A mechanistic study of thiol addition to N-acryloylpiperidine

Abstract

Nucleophilic cysteine (Cys) residues are present in many enzyme active sites and represent the target of many different irreversible enzyme inhibitors. Given its fine balance between aqueous stability and thiolate reactivity, the acrylamide group is a particularly popular warhead pharmacophore among inhibitors designed for biological and therapeutic application. The acrylamide group is well known to undergo thiol addition, but the precise mechanism of this addition reaction has not been studied in as much detail. In this work we have focussed on the reaction of N-acryloylpiperidine (AcrPip), which represents a motif found in many targeted covalent inhibitor drugs. Using a precise HPLC-based assay, we measured the second order rate constants for the reaction of AcrPip with a panel of thiols possessing different pK_a values. This allowed construction of a Brønsted-type plot that reveals the relative insensitivity of the reaction to the nucleophilicity of the thiolate. By studying temperature effects, we were able to construct an Eyring plot from which the enthalpy and entropy of activation were calculated. Ionic strength and solvent kinetic isotope effects were also studied, informing on charge dispersal and proton transfer in the transition state. DFT calculations were also performed, providing information on the potential structure of the activated complex. Taken together, these data strongly support one cohesive addition mechanism that is the microscopic reverse of the E1cb elimination, and highly relevant to the intrinsic thiol selectivity of AcrPip inhibitors and their subsequent design.