Tetrazine-Enabled Peptide Macrocyclization and Bioorthogonal Cell Penetration Profiling

Abstract Cyclization of linear peptides into macrocyclic structures confers superior properties such as improved membrane permeability and proteolytic stability while maintaining high target affinity and specificity toward protein targets. Here, we present a cysteine-selective macrocyclization strategy that employs an alkylating motif of chloroethyl tetrazine to react with the thiol side chain in unprotected peptides. This approach efficiently yields cyclic peptides bearing bioorthogonal tetrazine handles, enabling precise late-stage modification via robust tetrazine ligations. We applied this method to synthesize and screen rofapitide tetraxetan analogues, identifying a novel peptide 2o, which exhibits potent binding to cancer-associated fibroblast activation protein (FAP) both in vitro and in vivo. Furthermore, we developed an innovative bioorthogonal cell penetration assay (BCPA) based on a “click-to-release” reaction between the tetrazine-containing peptide (Tz-Pep) and a trans-cyclooctene-caged Nile Blue dye (TCO-Nile Blue) to liberate fluorescent Nile Blue, allowing quantitative evaluation of peptide internalization in living cells. We anticipate that this tetrazine-mediated cyclization strategy and cell penetration assay will find wide applications in biological research and drug discovery.