Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.

A combination of transient kinetic and equilibrium titration methods has been used to show that both primer/template and nucleotide binding to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are two-step processes. In both cases, after initial formation of relatively weakly bound states, isomerization reactions lead to tightly bound states. In the case of deoxynucleotide binding to the reverse transcriptase-primer/template complex, the second step in the interaction is rate-limiting in the overall reaction during processive polymerization. Discrimination against incorrect nucleotides occurs both in the initial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucleoside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the affinity of the primer/template duplex is increased by at least a factor of 10. The major effect of nonnucleoside inhibitors is on the chemical step (nucleotide transfer).

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis.

Nuclei of digitonin-permeabilized cells that had been preloaded with a model transport substrate in a cytosol-dependent import reaction were subsequently incubated to investigate which conditions would result in export of transport substrate. We found that up to 80% of the imported substrate was exported when recombinant human Ran and GTP were present in the export reaction. Ran-mediated export was inhibited by nonhydrolyzable GTP analogs and also by wheat germ agglutinin but was unaffected by a nonhydrolyzable ATP analog. Moreover, a recombinant human Ran mutant that was deficient in its GTPase activity inhibited export. These data indicate that export of proteins from the nucleus requires Ran and GTP hydrolysis but not ATP hydrolysis. We also found that digitonin-permeabilized cells were depleted of their endogenous nuclear Ran, thus allowing detection of Ran as a limiting factor for export. In contrast, most endogenous karyopherin alpha was retained in nuclei of digitonin-permeabilized cells. Unexpectedly, exogenously added, fluorescently labeled Ran, although it accessed the nuclear interior, was found to dock at the nuclear rim in a punctate pattern, suggesting the existence of Ran-binding sites at the nuclear pore complex.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

The synthesis of a GSK 2nd generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I) and gold(I) catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I) catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

Electrocatalytic activity of Ni-doped nanoporous carbons in the electrooxidation of propargyl alcohol

Herein, we explore the immobilization of nickel on various carbon supports and their application as electrocatalysts for the oxidation of propargyl alcohol in alkaline medium. In comparison with massive and nanoparticulated nickel electrode systems, Ni-doped nanoporous carbons provided similar propargyl alcohol conversions for very low metallic contents. Nanoparticulated Ni on various carbon supports gave rise to the highest electrocatalytic activity in terms of product selectivity, with a clear dependence on Ni content. The results point to the importance of controlling the dispersion of the Ni phase within the carbon matrix for a full exploitation of the electroactive area of the metal. Additionally, a change in the mechanism of the propargyl alcohol electrooxidation was noted, which seems to be related to the physicochemical properties of the carbon support as well. Thus, the stereoselectivity of the electrooxidative reaction can be controlled by the active nickel content immobilized on the anode, with a preferential oxidation to (Z)-3-(2-propynoxy)-2-propenoic acid with high Ni-loading, and to propiolic acid with low loading of active Ni sites. Moreover, the formation of (E)-3-(2-propynoxy)-2-propenoic acid was discriminatory irrespective of the experimental conditions and Ni loadings on the carbon matrixes.

Palladium(II) pincer complexes of α-amino acids: towards the synthesis of catalytically active artificial peptides

NCN palladium(II) complexes have been covalently attached to the N- and C-terminus of the dipeptide L-Phe-L-Va-OMe. Remarkably, the hydrolysis of the NCN-Pd(II) L-Val-OMe afforded the corresponding, palladated free amino acid without affecting the metal site. This deprotected amino acid could be coupled to any protein, enzyme or peptidic chain by simple peptide chemistry. This bioorganometallic systems were active as catalysts in the aldol reaction between methyl isocianate and benzaldehyde.

Isotopic study of ceria-catalyzed soot oxidation in the presence of NOx

The ceria-catalyzed soot oxidation mechanism has been studied by a pulse technique with labeled O2 in the absence and presence of NO, using ceria–soot mixtures prepared in the loose contact mode. In the absence of soot, the ceria-catalyzed oxidation of NO to NO2 takes place with ceria oxygen and not with gas-phase O2. However, the oxygen exchange process between gas-phase O2 and ceria oxygen (to yield back O2, but with oxygen atoms coming from ceria) prevailed with regard to the ceria-catalyzed oxidation of NO to NO2. Gas-phase O2 did not react directly with soot when pulsed to a soot–ceria loose contact mixture. Instead, ceria oxygen is transferred to soot (this step does not require gas-phase molecular oxygen to be present), and gas-phase O2 fills up the vacancies created on the oxide in a further step. The transfer of oxygen between ceria and soot occurred directly in the absence of NO. However, in the presence of NO, NO2 is expected to be additionally generated by ceria oxygen oxidation, which also reacts with soot. The main reaction products of the ceria-catalyzed soot oxidation reaction with NO/O2 were CO2 and NO. Additionally, evidence of the reduction of NOx to N2 was found.

Low metal content Co and Ni alumina supported catalysts for the CO2 reforming of methane

Low metal content Co and Ni alumina supported catalysts (4.0, 2.5 and 1.0 wt% nominal metal content) have been prepared, characterized (by ICP-OES, TEM, TPR-H2 and TPO) and tested for the CO2 reforming of methane. The objective is to optimize the metal loading in order to have a more efficient system. The selected reaction temperature is 973 K, although some tests at higher reaction temperature have been also performed. The results show that the amount of deposited carbon is noticeably lower than that obtained with the Co and Ni reference catalysts (9 wt%), but the CH4 and CO2 conversions are also lower. Among the catalysts tested, the Co(1) catalyst (the value in brackets corresponds to the nominal wt% loading) is deactivated during the first minutes of reaction because CoAl2O4 is formed, while Ni(1) and Co(2.5) catalysts show a high specific activity for methane conversion, a high stability and a very low carbon deposition.

Beyond the H2/CO2 upper bound: one-step crystallization and separation of nano-sized ZIF-11 by centrifugation and its application in mixed matrix membranes

The synthesis of nano-sized ZIF-11 with an average size of 36 ± 6 nm is reported. This material has been named nano-zeolitic imidazolate framework-11 (nZIF-11). It has the same chemical composition and thermal stability and analogous H2 and CO2 adsorption properties to the conventional microcrystalline ZIF-11 (i.e. 1.9 ± 0.9 μm). nZIF-11 has been obtained following the centrifugation route, typically used for solid separation, as a fast new technique (pioneering for MOFs) for obtaining nanomaterials where the temperature, time and rotation speed can easily be controlled. Compared to the traditional synthesis consisting of stirring + separation, the reaction time was lowered from several hours to a few minutes when using this centrifugation synthesis technique. Employing the same reaction time (2, 5 or 10 min), micro-sized ZIF-11 was obtained using the traditional synthesis while nano-scale ZIF-11 was achieved only by using centrifugation synthesis. The small particle size obtained for nZIF-11 allowed the use of the wet MOF sample as a colloidal suspension stable in chloroform. This helped to prepare mixed matrix membranes (MMMs) by direct addition of the membrane polymer (polyimide Matrimid®) to the colloidal suspension, avoiding particle agglomeration resulting from drying. The MMMs were tested for H2/CO2 separation, improving the pure polymer membrane performance, with permeation values of 95.9 Barrer of H2 and a H2/CO2 separation selectivity of 4.4 at 35 °C. When measured at 200 °C, these values increased to 535 Barrer and 9.1.

Superior performance of gold supported on doped CeO2 catalysts for the preferential CO oxidation (PROX)

Herein, the preferential oxidation of CO in excess hydrogen (PROX reaction) was studied over Au catalysts supported on ceria and Y or Nb doped ceria. Both supports and catalysts have been extensively characterized by a number of advanced techniques; XRD, N2-adsortion, Raman spectroscopy, XPS, and H2-TPR. The catalytic results showed that when an ideal mixture of H2 and CO is used for the PROX reaction the gold supported on pure ceria behaves better than the others samples. However, when a typical reformate gas composition containing CO2 and H2O is used, the gold supported on Nb doped sample behaves better than gold supported in pure ceria. It is suggested that niobium hampers the strong adsorption of CO2 and H2O in the active sites, thus improving the catalytic performance in real reformate gas.

Insights into the Active Species of Nanoparticle-Functionalized Hierarchical Zeolites in Alkylation Reactions

Supported iron oxide nanoparticles have been incorporated onto hierarchical zeolites by microwave-assisted impregnation and mechanochemical grinding. Nanoparticle-functionalised porous zeolites were characterised by a number of analytical techniques such as XRD, N2 physisorption, TEM, and surface acidity measurements. The catalytic activities of the synthesised nanomaterials were investigated in an alkylation reaction. The results pointed to different species with varying acidity and accessibility in the materials, which provided essentially different catalytic activities in the alkylation of toluene with benzyl chloride under microwave irradiation, selected as the test reaction.

Primary Amine–2-Aminopyrimidine Chiral Organocatalysts for the Enantioselective Conjugate Addition of Branched Aldehydes to Maleimides

Chiral primary amines containing the (R,R)- and (S,S)-trans-cyclohexane-1,2-diamine scaffold and a pyrimidin-2-yl unit are synthesized and used as general organocatalysts for the Michael reaction of α-branched aldehydes to maleimides. The reaction takes place with 10 mol% organocatalyst loading and hexanedioic acid as cocatalyst in aqueous N,N-dimethylformamide at 10 °C affording the corresponding succinimides in good yields and enantioselectivities. DFT calculations support the stereochemical results and the role played by the solvents.

Enzyme mediated synthesis of polypyrrole in the presence of chondroitin sulfate and redox mediators of natural origin

Polypyrrole (PPy) was synthesized by enzyme mediated oxidation of pyrrole using naturally occurring compounds as redox mediators. The catalytic mechanism is an enzymatic cascade reaction in which hydrogen peroxide is the oxidizer and soybean peroxidase, in the presence of acetosyringone, syringaldehyde or vanillin, acts as a natural catalysts. The effect of the initial reaction composition on the polymerization yield and electrical conductivity of PPy was analyzed. Morphology of the PPy particles was studied by scanning electron microscopy and transmission electron microscopy whereas the chemical structure was studied by X-ray photoelectron and Fourier transformed infrared spectroscopic techniques. The redox mediators increased the polymerization yield without a significant modification of the electronic structure of PPy. The highest conductivity of PPy was reached when chondroitin sulfate was used simultaneously as dopant and template during pyrrole polymerization. Electroactive properties of PPy obtained from natural precursors were successfully used in the amperometric quantification of uric acid concentrations. PPy increases the amperometric sensitivity of carbon nanotube screen-printed electrodes toward uric acid detection.