Late‐Stage Annulative Imidazolation for Expanding Histidine‐Analogous Side Chains in Peptides and Proteins

ABSTRACT Histidine (His) plays indispensable roles in peptides and proteins owing to the unique reactivity and ionization properties of its imidazole side chain. However, the chemical space of imidazole‐containing side chains remains remarkably underexplored, largely due to the synthetic challenges associated with the selective installation and diversification of imidazole motifs on complex biomolecules. Classical reactions for forming or modifying imidazoles typically require harsh conditions, limiting their applicability in sensitive biomolecular settings. Here, we report a mild and modular late‐stage multi‐component annulative imidazolation strategy for the direct installation of histidine‐analogous side chains in peptides and proteins. In contrast to classical Debus–Radziszewski protocols, the reaction proceeds efficiently at ambient temperature under near‐neutral to basic aqueous conditions, with water as the optimal cosolvent. The method enables rapid access to structurally diverse imidazole motifs with high chemoselectivity and biomolecular compatibility. Beyond providing facile access to noncanonical histidine analogues, this platform offers a programmable approach for tuning peptide electrostatics and function through late‐stage side‐chain modification.