ENZYMATIC SYNTHESIS OF L-[15N]-VALINE AND L-[15N]-LEUCINE

Maria Chiriac1, Iulia Lupan2, O.Popescu2, N. Palibroda1, S. Fărcaş1

1National Institute for Research and Development of Isotopic and Molecular Technologies; P.O. BOX 700, 400293 Cluj‑Napoca, Romania.
2Molecular Biology Center, Institute for Interdisciplinary Experimental Research, Babeş-Bolyai University, Cluj-Napoca, Romania.

	Stable isotope-labeled amino acids are useful for biological studies of amino acid metabolism, for examining the tertiary structure of proteins by NMR spectroscopic techniques, in medical and agricultural researches to study biochemical and nutritional problems.
L-amino acids labelled with 15N are frequently either commercially unavailable or prohibitively expensive. Chemical synthesis of the amino acids usually produces a racemic product, although there is occasionally a strong bias towards a single isomer.
Enzymatic synthesis however can be made to produce the L-isomer alone. Such enzymatic synthesis has been carried out by conventional fermentation techniques, or by biosynthesis using either isolated cells, or enzyme extracts.
In this paper we report a simple enzymatic procedure for preparation of L-[15N]-valine and L-[15N]-leucine from corresponding L-beta acids as substrate using bacterial NAD-dependent amino acids dehydrogenase: leucine dehydrogenase (LeuDH), as a catalyst.
The reaction mixtures included glucose dehydrogenase (GlucDH) for NADH regeneration with glucose as a electron donor.
As isotopically  labeled material we used 15NH4Cl 99 at. % 15N produced at National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania.    
The reactions are simple and efficient for producing  gram quantities of 15N –labelled amino acids, with high yield  (  90%).
The structure of L-valine and L-leucine labelled with 15N was confirmed using mass spectrometry combined with  gas chromatography (GC / MS) and NMR Spectroscopy, and the isotopic label was determined by MS on the molecular compounds.

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