pH-Controlled Stepwise Cyclization Enables Phage Display of Heterobridged Bicyclic Peptide Libraries

Phage-displayed bicyclic peptides offer access to conformationally constrained ligands with high affinity and specificity; however, current library designs are restricted to chemically identical crosslinkers, limiting structural and functional diversity. Here, we introduce a pH-controlled stepwise cyclization strategy that enables orthogonal installation of two distinct chemical crosslinkers within a single bicyclic peptide scaffold. Under mildly acidic conditions, kinetic suppression of thiol reactivity enables selective N-terminal cysteine modification with an N-terminus-specific linker while preserving internal cysteine thiols. Subsequent bicyclization with a second crosslinker affords heterobridged bicyclic peptides with defined and predictable topologies. This method is readily integrated with the phage display system to construct bicyclic peptide libraries. Screening of these libraries yielded submicromolar ligands targeting PD-L1 and FAP and revealed that binding activity critically depends on the heterobridged configuration. This work establishes a robust and generalizable chemical approach for introducing linker diversity into phage-displayed bicyclic peptide libraries, thereby offering a versatile platform for the discovery of structurally and functionally diverse peptide ligands.