Experimental and Computational Evidence for a Loose Transition State in Phosphoroimidazolide Hydrolysis

Abstract

Phosphoroimidazolides play a critical role in several enzymatic phosphoryl transfer reactions and have been studied extensively as activated monomers for nonenzymatic nucleic acid replication, but the detailed mechanisms of these phosphoryl transfer reactions remain elusive. Some aspects of the mechanism can be deduced by studying the hydrolysis reaction, a simpler system that is amenable to a thorough mechanistic treatment. Here we characterize the transition state of phosphoroimidazolide hydrolysis by kinetic isotope effect (KIE) and linear free energy relationship (LFER) measurements, and theoretical calculations. The KIE and LFER observations are best explained by calculated loose transition structures with extensive scissile bond cleavage. These three-dimensional models of the transition state provide the basis for future mechanistic investigations of phosphoroimidazolide reactions.

Scheme 1. Model Systems for the Study of Nonenzymatic RNA Replication

Figure 1

Isotopologues and their kinetic isotope effects. (a) The reaction scheme for hydrolysis of isotopologues. (b) KIE measurement for 1a vs 1b. (c) KIE measurement for 1a vs 1c. The KIE was determined by measuring R_S, the enrichment of the heavy to light isotopologues of the substrate ImpG, over the reaction course. R_S is normalized by the initial ratio R₀. L is the integrated intensity of the light isotopologue, and it is normalized by the initial intensity L₀. The solid and dashed lines in (b) and (c) are fits using KIEs of the mean and the limits of 95% confidence interval (n = 8).

Scheme 2. Summary of Calculated A_ND_N Mechanisms; Calculated P–N Bond Lengths Are Labeled in Å

Figure 2

Representative transition structure 2 and intermediate 3 of the loose A_ND_N mechanism. Key distances are labeled in Å. Gibbs free energies are relative to reactants. In the intermediate, the leaving group is separated infinitely from 3. Water molecules, except for the nucleophile, are shown in cyan.

Figure 3

Dependence of the hydrolysis rates on the leaving group pK_a for phosphoroimidazolides. The red line is the least-squares fit to the data. Error bars show one standard deviation (n = 5).