Unambiguous Assignment of the H3S and H3R Deuterations of Cerebral (2-13C) Glutamate by 13C NMR at 18.8 Tesla

We used high-field C-13 NMR (18.8 T) to assign unambiguously the isotopic shifts induced by the deuterium substitutions of the H3(proR) and H3(proS) hydrogens of (2-C-13) glutamate in extracts of the brain from deuterated animals. Monodeuterated H3R or H3S glutamate diastereoisomers were produced stereospecifically either by chemical synthesis or by coupling the reactions of isocitrate dehydrogenase and aspartate aminotransferase in deuterated medium, respectively. We show that the (3S-H-2) or (3R-H-2) deuterations induce characteristic small (Delta(2) = -0.058 parts per million (ppm)) or large (Delta(2) = -0.071 ppm) vicinal isotopic shifts upfield of the perprotonated (2-C-13) glutamate resonance (at 55.5 ppm). Isotopically shifted (2-C-13, 3S-H-2) or (2-C-13, 3R-H-2) glutamate singlets are conveniently observed by high-field C-13 NMR in brain extracts from deuterated rats. Since the (3S-H-2) or (3R-H-2) glutamate diastereoisomers are produced stereospecifically by the cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, our results will facilitate the C-13 NMR investigation of these enzymatic activities and their role in subcellular glutamate trafficking. Magn Reson Med 63:1088-1091, 2010. (C) 2010 Wiley-Liss, Inc.