A series of 2,3-diarylindoles were synthesized from 2-iodoaniline and unsymmetrical diarylacetylenes using the Larock heteroannulation. Diarylacetylenes bearing electron-withdrawing substituents lead to 2,3-diarylindoles with substituted phenyl moieties at the 2-position as major products, while those with electron-donating groups preferably yield indole products with substituted phenyl moieties at the 3-position. The regioisomeric product ratios exhibit a clear correlation with Hammett ?p values. DFT calculations reveal the origin of this effect, displaying smaller activation energy barriers for those pathways leading to the major regioisomer.
Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNA(Asp) and tRNA(Asn) with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of ?-helix and ?-sheet secondary structure at 20°C and predominantly ?-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and (1)H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein.
Twenty-six novel naphthoquinone aliphatic esters were synthesized by esterification of 1,4-naphthoquinone alcohols with various aliphatic acids. The 1,4-naphthoquinone alcohols were prepared from 1-hydroxy-2-naphthoic acid in nine steps with excellent yields. Twenty-four of the novel synthetic naphthoquinone esters showed significant antimalarial activity with IC(50) values in the range of 0.03-16.63 microM. The length of the aliphatic chain and the presence of C-2 substituents on the propyl chain affected the activity. Interestingly, compounds 31 and 37 showed very good antimalarial activity and were not toxic to normal Vero cells, and the PTI values of 31 (>1990.38) and 37 (1825.94) are excellent. Both 31 and 37 showed potent inhibition against P. falciparum 3D7 cyt bc(1) and no inhibition on rat cyt bc(1). They showed IC(50) values in the nanomolar range, providing full inhibition of cyt bc(1) with one molecule inhibitor bound per cyt bc(1) monomer at the Q(o) site.
Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln). In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNA(Asn) from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNA(Asn). Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a non-discriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNA(Asn) for assembly. Hp0100 also enhances the capacity of AdT to convert Asp-tRNA(Asn) into Asn-tRNA(Asn) by ?35-fold. Our results demonstrate that bacteria have adopted multiple divergent methods for transamidosome assembly and function.
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