Hydrolytic cleavage of an RNA-model phosphodiester catalyzed by a highly negatively charged polyoxomolybdate [Mo7O24]6- cluster.

Hydrolysis of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP), a commonly used RNA model substrate, was examined in molybdate solutions by means of (1)H, (31)P, and (95)Mo NMR, Raman, and Mo K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. (1)H and (31)P NMR spectroscopy indicate that at 50 degrees C and pD 5.9 the cleavage of the phosphodiester bond in HPNP proceeds with a rate constant of 6.62 x 10(-6) s(-1), giving a cyclic phosphate ester and p-nitrophenol as the only products of hydrolysis. 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 phosphodiester 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. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed at pD 5.9. Comparison of the rate profile with the concentration profile of polyoxomolybdates shows a striking overlap of the k(obs) profile with the concentration of heptamolybdate, suggesting that the highly negatively charged [Mo(7)O(24)](6-) is the hydrolytically active species. Kinetic experiments at pD 5.9 using a fixed amount of [Mo(7)O(24)](6-) and increasing amounts of HPNP revealed slight signs of curvature at 25 molar excess of HPNP. The data fit the general Michaelis-Menten reaction scheme, permitting the calculation of the catalytic rate constant k(2) (3.02 x 10(-4) s(-1)) and K(m) (1.06 M). Variable temperature (31)P NMR spectra of a reaction mixture revealed broadening of the HPNP (31)P resonance upon increase of temperature, implying the dynamic exchange process between free and bound HPNP at higher temperatures. Addition of salts resulted in the inhibition of HPNP hydrolysis, as well as addition of dimethyl phosphate, suggesting competition for the binding to [Mo(7)O(24)](6-). The hydrolysis of 10 equiv of HPNP could be achieved in the presence of 1 equiv of [Mo(7)O(24)](6-), and the multiple turnovers demonstrate that the reaction is catalytic. (31)P NMR and Mo K-edge EXAFS spectra measured during different stages of the hydrolysis indicated that under catalytic conditions a partial conversion of [Mo(7)O(24)](6-) into [P(2)Mo(5)O(23)](6-) occurs.

Phosphoesterase activity of polyoxomolybdates: diffusion ordered NMR spectroscopy as a tool for obtaining insights into the reactivity of polyoxometalate clusters.

Diffusion ordered NMR spectroscopy (DOSY NMR) is shown to be an excellent tool for observing reactive transients in the hydrolysis of the phosphatase model substrate (p-nitrophenyl)phosphate (NPP) promoted by polyoxomolybdate.

Structural characterization and reactivity of gamma-octamolybdate functionalized by proline.

The reaction of molybdate and dl-proline at pH 3.4 results in the formation of a Na(4)[Mo(8)O(26)(proO)(2)] x 22H(2)O complex (pro=proline) in which two proline ligands are attached to molybdenum(VI) ions via monodentate coordination of the carboxylate groups. The structure of the complex was determined by single crystal X-ray diffraction and by combination of (1)H, (13)C and (95)Mo NMR spectroscopy techniques in solution. The structure of the complex is strongly dependent on the pH. At native pH 3.4 the octamolybdate-type structure seems to be present in solution, but the increase of pH to 5.8 resulted in a rearrangement of the structure to a heptamolybdate-type structure. At physiological pH, the polyoxometalate framework was completely dissociated into the monomeric MoO(4)(2-) unit. The reactivity of the Na(4)[Mo(8)O(26)(proO)(2)] x 22H(2)O towards the hydrolysis of ATP was tested at different pH values. While in solution at pH 3.4 the hydrolysis proceeded to yield AMP (adenosine monophosphate) and ADP (adenosine diphosphate) in nearly equal amounts, reaction mixture at pH 5.8 gave ADP as the only product of hydrolysis after 24h of reaction. At neutral pH, the hydrolysis of ATP was slower, but it proceeded to yield 75% of ADP after 48 h of reaction.

Questioning the paradigm of metal complex promoted phosphodiester hydrolysis: [Mo7O24](6-) polyoxometalate cluster as an unlikely catalyst for the hydrolysis of a DNA model substrate.

The first example of a phosphodiester bond cleavage promoted by a highly negatively charged polyoxometalate cluster has been discovered: the hydrolysis of the phosphodiester bond in a DNA model substrate bis(p-nitrophenyl)phosphate (BNPP) is promoted by the heptamolybdate anion [Mo7O24](6-) with rates which represent an acceleration of nearly four orders of magnitude compared to the uncatalyzed cleavage.