Site-specific isotope fractionation of hydrogen in the biosynthesis of plant fatty acids

The overall deuterium content of plant lipids has been investigated by isotope ratio mass spectrometry (IRMS), and the site-specific natural isotope fractionation of hydrogen has been studied by ²H-NMR at natural abundance (SNIF-NMR). An analytical strategy has been developed in order to exploit the isotopomeric composition determined in clusters associated with different chemical sites of one or several fatty acid components. The method, which combines spectrometric and chromatographic data, enables isotopic criteria to be directly derived from raw vegetable oils containing in general two saturated and two unsaturated fatty acids. These results provide new information on isotopic fractionation caused by biochemical, physiological and natural environmental effects. Some alternation in the molecular deuterium distribution has been detected which may be related to the mechanism of fatty acid elongation. The successive methylene groups introduced through malonyl CoA are the subjects of different kinetic isotope effects since one of them is exclusively derived from NADH whereas the other has a contribution from pyruvate. A discriminant analysis of the cluster isotopic parameters enables several kinds of botanical precursors to be distinguished. The authenticating performances can be improved by taking into account the influence of climatic effects related to the region in which the plant grew.     

Site-specific natural isotope fractionation of hydrogen in plant products studied by nuclear magnetic resonance

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.     

The fellowship of natural abundance 2H-isotopomers of monoterpenes

Site-specific natural abundance hydrogen isotope ratios have been measured by deuterium-NMR in a wide variety of monoterpenes from numerous kinds of plants grown in different environments. Once the NMR signals have been assigned to the whole sets of isotopomers in the different molecules and schemes of connections to the parent isotopomers in the geranyl diphosphate (GPP) precursor have been defined, a very consistent set of isotopic profiles is evidenced. The results, which are incompatible with the mevalonate pathway, can be satisfactorily interpreted by considering the deoxyxylulose pathway (DOXP), which is now recognized as the usual route for monoterpene biosynthesis in plants. Strong deuterium depletion at ex-site 2 of GPP, accompanied by high isotope ratio values at site ex-6, are consistent with synthesis of GPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) molecules independently produced by the DOXP pathway. However, for a given molecular species, significant differences are observed as a function of the plant source, in particular at site ex-6 of GPP. Thus, monoterpenes from plants with a C3 metabolism are mostly characterized by relatively high values of (D/H)6, whereas C4 plants tend to show much lower values. This behavior may be attributed to more or less significant contributions of GPP resulting from the condensation of IPP with DMAPP produced by isomerization. The isotopic profile therefore enables the role of physiological and environmental factors on the relative importance of the "independent" and "isomerized" model to be estimated. More generally, isotope ratios at individual sites in geraniol can be traced back to the corresponding sites in GPP, then to sites of the IPP and DMAPP building blocks, then to the pyruvate and glyceraldehyde 3-phosphate DOXP active molecules, and finally to the carbohydrate photosynthetic precursor. Furthermore, the methylenic hydrogen atoms, which are enantiotopic in geraniol, become diastereotopic in chiral, and more specially in cyclic, monoterpenes. This provides an isotopic verification for the complete stereochemical chain of affiliation, and a way of estimating enantiomeric purity and whether intermolecular exchanges have taken place.     

Multi-element and multi-site isotopic analysis of nicotine from tobacco leaves

The carbon, nitrogen and hydrogen stable-isotope contents of nicotine extracted from tobacco leaves were determined by Isotope Ratio Mass Spectrometry (overall ²H, ¹³C and ¹⁵N contents) and by the SNIF-NMR method (site-specific deuterium content). In addition, nicotine was chemically degraded into nicotinic acid so that the intramolecular distribution of carbon and nitrogen isotopes could be studied. A prerequisite for reliable measurements is the use of experimental procedures free of isotopic fractionation. Therefore, it was ensured that isotopic integrity was maintained throughout extraction, purification and chemical degradation steps. The multi-element and multi-site information provided is tentatively interpreted in terms of biochemical isotopic effects on intramolecular distribution patterns and of the influence of environmental factors on nicotine isotopic parameters.