Synthesis and purification of deoxyribose analogues of NAD+ by affinity chromatography and strong-anion-exchange high-performance liquid chromatography.

Two different chemical analogues of NAD+, containing either 2'-deoxyribose (2'-dNAD+) or 3'-deoxyribose (3'-dNAD+) were synthesized enzymatically with oxidized nicotinamide mononucleotide (NMN+)-adenyl transferase (E.C. 2.7.7.18). These compounds were efficiently purified by affinity chromatography on a boronate gel, followed by strong-anion-exchange high-performance liquid chromatography under isocratic conditions. These chromatographic steps resulted in the elimination of unreacted deoxyadenosine triphosphates (dATP) and NMN+, respectively. The purified dNAD+ isomers were examined as possible substrates of homogeneous poly(ADP-ribose) polymerase, purified from calf thymus. 3'-dNAD+ was effectively utilized as a substrate by the polymerase, which catalyzed the formation of protein-bound poly(3'-dADP-ribose) during automodification. However, 2'-dNAD+ was not a substrate for the automodification reaction catalyzed by this DNA-dependent enzyme. Instead, 2'-dNAD+ was a potent non-competitive inhibitor of NAD+ in the elongation reaction.