Accurate determination of 13C enrichments in nonprotonated carbon atoms of isotopically enriched amino acids by 1H nuclear magnetic resonance.

A method for the accurate determination of 13C enrichments in nonprotonated carbon atoms of organic compounds that makes use of unresolved 13C satellites of proton(s) bonded to the vicinal carbon atom was developed. Using glutamate as a model molecule, this 1H nuclear magnetic resonance (NMR) inverse spin-echo difference spectroscopy method was calibrated for inversion efficiency and relaxation effects which were then shown to cause only a minor loss of the measured 13C satellite amplitude (2% for glutamate C-1 and 7% for glutamate C-5). The determination of 13C enrichments in nonprotonated glutamate carbon atoms by this method was shown to be more precise than 13C NMR. As a first application, a [5-13C]glucose labeling experiment with Corynebacterium glutamicum ASK1 was performed. The labeling patterns of glutamate and arginine extracted from cellular protein were determined using the newly developed method and standard 1H NMR with and without broadband 13C decoupling. Determination of the 13C enrichment in C-5 of glutamate and arginine, respectively, by the two methods showed good agreement. From the deduced labeling pattern of 2-oxoglutarate, an in vivo carbon flux distribution within the central metabolism of C. glutamicum ASK1 was calculated. Thus, the relative flux toward oxaloacetate via the tricarboxylic acid cycle enzyme malate dehydrogenase was determined as 45%, whereas that via anaplerotic C3 carboxylation was determined as 55%.