Tyrosinase-Catalyzed Tyr–Aniline Coupling Enables Peptide Editing, Macrocyclization, and Phage-Display-Based Selection

Abstract Tyrosine-selective peptide modification provides a valuable route to late-stage diversification, yet current methods remain largely limited to site-specific labeling and rarely extend to macrocyclization or ligand discovery within a unified platform. Here, we report a tyrosinase-catalyzed tyrosine–aniline coupling platform that enables peptide editing, conjugation, macrocyclization, and phage-display-based ligand discovery within a single reaction manifold. Under mild aqueous conditions, this enzymatic oxidative coupling proceeds rapidly within minutes and exhibits broad compatibility with unprotected peptide substrates containing diverse proteinogenic side chains. Structurally diverse and readily accessible arylamines can be directly introduced onto unprotected peptides to install functional, fluorophore, bioactive, and bifunctional modules. Moreover, bifunctional arylamines enable tyrosine-directed peptide conjugation and macrocyclization in a modular manner. Importantly, this chemistry is compatible with phage display and enables post-translational diversification of genetically encoded peptide libraries through on-phage macrocyclization. The resulting platform was applied to dual-tyrosine randomized peptide libraries for target-directed biopanning, yielding cyclic peptide ligands against two independent protein targets, Keap1 and EphA4. The lead cyclic peptide CM1 bound Keap1 with a Kd of 211 ± 60 nM, while the independently identified EphA4 ligand CN1 further demonstrated the transferability of the platform beyond a single-target system. Alanine-scanning analysis and 1H–15N-heteronuclear single quantum coherence (HSQC) titration NMR experiments further supported direct and sequence-dependent recognition of CM1 toward Keap1. Collectively, this work establishes a generalizable tyrosine-based chemical platform that connects residue-selective peptide modification, macrocycle construction, and genetically encoded ligand discovery, providing a versatile strategy for expanding the chemical and functional diversity of peptide libraries.