Phage-encoded bismuth bicycles enable instant access to targeted bioactive peptides.

Genetically encoded libraries play a crucial role in discovering structurally rigid, high-affinity macrocyclic peptide ligands for therapeutic applications. Bicyclic peptides with metal centres like bismuth were recently developed as a new type of constrained peptide with notable affinity, stability and membrane permeability. This study represents the genetic encoding of peptide-bismuth and peptide-arsenic bicycles in phage display. We introduce bismuth tripotassium dicitrate (gastrodenol) as a water-soluble bismuth(III) reagent for phage library modification and in situ bicyclic peptide preparation, eliminating the need for organic co-solvents. Additionally, we explore arsenic(III) as an alternative thiophilic element that is used analogously to our previously introduced bicyclic peptides with a bismuth core. The modification of phage libraries and peptides with these elements is instantaneous and entirely biocompatible, offering an advantage over conventional alkylation-based methods. In a pilot display screening campaign aimed at identifying ligands for the biotin-binding protein streptavidin, we demonstrate the enrichment of bicyclic peptides with dissociation constants two orders of magnitude lower than those of their linear counterparts, underscoring the impact of structural constraint on binding affinity.