnais) in order to ‘zoom’ the approach to smaller production areas. An exhaustive statistical evaluation of the 165 ± 16 data set was carried out by analysis of variance and principal component analysis. To conclude this study, the classification of the samples in typical Appellations was carried out by discriminant analysis with a nearly 100% degree of achievement.

The overall 13C or 2H content of the organic matter of marine algae is an interesting source of information on metabolic and environmental effects experienced by aquatic plants. However, in order to appraise the complexity of the phenomena which influence the isotopic distribution, access to more specific isotopic probes is desirable. Thus, determination of the overall and site-specific natural deuterium fingerprint of the galactose unit of carrageenans is expected to improve the characterization of these molecular species in terms of origin and history of the precursors. A strategy has been developed in order to render the galactose moiety accessible to the study of site-specific natural isotope fractionation of hydrogen by nuclear magnetic resonance (SNIF-NMR). Extraction and derivatization procedures have been elaborated and the possible occurrence of fractionation effects in the course of the physical and chemical transformations has been examined. By combining mass spectrometric determination of the overall isotope ratio of the galactose skeleton and analysis of the cluster signals observed in the 2H-NMR spectra of appropriate galactose derivatives, several groups of site-specific isotope contents can be obtained. Significant deviations with respect to a statistical distribution of deuterium in the galactose unit have been determined and the isotopic parameters of galactoses from carrageenans have been shown to differ greatly from those of galactose extracted from milk. Moreover, significant variations are observed among the different sources of carrageenans. This behaviour is consistent with the large range of overall isotope contents measured in cellulose from aquatic plants.

The site-specific natural isotope fractionation of hydrogen in glucose was investigated by nuclear magnetic resonance spectroscopy (SNIF-NMR). Several glucose samples obtained from plants undergoing either C, (potato, barley, sunflower, wheat) or C4 (maize, sorghum) photosynthetic metabolism were investigated. Since the glucose molecule is not suitable for direct SNIF-NMR analysis, several kinds of derivatives were synthesized. Three clusters of isotopomers were identified in glucose penta-acetate which is easily prepared. Up to 5-6 sites could be distinguished in 6 other derivatives. An integrated statistical analysis of the whole sets of cluster lines was performed in order to determine the individual isotope contents in the carbon-bound hydrogen positions of the glucose skeleton. Both the mole fractions of the monodeuterated isotopomers and the deuterium/hydrogen ratios of the individual sites thus become accessible. Using this approach, it is shown that similar trends in the isotopic distribution are exhibited within a given photosynthetic C3 or C4 family. Larger deviations with respect to a statistical distribution of deuterium are observed in glucose from C3 plants as compared to C4 plants. The methylenic sites 6,6' in particular are significantly depleted in C, glucose as compared to C4 glucose.

Cyanide is one of the common poisons in murders. When cyanide has been used, to identify the origin of cyanide may be necessary in the forensic investigation. We have examined the possibility of distinguishing different commercial cyanide samples through the δ 13 C and δ 15 N values and developed a protocol for the isotope analysis of cyanide extracted from several matrices as food and medicine. Several cyanide precipitates were tested for the isotope analysis. The results show that cupric ferrocyanide Cu 2 [Fe(CN) 6 ] is the most appropriate precipitate for the analysis. Thirteen batches of KCN and nine batches of NaCN chemicals were randomly chosen from different suppliers. The cyanides were converted to cupric ferrocyanide and then analysed by isotope ratio mass spectrometry coupled to elemental analysis (EA-IRMS). The isotopic signature of the commercial samples varied from −51.96 to −25.77‰ for δ 13 C and from −4.51 to +3.81‰ for δ 15 N, highlighting the potential of applying EA-IRMS technique to identify cyanide from different batches and sources. The influence of the cyanide extraction and isolation from spiked matrix on the isotopic analysis was also studied. Three matrices: orange juice, yogurt drink and a medicine were tested. In many cases, the isotopic analysis results obtained from the original cyanides precipitates and those isolated from the matrices showed a good accordance, especially for δ 15 N. In some matrices, the 13 C analysis was interfered by co-precipitates. With carefully elaborated working protocol, determining the isotope ratio of N and C in cyanide by EA-IRMS is a promising method for forensic investigations.

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.

NMR determination of site-specific hydrogen isotope ratios at natural deuterium abundance (SNIF-NMR) provides the basis for simultaneous access to primary and secondary thermodynamic fractionation factors in exchange reactions and avoids the need for selective isotope labelling of the reagents. The method was applied to the measurement of fractionation parameters involving OH, CH2, CH3 and =CH groups in keto–enol tautomeric equilibria. The fractionation factors relating the =CH and OH sites of the enol species are simply derived from 2H NMR spectra whereas the determination of isotope parameters which relate keto and enol positions exploits a combination of 2H and 1H NMR experiments. Since only monolabelled isotopomers have to be considered at natural abundance, the method also offers the advantage of avoiding the occurrence of complex equilibria associated with multi-labelled species possibly introduced by deuterium enrichment. The primary equilibrium isotope effects illustrate a preference of deuterium for the methylene fragment of the keto form with respect to the ethylenic position of the enol tautomer. Since the enol species is itself engaged in a fast tautomeric equilibrium associated with a symmetric or unsymmetric double minimum potential, the thermodynamic parameters are averaged over the exchanging partners. It is shown that the average thermodynamic fractionation factor relating the OH and =CH hydrogens of the enol are significantly influenced by the nature of the substituents at both carbonyl positions of the β-diketones. Moreover, methyl and chlorine substitution increases by a factor of about 1·1 the thermodynamic isotopic fractionation factor relating the –COCHCO– position of the keto form to the hydroxyl position of the enol.

Deuterium NMR at the natural abundance was used to determine the site-specific isotope ratios (D/H)i of the non-equivalent isotopomers of various chemical species which exist in plant products. The deuterium distribution in glucose, galactose and mannitol samples from different botanical and compartmental origins is discussed in terms of the influence of plant metabolism and environmental factors. Particular emphasis is given to the potential versatility of deuterium NMR in the study of natural isotopic distribution in pro-chiral situations. Typical examples of chiral recognition are given in the field of glycolysis metabolites (ethanol, amino-acids) and of monoterpene biosynthesis.

A great variety of techniques has been used to study deuterium-protium partitioning in exchanging systems at equilibrium. In this context it is shown that the determination of site-specific isotope contents by {sup 2}H-NMR at natural abundance provides simple and direct access to thermodynamic isotope fractionation factors. This method avoids the recourse to deuterated species and therefore the approximations inherent in the analysis of complex isotopomeric mixtures. A number of slowly exchanging systems involving OH, NH, or SH groups and water offer suitable conditions (large chemical shift differences, moderate line widths,{hor_ellipsis}) for the relatively accurate determination of thermodynamic fractionation factors. Moreover multiple fractionations can be observed and compared in ternary mixtures. 25 refs., 2 tabs.

International audience; The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative H-2 NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C-f) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C-f- 1-R: C-f-1-S ratio is 2.1, whereas the Cf-3-R: Cf-3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH- and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.

The hydrogen transfer mechanism of cofactor reduction and recycling processes in the yeast reduction of alpha,beta-unsaturated ketone was studied by using quantitative isotope tracing close to natural abundance measured by (2)H NMR. In the reaction, the active cofactor is NADPH. The cofactor-transferred hydride attacks the beta sp(2) carbon of the enone carbonyl while water hydrogen is transferred to the alpha position. The reductant involved in the reaction depends on the quantity of yeast. When the amount of yeast is very large, the enzymes use preferentially certain unidentified substance stored in the yeast cells rather than the added glucose as electron donor. In this case, the hydrogen transferred by the cofactor is mainly of water origin. When the yeast amount is low, the added glucose is more efficiently used by the enzymes as electron donor and its hydrogen atoms bound to C-1 and C-3 are delivered to the substrate.

A laboratory procedure for the analysis of the oxygen-18/oxygen-16 isotope ratios of ethanol derived from sugars and fruit juices by pyrolysis-isotope ratio mass spectrometry (IRMS) has been applied to the study of isotopic fractionation induced by the isotope effects of fermentation and distillation. For both processes, an experimental model has been established to describe and explain the observed fractionation phenomena. It is shown that reproducible results can be obtained when appropriate analytical conditions are used. Moreover, the ability of ethanol to act as a reliable indicator of the (18)O/(16)O ratio of sugars in orange juice (and therefore to be used as an internal reference for detecting water addition) is demonstrated both in theory and in practice.

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.

Pure d (−) and l (+) enantiomers of lactic acid were prepared by fermentation reactions with specific bacteria. In addition, naturally deuterated ethanol was prepared and converted into diastereoisomers using mandelic acid. Various sugars and nutrients were fermented into lactic acid in water having different deuterium contents and ethanol samples were obtained from yeast fermentation of sugars from different botanical origins. The methine and methylene groups in lactic acid and ethanol respectively show similar deuterium contents which are related to that found in the fermentation water. However, the methyl groups of both molecules are significantly different whatever the botanical origin of the carbon source in the fermentation medium.

Internal and external reference methods are introduced for the study of kinetic deuterium isotope effects (KDIEs) by natural abundance 2H NMR. These methods take account of both inter- and intra-molecular competive reactions of fully protonated and naturally monodeuteriated molecules of the substrate in the reaction. Specifically deuteriated compounds are not necessary using these methods. These techniques have further advantages over other methods; for example, primary and secondary KDIEs of different types can be measured simultaneously. The methodology is summarized and examples of its application are presented.

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.

A set of 4-monosubstituted cyclopentenes, , were synthesized and their relative rates (kX/kH) for bromination and chlorination were determined in methanol, ethanol and acetic acid at 25 °C by competitive method. log(kX/kH) for most of the substituents can be correlated by means of Taft's equation, log(kX/kH) = ρI σI + C. In methanol ρ = −2·91, ρ = −0·49, in ethanol ρ = −3·07, ρ = −0·70 and in acetic acid ρ = −1·64, ρ = −0·65. The presence of C(

Le role nucleophile du solvant dans l'etape d'ionisation de bromation via des intermediaires bromonium n'est pas negligeable. Ce resultat est deduit de la comparaison des effets polaires et steriques de groupes alkyles dans le methanol et l'acide acetique

Site-specific natural isotope fractionation measured by deuterium NMR (SNIF-NMR) was used for investigating the deuterium transfers occurring in the fermentation of sugars into ethanol. In contrast to carbon-13, which is usually assumed to be randomly distributed within the glucose skeleton, very large deviations with respect to a statistical repartition are determined for deuterium. By transforming glucose samples from different origins into acetates and nitrates, the absolute values of the D/H ratios in the nonexchangeable sites were obtained. The hydroxyl sites were considered to contribute to the isotope content of the starting water medium. No direct connection is found between glucose and the methylene site which is only sensitive, with a strong discriminating effect against deuterium, to the isotope content of water. A redistribution coefficient slightly less than unity (0.96 for a concentration of sugar of 100 g L/sup -1/) is found between the isotope ratio of the end and starting water media. The site-specific natural isotope parameters of ethanol constitute a faithful and powerful probe for investigating the physiological biochemical and climatological effects which have governed the photosynthesis of sugars in natural conditions.

Naturel abundance deuterium NMR has been applied to the quantitative determination of natural methylene chirality in amino acids of different configurations and in ethanols of different origins. An enantiomeric enrichment is detected for aspartic acid of the L series obtained from fermentation and the technique therefore provides an absolute method for identifying the natural origin of a given substance. In ethanols of different origins the pro-R and pro-S positions appear as naturally equally populated at the present level of precision of the experiments. These results provide a new source of information about reaction mechanisms in the natural conditions.

illus. 12 ref.