Evidence for direct attack by hydroxide in phosphodiester hydrolysis

Phosphodiester hydrolysis has been the subject of intense study due to its importance in biology. Despite the numerous significant analyses of phosphodiester cleavage mechansim, comparatively little is known about the nucleophiles in these reactions. To determine whether hydroxide acts as a nucleophile or a general base in the hydrolysis of thymidine-5′-p-nitrophenyl phosphate,we determined solvent deuterium isotope effects (D2Ok), ionic strength effects, and 18O isotope effects on the solvent nucleophile (18knuc). The D2Ok for hydroxide-catalyzed phosphodiester hydrolysis is slightly inverse (0.9 ± 0.1), suggesting that a proton transfer does not occur in the transition state. A significant α effect is observed with hydroperoxide, demonstrating that oxyanions can act as nucleophiles in the reaction. Additionally, the ionic strength dependencies of hydroxide and hydroperoxide catalysis are indistinguishable, suggesting that they act by the same mechanism. Finally, the 18knuc for the hydroxide-catalyzed reaction is 1.068 ± 0.007, well in excess of the equilibrium 18O isotope effect between water and hydroxide (1.040 ± 0.003). Together, the data are most consistent with direct nucleophilic attack by hydroxide. From the observed 18knuc and the known equilibrium component, the kinetic component of the isotope effect was calculated to be 1.027 ± 0.010. This large kinetic component suggests that little bond order to the nucleophile occurs in the transition state. Copyright © 2002 American Chemical Society.