3-Aminopyridine 1, N6-ethenoadenine dinucleotide phosphate: A fluorescent reagent for NADP-requiring enzymes

Abstract

3-Aminopyridine 1, N 6-ethenoadenine dinucleotide phosphate (ϵ-AADP) was synthesized from 3-aminopyridine adenine dinucleotide phosphate by reaction with chloroacetaldehyde. The reaction was monitored by high-pressure liquid chromatography. The product, ϵ-AADP, was purified by ion-exchange chromatography followed by a Sephadex G-10 desalting process. Spectrophotometric and fluorimetric properties of ϵ-AADP were obtained at neutral pH. Diazotization of ϵ-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, ϵ-AADP was demonstrated to be an effective NADP-competitive inhibitor of NADP-requiring dehydrogenases, reductases, and an NAD glycohydrolase.