11] Preparation of 3-aminopyridine adenine dinucleotide and 3-aminopyridine adenine dinucleotide phosphate

Abstract

This chapter discusses the methods for the preparation of 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP). For preparing AAD, a hypobromite solution is prepared in a hood by dissolving 0.077 ml of ice-cold bromine in 100 ml of vigorously stirred 0.5 M NaOH. This solution was added to 400 ml of water, containing 408 mg of β-NAD. The resulting mixture was stirred at room temperature for 180 min., at the time the reaction is complete and the reaction mixture can be adjusted to pH 7, with concentrated HCl. For the preparation of AADP, the pig brain NADase-catalyzed pyridine base-exchange reaction is used. In this study, analyses of purified AAD and AADP were performed to determine quantitatively the presence of 3-aminopyridine, adenine, phosphate, and ribose. Both AAD and AADP can be diazotized in nitrous acid, and the resulting diazonium chlorides react at low pH, with arylamines and phenols to form azodyes. At neutral pH, diazotized AAD(P) does not react with arylamines and phenols; however, rapid reactions, with sulfhydryl compounds, are observed. The reactions, with sulfhydryl compounds to produce diazomercaptides or thioethers, form the basis for the application of the diazotized derivatives as site-labeling reagents for pyridine nucleotide-dependent enzymes.