Incubation of protonated NADH or NADPH with ortho-aminobenzaldehyde generates a novel fluorescent nicotinamide dihydroquinazoline condensate.

ABSTRACT

Incubation of reduced nicotinamide adenine dinucleotide (NADH) but not oxidized NAD+ with ortho-aminobenzaldehyde (oABA) generated an uncharacterized chromophore with an absorption peak characteristic of a dihydroquinazoline condensate. This chromophore is responsible for a non-specific signal in a diamine oxidase (DAO) activity assay based on the generation of fluorescent dihydroquinazoline structures directly from DAO substrates. Herein we show that at pH values below 3.0 the glycosidic bond of NADH/NADPH is broken releasing double protonated dihydro-nicotinamide (dihydro-NAM), which consequently condensates with oABA to a novel dihydroquinazoline chromophore and fluorophore, namely the 6- or 8-carbamoyl-5H,7H,8H,9H-10λ5-pyrido[2,1-b]quinazolin-10-ylium isomer (CMPQ). The second protonation event closely correlates with the pKa of the N1 nitrogen of C5-protonated dihydro-NAM and fluorophore stability. The fusion partner of oABA is likely the iminium of the primary acid product of dihydro-NAM after glycosidic bond hydrolysis and before irreversible cyclization. Trapping of protonated dihydro-NAM from NADH or NADPH with oABA allows quantification of these dinucleotides. Despite almost a century of research studying acid-catalyzed molecular rearrangements of NADH and NADPH, new and surprising details can be discovered.