Fluoromethylketone‐Fragment Conjugates Designed as Covalent Modifiers of EcDsbA are Atypical Substrates

Abstract

AbstractDisulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram‐negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment‐based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and “clickable” ethynylfluoromethylketone was designed for reaction with azide‐functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol‐disulfide oxidoreducatases.