ATP formation from adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analog.

The purity of several preparations of adenyl-5'-yl imidodiphosphate (AMP-PNP) was analyzed using thin layer chromatography and the luciferin-luciferase assay. Three contaminants were identified: adenyl-5'-yl phosphoramidate, phosphorylated AMP-PNP, and ATP. The level of ATP contamination ranged from 0.02% to 0.3% in commercially obtained AMP-PNP preparations, and rose to 10% following incubation of AMP-PNP at 37 degrees C for 3 weeks in aqueous solution. The chemistry of the phosphoramidate bond is reviewed briefly, and evidence for a simple mechanism for the spontaneous formation of ATP from AMP-PNP is presented.