Isotopic criteria in the characterization of aromatic molecules. 1. Hydrogen affiliation in natural benzenoid/phenylpropanoid molecules.

The site-specific isotope ratios of several families of aromatic molecules are analyzed in terms of hydrogen affiliation and discriminating potential. Among the aromatic molecules produced by plants, many are biosynthesized by the shikimate pathway, but the terpenic pathway also forms some compounds with a benzenic ring. In compounds of the phenylpropanoid family, specific hydrogen connections are determined with cinnamic acid, a key intermediate in the formation of a large number of aromatic molecules. Then affiliations through the phenylalanine precursor, back to the parent d-erythrose 4-phosphate and phosphoenolpyruvate molecules and finally to glucose, are considered. Typical isotopic profiles of the benzenic ring in natural, as compared to non-natural, molecules are defined. The dispersion observed in the (D/H)i ratios of the lateral chains is illustrative of diverse mechanistic responses and the role of exchange phenomena. The isotopic patterns of aromatic molecules pertaining to the terpenic family are drastically different from those of the shikimate descendants, and they exhibit much less variability. They enable the stereochemical affiliation of individual hydrogen atoms to be traced back first to the parent atoms in the common intermediate, geranyl diphosphate, then to the glyceraldehyde 3-phosphate and pyruvate couple involved in the DOXP pathway, and ultimately to the glucose precursor. The results illustrate the aptitude of the site-specific isotope ratios not only to authenticate natural with respect to chemical molecules but also to characterize different metabolic pathways and to reveal differences associated with the nature of the plant precursor.

Isotopic criteria in the characterization of aromatic molecules. 2. Influence of the chemical elaboration process.

Most valued natural aromatic molecules can be substituted by their low-cost chemical counterparts. Isotopic methods, which offer the most powerful tool to infer the origin of a molecule, are applied to the characterization of a large number of chemical aromatic species. Isotopic affiliation between precursors and products is investigated in several types of reactions: oxidation of benzyl chloride and benzyl alcohol and hydrolysis of benzylidene chloride and cinnamaldehyde. The isotopic parameters strongly depend not only on the type of process but, for a given process, on the experimental conditions of the reaction. Kinetic isotope effects occurring in several formylation reactions are estimated. It is shown that, in the drastic experimental conditions of many industrial processes, the benzenic hydrogen atoms may be affected by exchange phenomena. Consequently, the site-specific isotopic parameters of the ring fragment of chemical species are usually much less stable than those of the corresponding natural molecules biosynthesized in mild environments. The isotope ratios of substituents such as CH3, CH2Cl, and CHO are more resistant to exchange and provide useful criteria for characterizing both the raw materials and the process. It is shown in particular that radical hydrogen abstraction in toluene to produce benzyl chloride induces relatively moderate fractionation effects. In contrast, oxidation reactions frequently produce strong fractionation effects. In particular, industrial direct oxidation of toluene into benzaldehyde is characterized by deuterium enrichments at the formyl site, which may exceed 900 ppm. Taking into account the large magnitude and high variability of many fractionation effects occurring in chemical reactions, the isotopic fingerprint may provide unambiguous criteria, not only for excluding a natural origin and characterizing the type of process, but also for differentiating molecules synthesized by a given process in different industrial contexts. The isotopic fingerprint may therefore be used by manufacturers as a powerful label for characterizing their production batches.

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.

Hydrogen isotopic profile in the characterization of sugars. Influence of the metabolic pathway.

The site-specific natural hydrogen isotope ratios of plant metabolites determined by 2H nuclear magnetic resonance (SNIF-NMR method) can provide powerful criteria for inferring mechanistic and environmental effects on biosynthetic pathways. This work examines the potential of isotopic profiles for the main constituents of carbohydrates, glucose and fructose, to distinguish different photosynthetic pathways. An appropriate analytical strategy, involving three suitable isotopic probes, has been elaborated with a view to measuring simultaneously, in conditions devoid of isotopic perturbations, all (or nearly all) of the carbon-bound hydrogen isotope ratios. It is shown that the type of photosynthetic metabolism, either C3 (sugar beet, orange, and grape), C4 (maize and sugar cane), or CAM (pineapple), and the physiological status of the precursor plant exert strong influences on the deuterium distribution in the sugar molecules. Consequently, this isotopic fingerprint may be a rich source of information for the comparison of mechanisms in metabolic pathways. In addition, it can provide complementary criteria to ethanol as a probe for the origin of sugars.