Genetic Incorporation of Seven ortho-Substituted Phenylalanine Derivatives

[Image: see text] Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNA(CUA)(Pyl). Ortho substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications. Specifically, we demonstrated that o-cyano-phenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For six of these compounds this constitutes the first report of their genetic incorporation in living cells. With these compounds the total number of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain aromatic position as a substrate. To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.