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

Abstract Rapid, aqueous macrocyclization strategies that proceed in high conversion are valuable for ultralarge macrocyclic peptide (MP) 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. MPs ranging from 15 to 52 membered rings bearing diverse amino acids were synthesized efficiently. The on-DNA conditions readily translate off DNA to furnish sulfonate and sulfonamide linked MPs. NMR analyses showed SuFEx-derived biaryl linkers act as conformational tuner: sequential changes in aryl substitution and linker length shift backbone conformations from extended strands to rigid turns. Leveraging this chemistry, we designed and synthesized ultralarge MP libraries via DEL technology. DEL screening followed by off-DNA hit validation identified a potent, de novo MP inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1). Collectively, these findings establish SuFEx cyclization as a robust, DEL-compatible strategy for programmable macrocycle design and drug discovery.