Normal mode analysis of oligomeric proteins: Reduction of the memory requirement by consideration of rigid geometry and molecular symmetry

A method is presented to reduce the memory requirement of normal mode analysis applied to systems containing two or more large proteins when these systems exhibit symmetry properties. We use a rigid geometry model (i.e., only the dihedral angles of the polypeptide chain are considered as variables). This model allows a reduction by a factor of 8 on average of the number of variables with a concomitant freezing of the high-frequency modes. The symmetry properties of the system are used to reduce further the number of variables that must be considered in the computation. Application of group theory leads to a factorization of the matrices of interest (the coefficient and the Hessian matrices) into independent blocks along the diagonal. The initial, reducible representation is thus transformed into a number of irreducible representations of smaller dimensions. In the case of the C₂ symmetry group, the method leads to a reduction of the size of the matrices that must be manipulated during the computation (coefficient matrix, Hessian matrix, and eigenvectors matrix) by a factor of 256 compared with the usual normal mode analysis in Cartesian coordinate space. The method is particularly well adapted to the study of the dynamics of oligomeric proteins because these proteins often display symmetry properties (e.g., virus coat proteins, immunoglobulins, hemoglobin, etc.). In favorable cases, in conjunction with X-ray diffuse scattering data, the study of systems showing allosteric properties might be considered. © 1994 by John Wiley & Sons, Inc.     

Development and validation of a high-performance liquid chromatographic method for the determination of clomazone residues in surface water

A method is described for the determination of clomazone residues in surface water by high-performance liquid chromatography with UV detection. The method involves solid-phase extraction with C18 extraction tubes. Clomazone was separated on a C18 column with a mobile phase of methanol-water (65:35, v/v) at pH 4.0 and a flow-rate of 1.0 ml/min. After optimization of the extraction and separation conditions, the method was validated. The method developed can be used for determination of clomazone in surface water, at the limit of 0.1 mcirog/l set by the European Union drinking water directive, with a 400-fold preconcentration.     

Supercritical fluid extraction of flumetralin in tobacco

Summary This work evaluates the level of Flumetralin residues in real tobacco samples. Conventional extraction methods were compared with SFE methods in both static and dynamic modes using CO₂ and CO₂ with modifier. Additionally, in the dynamic SFE mode, different collection approaches, such as collection at room temperature without solvent, collection at room temperature in the presence of solvent (hexane) and collection in an ice bath, without solvent, were studied. SFE showed itself to be a promising extraction technique for pesticide residues in tobacco samples.     

Competitive adsorption and desorption of a bi-solute mixture: effect of activated carbon type

This study aims to clarify the effects of carbon activation type and physical form on the extent of adsorption capacity and desorption capacity of a bi-solute mixture of phenol and 2-chlorophenol (2-CP). For this purpose, two different PACs; thermally activated Norit SA4 and chemically activated Norit CA1, and their granular countertypes with similar physical characteristics, thermally activated Norit PKDA and chemically activated Norit CAgran, were used. The thermally activated carbons were better adsorbers for phenol and 2-CP compared with chemically activated carbons, but adsorption was more reversible in the latter case. 2-CP was adsorbed preferentially by each type of activated carbon, but adsorption of phenol was strongly suppressed in the presence of 2-CP. The simplified ideal adsorbed solution (SIAS) model underestimated the 2-CP loadings and overestimated the phenol loadings. However, the improved and modified forms of the SIAS model could better predict the competitive adsorption. The type of carbon activation was decisive in the application of these models. For each activated carbon type, phenol was desorbed more readily in the bi-solute case, but desorption of 2-CP was less compared with single-solute. This was attributed to higher energies of 2-CP adsorption.     

Redox behaviour of cysteine in the presence of ammonium trioxovanadate(V)

The interaction of ammonium trioxovanadate(V) with cysteine in aqueous solution was studied by cyclic voltammetry and absorption spectroscopy techniques. In the absence of cysteine, the cyclic voltammogram (CV) of ammonium trioxovanadate(V) solution in 0.1 M phosphate buffer (pH 7) gave two peaks at -0.130 V (reversible) and -0.400 V (irreversible). These peaks (-0.130 V, -0.400 V) can be attributed to V(V)/V(IV) and V(IV)/V(III) redox processes, respectively. In the presence of cysteine at low scan rate (40 mV/s), the peak at -0.780 V, which is assigned to the irreversible reduction of free cystine, was observed. In addition, the reduction peak of the disulfidic anion S(2)(2-) was seen at -0.650 V. Under aerobic conditions, the peaks of the disulfidic anion S(2)(2-) and free cystine are well separated. From electronic spectra of ammonium trioxovanadate(V) and cysteine mixtures, LMCT transition associated with V(V)-cyteine complex was obtained at 743 nm. The stoichiometry (ML(2)) and stability constant (log beta(1:2)=6.67) of V(V)-cysteine complex were determined by means of mole ratio method.     

Enantioselective Preparation of 2‐Aminomethyl Carboxylic Acid Derivatives: Solving the β2‐Amino Acid Problem with the Chiral Auxiliary 4‐Isopropyl‐5,5‐diphenyloxazolidin‐2‐one (DIOZ). Preliminary Communication

Multigram amounts of suitably protected β²‐amino acids with 17 of the 20 proteinogenic side chains are prepared by diastereoselective reactions of Li, B, or Ti enolates of the corresponding 3‐acyl‐4‐isopropyl‐5,5‐diphenyloxazolidin‐2‐ones (acyl‐DIOZ; 1 ) with appropriate electrophiles (amidomethylation, hydroxyalkylation, (benzyloxycarbonyl)methylation) in yields of 55–90% and with diastereoselectivities of 80 to >97% (Scheme). The primary products 2 – 8 thus obtained are converted to protected β²‐amino acids by standard procedures (Table 1). Many of the DIOZ derivatives are highly crystalline compounds (31 X‐ray crystal structures in Table 2). The chiral auxiliary DIOZ, readily prepared in either enantiomeric form, is recovered with high yield.     

Polyelectrolyte multilayers and degradable polymer layers as multicompartment films

Polyelectrolyte multilayers are now a well established concept with numerous potential applications in particular as biomaterial coatings. To timely control the biological activity of cells in contact with a substrate, multicompartment films made of different polyelectrolyte multilayers deposited sequentially on the solid substrate constitute a promising new approach. In a first paper (Langmuir 2004, 20, 7298) we showed that such multicompartment films can be designed by alternating exponentially growing polyelectrolyte multilayers acting as reservoirs and linearly growing ones acting as barriers. In the present study, we first demonstrate however that these barriers composed of synthetic polyelectrolytes are not degraded despite the presence of phagocytic cells. We propose an alternative approach where exponentially growing poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers, used as reservoirs, are alternated with biodegradable polymer layers consisting in poly(lactic-co-glycolic acid) (PLGA) and acting as barriers for PLL chains that diffuse within the PLL/HA reservoirs. We first show that these PLGA layers can be deposited alternatively with PLL/HA multilayers leading to polyelectrolyte multilayer/hydrolyzable polymeric layer films and acting as a reservoirs/barriers system. Bone marrow cells seeded on these films ending by a PLL/HA reservoir rapidly degrade it and internalize the PLL chains confined in this reservoir. Then the cells degraded locally the PLGA barrier and internalize the PLL localized in a lower (PLL/HA) compartment after 5 days of seeding. By changing the thickness of the PLGA layer, we hope to be able to tune the time delay of degradation. Such mixed architectures made of polyelectrolyte multilayers and hydrolyzable polymeric layers could act as coatings allowing us to induce a time scheduled cascade of biological activities. We are currently working on the use of comparable films with compartments filled by proteins or peptides and in which the degradation of the barriers results from a hydrolysis over tunable time scales.     

Synthesis of models of metabolites: Oxidation of variously substituted chromenes including acronycine,by a porphyrin catalytic system

The influence of chemical neighbouring on oxidation of substituted 2,2‐dimethylchromenes derivatives 5‐8 by a biomimetic catalytic system was first studied. It was then applied to acronycine an anti‐cancer drug in order to obtain in one single step oxidized products resulting from the reactivity of the 1,2‐double bond in the pyranic D‐ring. These 2,2‐dimethylchromenes constitute the structural moiety responsible for the activity of acronycine. This oxidation showed the sensitivity of the ethylenic bond, leading to the formation of the corresponding epoxides, diols and/or ketoalcohol. In the case of 5‐dimethylamino‐2,2‐dimethylchromene 8 , the double bond was not sensitive to oxidation, but the N‐methyl groups reacted to lead to the formamide derivative 16 and an imino‐alcohol 17 . This methodology applied to acronycine molecule 1 , allowed to obtain in one step, two oxidized compounds, a trans‐diol 3 and a ketoalcohol 4 under preparative conditions.     

Dendrophanes: Water-soluble dendritic receptors as models for buried recognition sites in globular proteins

Water-soluble dendritic cyclophanes (dendrophanes) of first ( 1 , 4 ), second ( 2 5 ), and third generation ( 3 6 ) with poly(ether amide) branching and 12, 36, and 108 terminal carboxylate groups, respectively, were prepared by divergent synthesis, and their molecular recognition properties in aqueous solutions were investigated. Dendrophanes 1 – 3 incorporate as the initiator core a tetraoxa[6.1.6.1]paracyclophane 7 with a suitably sized cavity for inclusion complexation of benzene or naphthalene derivatives. The initiator core in 4 – 6 is the [6.1.6.1]cyclo-phane 8 shaped by two naphthyl(phenyl) methane units with a cavity suitable for steroid incorporation. The syntheses of 1 – 6 involved sequential peptide coupling to monomer 9 , followed by ester hydrolysis (Schemes 1 and 4), Purification by gel-permeation chromatography (GPC; Fig. 3) and full spectral characterization were accomplished at the stage of the intermediate poly(methyl carboxylates) 10 – 12 and 23 – 25 , respectively. The third-generation 108-ester 25 was also independently prepared by a semi-convergent synthetic strategy, starting from 4 (Scheme 5). All dendrophanes with terminal ester groups were obtained in pure form according to the ¹³C-NMR spectral criterion (Figs, 1 and 5). The MALDI-TOF mass spectra of the third-generation derivative 25 (mol. wt. 19328 D) displayed the molecular ion as base peak, accompanied by a series of ions [M – n(1041 ± 7)]⁺, tentatively assigned as characteristic fragment ions of the poly(ether amide) cascade. A similar fragmentation pattern was also observed in the spectra of other higher-generation poly(ether amide) dendrimers. Attempts to prepare monodisperse fourth-generation dendrophanes by divergent synthesis failed. ¹H-NMR and fluorescence binding titrations in basic aqueous buffer solutions showed that dendrophanes 1 – 3 complexed benzene and naphthalene derivatives, whereas 4 – 6 bound the steroid testosterone. Complexation occurred exclusively at the cavity-binding site of the central cyclophane core rather than in fluctuating voids in the dendritic branches, and the association strength was similar to that of the complexes formed by the initiator cores 7 and 8 , respectively (Tables 1 and 3). Fluorescence titrations with 6-(p-toluidino)naphthalene-2-sulfonate as fluorescent probe in aqueous buffer showed that the micropolarity at the cyclophane core in dendrophanes 1 - 3 becomes increasingly reduced with increasing size and density of the dendritic superstructure; the polarity at the core of the third-generation compound 3 is similar to that of EtOH (Table 2). Host-guest exchange kinetics were remarkably fast and, except for receptor 3 , the stabilities of all dendrophane complexes could be evaluated by ¹H-NMR titrations. The rapid complexation-decomplexation kinetics are explained by the specific attachment of the dendritic wedges to large, nanometer-sized cyclophane initiator cores, which generates apertures in the surrounding dendritic superstructure.     

Layer by layer buildup of polysaccharide films: physical chemistry and cellular adhesion aspects

The formation ofpolysaccharide films based on the alternate deposition of chitosan (CHI) and hyaluronan (HA) was investigated by several techniques. The multilayer buildup takes place in two stages: during the first stage, the surface is covered by isolated islets that grow and coalesce as the construction goes on. After several deposition steps, a continuous film is formed and the second stage of the buildup process takes place. The whole process is characterized by an exponential increase of the mass and thickness of the film with the number of deposition steps. This exponential growth mechanism is related to the ability of the polycation to diffuse "in" and "out" of the whole film at each deposition step. Using confocal laser microscopy and fluorescently labeled CHI, we show that such a diffusion behavior, already observed with poly(L-lysine) as a polycation, is also found with CHI, a polycation presenting a large persistence length. We also analyze the effect of the molecular weight (MW) of the diffusing polyelectrolyte (CHI) on the buildup process and observe a faster growth for low MW chitosan. The influence of the salt concentration during buildup is also investigated. Whereas the CHI/HA films grow rapidly at high salt concentration (0.15 M NaCl) with the formation of a uniform film after only a few deposition steps, it is very difficult to build the film at 10(-4) M NaCl. In this latter case, the deposited mass increases linearly with the number of deposition steps and the first deposition stage, where the surface is covered by islets, lasts at least up to 50 bilayer deposition steps. However, even at these low salt concentrations and in the islet configuration, CHI chains seem to diffuse in and out of the CHI/HA complexes. The linear mass increase of the film with the number of deposition steps despite the CHI diffusion is explained by a partial redissolution of the CHI/HA complexes forming the film during different steps of the buildup process. Finally, the uniform films built at high salt concentrations were also found to be chondrocyte resistant and, more interestingly, bacterial resistant. Therefore, the (CHI/HA) films may be used as an antimicrobial coating.