Zirconocenes as models for homogeneous Ziegler-Natta olefin polymerization catalysts

Using density functional theory, we studied the fundamental steps of olefin polymerization for zwitterionic and cationic Group IV ansa-zirconocenes and a neutral ansa- yttrocene. Complexes [H₂E(C₅H₄)₂ZrMe]ⁿ (n = 0: E = BH₂ (1), BF₂ (2), AlH₂(3); n = +: E = CH₂(4), SiH₂(5)) and H₂Si(C₅H₄)₂YMe were used as computational models. The largest differences among these three classes of compounds were the strength of olefin binding and the stability of the β-agostic alkyl intermediate towards β-hydrogen elimination. We investigated the effect of solvent on the reaction energetics for land 5. We found that in benzene the energetics became very similar except that a higher olefin insertion barrier was calculated for 1. The calculated anion affinity of [CH₃BF₃]^- was weaker towards 1 than 5. The calculated olefin binding depended primarily on the charge of the ansa linker, and the olefin insertion barrier was found to decrease steadily in the following order: [H₂C(C₅H₄)₂ZrMe]⁺ > [F₂B(C₅H₄)₂ZrMe] ≈ [H₂B(C₅H₄)₂ZrMe] > [H₂Si(C₅H₄)₂ZrMe]⁺ > [H₂Al(C₅H₄)₂ZrMe].

We prepared ansa-zirconocene dicarbonyl complexes Me₂ECp₂Zr(CO)₂ (E = Si, C), and t-butyl substituted complexes (t-BuCp)₂Zr(CO)₂, Me₂E(t-BuCp)₂Zr(CO)₂ (E = Si, C), (Me₂Si)₂(t-BuCp)₂Zr(CO)₂ as well as analogous zirconocene complexes. Both the reduction potentials and carbonyl stretching frequencies follow the same order: Me₂SiCp₂ZrCl₂> Me₂CCp₂ZrCl₂> Cp₂ZrCl₂> (Me₂Si)₂Cp₂ZrCl₂. This ordering is a result of both the donating abilities of the cyclopentadienyl substituents and the orientation of the cyclopentadiene rings. Additionally, we prepared a series of analogous cationic zirconocene complexes [LZrOCMe₃][MeB(C₆F₅)₃] (L = CP₂, Me₂SiCp₂, Me₂CCP₂, (Me₂Si)₂Cp₂) and studied the kinetics of anion dissociation. We found that the enthalpy of anion dissociation increased from 10.3 kcal•mol^-1 to 17.6 kcal•mol^-1 as exposure of the zirconium center increased.

We also prepared series of zirconocene complexes bearing 2,2-dimethyl-2-sila-4-pentenyl substituents (and methyl-substituted olefin variants). Methide abstraction with B(C₆F₅) results in reversible coordination of the tethered olefin to the cationic zirconium center. The kinetics of olefin dissociation have been examined using NMR methods, and the effects of ligand variation for unlinked, singly [SiMe₂]-linked and doubly [SiMe₂]-linked bis(cyclopentadienyl) arrangements has been compared (ΔG‡ for olefin dissociation varies from 12.8 to 15.6 kcal•mol^-1). Methide abstraction from 1,2-(SiMe₂)₂(η⁵-C₅H₃)₂Zr(CH₃)-(CH₂CMe₂CH₂CH = CH₂) results in rapid β-allyl elimination with loss of isobutene yielding the allyl cation [{1,2-(SiMe₂)₂(η⁵-C₅H₃)₂Zr(η³-CH₂CH=CH₂)]⁺.

Optimal feedback control of two-photon fluorescence based on genetic algorithm

An optimal feedback control of two-photon fluorescence in the ethanol solution of 4-dicyanomethylene-2-methyl-6-p-dimethyl-amiiiostryryl-4H-pyran (DCM) using pulse-shaping technique based on genetic algorithm is demonstrated experimentally. The two-photon fluorescence of the DCM ethanol solution is enhanced in intensity of about 23%. The second harmonic generation frequency-resolved optical gating (SHG-FROG) trace indicates that the effective population transfer arises from the positively chirped pulse. The experimental results appear the potential applications of coherent control to the complicated molecular system.

Ancillary ligand effects : from zirconium(IV)-catalyzed homogeneous propylene polymerization to platinum(II)-mediated C-H bond activation

A series of C_s- and C₁-symmetric doubly-linked ansa-metallocenes of the general formula {1,1'-SiMe₂-2,2'-E-('ƞ⁵-C₅H₂-4-R¹)-(ƞ⁵-C₅H-3',5'-(CHMe₂)₂)}ZrC₂ (E = SiMe₂ (1), SiPh₂ (2), SiMe₂ -SiMe₂ (3); R¹ = H, CHMe₂, C₅H₉, C₆H₁₁, C₆H₅) has been prepared. When activated by methylaluminoxane, these are active propylene polymerization catalysts. 1 and 2 produce syndiotactic polypropylenes, and 3 produces isotactic polypropylenes. Site epimerization is the major pathway for stereoerror formation for 1 and 2. In addition, the polymer chain has slightly stronger steric interaction with the diphenylsilylene linker than with the dimethylsilylene linker. This results in more frequent site epimerization and reduced syndiospecificity for 2 compared to 1.

C₁-Symmetric ansa-zirconocenes [1,1 '-SiMe₂-(C₅H₄)-(3-R-C₅H₃)]ZrCl₂ (4), [1,1 '-SiMe₂-(C₅H₄)-(2,4-R₂-C₅H₂)]ZrCl₂ (5) and [1,1 '-SiMe₂-2,2 '-(SiMe₂-SiMe₂)-(C₅H₃)-( 4-R-C₅H₂)]ZrCl₂ (6) have been prepared to probe the origin of isospecificity in 3. While 4 and 3 produce polymers with similar isospecificity, 5 and 6 give mostly hemi-isotactic-like polymers. It is proposed that the facile site epimerization via an associative pathway allows rapid equilibration of the polymer chain between the isospecific and aspecific insertion sites. This results in more frequent insertion from the isospecific site, which has a lower kinetic barrier for chain propagation. On the other hand, site epimerization for 5 and 6 is slow. This leads to mostly alternating insertion from the isospecific and aspecific sites, and consequently, a hemi-isotactic-like polymers. In comparison, site epimerization is even slower for 3, but enchainment from the aspecific site has an extremely high kinetic barrier for monomer coordination. Therefore, enchainment occurs preferentially from the isospecific site to produce isotactic polymers.

A series of cationic complexes [(ArN=CR-CR=NAr)PtMe(L)]⁺[BF₄]⁺ (Ar = aryl; R = H, CH₃; L = water, trifluoroethanol) has been prepared. They react smoothly with benzene at approximately room temperature in trifluoroethanol solvent to yield methane and the corresponding phenyl Pt(II) cations, via Pt(IV)-methyl-phenyl-hydride intermediates. The reaction products of methyl-substituted benzenes suggest an inherent reactivity preference for aromatic over benzylic C-H bond activation, which can however be overridden by steric effects. For the reaction of benzene with cationic Pt(II) complexes, in which the diimine ligands bear 3,5-disubstituted aryl groups at the nitrogen atoms, the rate-determining step is C-H bond activation. For the more sterically crowded analogs with 2,6-dimethyl-substituted aryl groups, benzene coordination becomes rate-determining. The more electron-rich the ligand, as reflected by the CO stretching frequency in the IR spectrum of the corresponding cationic carbonyl complex, the faster the rate of C-H bond activation. This finding, however, does not reflect the actual C-H bond activation process, but rather reflects only the relative ease of solvent molecules displacing water molecules to initiate the reaction. That is, the change in rates is mostly due to a ground state effect. Several lines of evidence suggest that associative substitution pathways operate to get the hydrocarbon substrate into, and out of, the coordination sphere; i.e., that benzene substitution proceeds by a solvent- (TFE-) assisted associative pathway.

Theoretical and experimental investigations of the flocculation of charged particles in aqueous solutions by polyelectrolytes of opposite charge

An electrostatic mechanism for the flocculation of charged particles by polyelectrolytes of opposite charge is proposed. The difference between this and previous electrostatic coagulation mechanisms is the formation of charged polyion patches on the oppositely charged surfaces. The size of a patch is primarily a function of polymer molecular weight and the total patch area is a function of the amount of polymer adsorbed. The theoretical predictions of the model agree with the experimental dependence of the polymer dose required for flocculation on polymer molecular weight and solution ionic strength.

A theoretical analysis based on the Derjaguin-Landau, Verwey- Overbeek electrical double layer theory and statistical mechanical treatments of adsorbed polymer configurations indicates that flocculation of charged particles in aqueous solutions by polyelectrolytes of opposite charge does not occur by the commonly accepted polymerbridge mechanism.

A series of 1, 2-dimethyl-5 -vinylpyridinium bromide polymers with a molecular weight range of 6x10^3 to 5x10^6 was synthesized and used to flocculate dilute polystyrene latex and silica suspensions in solutions of various ionic strengths. It was found that with high molecular weight polymers and/or high ionic strengths the polymer dose required for flocculation is independent of molecular weight. With low molecular weights and/or low ionic strengths, the flocculation dose decreases with increasing molecular weight.

Mechanism and applications of the photochemistry of Bis(η^5-cyclopentadienyl)titanacyclobutanes

The photochemically induced reductive elimination of cyclopropanes from bis(η⁵-cyclopentadienyl)titanacyclobutanes has been examined. Stereochemical labelling studies indicate that the cyclopropane is initially formed in a 6±1:1, ratio favoring retention of stereochemistry. The starting titanacyclobutane is isomerized during the course of the reaction. The isomerization of the starting material results from metal-carbon bond homolysis to yield a 1,4-biradical, which can either close to give the starting material or generate cyclopropane. The 1,4-biradical can be observed through a cyclopropyl carbinyl rearrangement employing 2-bis(η⁵- cyclopentadienyl)titana-5,5-dimethylbicyclo[2.1.0]pentane, to give the titanium alkylidene, 1-bis(η⁵-cyclopentadienyl)titana-3,3-dimethyl-1,4- pentadiene, which can be observed directly by NMR at low temperature.

The oxidation of titanacyclobutanes by chemical and electrochemical methods also yields cyclopropanes. Reduction of the metal center does not yield cyclopropanes. Depending on the oxidant, stereochemically labelled titanacyclobutanes yield cyclopropanes that are between 7:1 and 100:1 retention:isomerization. The fragmentation reaction resembles the photochemically induced reductive elimination. Both result from formal oxidation of a metal-carbon bond, which then results in very rapid formation of cyclopropane.

The titanocene generated photochemically reacts with a variety of substrates even at low temperature. Titanocene can be generated in a glass at 77 K. The titanocene can be trapped in noncoordinating solvents in high yield with bulky internal acetylenes to give monoacetylene adducts of titanocene. Less bulky acetylenes give the titanacyclopentadienes. The titanocene can be trapped with olefins to give less stable adducts, which appear by NMR analysis to be intermediate in structure between a titanacyclopropane and an η² olefin adduct of titanocene. Reaction of titanocene with butadiene gives a stable product, which appears to be the s-trans butadiene adduct of titanocene. It does not isomerize on heating. Titanocene reacts with epoxides to give titanocene-µ-oxo polymer and olefin. Stereochemically labelled epoxides and episulfides yield isomerized olefin upon deoxygenation by titanocene. The observations are rationalized as a result of a 1,4-biradical formed by stepwise insertion of titanocene into a carbon-oxygen bond.

Development and Application of Embedding Methods for the Simulation of Large Chemical Systems

The high computational cost of correlated wavefunction theory (WFT) calculations has motivated the development of numerous methods to partition the description of large chemical systems into smaller subsystem calculations. For example, WFT-in-DFT embedding methods facilitate the partitioning of a system into two subsystems: a subsystem A that is treated using an accurate WFT method, and a subsystem B that is treated using a more efficient Kohn-Sham density functional theory (KS-DFT) method. Representation of the interactions between subsystems is non-trivial, and often requires the use of approximate kinetic energy functionals or computationally challenging optimized effective potential calculations; however, it has recently been shown that these challenges can be eliminated through the use of a projection operator. This dissertation describes the development and application of embedding methods that enable accurate and efficient calculation of the properties of large chemical systems.

Chapter 1 introduces a method for efficiently performing projection-based WFT-in-DFT embedding calculations on large systems. This is accomplished by using a truncated basis set representation of the subsystem A wavefunction. We show that naive truncation of the basis set associated with subsystem A can lead to large numerical artifacts, and present an approach for systematically controlling these artifacts.

Chapter 2 describes the application of the projection-based embedding method to investigate the oxidative stability of lithium-ion batteries. We study the oxidation potentials of mixtures of ethylene carbonate (EC) and dimethyl carbonate (DMC) by using the projection-based embedding method to calculate the vertical ionization energy (IE) of individual molecules at the CCSD(T) level of theory, while explicitly accounting for the solvent using DFT. Interestingly, we reveal that large contributions to the solvation properties of DMC originate from quadrupolar interactions, resulting in a much larger solvent reorganization energy than that predicted using simple dielectric continuum models. Demonstration that the solvation properties of EC and DMC are governed by fundamentally different intermolecular interactions provides insight into key aspects of lithium-ion batteries, with relevance to electrolyte decomposition processes, solid-electrolyte interphase formation, and the local solvation environment of lithium cations.

I. The effect of N,N,N',N' -tetramethylethylenediamine on the Schlenk Equilibrium. II. The nature of the Di-Grignard reagent

I. The influence of N,N,N’,N’-tetramethylethylenediamine on the Schlenk equilibrium

The equilibrium between ethylmagnesium bromide, diethylmagnesium, and magnesium bromide has been studied by nuclear magnetic resonance spectroscopy. The interconversion of the species is very fast on the nmr time scale, and only an averaged spectrum is observed for the ethyl species. When N,N,N’,N’-tetramethylethylenediamine is added to solutions of these reagents in tetrahydrofuran, the rate of interconversion is reduced. At temperatures near -50°, two ethylmagnesium species have been observed. These are attributed to the different ethyl groups in ethylmagnesium bromide and diethylmagnesium, two of the species involved in the Schlenk equilibrium of Grignard reagents.

II. The nature of di-Grignard reagents

Di-Grignard reagents have been examined by nuclear magnetic resonance spectroscopy in an attempt to prove that dialkylmagnesium reagents are in equilibrium with alkylmagnesium halides. The di-Grignard reagents of compounds such as 1,4-dibromobutane have been investigated. The dialkylmagnesium form of this di-Grignard reagent can exist as an intramolecular cyclic species, tetramethylene-magnesium. This cyclic form would give an nmr spectrum different from that of the classical alkylmagnesium halide di-Grignard reagent. In dimethyl ether-tetrahydrofuran solutions of di-Grignard reagents containing N N,N,N’,N’-Tetramethylethylenediamine, evidence has been found for the existence of an intramolecular dialkylmagnesium species. This species is rapidly equilibrating with other forms, but at low temperatures, the rates of interconversion are reduced. Two species can be seen in the nmr spectrum at -50°. One is the cyclic species; the other is an open form.

Inversion of the carbon at the carbon-magnesium bond in di-Grignard reagents has also been studied. This process is much faster than in corresponding monofunctional Grignard reagents.

I. Efforts toward the synthesis of aliphatic iodonium salts. II. Flourine-19 nuclear magnetic resonance spectroscopy of cyclic and bicyclic systems

The synthesis of iodonium salts of the general formula [C₆H₅IR]⁺X^-, where R is an alkyl group and x- is a stabilizing anion, was attempted. For the choice of R three groups were selected, whose derivatives are known to be sluggish in S_N1 and S_N2 substitutions: cyclopropyl, 7, 7 -dimethyl-1-norbornyl, and 9 -triptycyl. The synthetic routes followed along classical lines which have been exploited in recent years by Beringer and students. Ultimately, the object of the present study was to study the reactions of the above salts with nucleophiles. In none of the three cases, however, was it possible to isolate a stable salt. A thermodynamic argument suggests that this must be due to kinetic instability rather than thermodynamic instability. Only iodocyclopropane and 1-iodoapocamphane formed isolable iododichlorides.

Several methylated 2, 2-difluoronorbornanes were prepared with the intent of correlating fluorine -19 chemical shifts with geometric features in a rigid system. The effect of a methyl group on the shielding of a β -fluorine is dependent upon the dihedral angle; the maximum effect (an upfield shift of the resonance) occurs at 0° and 180°, whereas almost no effect is felt at a dihedral angle of 120°. The effect of a methyl group on a γ -fluorine is to strongly shift the resonance downfield when fluorine and methyl group are in a 1, 3 - diaxial-like relationship. Molecular orbital calculations of fluorine shielding in a variety of molecules were carried out using the formalism developed by Pople; the results are, at best, in modest agreement with experiment.

I. Configurational stability and redistribution equilibria in organomagnesium compounds. II. Redistribution equilibria in organocadmium compounds

I. CONFIGURATIONAL STABILITY AND REDISTRIBUTION EQUILIBRIA IN ORGANOMAGNESIUM COMPOUNDS

The dependence of the rate of inversion of a dialkylmagnesium compound on the solvent has been studied.

Examination of the temperature dependence of the nuclear magnetic resonance spectrum of 1-phenyl-2-propylmagnesium bromide in diethyl ether solution indicates that inversion of configuration at the methylene group of this Grignard reagent occurs with an approximate rate of 2 sec^-1 at room temperature. This is the first example of a rapid inversion rate in a secondary Grignard reagent.

The rates of exchange of alkyl groups between dineopentylmagnesium and di-s-butylmagnesium, bis-(2-methylbutyl)-magnesium and bis-(4, 4-dimethyl-2-pentyl)-magnesium respectively in diethyl ether solution were found to be fast on the nmr time scale. However, the alkyl group exchange rate was found to be slow in a diethyl ether solution of dineopentylmagnesium and bis-(2-methylbutyl)-magnesium containing N, N, N', N'-tetramethylethylenediamine. The unsymmetrical species neopentyl-2-methylbutyl-magnesium was observed at room temperature in the nmr spectrum of the solution containing the diamine.

II. REDISTRIBUTION EQUILIBRIA IN ORGANOCADMIUM COMPOUNDS

The exchange of methyl groups in dimethylcadmium has been studied by nuclear magnetic resonance spectroscopy. Activation parameters for the methyl group exchange have been measured for a neat sample and for a solution in tetrahydrofuran. The exchange is faster in the basic solvent tetrahydrofuran relative to the neat sample and in tetrahydrofuran solution is retarded by the solvating agent N, N, N’, N’-tetramethylethylenediamine and greatly increased by cadmium bromide. The addition of methanol to a solution of dimethylcadmium in tetrahydrofuran appears to have very little effect on the rate of exchange. The exchange was found to proceed with retention of configuration. The rate-limiting step for the exchange of methyl groups in a basic solvent appears to be the dissociation of coordinating solvent from dimethylcadmium.

The equilibrium between methylcadmium bromide, dimethylcadmium and cadmium bromide in tetrahydrofuran solution has also been studied. At room temperature the interconversion of the species is very fast on the nmr time scale but at -100° distinct absorptions for methylcadmium bromide and imethylcadmium are observed.

The species ethylmethylcadmium has been observed in the nmr spectrum.

The rate of exchange of vinyl groups in a solution of divinylcadmium in tetrahydrofuran has been found to be fast on the nmr time scale.

Rydberg states of diatomic and polyatomic molecules using model potentials

A simple model potential is used to calculate Rydberg series for the molecules: nitrogen, oxygen, nitric oxide, carbon monoxide, carbon dioxide, nitrogen dioxide, nitrous oxide, acetylene, formaldehyde, formic acid, diazomethane, ketene, ethylene, allene, acetaldehyde, propyne, acrolein, dimethyl ether, 1, 3-butadiene, 2-butene, and benzene. The model potential for a molecule is taken as the sum of atomic potentials, which are calibrated to atomic data and contain no further parameters. Our results agree with experimentally measured values to within 5-10% in all cases. The results of these calculations are applied to many unresolved problems connected with the above molecules. Some of the more notable of these problems are the reassignment of states in carbon monoxide, the first ionization potential of nitrogen dioxide, the interpretation of the V state in ethylene, and the mystery bands in substituted ethylenes, the identification of the R and R’ series in benzene and the determination of the orbital scheme in benzene from electron impact data.

Modificação química de amostras de poli(metacrilato de metila) comerciais com aminas funcionalizadas assistidas por micro-ondas

Este trabalho avaliou a reação de amidação sob catálise de DBU/NaCN de amostras comerciais de poli(metacrilato de metila), (PMMA), massa molar 60.000 u.m.a. com as aminas, alilamina, 2-morfolilaminoetano, 1-(3-aminopropil)-imidazol e N,N-dimetil-trimetilenodiamina com ativação por reator de micro-ondas. O estudo com alilamina e 2-morfolilaminoetano se mostrou eficaz, apresentando percentuais de derivatização significativos (27% e 37% , respectivamente). A quantidade de material polimérico obtido na purificação foi de cerca de 36 % para as duas aminas. O estudo evidenciou a importância dos catalisadores DBU/NaCN, pois sem eles a reação não ocorre e quando utilizados isoladamente, o grau de incorporação é muito menor. Já o estudo com as aminas 1-(3-aminopropil)-imidazol e N,N-dimetil-trimetilenodiamina mostrou-se ineficaz devido à dificuldade de purificação dos produtos. Tentativas de purificação por precipitação com metanol e soluções hidrometanólicas de diferentes concentrações mostraram-se ineficazes. Os novos PMMA modificados foram caracterizados por FT-IR, RMN-1H e análise elementar. Foram observadas alterações na taticidade do PMMA em reações ativadas por micro-ondas

Mapeamento de proteínas alvo para novos antifúngicos na fração microssomal e citosólica do patógeno humano Aspergillus fumigatus

A incidência de infecções fúngicas invasivas vem aumentando nos últimos anos. Estas infecções, em geral, apresentam altas taxas de mortalidade. A profilaxia com antifúngicos ainda é a estratégia mais comum na contenção da mortalidade e prevenção contra infecções fúngicas invasivas, porém, apresenta baixa eficiência, e relatos de resistência às drogas. Além disso, a terapia antifúngica é limitada a um pequeno grupo de drogas, como os polienos, azóis e equinocandinas. Desta forma, a busca de novos alvos de drogas é fundamental para o desenvolvimento de novos antifúngicos. Estudos in silico indicaram quatro genes como potenciais alvo de drogas em fungos patogênicos. Neste contexto, o objetivo deste trabalho foi verificar a expressão das proteínas codificadas por dois destes possíveis genes alvo, a proteína erg6, na fração microssomal, e trr1, na fração citosólica, em hifas de A. fumigatus. Visando alcançar este objetivo, foram primeiramente padronizadas todas as etapas de fracionamento celular visando isolar estas duas subfrações celulares de A. fumigatus. Posteriormente, foi otimizado o protocolo de extração e reidratação de proteínas microssomais bem como reidratação de proteínas citosólicas. Estes extratos foram submetidos a diferentes protocolos de fracionamento proteico em um sistema de eletroforese OFFGEL (OGE). Os resultados de Western immunoblot mostraram que estas duas proteínas, erg6 e trr1, são de fato expressas na fase filamentosa de A. fumigatus. O extrato proteico da fração microssomal submetido ao OGE em doze subfrações apresentou três subunidades da proteína erg6, reconhecidas pelo anticorpo monoclonal, com massas moleculares e pI distintos: uma subunidade de aproximadamente 79 kDa com pI entre 5,91 e 6,49, e outras duas subunidades de aproximadamente 35 kDa e 32 kDa, ambas com pI entre 6,49 e 7,08. A enzima erg6 foi descrita como um homotetrâmero em outros fungos. Porém, nossos resultados sugerem que, em A. fumigatus, a erg6 possui uma estrutura heterotetramérica. Quanto à proteína trr1, tanto no extrato total quanto nas frações resultantes do fracionamento em OGE, uma banda única de aproximadamente 40 kDa, com pI na faixa de 4,79 e 5,33, foi reconhecida pelo anticorpo policlonal. Desta forma, esta proteína parece ter uma estrutura homodimérica, assim como descrito em outros micro-organismos.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

Investigação da atividade toxicológica de análogos de estatinas em modelos experimentais in vitro

As estatinas são fármacos inibidores competitivos da enzima hidroxi-3-metil-glutaril Coenzima A (HMGCoA) redutase, amplamente utilizados para o controle da hipercolesterolemia total e, em especial, para a redução dos níveis séricos de LDLc (Low Density Lipoprotein cholesterol). Além do efeito primário, esses fármacos apresentam vários efeitos secundários, chamados de efeitos pleiotrópicos, envolvendo atividade anti-inflamatória, antitumoral e antiparasitária. Para o desenvolvimento de inovações na área de química medicinal é imprescindível avaliar o risco de efeitos adversos para saúde ou, em outras palavras, a segurança terapêutica do novo produto nas condições propostas de uso. Nesse sentido, o objetivo desse trabalho foi investigar a genotoxicidade de quatro análogos inibidores da biossíntese de lipídios, da classe das estatinas, em modelos experimentais in vitro, testados previamente contra o clone W2 de Plasmodium falciparum a fim de se obter o IC50 dessas moléculas frente ao patógeno. Foram desenvolvidas quatro novas moléculas (PCSR02.001, PCSR09.001, PCSR08.002 e PCSR10.002). Para a avaliação da toxicidade, foram realizados o teste de mutagenicidade bacteriana (teste de Ames), o ensaio de viabilidade celular utilizando o reagente WST-1 e o ensaio de indução de micronúcleos, ambos utilizando uma linhagem ovariana (CHO-K1) e uma linhagem hepática (HepG2). Levando em conta o fato de nenhuma das amostras ter induzido efeitos mutagênicos nas linhagens de S. enterica sorovar Typhimurium, e PCSR10.002 ter apresentado citotoxicidade sugere-se então que este composto seja o mais tóxico. Comparativamente, PCSR10.002 foi mais genotóxico e citotóxico para a linhagem CHO-K1 do que para a linhagem HepG2. PCSR02.001 apresentou elevado potencial genotóxico para células ovarianas, mas não foi capaz de induzir a formação de micronúcleos em células hepáticas, apresentando, portanto um perfil similar ao observado em PCSR10.002. Assim como a atorvastatina, PCSR09.001 apresentou elevado potencial pró-apoptótico para a linhagem de hepatócitos. Já PCSR08.002, apresentou aumento na apoptose de CHO-K1. A indução de apoptose não é necessariamente um evento negativo, já que é pouco lesiva e responsável pela eliminação de células danificadas. Porém, as respostas de apoptose induzidas por esse composto foram muito inferiores àquelas induzidas pela atorvastatina (cerca de 4 vezes menor que a atorvastatina). PCSR08.002 foi aquele se mostrou menos tóxico e essa amostra foi a que teve menor risco relativo, em uma análise global das respostas de citotoxicidade e não demonstrou ter potencial genotóxico para as linhagens utilizadas nesse estudo. Conclui-se, portanto, que a análise da atividade toxicológica utilizando modelos experimentais in vitro dessas estatinas constitui um importante passo para o estabelecimento de novos candidatos à fármacos com maior segurança.

小麦肉桂酰辅酶A还原酶（CCR）基因的分离和功能分析

作为植物界广泛存在的一类酚类聚合物，木质素是陆生植物正常生长发育过程中非常重要的生物大分子，而且与人类的生活息息相关。利用分子生物学手段和基因工程方法，从小麦中分离木质素生物合成途径的关键酶-肉桂酰辅酶A还原酶基因（CCR），研究肉桂酰辅酶A还原酶基因在木质素代谢途径中的调控规律，从其催化的限速步骤入手，来调控木质素的合成，有效的改变木质素的组成、含量和结构，是改善木质素在植物生长发育中的作用乃至开发木质素资源的关键所在。本文就小麦肉桂酰辅酶A还原酶基因的分离、表达特征及其在木质素合成途径中的作用开展了研究工作。 首先用RACE方法从小麦中克隆了CCR的两个cDNA的部分序列，序列分析表明它们编码的蛋白具有CCR的典型特点，GC含量高于均60%，两者在核酸水平和蛋白水平的同源性为76%和 69%，证明在小麦中至少存在着两个CCR基因。通过 RT-PCR和Northern 杂交确定W-cr6和W-cr19在小麦的发育中具有不同的表达特征，W-cr6主要在茎中表达，而W-cr19的表达集中在根中。以W-cr6为探针，从cDNA文库中筛选到一个全长1317bp的cDNA，命名为TaCCR1。TaCCR1包括开放阅读框 (ORF) 1047bp、5′端侧翼 72bp和3′端侧翼198bp的非翻译序列。TaCCR1能够编码由349个氨基酸组成的蛋白质，预期的分子量为37.4kD。同源性比较显示TaCCR1基因在核酸水平和蛋白质水平与其他物种的CCR基因的同源性高于60%。 为了分析CCR在木质素合成中的作用，用TaCCR1构建了用于转化烟草的正义和反义表达载体pStCCR和pAtCCR、用于转化小麦的正义和反义表达载体pBSC1和pBAC1。通过农杆菌介导得到了30株反义转基因烟草和12株正义转基因烟草。由于外源基因的抑制作用，转基因烟草在形态、木质素组成和含量、木质部显微结构上都程度不同的发生了变化。正义和反义的转基因株系呈现出株型矮化、木质素含量下降、木质部导管细胞壁受到破坏等现象。同时利用花粉管通道法转化小麦种子5000多粒，部分处理经过初步的PCR和 Southern分子鉴定获得了1株转基因株系，需要对其遗传、生理和形态特征做进一步的研究。 本文还对木质素对小麦茎杆的机械强度的影响做了初步的探讨，得到的结果是小麦茎杆的木质素含量、维管束的数量、茎杆有效的横界面积与其最大弯曲应力存在着正相关，而维管束的结构、密度对茎杆的最大弯曲应力没有明显的影响，从而为通过CCR基因来改善小麦茎杆的抗倒特性建立了生理学基础。