Genetic Encoding of Cyanopyridylalanine for In‐Cell Protein Macrocyclization by the Nitrile–Aminothiol Click Reaction

Cyanopyridylalanines are non‐canonical amino acids that react with aminothiol compounds under physiological conditions in a biocompatible manner without requiring added catalyst. Here we present newly developed aminoacyl‐tRNA synthetases for genetic encoding of meta‐ and para‐cyanopyridylalanine to enable the site‐specific attachment of a wide range of different functionalities. The outstanding utility of the cyanopyridine moiety is demonstrated by examples of i) post‐translational functionalization of proteins, ii) in‐cell macrocyclization of peptides and proteins, and iii) protein stapling. The biocompatible nature of the protein ligation chemistry enabled by the cyanopyridylalanine amino acid opens a new path to specific in vivo protein modifications in complex biological environments. Genetic encoded cyanopyridylalanines are presented as convenient and highly selective reactive groups for in vivo functionalization, cyclization and stapling of polypeptides. The non‐toxic compounds can be genetically incorporated into proteins with high yields and their biocompatible reaction with amino‐thiol moieties proceeds under physiological conditions to near completion without requiring a catalyst.