Abstract
Carboxylate side chains in aspartic and glutamic acids play critical roles in protein structure and function due to their polarity and negative charge. These acidic residues, which are abundant in high-value drug targets, represent attractive yet underexplored hotspots for covalent inhibitor discovery. In this study, we introduce N-aryl aziridines as a systematically tunable, chemoselective scaffold for covalent targeting of carboxylates across the proteome. Using a library of N-pyridinium aziridine-based fragments combined with chemoproteomics-enabled target deconvolution, we identified lead hits for aspartates and glutamates in proteins such as mitochondrial carrier homologue 2 (MTCH2), RUN and FYVE domain-containing protein 1 (RUFY1), and delta(24)-sterol reductase (DHCR24). Modular build-and-couple synthetic logic enabled fragment evolution via Ni-catalyzed cross-coupling to access N-aryl aziridines with enhanced affinities for MTCH2 and RUFY1. Notably, N-aryl aziridine 5b selectively modified RUFY1 at E502, disrupting its interactions within the endosomal trafficking network and impairing receptor recycling. This work establishes N-aryl aziridines as versatile carboxylate-targeting covalent inhibitor scaffolds, broadening the scope of covalent ligand discovery.