A Modular PMDA Linker Enables Lysine-Selective Cyclization of Unprotected Peptides and Automated Macrocycle Assembly

Macrocyclic peptide stapling is a powerful strategy to enhance conformational stability and functional performance, yet existing approaches often suffer from limited chemoselectivity, constrained modularity, or poor compatibility with automated synthesis. Here we report a pyromellitic dianhydride (PMDA)-based Lys-Lys stapling platform that enables highly chemoselective intramolecular bis-amidation of fully unprotected peptides under mild conditions. Distinct from conventional bifunctional linkers, PMDA operates as a tetrafunctional scaffold, simultaneously inducing efficient macrocyclization while installing two carboxylate handles for downstream diversification. The reaction exhibits exceptional selectivity for lysine ε-amines, tolerating all other nucleophilic side chains, including cysteine, across a broad range of peptide sequences and linker spacings. The resulting macrocycles support stepwise bicyclization and modular postcyclization conjugation with nucleic acids, small-molecule drugs, and affinity tags. Application to antimicrobial peptides demonstrates enhanced conformational constraint and markedly improved proteolytic stability. Importantly, the chemistry is intrinsically compatible with solid-phase peptide synthesis and fully integrated, end-to-end automated workflows without hardware modification. This PMDA-mediated stapling strategy establishes a versatile, automation-ready linker platform for rapid assembly and functional diversification of macrocyclic peptides.