α‐Lactam Electrophiles for Covalent Chemical Biology**

Abstract

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.

N‐Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1

“…The transition from 5‐membered pyrrolidines to 6‐membered heterocycles markedly improved compound specificity by eliminating binding to the protein deglycase PARK7 and by dampening reactivity with other DUBs, both in cells and in vitro. The data are consistent with other cyclic electrophilic moieties, for which target selectivity is encoded by ring‐size [43,44] . Moreover, the transition introduced an unexpected and context‐dependent reversibility of the isothiourea linkage of the compound to the catalytic cysteine of UCHL1, preventing efficient functionalisation of the probe through copper‐catalysed click chemistry.…”

“…The data are consistent with other cyclic electrophilic moieties, for which target selectivity is encoded by ring-size. [43,44] Moreover, the transition introduced an unexpected and…”

N‐Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1

“…The data are consistent with other cyclic electrophilic moieties, for which target selectivity is encoded by ring-size. [43,44] Moreover, the transition introduced an unexpected and context-dependent reversibility of the isothiourea linkage of the compound to the catalytic cysteine of UCHL1, preventing efficient functionalisation of the probe through coppercatalysed click chemistry. Our data highlight an important caveat for the investigation of covalent small-moleculeprotein isothiourea linkages.…”

Proteome-wide Ligand and Target Discovery by Using Strain-Enabled Cyclopropane Electrophiles

“…For example, Poulsen et al found that cyclopropene-and α-lactam-based electrophiles exhibit a strong capability to conjugate protein targets within cells. 33,34 Notably, protein targets such as GSTO1 and CES1/2 were effectively conjugated by these electrophiles (Figure 1a,b). 33,34 Chang et al demonstrated the effectiveness of oxaziridine-based reagents as a methionine-targeting warhead for the synthesis of antibody-drug conjugates and the identification of reactive methionine residues in whole proteomes (Figure 1c).…”

“…33,34 Notably, protein targets such as GSTO1 and CES1/2 were effectively conjugated by these electrophiles (Figure 1a,b). 33,34 Chang et al demonstrated the effectiveness of oxaziridine-based reagents as a methionine-targeting warhead for the synthesis of antibody-drug conjugates and the identification of reactive methionine residues in whole proteomes (Figure 1c). 35 We found that 3-phenyl-2H-azirine can selectively bioconjugate with carboxylic acid residues (Glu/ Asp) in proteins within live cells (Figure 1d).…”