The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA EcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli -based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNA CUA EcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNA CUA EcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.

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The first organocatalytic enantioselective synthesis of bridged O,O‐ketals embedded with spirooxindoles has been developed. Dioxindoles and ortho‐hydroxy‐benzylidene acetones were engaged as the reaction partners in this method. The desired products were obtained via epi‐cinchonine primary amine catalyzed Michael reaction followed by ketal formation with TFA.

The E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform to genetically encode new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed for its engineering, has impeded its success in the past. Recently, we showed that EcTyrRS can be engineered using a facile E. coli based selection system, in a strain where the endogenous tyrosyl pair has been substituted with an archaeal counterpart. However, a significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems using this tRNA enabled efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide-dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporate useful ncAAs in mammalian cells, including photo-affinity probes, bioconjugation handles, and a non-hydrolyzable mimic of phosphotyrosine.