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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Project description:While 20 canonical amino acids are used by most organisms for protein synthesis, the creation of cells that can use noncanonical amino acids (ncAAs) as additional protein building blocks holds great promise for preparing novel medicines and for studying complex questions in biological systems. However, only a small number of biosynthetic pathways for ncAAs have been reported to date, greatly restricting our ability to generate cells with ncAA building blocks. In this study, we report the creation of a completely autonomous bacterium that utilizes 3,4-dihydroxy-L-phenylalanine (DOPA) as its 21st amino acid building block. Like canonical amino acids, DOPA can be biosynthesized without exogenous addition and can be genetically incorporated into proteins in a site-specific manner. Equally important, the protein production yields of DOPA-containing proteins from these autonomous cells are greater than those from cells exogenously fed with 9 mM DOPA. The unique catechol moiety of DOPA can be used as a versatile handle for site-specific protein functionalizations via either oxidative coupling or strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition reactions. We further demonstrate the use of these autonomous cells in preparing fluorophore-labeled anti-human epidermal growth factor 2 (HER2) antibodies for the detection of HER2 expression on cancer cells.
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Project description:Cyanine dyes represent a family of organic fluorophores with widespread utility in biological-based applications ranging from real-time PCR probes to protein labeling. One burgeoning use currently being explored with indodicarbocyanine (Cy5) in particular is that of accessing exciton delocalization in designer DNA dye aggregate structures for potential development of light-harvesting devices and room-temperature quantum computers. Tuning the hydrophilicity/hydrophobicity of Cy5 dyes in such DNA structures should influence the strength of their excitonic coupling; however, the requisite commercial Cy5 derivatives available for direct incorporation into DNA are nonexistent. Here, we prepare a series of Cy5 derivatives that possess different 5,5'-substituents and detail their incorporation into a set of DNA sequences. In addition to varying dye hydrophobicity/hydrophilicity, the 5,5'-substituents, including hexyloxy, triethyleneglycol monomethyl ether, tert-butyl, and chloro groups were chosen so as to vary the inherent electron-donating/withdrawing character while also tuning their resulting absorption and emission properties. Following the synthesis of parent dyes, one of their pendant alkyl chains was functionalized with a monomethoxytrityl protective group with the remaining hydroxyl-terminated N-propyl linker permitting rapid, same-day phosphoramidite conversion and direct internal DNA incorporation into nascent oligonucleotides with moderate to good yields using a 1 μmole scale automated DNA synthesis. Labeled sequences were cleaved from the controlled pore glass matrix, purified by HPLC, and their photophysical properties were characterized. The DNA-labeled Cy5 derivatives displayed spectroscopic properties that paralleled the parent dyes, with either no change or an increase in fluorescence quantum yield depending upon sequence.
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Project description:The activity and function of proteins can be improved by incorporation of non-canonical amino acids (ncAAs). To avoid the tedious synthesis of a large number of chiral phenylalanine derivatives, we synthesized the corresponding phenylpyruvic acid precursors. Escherichia coli strain DH10B and strain C321.ΔA.expΔPBAD were selected as hosts for phenylpyruvic acid bioconversion and genetic code expansion using the MmPylRS/pyltRNACUA system. The concentrations of keto acids, PLP and amino donors were optimized in the process. Eight keto acids that can be biotransformed and their coupled genetic code expansions were identified. Finally, the genetic encoded ncAAs were tested for incorporation into fluorescent proteins with keto acids.
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Project description:Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.
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