Evaluation of capillary isoelectric focusing in glycerol-water media with a view to hydrophobic protein applications

Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.     

Study of poly(bisphenol A carbonate) relaxation kinetics at the glass transition temperature

In this work, the variations of the relaxation times are investigated above and below the glass transition temperature of a model amorphous polymer, the polycarbonate. Three different techniques (calorimetric, dielectric and thermostimulated currents) are used to achieve this goal. The relaxation time at the glass transition temperature was determined at the temperature dependence convergence of the relaxation times calculated with dynamic dielectric spectroscopy (DDS) for the liquid state and thermostimulated depolarisation currents (TSDC) for the vitreous state. We find a value of τ(T_g) = 110 s for PC samples. The knowledge of the temperature dependence, τ(T), and the value τ(T_g) enables to determine the glass-forming liquid fragility index, m. We find m = 178 ± 5.     

Monte Carlo versus molecular dynamics simulations in heterogeneous systems: an application to the n-pentane liquid-vapor interface

The Monte Carlo (MC) and molecular dynamics (MD) methodologies are now well established for computing equilibrium properties in homogeneous fluids. This is not yet the case for the direct simulation of two-phase systems, which exhibit nonuniformity of the density distribution across the interface. We have performed direct MC and MD simulations of the liquid-gas interface of n-pentane using a standard force-field model. We obtained density and pressure components profiles along the direction normal to the interface that can be very different, depending on the truncation and long range correction strategies. We discuss the influence on predicted properties of different potential truncation schemes implemented in both MC and MD simulations. We show that the MD and MC profiles can be made in agreement by using a Lennard-Jones potential truncated via a polynomial function that makes the first and second derivatives of the potential continuous at the cutoff distance. In this case however, the predicted thermodynamic properties (phase envelope, surface tension) deviate from experiments, because of the changes made in the potential. A further readjustment of the potential parameters is needed if one wants to use this method. We conclude that a straightforward use of bulk phase force fields in MD simulations may lead to some physical inconsistencies when computing interfacial properties.     

Analysis of multiple pesticide residues in tobacco using pressurized liquid extraction, automated solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry

A new method was developed for the analysis of pesticide residues in tobacco. The objective was to significantly increase the number of samples that can be processed by the laboratory and to enable the extension of the current coverage to additional pesticides. A new analytical approach was therefore defined based on two main axes, the automation of the sample preparation and the selectivity of the analyte detection using tandem mass spectrometry. This latter aspect reduces the stringency of the requirements placed on the clean-up of the extracts and on the chromatographic resolution when less selective detectors are used. The extraction of the analytes from the matrix is performed using the pressurized liquid extraction technique. Tobacco samples are extracted at elevated temperature and pressure (100 C and 100 atm; 1 atm = 101,325 Pa) using acetone as an extraction solvent. The resulting extract is then concentrated using a Vortex evaporator. Three different solid-phase extraction (SPE) procedures, adjusted to the chemical properties of the different active ingredients to be measured, are applied to the concentrated extract, thus leading to three extract fractions. The first fraction contains such main classes of active ingredients as organohalogenated and 2,6-dinitroaniline compounds while the second one collects the organophosphorus and acylalanines residues; these two fractions are analyzed by capillary gas chromatography coupled to tandem mass spectrometry using negative chemical ionization and electron impact ionization in the positive mode, respectively. The third extract fraction gathers the N-methylcarbamates residues which are analyzed by HPLC with post-column derivatization and fluorescence detection. The different sample preparation stages from extraction to SPE clean-up have been automated through the use of recent analytical technologies. In combination with the analysis by tandem mass spectrometry, this provided a potential for a high sample throughput.     

A systematic study of the interactions between chemical partners (metal, ligands, counterions, and support) involved in the design of Al2O3-supported nickel catalysts from diamine-Ni(II) chelates

1.5 Ni wt %/Al2O3 catalysts have been prepared by incipient wetness impregnation using [Ni(diamine)x(H2O)(6-2x)]Y2 precursors (diamine = 1,2-ethanediamine (en) and trans-1,2-cyclohexanediamine (tc); x = 0, 1, and 2; Y = NO3- and Cl-), to avoid the formation, during calcination, of difficult-to-reduce nickel aluminate. N2 was chosen for thermal treatment to help reveal and take advantage of the reactions occurring between Ni2+, ligands, counterions, and support. In the case of [Ni(en)2(H2O)2]Y2 salts used as precursors, in situ UV-vis and DRIFT spectroscopies show that after treatment at 230 degrees C Ni(II) ions are grafted to alumina via two OAl bonds and that the diamine ligands still remain coordinated to grafted nickel ions but in a monodentate way, bridging the cation with the alumina surface. With Y = Cl-, the chloride counterions desorb as hydrogen chloride, and hydrogen released upon decomposition of the en ligands is able to reduce a fraction of nickel ions into metal as evidenced by XPS. In contrast, with Y = NO3-, compounds such as CO or NO are formed during thermal treatment, indicating that nitrate ions burn the en ligands. After thermal treatment at 500 degrees C, a surface phase containing Ni(II) ions forms, characterized by XPS and UV-vis spectroscopy. Temperature-programmed reduction shows that these ions can be quantitatively reduced to the metallic state at 500 degrees C, in contrast with the aluminate obtained when the preparation is carried out from [Ni(H2O)6]2+, which is reduced only partly at 950 degrees C. On the other hand, a total self-reduction of nickel complexes leading to 2-5-nm metal particles is obtained upon thermal treatment via the hydrogen released by a hydrogen-rich ligand such as tc, whatever the Y counterion. An appropriate choice of the ligand and the counterion allows then to obtain selectively Ni(II) ions or a dispersed reduced nickel phase after treatment in N2, as a result of the reactions occurring between the chemical partners present on alumina.     

Spectroscopic and electrochemical characterization of an aqua ligand exchange and oxidatively induced deprotonation in diiron complexes

Reaction of the unsymmetrical phenol ligand 2-((bis(2-pyridylmethyl)amino)methyl)-6-(((2-pyridylmethyl)benzylamino)methyl)-4-methylphenol (HL-Bn) or its 2,6-dichlorobenzyl analogue (HL-BnCl(2)) with Fe(H(2)O)(6)(ClO(4))(2) in the presence of disodium m-phenylenedipropionate (Na(2)(mpdp)) followed by exposure to atmosphere affords the diiron(II,III) complexes [Fe(2)(L-Bn)(mpdp)(H(2)O)](ClO(4))(2) and [Fe(2)(L-BnCl(2))(mpdp)(CH(3)OH)](ClO(4))(2), respectively. The latter complex has been characterized by X-ray crystallography. It crystallizes in the monoclinic system, space group P2(1)/n, with a = 13.3095(14) A, b = 20.1073(19) A, c = 19.4997(19) A, alpha = 90 degrees, beta = 94.471(2) degrees, gamma = 90 degrees, V = 5202.6(9) A(3), and Z = 4. The structure of the compound is very similar to that of [Fe(2)(L-Bn)(mpdp)(H(2)O)](BPh(4))(2) determined earlier, except for the replacement of a water by a methanol on the ferrous site. Magnetic measurements of [Fe(2)(L-Bn)(mpdp)(H(2)O)](BPh(4))(2) reveal that the two high-spin Fe ions are moderately antiferromagnetically coupled (J = -3.2(2) cm(-)(1)). Upon dissolution in acetonitrile the terminal ligand on the ferrous site is replaced by a solvent molecule. The acetonitrile-water exchange has been investigated by various spectroscopic techniques (UV-visible, NMR, M?ssbauer) and electrochemistry. The substitution of acetonitrile by water is clearly evidenced by M?ssbauer spectroscopy by a reduction of the quadrupole splitting value from 3.14 to 2.41 mm/s. In addition, it causes a 210 mV downshift of the oxidation potential of the ferrous site and a similar reduction of the stability domain of the mixed-valence state. Exhaustive electrolysis of a solution of [Fe(2)(L-Bn)(mpdp)(H(2)O)](2+) shows that the aqua diferric species is not stable and undergoes a chemical reaction which can be partly reversed by reduction to the mixed-valent state. This and other electrochemical observations suggest that upon oxidation of the diiron center to the diferric state the aqua ligand is deprotonated to a hydroxo. This hypothesis is supported by M?ssbauer spectroscopy. Indeed, this species possesses a large quadrupole splitting value (DeltaE(Q) >or= 1.0 mm.s(-)(1)) similar to that of analogous complexes with a terminal phenolate ligand. This study illustrates the drastic effects of aqua ligand exchange and deprotonation on the electronic structure and redox potentials of diiron centers.     

Face Selectivity of the Diels-Alder Additions of Sulfur-Substituted Dienes and Tetraenes Grafted onto 7-Oxabicyclo[2.2.1]heptanes

Stereoselective synthesis of 2-methylidene-3-[(Z)-(2-nitrophenylsulfenyl)methylidene]-7-oxabicyclo[2.2.1]-heptane ( 16 ), 1,4-epoxy-1,2,3,4-tetrahydro-5,8-dimethoxy-2-methylidene-3-[(Z)-(2-nitrophenylsulfenyl)methylidene]anthracene ( 18 ), and 1,4-epoxy-1,2,3,4-tetrahydro-5,8-dimethyoxy-2-methylidene-3-[(Z)-(phenylsulfenyl)-methylidene]anthracene ( 19 ) are presented. The Diels-Alder additions of these S-substituted dienes and those of 2,5-dimethylidene-3,6-bis{[(Z)-(2-nitrophenyl)sulfenyl]methylidene}-7-oxabicyclo[2.2.1]heptane ( 17 ) have been found to be face selective and ‘ortho’ regiospecific. The face selectivity depends on the nature of the dienophile. It is exo-face selective with bulky dienophiles such as ethylene-tetracarbonitrile (TCNE) and 2-nitro-1-butene and endo-face selective with methyl vinyl ketone, methyl acrylate, and 3-butyn-2-one. In the presence of a Lewis acid, the face selectivity of the Diels-Alder reaction can be reversed. The addition of the first equivalent of a dienophile to tetraene 17 is at least 100 times faster than the addition of the second equivalent of the same dienophile to the corresponding mono-adduct. The X-ray structure of the crystalline bis-adduct 43 , a 7-oxabicyclo[2.2.1]hepta-2,5-diene system annellated to two cyclohexene rings, resulting from the successive additions of methyl acrylate and methyl vinyl ketone to tetraene 17 is presented. Only one of the two endocyclic double bonds of the 7-oxabicyclo[2.2.1]hepta-2,5-diene deviates from planarity, the substituents bending towards the endo face by 5.7°.     

Molecular simulation of the homogeneous crystal nucleation of carbon dioxide

We report on a molecular simulation study of the homogeneous nucleation of CO2 in the supercooled liquid at low pressure (P = 5 MPa) and for degrees of supercooling ranging from 32% to 60%. In all cases, regardless of the degree of supercooling, the structure of the crystal nuclei is that of the Pa3 phase, the thermodynamically stable phase. For the more moderate degree of supercooling of 32%, the nucleation is an activated process and requires a method to sample states of high free energy. In this work, we apply a series of bias potentials, which promote the ordering of the centers of mass of the molecules and allow us to gradually grow crystal nuclei. The reliability of the results so obtained is assessed by studying the evolution of the nuclei in the absence of any bias potential, and by determining their probability of growth. We estimate that the size of the critical nucleus, for which the probability of growth is 0.5, is approximately 240 molecules. Throughout the nucleation process, the crystal nuclei clearly exhibit a Pa3 structure, in apparent contradiction with Ostwald's rule of stages. The other polymorphs have a much larger free energy. This makes their formation highly unlikely and accounts for the fact that the nucleation of CO2 proceeds directly in the stable Pa3 structure.     

Vinyl nosylates as partner in copper and silver co-catalyzed Sonogashira cross-coupling reactions

Vinyl nosylates, readily obtained from β-dicarbonyl derivatives, could be efficiently engaged in Sonogashira cross-coupling reactions, either cocatalyzed by copper or silver salts. The para-nitrobenzenesulfonate (nosylate) group allows this coupling to be performed under very mild conditions (room temperature). These new leaving group and mild conditions could be applied to the synthesis of acetylenic coumarinyl derivatives and to the total synthesis of an acetylenic monoterpene natural product, named cleviolide.