Polyoxomolybdate Promoted Hydrolysis of a DNA-Model Phosphoester Studied by NMR and EXAFS Spectroscopy

Hydrolysis of (p-nitrophenyl)phosphate (NPP), a commonly used phosphatase model substrate, was examined in molybdate solutions by means of H-1, P-31, and Mo-95 NMR spectroscopy and Mo K-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. At 50 degrees C and pD 5.1 the cleavage of the phosphoester bond in NPP proceeds with a rate constant of 2.73 x 10(-5) s(-1) an acceleration of nearly 3 orders of magnitude as compared to the hydrolysis measured in the absence of molybdate. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed in solutions where [Mo7O24](6-) is the major species in solution. Mixing of NPP and [Mo7O24](6-) resulted in formation of these two intermediate complexes that were detected by P-31 NMR spectroscopy. Complex A was characterized by a P-31 NMR resonance at -4.27 ppm and complex B was characterized by a P-31 NMR resonance at -7.42 ppm. On the basis of the previous results from diffusion ordered NMR spectroscopy, performed with the hydrolytically inactive substrate phenylphosphonate (PhP), the structure of these two complexes was deduced to be (NPP)(2)Mo5O214- (complex A) and (NPP)(2)Mo12O36(H2O)(6)(4-) (complex B). The pH studies point out that both complexes are hydrolytically active and lead to the hydrolysis of phosphoester bond in NPP. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), and indicating that the cleavage of the phosphomonoester bond is purely hydrolytic. The Mo K-edge XANES region also did not show any sign of Mo(VI) to Mo(V) reduction during the hydrolytic reaction. Mo-95 NMR and Mo K-edge EXAFS spectra measured during different stages of the hydrolytic reaction showed a gradual disappearance of [Mo7O24](6-) during the hydrolytic reaction and appearance of [P2Mo5O23](6-), which was the final complex observed at the end of hydrolytic reaction.