Identification of an Allene Warhead That Selectively Targets a Histidine Residue in the <i>Escherichia coli</i> Oxidoreductase Enzyme DsbA

Small molecules that covalently modify proteins typically contain an electrophile that selectively reacts with nucleophilic residues in a protein target, such as cysteine, serine, and threonine. Targeting other amino acids is an emerging strategy in covalent probe design. This study reports the discovery and characterization of the covalent reaction between a novel allene warhead and a histidine residue in the active site of the bacterial thiol-disulfide oxidoreductase enzyme <i>Escherichia coli</i> DsbA (<i>Ec</i>DsbA). Allenes have not been widely reported for their use as covalent warheads. The interaction was characterized 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 <i>Ec</i>DsbA. Thus, the allene represents a novel histidine-modifying warhead.