Genetically Encoded Lysine-Selective Photocyclization Enables Phage Display Selection of Cyclic Peptide Binders

Genetically encoded macrocyclization strategies have expanded the cyclic peptide chemical space accessible to phage display but remain constrained by reliance on cysteine-based reactivity or hydrolytically unstable lysine-targeted reagents. Here, we introduce a lysine-selective and proximity-induced photocyclization platform enabled by the noncanonical amino acid o-nitrobenzyl alcohol lysine (o-NBAK), incorporated into phage-displayed peptides and activated using the mild and fully biocompatible PANAC photoclick reaction. Photocyclization proceeded efficiently on both purified pIII fusion proteins and intact phage particles with no detectable loss of infectivity, driven by intramolecular proximity between o-NBAK and a neighboring lysine, yielding well-defined stable indazolone macrocycles. Screening PANAC-cyclized libraries against disease-relevant proteins produced cyclic ligands with nanomolar to low-micromolar affinities, and cyclization enhanced proteolytic stability by more than an order of magnitude. These findings establish the first lysine-selective cyclic peptide library constructed on phage through ncAA incorporation and demonstrate a robust, chemoselective, and broadly applicable strategy for discovering bioactive cyclic peptides.