Genetic incorporation of seven ortho-substituted phenylalanine derivatives.
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ABSTRACT:
SUBMITTER: Tharp JM
PROVIDER: S-EPMC3997995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:When coexpressed with its cognate amber suppressing tRNACUAPyl(CUA), a pyrrolysyltRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro,ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone,alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.
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Project description:To explore the effect of the nature of substitutions on the structural parameters and hydrogen-bond inter-actions in N-acyl-hydrazone derivatives, the crystal structures of three ortho-substituted N-acyl-hydrazone derivatives, namely (E)-N-{2-[2-(2-chloro-benzyl-idene)hydrazin-yl]-2-oxoeth-yl}-4-methyl-benzene-sulfon-amide, C16H16ClN3O3S (I), (E)-N-{2-[2-(2-methyl-benzyl-idene)hydrazin-yl]-2-oxoeth-yl}-4-methyl-benzene-sulfonamide, C17H19N3O3S (II), and (E)-N-{2-[2-(2-nitro-benzyl-idene)hydrazin-yl]-2-oxoeth-yl}-4-methyl-benzene-sulfonamide, C16H16N4O5S (III), have been determined. The structures of the three compounds display similar mol-ecular conformations and hydrogen-bond patterns. The hydrazone part of the mol-ecule, C-C-N-N=C, is almost planar in all the compounds, with the C-C-N-N and C-N-N=C torsion angles being 179.5?(3) and 177.1?(3)°, respectively, in (I), -179.4?(2) and -177.1?(3)° in (II) and -179.7?(2) and 173.4?(2)° in (III). The two phenyl rings on either side of the chain are approximately parallel to each other. In the crystal, the mol-ecules are linked to each other via N-H?O hydrogen bonds, forming ribbons with R22(8) and R22(10) ring motifs. The introduction of electron-withdrawing groups (by a chloro or nitro group) to produce compounds (I) or (III) results in C-H?O hydrogen-bonding inter-actions involving the sulfonyl O atoms of adjacent ribbons, forming layers parallel to the ab plane in (I) or a three-dimensional network in (III). In (III), one O atom of the nitro group is disordered over two orientations with refined occupancy ratio of 0.836?(12):0.164?(12).
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