Halogen Bonding as a Molecular Recognition Strategy for Genetic Code Expansion

Aminoacyl-tRNA synthetases (aaRSs) catalyze the attachment of amino acids (AAs) to their cognate tRNAs during protein synthesis. As aaRSs possess highly selective amino acid-binding sites, a distinct enzyme is generally required for each amino acid in the genetic code. Recently, genetic code expansion (GCE) has emerged as a powerful strategy for incorporating non-canonical amino acids (ncAAs) during ribosomal translation, enabling the production of proteins with novel structures and functions. In this study, we propose that aaRSs can recognize halogenated ncAAs through halogen bonding (XB), and that the stronger XB-forming ability of iodine, compared with chlorine or bromine, enables the selective incorporation of 3,5-diiodo-l-tyrosine into proteins. We demonstrate the successful generation of aaRSs that specifically recognize 3,5-diiodo-l-tyrosine during translation, even in the presence of closely related amino acids such as 3,5-dichloro-, 3,5-dibromo-, and 3,5-dimethyl-l-tyrosine. These results confirm that ncAA recognition by aaRSs occurs through XB. Overall, this study not only shows that XB can serve as a driving force for expanding the genetic code with ncAAs, but also demonstrates that aaRSs can be engineered to discriminate among structurally similar ncAAs that differ by only a single atom.