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

Abstract 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 wide range different functionalities. The outstanding utility cyanopyridine moiety is demonstrated by examples i) post‐translational functionalization proteins, ii) in‐cell macrocyclization peptides iii) protein stapling. nature ligation chemistry enabled cyanopyridylalanine acid opens new path specific vivo modifications complex biological environments.