Synthesis and properties of (4‐¹³C)NAD⁺

Abstract

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