MODELS FOR NUCLEOSIDE GLYCOSYLASE ENZYMES - EVIDENCE THAT THE HYDROLYSIS OF BETA-D-RIBOFURANOSIDES REQUIRES A BACKSIDE PREASSOCIATION NUCLEOPHILE

We have compared the pH-independent rates of glycosidic hydrolysis in a series of four fluorenone ketal derivatives of p-nitrophenyl β-D-ribofuranoside with each other and with that of p-nitrophenyl β-D-ribofuranoside itself. A syn-oriented carboxylate group clearly affords catalysis in the reaction and is more effective than identically oriented amide and ester groups by factors of at least 100-and 240-fold, respectively. The effect of the carboxylate can be viewed in three different ways: it provides a 3.2-fold acceleration as compared to underivatized p-nitrophenyl β-D-ribofuranoside, an approximately 30-fold acceleration when the decelerating effect of the ketal group is considered, and an 860-fold acceleration as compared to the compound that models an Asp-52 → Ala-52 mutant lysozyme. Most strikingly, the hydrolysis of a reference compound with impeded backside solvation of the oxocarbonium ion is very slow and provides a direct experimental verification for the importance of backside solvent participation in the hydrolysis reaction of a nucleoside analogue. These kinetic results do not provide any evidence concerning two potential explanations for the role of carboxylate in acetal hydrolysis: electrostatic destabilization of the ES complex by binding-induced desolvation of the carboxylate ion and lone pair exchange repulsion. The results are consistent with two other proposed carboxylate roles: electrostatic stabilization of an oxocarbonium ion like transition structure and anchimeric assistance by the carboxylate. While the most likely role for carboxylate in acetal hydrolysis is as a nucleophile, the low reactivity of the endo-amide and the unexceptional reactivity of the syn-oriented endo-carboxylate more directly support the electrostatic stabilization mechanism for the hydrolyses of these riboside derivatives. © 1990, American Chemical Society. All rights reserved.