Isothiocyanate Enabled Versatile Cyclizations of Phage Dis-played Peptides for the Discovery of Macrocyclic Binders

Cyclic peptides exhibit advantages in binding protein targets with high affinity and competency in inhibiting protein-protein interactions (PPIs). Cyclic peptide phage display with over a billion variants is an invaluable tool in drug discovery. However, achieving efficient peptide cyclization on phages remains a challenge due to the limited availability of reaction sites, which also restricts scaffold diversity. Here, we report an isothiocyanate-derived crosslinker featuring dual reactive groups: a bro-mide that covalently attaches to cysteine thiols, and a thiocyanogen that selectively forms a thiourea bridge with either the N-terminal amino group or ε-amines of lysine, depending on pH. This strategy enables pH-modulated cyclization, with head-to-side chain cyclization at pH 6.5 and side chain-to-side chain ligation at pH 9.5, simultaneously generating thiourea scaffolds. To demonstrate the versatility and biocompatibility of this approach, we constructed cyclic peptide libraries using both cy-clization methods and successfully selected binders for several targets, including Cyp D, TNF, MDM2, and Keap1, with disso-ciation constants (KD) ranging from micromolar to nanomolar. Given the broad pharmacological potential of the thiourea moiety, this phage display library opens new chemical space with high scaffold diversity and the integration of a proven pharmacophore for the development of cyclic peptide therapeutics.