3-aminopyridine 1,N6-ethenoadenine dinucleotide phosphate: a fluorescent reagent for NADP-requiring enzymes

3-Aminopyridine 1,N6-ethenoadenine dinucleotide phosphate (epsilon-AADP) was synthesized from 3-aminopyridine adenine dinucleotide phosphate by reaction with chloroacetaldehyde. The reaction was monitored by high-pressure liquid chromatography. The product, epsilon-AADP, was purified by ion-exchange chromatography followed by a Sephadex G-10 desalting process. Spectrophotometric and fluorimetric properties of epsilon-AADP were obtained at neutral pH. Diazotization of epsilon-AADP was documented by azodye formation at pH values below 3 and the diazotized derivative was shown to react rapidly with sulfhydryl compounds at neutrality. As an analog of NADP, epsilon-AADP was demonstrated to be an effective NADP-competitive inhibitor of NADP-requiring dehydrogenases, reductases, and an NAD glycohydrolase.