Hydrolysis of the RNA model substrate catalyzed by a binuclear ZrIV-substituted Keggin polyoxometalate

Abstract

The reactivity and solution behaviour of the binuclear Zr^IV-substituted Keggin polyoxometalate (Et₂NH₂)₈[{α-PW₁₁O₃₉Zr(μ-OH)(H₂O)}₂]·7H₂O (ZrK 2 : 2) towards phosphoester bond hydrolysis of the RNA model substrate 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP) was investigated at different reaction conditions (pD, temperature, concentration, and ionic strength). The hydrolysis of the phosphoester bond of HPNP, followed by means of ¹H NMR spectroscopy, proceeded with an observed rate constant, k_obs = 11.5(±0.42) × 10⁻⁵ s⁻¹ at pD 6.4 and 50 °C, representing a 530-fold rate enhancement in comparison with the spontaneous hydrolysis of HPNP. ¹H and ³¹P NMR spectra indicate that at these reaction conditions the only products of hydrolysis are p-nitrophenol and the corresponding cyclic phosphate ester. The pD dependence of k_obs exhibits a bell-shaped profile, with the fastest rate observed at pD 6.4. The formation constant (K_f = 455 M⁻¹) and catalytic rate constant (k_c = 42 × 10⁻⁵ s⁻¹) for the HPNP–ZrK 2 : 2 complex, activation energy (E_a) of 63.35 ± 1.82 kJ mol⁻¹, enthalpy of activation (ΔH^‡) of 60.60 ± 2.09 kJ mol⁻¹, entropy of activation (ΔS^‡) of −133.70 ± 6.13 J mol⁻¹ K⁻¹, and Gibbs activation energy (ΔG^‡) of 102.05 ± 0.13 kJ mol⁻¹ at 37 °C were calculated from kinetic experiments. Binding between ZrK 2 : 2 and the P–O bond of HPNP was evidenced by the change in the ³¹P chemical shift and signal line-broadening of the ³¹P atom in HPNP upon addition of ZrK 2 : 2. Based on ³¹P NMR experiments and isotope effect studies, a mechanism for HPNP hydrolysis in the presence of ZrK 2 : 2 was proposed.