Identification of an Allene Warhead that Selectively Targets a Histidine Residue in the Escherichia coli Oxidoreductase Enzyme DsbA

Covalent modification of protein targets has application in both protein activity profiling and in drug discovery. Covalent warheads typically contain an electrophile that selectively reacts with nucleophilic residues in a protein target, such as cysteine, serine and threonine. Expanding this to other amino acids is an emerging strategy in covalent probe design. Allenes, while known in synthetic chemistry, have not been widely reported for their use as covalent warheads. This study reports the discovery and characterisation of the covalent reaction between a novel allene warhead and a histidine residue in the active site of the bacterial thiol-disulfide oxidoreductase enzyme Escherichia coli DsbA (EcDsbA). The covalent interaction was characterised by X-ray crystallography, nuclear magnetic resonance spectroscopy, and mass spectrometry. This analysis provided insights into the structure, reaction rate and selectivity of the allene. Investigation of the reactivity with nucleophilic amino acids revealed that the reaction with the allene warhead shows some specificity for the histidine in the active site of EcDsbA. This discovery expands the tools of chemical biology by identifying a novel warhead that covalently modifies a histidine residue. This may offer new avenues for targeted protein modification.