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Analytical and Bioanalytical Chemistry

, Volume 399, Issue 3, pp 1187–1200 | Cite as

Recent advances in the analysis of the site-specific isotopic fractionation of metabolites such as fatty acids using anisotropic natural-abundance 2H NMR spectroscopy: application to conjugated linolenic methyl esters

  • Philippe LesotEmail author
  • Zeinab Serhan
  • Isabelle Billault
Original Paper

Abstract

The full elucidation of the enzymatic mechanisms leading to polyunsaturated ω-3 to ω-5 fatty acids (PUFAs) occurring in plants or microorganisms by analyzing their site-specific isotopic fractionation profiles is a challenging task. Isotropic SNIF-NMR® method is an historical, powerful tool for the determination of (2H/1H) ratios. However, the absence of accessible isotopic data on the enantiotopic hydrogen sites (CH2 groups) prevents the study of the enzymatic reaction stereoselectivity. Natural-abundance deuterium (NAD) 2D NMR experiment using chiral liquid crystals (CLC) as solvent is a new tool in this field, overcoming this limitation. In this work, we have explored various possibilities for optimizing the enantio-discrimination properties of CLC by changing the nature of the polypeptide and/or increasing the polarity of the organic co-solvents. We report also the first applications of TMU as co-solvent for preparing enantio-discriminating, homogenous polypeptide mesophases. The various experimental NAD NMR results recorded at an optimal sample temperature are discussed and compared in terms of number of discriminated 2H sites and magnitude of spectral separation for different PUFAs such as the linoleic and linolenic acids. The comparison of all NMR results shows that optimal results are obtained when CLC mixtures made of poly-γ-benzyl-l-glutamate (PBLG) and high polarity co-solvents are used. As new challenging examples of applications, we report the preliminary analytical results obtained from two ω-5 conjugated linolenic acids: the α-eleostearic acid (9Z, 11E, 13E) and the punicic acid (9Z, 11E, 13Z). NMR data are discussed in terms of molecular orientational ordering parameters and isotopic distribution.

Figure

The new challenges of the site-specific isotopic fractionation analysis of fatty acids using the natural-abundance deuterium NMR in polypeptide aligned media

Keywords

Polyunsaturated fatty acids Site-specific isotopic fractionation 2H NMR Quadrupolar splittings PBLG Chirality Ordering behavior 

Notes

Acknowledgments

P. L. gratefully acknowledges Profs. M. Longeri and A. Loewenstein as well as Dr. P. Judeinstein for scientific discussions and/or their help during the preparation of this manuscript. The authors also thank CNRS for its financial support. Z. S. thanks the MNERS for her Ph.D granting.

Supplementary material

216_2010_4394_MOESM1_ESM.pdf (2 mb)
ESM1 (PDF 2.01 mb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Philippe Lesot
    • 1
    Email author
  • Zeinab Serhan
    • 1
  • Isabelle Billault
    • 2
  1. 1.Université de Paris Sud 11, CNRS UMR 8182 ICMMO, RMN en Milieu OrientéOrsay CedexFrance
  2. 2.Université de Nantes, Elucidation of Biosynthesis by Isotopic Spectrometry Group, CEISAM, UMR CNRS 6230, CNRS UMR 6230Nantes CedexFrance

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