Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/Protein

Protocols have been achieved that permit facile introduction of s-tetrazine into unprotected peptides and the protein, thioredoxin, between two cysteine sulfhydryl groups (i.e., staple), followed by photochemical release (i.e., unstaple) and regeneration of the peptide/protein upon removal of the cyano groups from the derived bisthiocyanate. The S,S-tetrazine macrocycles in turn provide a convenient handle for probe introduction by exploiting the inverse electron demand Diels–Alder reactivity of the tetrazine.