Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3‐membered N‐heterocyclic compounds—such as aziridines, azirines, and oxaziridines—possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α‐lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α‐lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo‐ and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α‐lactam‐based electrophilic probes in covalent chemical biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry, constituting the anchor point for the design of targeted covalent inhibitors, the identification of novel therapeutic targets, and the development of a wide diversity of bioconjugation and proteomic profiling techniques. Naturally, there is great interest in the discovery of new types of biologically relevant electrophiles with potent action and broad applicability. For instance, the group of 3-membered N-heterocyclic compounds - such as aziridines, azirines, and oxaziridines – combine unique electronic properties and structural strain to trigger their respective potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, but, until this point, their utility within the field has been unexplored. Here, we demonstrate an α-lactam reagent that can be efficiently employed in the framework of bioconjugation and proteomic profiling. We have designed and synthesised a stable α-lactam, AM2, that is compatible with aqueous buffers, and have extensively characterised its properties and reactivity. In simple settings, AM2 was e.g. found to be reactive towards both thiols and amines, with the former reacting significantly faster. We further demonstrated that AM2 undergoes conjugation to free cysteine residues in peptides suggesting applications for bioconjugation. Interestingly, liver carboxylesterase 1 (CES1), a serine hydrolase with key roles in both endo- and xenobiotic metabolism, was found as a highly selective covalent target for AM2 in HepG2 liver cancer cells. All in all, our study constitutes the starting point for the further development of α-lactam-based electrophilic probes and exploration of their use in covalent chemical biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3‐membered N‐heterocyclic compounds—such as aziridines, azirines, and oxaziridines—possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α‐lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α‐lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo‐ and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α‐lactam‐based electrophilic probes in covalent chemical biology.