Disulfide bond formation is an essential process for the folding and biological activity of most extracellular proteins; however, it may become the limiting step when the production of these proteins is attempted in heterologous hosts such as Escherichia coli. The rearrangement of incorrect disulfide bonds between cysteines that do not normally interact in the native structure of a protein is carried out by disulfide isomerase enzymes. The disulfide isomerase present in the bacterial secretory compartment (the periplasmic space) is the homodimeric enzyme DsbC. The objective of this dissertation was to understand the key features of how DsbC catalyzes disulfide bond isomerization. Chimeric disulfide isomerases comprising of protein domains that share a similar function, or are homologous to domains of DsbC were constructed in an effort to understand the effect of the domain orientation in the dimeric protein, and the need for a substrate binding region in disulfide isomerases. We successfully created a series of fusion enzymes, FkpA-DsbAs, which catalyze in vivo disulfide isomerization with comparable efficiency to DsbC. These enzymes comprise of the peptide binding region of the periplasmic chaperone FkpA, which is functionally and structurally similar to the binding domain of DsbC but share no amino acid homology with it, fused to the bacterial oxidase DsbA. In addition, these chimeric enzymes were shown to assist in the initial formation of disulfide bonds, a function that is normally exhibited only by DsbA. Directed evolution of the FkpA-DsbA proteins conferred improved resistance to CuCl₂, a phenotype dependent on disulfide bond isomerization and highlighted the importance of an optimal catalytic site. The bacterial disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers and two opposing catalytic domains. The functional significance of the existence of two catalytic domains of DsbC is not well understood yet. The fact that identical subunits naturally dimerize to generate DsbC has so far limited the study of the individual catalytic sites in the homodimer. In chapter 3 we discuss the engineering, in vivo function, and biochemical characterization chapter 3 we discuss the engineering, in vivo function, and biochemical characterization of DsbC variants covalently linked via (Gly3Ser) flexible linkers. We have either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Our results support the hypotheses that dual catalytic domains in DsbC are not necessary for disulfide bond isomerization, but are important in terms of increasing the effective concentration of catalytic equivalents, and that the availability of a substrate binding region is a determining feature in isomerization. Finally, we have carried out initial studies to map the residues and sequence motifs that are recognized in substrate proteins that interact with DsbC. Although the main putative binding region of DsbC has been localized within the limits of the hydrophobic cleft that emerges from the interaction of the N-terminal domains of this enzyme, and, a few native substrates have already been identified, no information on the features of substrate proteins that are recognized by the enzyme has been reported. To address this problem, we have screened two different, 15 amino-acid random peptide libraries for binding to DsbC. We have successfully isolated several peptides with high affinity for the enzyme. Possible consensus binding motifs were identified and their significance in substrate recognition will be examined in future studies. / text

The chemical catalysis of the cellulose conversion reaction via hydrolysis / solubilization and degradation and conversion of glucose via dehydration, the basis of heterogeneous solid acid catalysts has aroused great interest due to the great potential in terms of activity and selectivity or the possibility of development of chemical products and / or processes (Green chemistry) associated with the use of clean technologies. Thus, this work proposes the synthesis and characterization of TiO2 - based catalytic systems followed by wet impregnation of molybdenum oxide and catalytic tests against the conversion of cellulose and glucose in order to obtain fermentable sugars and other products of interest in fine chemistry. The work was divided in two topics where the first study the variation of the concentration of MoO3 in TiO2. Initially, TiO2 catalysts were synthesized by the sol-gel method and MoO3/TiO2 (with different concentrations of molybdenum oxide) by the wet impregnation method and calcined at 550 ° C for 4 hours under atmospheric conditions. Subsequently, different characterizations were made in order to know the structure and the acid nature of these catalysts. These catalysts were used in reaction tests of cellulose conversion under the conditions of 190 ° C, at 1, 2 and 4 hours and with varying amount of catalyst. The catalysts showed catalytic activity against cellulose conversion, with the catalyst having 25% MoO3/TiO2, which presented the highest conversion percentage (21%) in the test at 190 ° C / 4 h. The higher conversion observed when using the 25% MoO3/TiO2 catalyst may be associated with a greater amount of available Lewis and Brönsted acidic sites and their available acid strength as shown by absorption spectroscopy analysis in the infrared region with adsorbed pyridine and programmed ammonia desorption temperature, respectively. Therefore, this catalyst was chosen for reuse studies and did not present activity after the first test under the condition of 190 ° C, at 2 hours. As well as, alteration of the calcination temperature to see its influence on the structural properties, and consequently, on the catalytic activity of the same. This will be investigated in the second topic of this paper. In the second topic, new catalysts (TiO2-300 and 25% MoO3/TiO2-300) were synthesized without MoO3 anchoring and another with 25% MoO3 anchored to TiO2, both calcined at 300 ° C under the same synthetic methods. They were characterized by different techniques and carried out tests of conversion of cellulose and glucose, in order to observe the evolution of the formation of the products. However, the change from the anatase phase to rutile of TiO2, predominantly, and consequent alteration in the catalytic activity was observed. Therefore, the catalyst with an anatase phase based on TiO2 calcined at 300 ° C showed higher activity than the rutile phase with a TiO2 base calcined at 550 ° C. In this context, it is important to note that after the impregnation of molybdenum, changes in the texture properties occurred, as well as the change in the calcination temperature altered the morphological properties of the catalysts, presenting a variation in the crystalline phases of MoO3 (orthorhombic and monoclinic). In this context, it is important to note that the relationship between Lewis acid sites and the Bronsted acid sites was verified, where a higher amount of Lewis acid sites was verified for calcined at 300 ° C. Thus, the catalysts that presented higher relative acidity provided higher conversions of cellulose and glucose with formation of sugars and acetic acid, respectively. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A catálise química da reação de conversão da celulose e derivados, via hidrólise, solubilização e degradação, a base de catalisadores heterogêneos sólidos ácidos tem despertado grande interesse, devido ao potencial em termos de atividade e seletividade, ou em função da possibilidade de desenvolvimento de produtos e/ou processos químicos que estejam associados ao uso de tecnologias limpas. Assim, este trabalho propõe a síntese e caracterização de sistemas catalíticos a base de TiO2 seguido da impregnação úmida do óxido de molibdênio e testes catalíticos frente a conversão da celulose e glicose com o intuito de obter açúcares fermentáveis e outros produtos de interesse da química fina. O trabalho foi dividido em dois tópicos, em que o primeiro estuda a variação da concentração de MoO3 em TiO2. Inicialmente, sintetizou-se os catalisadores TiO2 pelo método sol-gel e o MoO3/TiO2 (com diferentes concentrações de óxido de molibdênio) pelo método de impregnação úmida e calcinados a 550°C por 4 horas em condições atmosféricas. Logo em seguida, foram feitas diferentes caracterizações a fim de conhecer a estrutura e a natureza ácida desses catalisadores. Esses catalisadores foram usados em testes reacionais de conversão da celulose sob as condições de 190 °C, a 1, 2 e 4 horas e com variação na quantidade de catalisador. Os catalisadores apresentaram atividade catalítica frente a conversão da celulose, sendo o catalisador 25 % MoO3/TiO2 o que apresentou maior porcentagem de conversão (21 %) no teste a 190 °C /4 h. A maior conversão observada quando do emprego do catalisador 25 % MoO3/TiO2 pode ser associada a maior quantidade de sítios ácidos de Lewis e Brönsted e sua força ácida, disponíveis, como mostra a análise de espectroscopia de absorção na região do infravermelho com piridina adsorvida e temperatura programada de dessorção de amônia, respectivamente. Logo, esse catalisador foi escolhido para estudos de reuso e não apresentou atividade após o primeiro teste sob a condição de 190 °C, a 2 horas. Assim como, alteração da temperatura de calcinação para ver sua influência sobre as propriedades estruturais, e consequentemente, na atividade catalítica do mesmo. Isso será investigado no segundo tópico desse trabalho. Posteriormente, no segundo tópico foram sintetizados novos catalisadores (TiO2-300 e 25% MoO3/TiO2-300) sem a ancoragem de MoO3 e outro com 25 % de MoO3 ancorado ao TiO2, ambos calcinados a 300 °C, sob os mesmos métodos de síntese. Foram caracterizados por diferentes técnicas e realizados testes de conversão da celulose e glicose, a fim de observar a evolução da formação dos produtos. Contudo, foi observada a mudança da fase anatase para rutilo do TiO2, predominantemente, e consequente alteração na atividade catalítica. Portanto, o catalisador com fase anatase a base de TiO2 calcinado a 300 ºC apresentou atividade superior a com fase rutilo a base de TiO2 calcinado a 550 ºC. Nesse âmbito, é imprescindível destacar que após a impregnação do molibdênio ocorreram alterações nas propriedades texturais, assim como, a mudança na temperatura de calcinação alterou as propriedades morfológicas dos catalisadores, apresentando uma variação nas fases cristalinas do MoO3 (ortorrômbica e monoclínica). Nesse contexto, é importante destacar que foi feita a relação entre sítios ácidos de Lewis e os sítios ácidos de Bronsted, e foi verificado uma maior quantidade de sítios ácidos de Lewis para àqueles calcinados a 300 °C. Sendo assim, os catalisadores que apresentaram maior acidez relativa proporcionaram maiores conversões da celulose e glicose com formação de açúcares e ácido acético, respectivamente.

Biofuels stand out as an alternative to stop the current process of climate change caused by high greenhouse gas emissions. Biodiesel is figure as a renewable source of expansion in Brazil, which an important parameter is the type of applied catalysis, a key point for both kinetic and economic aspects regarding production costs. The aim of this study is the application of magnetic ceramics with catalytic potential for biodiesel production. The magnetic ceramics tested were: NiFe2O4, Ni0,5Zn0,5Fe2O4, Ni0,5Cu0,5Fe2O4 and Ni0,5Co0,5Fe2O4 which were characterized regard the morphology, texture, thermal and magnetic structure. The results pointed that the ceramic showed the characteristic inverse spinel phase, with a crystallite size of 20-93 nm and the chemical composition of the oxides in agreement with the expected stoichiometry. It was observed that doping with Zn2+ and Cu2+ in the NiFe2O4 sample did not change the morphology, and it consists of large agglomerates in the form of irregular blocks, non-porous and fragile aspect except the sample doped with Co2+ which showed dense blocks with formation of irregular pre-sintered particles. All samples showed the characteristic behavior of soft ferromagnetic materials, however, the ferrite containing Zn2+ and containing Cu2+ showed an increase in 5 times in saturation magnetization and a decrease in coercivity about 72 and 61% respectively, when compared to the NiFe2O4. The samples had surface area values of 32.6 to 83.7 m2.g-1, the classification of pores in the mesoporous range, exhibit good thermal stability and indicated by low weight loss during thermogravimetric analysis. The catalytic tests showed catalytic activity around 10% in the transesterification reaction conditions proposed. In the esterification samples showed promising results, the highest conversion was observed for obtaining ceramic Ni0,5Zn0,5Fe2O4 about 15% within 1 hour of reaction. The catalytic activity both in transesterification as for esterification may be related to the low acidity of lewis sites in these materials, in addition to the restrictions on conversion of the products by the reactants diffusion hindrance in the internal structure. However, it was found that ceramics doped with Zn2+ ions had larger diameter portion (12.06 nm), which probably contributed to the satisfaction of fames of obtaining methyl estes of fatty acids (FAMEs). / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os biocombustíveis se destacam como uma alternativa para deter o atual processo de mudança climática causada pela elevada emissão de gases poluentes. O biodiesel figura-se como uma fonte renovável em expansão no Brasil, onde um parâmetro importante é o tipo da catálise aplicada, sendo um ponto chave tanto para aspectos cinéticos quanto aos econômicos no que tange custos de produção. O objetivo desse estudo é a aplicação de cerâmicas magnéticas com potencial catalítico para produção de biodiesel. As cerâmicas magnéticas testadas foram: NiFe2O4, Ni0,5Zn0,5Fe2O4, Ni0,5Cu0,5Fe2O4 e Ni0,5Co0,5Fe2O4 as quais foram caracterizadas quanto a estrutura morfológia, textural, térmica e magnética. Os resultados mostraram que as cerâmicas apresentaram a fase característica do espinélio inverso, com tamanho de cristalito entre 20 - 93 nm, e composição química dos óxidos em concordância com a estequiometria prevista. Observou-se que a dopagem com Zn2+e Cu2+ a amostra NiFe2O4 não alterou a morfologia, sendo constituída por aglomerados grandes, na forma de blocos irregulares, não porosos e de aspecto frágil, exceto a amostra dopada com Co2+ que apresentou blocos densos irregulares com formação de partículas pré-sinterizadas. Todas as amostras apresentaram comportamento característico de materiais ferrimagnéticos moles, contudo, a ferrita que contém Zn2+ e a que contém Cu2+, apresentaram aumento na ordem de 5 vezes na magnetização de saturação e redução na coercitividade cerca de 72 e 61% respectivamente, quando comparado com a NiFe2O4. As amostras apresentaram valores de área superficial entre 32,6-83,7 m2.g-1, com classificação dos poros na faixa mesoporosa, apresentam também boa estabilidade térmica indicada pela baixa perda de massa durante a análise termogravimetrica. Os testes catalíticos revelaram atividade catalítica em torno de 10% na transesterificação nas condições reacionais propostas. Na esterificação as amostras mostraram resultados promissores, sendo a maior conversão observada para a cerâmica Ni0,5Zn0,5Fe2O4 obtendo cerca de 15% em até 1 hora de reação. A atividade catalítica tanto em transesterificação quanto em esterificação pode estar relacionada à baixa acidez dos sítios de Lewis nestes materiais, além da restrição na conversão dos produtos por impedimento de difusão dos reagentes na estrutura interna. Contudo, verificou-se que a cerâmica dopada com íons Zn2+ apresentou maior diâmetro de poro (12,06 nm) o que possivelmente contribuiu de forma satisfatória na obtenção de estéres metílicos de ácidos graxos (FAMEs).

La réaction d’aziridination catalysée par les sels de cuivre permet de former des aziridines à partir d’oléfines en employant une source de nitrènes générée à partir de aryliminoiodanes.Le développement au laboratoire d’une version « one-pot » impliquant l’iodosylbenzène et un sulfonamide ainsi que l’emploi de l’iminoiodane N-Ses a rendu cette réaction plus attractive en synthèse.La première partie de ce travail a été consacrée à l’étude de la réactivité particulière des esters ω-insaturés dans les conditions d’aziridination. En effet, dans ces conditions, l’aziridine subit une ouverture intramoléculaire par le carbonyle de l’ester pour fournir une γ-lactone 5,5-disubstituée. Cette nouvelle réaction, baptisée réaction d’aminolactonisation, a été exemplifiée sur une série de dérivés styréniques 1-propanoates ou butanoates pour donner les γ-lactones correspondantes avec de très bons rendements. Cependant, sans doute pour des raisons stériques, les composés o-bromés et o-chlorés ont donné majoritairement le produit d’aziridination sans évoluer vers la forme lactonique. Par la suite, ces aziridines placées en milieu acide, ont conduit à la formation de γ-lactones ortho halogénées avec de bons rendements. Enfin, le champ d’application de cette méthodologie a été étendu à la formation d’hétérocycles gem-disubstitués, au départ d’alcools oléfiniques et de carbamates insaturés.La seconde partie de ce travail a été consacrée à l’élaboration d’hétérocycles fonctionnalisés à partir des produits issus de la réaction d’aminolactonisation. Ainsi, en exploitant la réactivité de la partie aromatique, il nous a été possible, notamment via des réactions de couplage croisé, d’obtenir différents motifs tels que des spiroindolines, des indolines gem-3,3-disubstituées ou encore des indolinobutyrolactones avec de très bons rendements. Par ailleurs, en usant de la réactivité de certaines γ-lactones, il nous a été possible d’élaborer deux autres motifs relativement intéressants tels qu’une benzospirolactone ou encore un spirohexahydropyrroloindole. Ce dernier composé a fait l’objet d’une étude approfondie dans le cadre de la synthèse d’un analogue spirolactone de la physostigmine, molécule utilisée notamment dans le traitement de la maladie d’Alzheimer.Enfin, dans le cadre d’une approche à la synthèse de la Fiscaline A, possédant un motif indoline 3,3-disubstituée au sein de sa structure, nos efforts se sont concentrés sur la fonctionnalisation de la position C-2 du noyau indoline. L’ensemble des stratégies élaborées a fait l’objet de la troisième partie de ce travail de thèse. / The reaction of an olefin with a nitrene generated from a phenyliminoiodane in the presence of a copper catalyst allows the direct formation of an aziridine.The versatility of this reaction was improved by a one-pot procedure developed in our laboratory mediated by iodosylbenzene and a sulfonamide and by the use of Ses iminoiodane. The first part of this thesis was devoted to the study of the unexpected reaction observed with ω-unsaturated esters under aziridination conditions in which an intramolecular ring-opening of the aziridine by the carbonyl oxygen of the ester function is observed providing a 5,5-disubstituted butyrolactone. This new reaction named aminolactonization reaction has been used on various styrene-derived 1-propanoates or butanoates for the synthesis of 5,5-disubstituted butyrolactones in high yields. Probably due to hindrance effects, o-bromo and o-chloro compounds selectively gave the aziridination product. The reactivity of these compounds, under acidic conditions, has been investigated and various ortho halogenated γ-lactones have been obtained in good yields. Finally, the field of application of this methodology has been extended to the formation of gem-disubstituted heterocycles starting from, for example, an olefinic alcohol and an unsaturated carbamate. The second part of this work was dedicated to the elaboration of functionalized heterocycles starting from aminolactonization products. To this end, the reactivity of the aromatic ring has been studied to give access to new scaffolds such as spiroindolines, gem-3,3-disubstituted indolines and indolinobutyrolactones in good yields. Furthermore, the reactivity of such γ-lactones allowed us to synthesize new interesting scaffolds such as benzospirolactone or spirohexahydropyrroloindole. The latter has been used as an intermediate for the synthesis of a spirolactone analogue of physostigmine used for the treatment of Alzheimer’s disease.Finally, this methodology was further exploited for an approach to the total synthesis of Fiscalin A, focusing on the C-2 functionalization of the indoline ring. The results are discussed in the third part of this work.

La distillation fractionnée des coupes pétrolières brutes se révèle insuffisante, pour répondre seule aux besoins en carburants et génère de fortes quantités de distillats à longue chaîne carbonée (C20-C50). Cette coupe d’hydrocarbures peu valorisable est ainsi transformée en molécules hydrocarbonées plus légères, par le biais de deux procédés : le craquage catalytique en lit fluidisé ou l’hydrocraquage. Ce dernier permet le craquage sélectif des hydrocarbures essentiellement en gazole et kérosène. Les catalyseurs acides utilisés sont « bifonctionnels » : une matrice zéolithique USY (zéolithe Y ultra-stabilisée) favorise le craquage des hydrocarbures, pendant qu’un sulfure mixte ou un métal noble greffé catalyse les réactions d’hydrogénation et de déshydrogénation. L’objectif de ce travail de thèse consiste à maximiser la sélectivité en distillats moyens, en diminuant le temps de séjour des molécules d’hydrocarbures dans la structure zéolithique, afin de réduire les réactions de surcraquage. Pour cela, il a été choisi de synthétiser directement des nanocristaux de zéolithe Y sans agent organique structurant, avec un rapport Si/Al le plus élevé possible. Cette propriété leur confère une meilleure stabilité lors des post-traitements, indispensables pour atteindre le rapport Si/Al des zéolithes USY actuellement utilisées. Grâce à l’élaboration d’une nouvelle stratégie de synthèse, des nanocristaux compris entre 20 et 90 nm avec un rapport Si/Al variant de 2,2 à 2,6 ont été obtenus. Puis, l’obtention de la forme protonée de ces nanocristaux (90 nm) a été étudiée et des mesures d’acidité ont également été effectuées. / The fractionated distillation of crude oil is not sufficient to cover all the fuel needs and produces large amounts of long chain carbon distillates (C20-C50). Thus, these hydrocarbons are transformed into lighter hydrocarbon molecules by two processes: fluid catalytic cracking or hydrocracking. The latter allows the selective cracking of hydrocarbons essentially in gas oil and kerosene. The acid catalysts used are “bifunctional”: a zeolite matrix USY (ultra-stabilized zeolite Y) performs the hydrocarbons cracking, at the same time a sulfide or a noble metal catalyzes hydrogenation and dehydrogenation reactions. The aim of this work is to maximize the selectivity to middle distillates by decreasing the residence time of hydrocarbon molecules in the zeolite framework. For this, it was chosen to directly synthesize zeolite Y nanocrystals with the highest possible Si/Al ratio, in a SDA-free medium. This property gives them a better stability during the post-treatments essential to reach the Si/Al ratio of the currently used USY zeolite. Thanks to a new synthesis strategy, nanocrystals between 20 and 90 nm with a Si/Al ratio varying from 2.2 to 2.6 were obtained. Then, the protonated form of these nanocrystals (90 nm) was studied and acidity measurements were also carried out.