Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli at an amber codon with high efficiency and selectivity using a previously reported, rationally designed, pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNAPylcua . 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 have demonstrated that ortho-cyanophenylalanine can be used as a selective sensor to probe the local environment of proteins, and we apply 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 aaRS with such a high substrate promiscuity.

Genetic incorporation of seven ?<i>ortho</i>?-substituted phenylalanine derivatives

247th National Meeting (2014)

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Genetic incorporation of seven ?ortho?-substituted phenylalanine derivatives