15N- and 17O-enriched samples of imidodiphosphate (PNP), its tetraethyl ester, and 5'-adenylyl imidodiphosphate (AMP-PNP) have been prepared. The 15N NMR spectra of both PNP and AMP-PNP reveal the presence of approximately 70-Hz 1H-15N coupling constants for the fully ionized samples, demonstrating an imido tautomeric structure in both cases. For AMP-PNP this coupling persists in the presence of a stoichiometric amount of Mg2+. The 17O NMR chemical shifts of the resonances associated with the phosphoryl oxygens have been assigned. The effect of pH on the resonances for PNP and AMP-PNP is similar to that observed for ATP (Gerlt, J. A.; Demou, P. C.; Mehdi, S. J. Am. Chem. Soc. 1982, 104, 2848) and its thiophosphate and phosphonate structural analogues (Gerlt, J. A.; Reynolds,. A.; Demou, P. C.; Kenyon, G. L. J. Am. Chem. Soc., preceding paper in this issue), indicating that protonation of the tetraanion of PNP occurs exclusively on the oxygens and that protonation of the tetraanion of AMP-PNP occurs exclusively on the γ-phosphoryl oxygens. Although nO NMR demonstrates that protonation of the monoanion of the tetraethyl ester of PNP occurs predominantly on nitrogen, the corresponding 15N NMR chemical shift change was only 2.50 ppm. Thus, 15N NMR chemical shift changes cannot be used reliably to ascertain the sites of protonation in imidodiphosphates.
ASJC Scopus subject areas
- Colloid and Surface Chemistry