Synthesis and properties of (4-13C)NAD+. Observation of its binding to yeast alcohol dehydrogenase by 13C-NMR spectroscopy.

Starting from (13C)formic acid, acetone and cyanoacetamide samples of (4-13C)nicotinic acid and (4-13C)-nicotinamide were synthesised in an overall and additive yield of 11%. 1H-NMR and mass spectroscopy showed 90% enrichment of 13C in the expected position. NADase-catalysed exchange between thionicotinamide-adenine dinucleotide and (4-13C)nicotinamide furnished (4-13C)NAD+ which was purified, characterized and quantified by 1H-NMR and 13C-NMR spectroscopy and by enzymic assay. The 13C-NMR signal of (4-13C)beta-NAD+ (146.09 ppm) was broadened and shifted (147.83 ppm) upon binding to yeast alcohol dehydrogenase.