Biocompatible SuFEx Cyclization Enables DNA-Encoded Macrocyclic Peptide Libraries for Drug Discovery

Rapid, aqueous macrocyclization strategies that proceed in high conversion are valuable for ultra-large macrocyclic peptide library synthesis via display and DNA-encoded library (DEL) technology. Here we report an on-DNA macrocyclization based on sulfur(VI) fluoride exchange (SuFEx) that unites above features. This approach embeds a phenol and an aryl sulfonyl fluoride within a DNA-tagged peptide to accelerate SuFEx and trigger intramolecular cyclization immediately upon dissolution in basic aqueous buffer. Macrocycles ranging from 15-to 52-membered rings bearing diverse amino acids were synthesized efficiently. The reaction integrates seamlessly into DEL workflows and enabled discovery of potent, de novo macrocyclic RIPK1 inhibitors. The on-DNA cyclization conditions readily translate off-DNA to furnish sulfonate-and sulfonamide-linked macrocycles. NMR analyses showed SuFEx-derived biaryl linkers act as conformational elements: changes in aryl substitution and linker length tune backbone conformations from extended strands to rigid turns. These findings establish SuFEx cyclization as a robust, biocompatible strategy for programmable macrocycle design and drug discovery.