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 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) prootein 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.

Programmable Synthesis of Biobased Materials Using Cell‐Free Systems

“…Meta‐ and para‐cyanopyridylalanine were introduced using tRNAPyl/PylRS from the archaeon ISO4‐G1 [ 142 ]…”

“…Glycosylation refers to an enzymatic reaction in which an oligosaccharide is covalently linked to the amino acid side-chains of a protein. This enzymatic reaction is the most abundant and were introduced using Mb d) tRNAPyl/PylRS [69,141] Meta-and para-cyanopyridylalanine were introduced using tRNAPyl/PylRS from the archaeon ISO4-G1 [142] AzidohomoAla AzidonorVal AzidonorLeu ProprgylGly [143] Bicyclic peptide generation by disulfide bond and click chemistry Ability for site-specific labeling O-nitrobenzyl-protected Tyr Mj b) tRNA/TyrRSnitropiperonyl TyrMj b) tRNA/TyrRS [144] Light-activable peptide synthesis Jedlitzke et al, 2020…”

Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids

“…The PylRS•tRNA Pyl pairs from M. barkeri and Methanosarcina mazei have been extensively studied [reviewed in 10,15,16,17,18,19,20,21]. Recently, by using the pairs of PylRS and tRNA Pyl from Methanomethylophilus alvus, the site-specific incorporations of non-canonical amino acids into proteins have been achieved in the methanogenic archaeon ISO4-G1, the methanogenic archaeon ISO4-H5, Methanomassiliicoccus intestinalis, and Methanomassiliicoccus luminyensis [22,23,24,25,26,27,28,29,30].…”

“…The mechanism by which ISO4-G1 PylRS recognizes tRNA Pyl and a variety of non-canonical amino acids in its active site must be elucidated for understanding its substrate specificity and orthogonality, and for achieving excellent genetic code expansion systems. Recently, the crystal structure of ISO4-G1 PylRS with the L124A, Y125L, V167A, Y204W, and A221S mutations for the incorporation of the non-canonical amino acid m-cyanopyridylalanine was determined in the apo form [29].…”

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase

“…Specifically, we reasoned that p -CNF-RS might tolerate electrophilic ncAAs, such as p - and/or m -cyanopyridylalanine ( p -CNpyrA and m -CNpyrA, respectively). These ncAAs have recently been employed for protein functionalization in cells and macrocyclization of synthetic peptides through condensation with N-terminal Cys residues for the formation of bridged pyridine–thiazolines (pyr–thn). , In contrast to the flexizyme-mediated thioether cyclization, this pyr–thn cyclization provides both (1) increased rigidity, which can drastically improve affinity by reducing the entropic cost to access the correct binding conformation, and (2) an aromatic heterocyclic system that has greater potential to engage in intermolecular contacts. We envisioned that p -CNF-RS might allow incorporation of this chemistry and the attendant formation of pyr–thn macrocyclic peptides into mRNA display libraries (Figure A).…”