Combined analysis of C-18 unsaturated fatty acids using natural abundance deuterium 2D NMR spectroscopy in chiral oriented solvents
Résumé
The quantitative determination of isotopic ((2)H/(1)H)(i) ratios at natural abundance using the SNIF-NMR protocol is a well-known method for understanding the enzymatic biosynthesis of metabolites. However, this approach is not always successful for analyzing large solutes and, specifically, is inadequate for prochiral molecules such as complete essential unsaturated fatty acids. To overcome these analytical limitations, we use the natural abundance deuterium 2D NMR (NAD 2D NMR) spectroscopy on solutes embedded in polypeptide chiral liquid crystals. This approach, recently explored for measuring ((2)H/(1)H)i ratios of small analytes (Lesot, P.; Aroulanda, C.; Billault, I. Anal Chem. 2004, 76, 2827-2835), is a powerful way to separate the (2)H signals of all nonequivalent enantioisotopomers on the basis both of the 2H quadrupolar interactions and of the 2H chemical shift. Two significant advances over our previous work are presented here and allow the complete isotopic analysis of four mono- and polyunsaturated fatty acid methyl esters: methyl oleate (1), methyl linoleate (2), methyl linolenate (3), and methyl vernoleate (4). The first consists of using NMR spectrometers operating at higher magnetic field strength (14. 11) and equipped with a selective cryoprobe optimized for deuterium nuclei. The second is the development of Q-COSY Fz 2D NMR experiments able to produce phased (2)H 2D maps after a double Fourier transformation. This combination of modern hardware and efficient NMR sequences provides a unique tool to analyze the ((2)H/(1)H)(i) ratios of large prochiral molecules (C-18) dissolved in organic solutions of poly(gamma-benzyl-L-glutamate) and requires smaller amounts of solute than previous study on fatty acids. For each compound (1-4), all (2)H quadrupolar doublets visible in the 2D spectra have been assigned on the basis of (2)H chemical shifts, isotopic data obtained from isotropic quantitative NAD NMR, and by an interspectral comparison of the anisotropic NAD spectra of four fatty acids. The NMR results are discussed in terms of ((2)H/(1)H)(i) isotopic distribution and molecular orientation in the mesophase. For the first time, we show that the investigation of natural isotopic fractionation of complete fatty acids is possible without the need of chemical modifications, hence providing an alternative method to probe the mechanisms of enzymes implied in the biosynthetic pathway of unsaturated fatty acids.