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

27 October 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


bioorthogonal reaction
genetic encoding
noncanonical amino acids
nitrile-aminothiol (NAT) click reaction
protein conjugation


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