Synthesis of 8-N3-[2'-32P]NADP(H), a new photoaffinity label for NADP(H)-specific enzymes

The synthesis of the photoaffinity label 8-N3-[2'-32P]NADP+ is described. The synthesis consists of the following steps: first 8-N3-AMP is synthesized from AMP or ATP and then the 8-N3-AMP is coupled with NMN, using DCCD as coupling reagent. Finally, the formed 8-N3-NAD+ is phosphorylated with [γ-32P]ATP, using NAD-kinase. The formed product is purified and analysed and is shown to react well with NADP-specific enzymes and not to react with NAD-specific enzymes. Additionally, it is proposed that in the NAD-kinase reaction 2 mol ATP are used for the formation of 1 mol NADP: one molecule of ATP is hydrolyzed to ADP and phosphate while the other delivers the phosphate for the phosphorylation of NAD+.