NMR Analysis of Carboxylate Isotopomers of C-Metabolites by Chemoselective Derivatization with N-Cholamine.

A substantial fraction of common metabolites contains carboxyl functional groups. Their C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group. N-cholamine has been shown to be a useful CS agent for carboxylates, producing a peptide bond that can be detected via N-attached H with high sensitivity in heteronuclear single quantum coherence experiments. Here, we report an improved method of derivatization and show how C-enrichment at the carboxylate and/or the adjacent carbon can be determined via one- and two-bond coupling of the carbons adjacent to the cholamine N atom in the derivatives. We have applied this method for the determination of C isotopomer distribution in the extracts of A549 cell culture and liver tissue from a patient-derived xenograft mouse.