The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved. In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase. In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches. In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance. In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. / QC20100629

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-07-28T18:13:32Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação de Mestrado Emmanuel Dias - ver Biblioteca 3.pdf: 5438340 bytes, checksum: 493d14db263ba9635f4d797c2d7f26cf (MD5) / Made available in DSpace on 2016-07-28T18:13:32Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação de Mestrado Emmanuel Dias - ver Biblioteca 3.pdf: 5438340 bytes, checksum: 493d14db263ba9635f4d797c2d7f26cf (MD5) Previous issue date: 2016-02-12 / CNPQ / Compostos orgânicos contendo dióis alílicos com configuração trans são substancias orgânicas muito úteis na área de síntese orgânica. Além disso, esses compostos podem ser encontrados na natureza, apresentando importantes atividades biológicas. Estes compostos também podem ser empregados como building blocks na preparação de moléculas bioativos de interesse. Dentre as várias metodologias existentes para preparar estes compostos, muitas empregam reagentes nocivos ao meio ambiente e/ou catalisadores metálicos, em rotas de síntese contendo numerosas etapas reacionais. Com isso, tivemos o interesse de desenvolver um método alternativo, visando à síntese destes compostos, em um processo mais limpo, de enantiosseletividade. A metodologia proposta consistiu no uso de lipases para obtenção dos compostos de forma seletiva. Com isso, após submeter os compostos em sua forma racêmica à resolução na presença de lipases, mono e diacetatos poderiam ser obtidos com alto grau de pureza óptica. A rota quimioenzimática proposta levou a preparação de uma biblioteca de (E)-dióis alílicos, com elevado excesso enantiomérico. / Organic compounds containing trans allylic diols are useful substances in organic synthesis. Furthermore, these compounds can be find in nature showing a wide range of biological activity. In addition, these compounds can be employed as building blocks in the preparation of bioactive compounds as target substances. Among several methodologies to prepare these compounds, the majority employs toxic reagents and/or metallic catalysts, in multistep pathway. Thereby, our interest was develop an alternative method aiming at the synthesis of these compounds, in a greener and enantioselective process. This proposal has consisted in employ lipases as a manner to achieve the related compounds selectively. Thus, after subjecting the racemic compounds to an enzymatic resolution, mono and bis acetates could be generated in high optical purity. The proposal chemo-enzymatic approach lead to the preparation of an enantioenriched library of (E)-allylic diols.

L'isosorbide est un dianhydrohexitol chiral obtenu énantiomériquement pur par double déshydratation du sorbitol, un des produits majoritaires de l'industrie amidonnière résultant de l'hydrogénation du glucose. L'intérêt croissant pour l'isosorbide ces deux dernières décennies s'explique notamment grâce à sa production industrielle par des entreprises comme Roquette (20 000 tonnes par an). Cette disponibilité a permis la valorisation de l isosorbide dans divers domaines : polymères (polyéthylène isosorbide téréphtalate), tensioactifs, solvants (diméthyl isosorbide) et pharmaceutique (isosorbide dinitrate). Les propriétés de l'isosorbide ont ouvert la voie à des applications en induction chirale comme ligand, auxiliaire ou catalyseur par transfert de phase. D'autres isohexides (isomannide et isoidide) ont été moins étudiés. Leur structure conformationnelle rigide offre une grande diversité de modifications afin de moduler leurs propriétés électroniques et stériques. Les isohexides sont des candidats prometteurs pour le développement d'organocatalyseurs biosourcés. Très récemment, l'alkylation énantiosélective d'indole catalysée par des dérivés thiourées basées sur le squelette isohexide a été décrite. Au cours de cette thèse, la fonctionnalisation des isohexides en dérivés aminés est rapportée. La synthèse d'une première série d'amino-alcools, amino-éthers et diamines est décrite. Une seconde génération d'α-amino-amides et 1,2-diamines est obtenue par amidation. Ces dérivés aminés sont engagés comme organocatalyseurs dans la synthèse asymétrique de la warfarine (avec des résultats atteignant 20% de rendement et 28% d'e.e.) et l'aldolisation de l'isatine (jusqu'à 73% de rendement et 33% d'e.e.) / Isosorbide is a chiral dianhydrohexitol obtained in enantiomerically pure form by double dehydration of sorbitol, a major product of the starch industry resulting from hydrogenation of glucose. Isosorbide has been increasingly studied these last twenty years, mainly thanks to industrial companies, such as Roquette (France) that implemented the production to 20,000 tons a year. This availability allowed the promotion of isosorbide applications in various fields: polymers (polyethylene isosorbide terephthalate), surfactants, solvents (dimethyl isosorbide) and pharmacy (isosorbide dinitrate). Isosorbide structural properties opened its investigation as a chiral inducer either as a ligand, an auxiliary or a phase transfer catalyst. Other isohexides (isomannide, isoidide) were much less studied. These diastereomeric conformationally rigid structures offer a large diversity of functionalizations to tune their electronic and steric properties. Isohexides are promising candidates for the development of biosourced organocatalysts. Very recently, Friedel-Crafts enantioselective alkylation of indole mediated by thiourea organocatalysts derived from isohexides has been described. In this thesis, the functionalization of isohexides into amino derivatives is reported. A first serie of amino-alcohols, amino-ethers and diamines is reported. A second generation of α-amino amides and 1,2-diamines is then obtained via amidation. These nitrogenfunctionalized derivatives are screened as organocatalysts for the asymmetric synthesis of warfarin (with results up to 20% of yield and 28% of e.e.) and for isatin aldolisation (with results up to 73% of yield and 33% of e.e.)

Les jasmonates de méthyle sont des oxylipines asymétriques impliquées dans les mécanismes de défense, de développement et de régulation des organismes photosynthétiques terrestres ou marins face à des stress biotiques et abiotiques. Parmi les quatre stéréoisomères, seuls l’épi-jasmonate et l’ent-épi-jasmonate de méthyle possèdent de bonnes propriétés organoleptiques mais aussi une forte activité phytohormonale permettant l’élicitation de métabolites secondaires bioactifs. En ciblant spécifiquement une hexokinase mitochondriale régulant le métabolisme des cellules cancéreuses, les jasmonates de méthyle constituent d’excellents candidats pour de nouveaux agents thérapeutiques. Dans la recherche constante de nouvelles molécules thérapeutiques issues du milieu naturel, notre laboratoire s’est donc focalisé sur la synthèse énantiosélective de l’épi-jasmonate et l’ent-épi-jasmonate de méthyle, au départ de diols bicycliques homochiraux monoprotégés dérivant du cyclooct-1,5-diène.Afin d’obtenir ces diols bicycliques énantiopurs, une stratégie innovante a consisté en l’étude de la réaction d’électrocyclisation du méso-époxyde dérivé du cyclooct-1,5-diène assistée par des ligands chiraux métallés diversement fonctionnalisés, les catalyseurs de Jacobsen. En mettant à profit notre savoir-faire sur les désymétrisations enzymatiques, une stratégie parallèle optant pour la résolution énantiosélective de diols homochiraux monocycliques, ou de diols bicycliques C2-symétriques nous a permis d’accéder à des silanyloxyindèn-5-ones chirales, précurseurs clé des cis-jasmonates de méthyle énantiopurs. Enfin, la synthèse racémique de deux jasmonoïdes clé, la (±)--jasmolactone, puis le (±)-épi-jasmonate de méthyle a été validée en 15 étapes à partir du cyclooct-1,5-diène. / Methyl jasmonates are asymmetric oxylipins involved in defensive, developmental and regulative mechanisms of terrestrial and marine photosynthetic organisms in response to biotic and abiotic challenges. Among the four stereoisomers, only methyl epi-jasmonate and ent-epi-jasmonate show good organoleptic properties but also phytohormonal activity allowing the elicitation of bioactive secondary metabolites. Because they specifically target a mitochondrial hexokinase regulating the metabolism of cancer cells, methyl jasmonates have become excellent candidates as new therapeutic agents. With a constant attention on new therapeutic agents derived from the natural environment, our laboratory has focused on the enantioselective synthesis of methyl epi-jasmonate and ent-epi-jasmonate using monoprotected homochiral diols derived from cyclooct-1,5-diene.In order to obtain these chiral bicyclic diols, an innovative strategy has involved the study of the chemo-stereoselective electrocyclization of the cyclooct-1,5-diene-derived meso-epoxide assisted by chiral metallated ligands known as Jacobsen’s catalysts. Taking advantage of our knowledge of enzymatic desymmetrization, a second strategy opting for the enantioselective resolution of monocyclic homochiral diols or C2-symmetric bicyclic diols led access to chiral silanyloxyinden-5-ones, key precursors to chiral methyl cis-jasmonates. Meanwhile, the racemic synthesis of (±)--jasmolactone and methyl (±)-epi-jasmonate was validated in 15 steps starting from cyclooct-1,5-diene.

La demande en composés chiraux est en plein essor ces dernières années. Pour accéder à leur synthèse, la biocatalyse, couplée à l'irradiation pourrait être une méthode innovante. Il existe en effet de nombreux cas dans la littérature où l'utilisation de micro-onde semble avoir un effet activateur sur l'efficacité enzymatique.Cependant, l'effet de l'irradiation micro-onde est mal compris et controversé. Le but de cette thèse était d'étudier l'impact de l'irradiation micro-onde sur des lipases, immobilisées ou non, en étudiant différentes réactions modèles, allant de la résolution d'alcools secondaires linéaires simples à la résolution de polyols complexes, et alcools polyfonctionalisés, par comparaison entre chauffage sous irradiation micro-onde (en conditions drastiques ou non) au chauffage classique. L'étude de l'irradiation micro-onde sur la stabilité enzymatique et sur paramètres intrinsèques de l'enzyme après modification des paramètres réactionnels a permis de mettre en évidence un rôle indéniable de l'irradiation micro-onde sur l'efficacité des réactions enzymatiques. Il a été possible d'une part de diminuer de façon importante les temps réactionnels, comparé au chauffage traditionnel,et d'autre part de contrôler efficacement l'énantio préférence et la sélectivité de la lipase pour l'obtention de molécules d'intérêt. Par des procédés innovants, l'impact de la puissance d'irradiation a été montré comme hautement dépendant du modèle réactionnel étudié. En optimisant les conditions réactionnelles pour obtenir les meilleures sélectivités et activités enzymatique sous irradiation micro-onde, la synthèse de a-hydroxyamides chiraux et de polyols parfaitement résolus a pu être entreprise de façon rapide, propre, tout en respectant les principes de chimie verte.

Different monomeric diols, with a central rigid FD unit connected with two aliphatic chains of various length, were prepared to react with aromatic mesogenic triad, TOBC. In this manner, thermotropic polyesters with possible low thermal transition temperatures (including Tmand Ti) and high solubility in organic solvent can be generated in view of the non-linear polymeric chain imposed by the rigid, bent FD moieties. In addition to the effect of the aliphatic chain length, polyesters of different molecular weight will be obtained by different synthesis approach (or fractionation of the resulting polyester product) and therefore, the influence of molecular weight on liquid crystalline properties can be evaluated.

Le diabète de type 2 est aujourd'hui une maladie de plus en plus répandu. En ce sens il est nécessaire de mettre au point de nouveau composé permettant d'inhiber la gluconéogenèse. C'est pourquoi nous nous sommes intéressé à la synthèse du FR225654 1, décaline présentant une activité hypoglycémiante in vivo après administration par voie orale et inhibiteur de la néoglucogenèse in vitro (IC50 = 1,1.10-7 M). Ce composé, isolé en 2005 du champignon Phoma sp N°00144 et jamais encore synthétisé à ce jour, possède un mécanisme d'action inconnu. La nécessité d'une synthèse par voie chimique convergente et flexible, permettant l'accès à des analogues, est donc évidente. La stratégie consistait à préparer une trans-décaline par le biais d'une réaction de Diels-Alder intramoléculaire à partir d'un triène précurseur. La combinaison de ces travaux a constitué une avancée importante dans le cadre de la synthèse du FR225654, un hypoglycémiant potentiel. La mise au point d'une synthèse convergente du précurseur de la réaction de Diels-Alder permettra notamment d'effectuer par la suite des modifications aisées en vue de la préparation d'une vaste gamme d'analogues simplifiés. A ce jour, le produit de cyclo-addition a été isolé et caractérisé, validant ainsi l'étape clé de la stratégie de synthèse. Ainsi, l'accès rapide au FR225654 est rendu possible et la synthèse d'analogues est maintenant envisageable. Les produits synthétisés feront l'objet d'une évaluation biologique, l'objectif ultime étant d'accéder à de nouveaux médicaments pour le traitement du diabète de type 2.

In this thesis, I report the gas phase infrared spectra of some polycyclic aromatic hydrocarbons (PAHs) namely 1, 5-, 1, 6-, and 2, 6-dimethyl naphthalenes (DMNs); 2, 4-, 2, 6-, 2, 7-, and 2,8-dimethyl quinolines (DMQs); and 1, 9-, 2, 4-, and 3, 9-dimethyl phenanthrenes (DMPs) and diols such as 1, 2-ethylene glycol (1,2-EG) and 1, 4-butanediol (1,4-BD). Assignment of the IR spectra has been done with the help of DFT calculations followed by scaled quantum mechanical force field calculations. Chapter 1 gives a brief introduction to PAHs and diols. A brief survey of literature pertinent to their sources, their importance, experimental techniques for identification of PAHs and different conformers of diols and intramolecular interactions between the two hydroxyl groups in the diols are presented. The scope and motivation of the present investigation have been described at the end of the chapter. In Chapter 2, I have discussed all the methods used in carrying out this work. The details of the experimental FT IR set-up and quantum-chemical calculations employed in this work have been elaborated in this chapter. The subsequent chapters 3 - 5 deal with the experimental and theoretical results obtained for DMNs, DMQs, and DMPs. In chapter 3, infrared spectra of 1,5-, 1,6-, and 2,6-DMN have been described. The spectra have been recorded using a multi-pass gas cell coupled with Nexus-870 spectrometer (Thermonicolet, US). Qualitatively, spectral assignments have been made with the help of calculated doubly scaled (one scaling factor for the C-H stretching and another factor for the non C-H stretching frequencies) harmonic frequencies at the B3LYP/6-31G* level of theory. The spectral features to distinguish three different isomers of DMN have been identified. In chapter 4, IR spectra of 2,4-, 2,6-, 2,7-, and 2,8-DMQ in the gas phase have been recorded using a high resolution Vertex-70 (Bruker Optics, Germany) FT-IR spectrometer. DFT calculations have been carried out in order to get harmonic and anharmonic frequencies and their intensities at the B3LYP/6-31G* level of theory. Unambiguous assignments of IR bands could not be made with the help of anharmonic or selectively scaled harmonic frequencies. Therefore, scaled quantum mechanical (SQM) force field calculations were performed where force fields in local coordinates were scaled for getting frequencies in close agreement with experiment. Potential energy distributions (PEDs) of the normal modes in terms of the local coordinates of the molecule using a modified UMAT program in the QCPE package were also obtained in order to identify the nature of the fundamental vibration modes. In chapter 5, I have presented the gas phase IR spectra of 1,9-, 2,4-, and 3,9-DMP. The harmonic and anharmonic frequencies and their intensities were obtained at the B3LYP/6-31G* and B3LYP/6-311G** levels of theory. I have carried out SQM calculations for the assignment of the experimental frequencies in a similar fashion as was done for the DMQs. In chapter 6, IR spectra of two diols namely 1,2-ethylene glycol and 1,4-butane diol are reported and discussed. DFT calculations have been carried out for the harmonic fundamental frequencies and intensities at the experimental temperatures with 10 unique chosen conformers of the diols at the B3LYP/6-311++G** level of theory. Gas phase equilibrium population analysis has been done in order to generate the theoretical spectrum with the weighted average contributions from the 10 conformers to match the experimental spectrum. The hydrogen bond enthalpy, strength and nature have been investigated in details. From this experimental and theoretical studies, it has been concluded that the intramolecular hydrogen bond is absent in 1,2-EG at ordinary temperatures whereas it is present in 1,4-BD. vi Chapter 7 is the concluding chapter where the main work done in this thesis is summarized and future direction is presented.

Les travaux de recherche présentés ici ont pour objectif la synthèse de fongicides innovants de la manière la plus éco-compatible qu’il soit. Pour cela, plusieurs axes de réflexion ont été étudiés. Tout d’abord, la synthèse d’un intermédiaire de fongicides bien connu, l’o-crésol1, a été réalisée à partir d’un substrat naturel, le salicylaldéhyde2. Les conditions expérimentales ont été éco-conçues afin de correspondre au mieux aux exigences de la chimie verte. Des solvants et des réactifs classés verts ont été utilisés, comme l’éthanol, l’eau et l’acide acétique3 par exemple, ainsi que des techniques alternatives comme l’activation par micro-ondes (Schéma 1). Dans un second temps, des synthèses éco-compatibles de nouveaux fongicides ont été réalisées. Le salicylaldéhyde, substrat naturel de référence, a été mis à réagir avec différents alcools afin de réaliser des réactions d’éthérifications réductrices. Les molécules obtenues sont composées d’une partie phénolique portant un groupement alcoxyle en position 2. Ce groupement alcoxyle se compose d’une longueur de chaîne variable ayant une influence sur les propriétés fongicides des produits synthétisés (Schéma 1). Troisièmement, des réactions d’homo- et d’hétéro-couplages pinacoliques ont été réalisées entre divers aldéhydes, aromatiques et aliphatiques, afin d’obtenir des diols vicinaux, appelés pinacols, symétriques ou non (Schéma 1). Dernièrement, les propriétés fongicides et anti-oxydantes de toutes les molécules obtenues de manière stable, référencées ou non, ainsi que les substrats de départ, ont été testés. Les tests microbiologiques ont été réalisés sur une gamme de diverses souches fongiques. / The purpose of this research is to synthesize innovative fungicides by the most ecocompatible way. Several hypotheses have been investigated. First, an extensively studied molecule, intermediate of fungicides, o-cresol1, has been achieved from a natural substrate, salicylaldehyde2. The experimental conditions have been eco-designed in the aim to best match green chemistry requirements. Green solvents and reactants have been used, as ethanol, water and acetic acid3 for example, as well as alternative technologies like microwaves activation (Schema 1). Secondly, novel fungicides green syntheses have been carried out. Salicylaldehyde, a natural substrate model, reacted with various alcohols in the aim to perform reductive etherification reactions. In this way, products were composed of phenolic part with an alkoxyl group at the 2nd position. These alcoxyl groups have various chain lengths, playing a key role in fungicidal ability (Schema 1). Then, homo and hetero pinacol coupling reactions have been achieved with various aromatic or aliphatic aldehydes to obtain symmetrical and asymmetrical vicinal diols, called pinacols (Schema 1). At last, fungidal and antioxidant properties of all stable obtained molecules, referenced or not, as well as starting materials, have been evaluated. Microbiological studies have been carried out with various fungal strains.

A busca por materiais derivados de fontes renováveis e com características como biocompatibilidade e biodegradabilidade tem crescido significativamente nos últimos anos. A utilização de enzimas na polimerização representa um grande passo para a obtenção destes, visto que possibilitam a produção de polímeros evitando a utilização de catalisadores tóxicos e, assim, melhorando sua biocompatibilidade. O presente trabalho descreve a utilização de monômeros funcionais derivados de fontes renováveis na produção de poliésteres hidrolisáveis via catálise enzimática. As sínteses de polímeros produzidos a partir de isosorbídeo e ácidos dicarboxílicos ou derivados - como seus ésteres alquílicos e vinílicos - foram feitas utilizando a lipase de Candida antarctica Fração B como catalisador. As polimerizações foram realizadas por policondensações em massa e em solução, utilizando-se diferentes solventes e diferentes técnicas para remoção de subprodutos de reação. A principal abordagem foi o estudo das diferentes condições reacionais realizadas, variando-se o tempo de reação, tipo do monômero, solvente utilizado (se for o caso) e tipo de técnica para remoção de subprodutos visando o aumento da massa molar dos polímeros. A condição que forneceu os materiais com maiores massas molares foi a policondensação em solução, utilizando a mistura cicloexano:benzeno como solvente. Tendo por objetivo investigar profundamente a condição ótima obtida, e estabelecer padrões de comparação com outros sistemas, foram estudados, nessa condição, parâmetros como tempo de reação, efeito do tamanho da cadeia carbônica do monômero, grupo de saída, solubilidade dos polímeros e diluição do sistema. Os materiais obtidos foram caracterizados por cromatografia por exclusão de tamanho (SEC), termogravimetria (TG), calorimetria exploratória diferencial (DSC), espectroscopia no infravermelho, difração de raios-X, e Ressonância Magnética Nuclear (RMN) de 1H e 13C. Através deste trabalho foi provado que, embora apresente uma cinética de reação lenta, a polimerização enzimática deste diol secundário estericamente impedido é possível, fornecendo poliésteres com massas molares similares às obtidas via catálise química. Todos os resultados obtidos neste trabalho são inéditos no que diz respeito à polimerização enzimática de dióis secundários impedidos, mais especificamente de isosorbídeo. / The search for materials derived from renewable sources, with characteristics such as biocompatibility and biodegradability has grown significantly in recent years. The use of enzymes in the polymerization is a major step for the attainment of these materials, since it allows the production of polymers while avoiding the use of toxic catalysts and thus improving its biocompatibility. This paper describes the use of functional monomers derived from renewable sources in the production of hydrolysable polyesters by enzyme catalysis The synthesis and characterization of polymers derived from isosorbide and dicarboxilic acids or derivatives - such as alkyl and vinyl esters - were carried out using the lipase from Candida antarctica - Fraction B as catalyst. The polymerizations were accomplished by polycondensations in bulk and in solution, using different solvents and different techniques for removal of reaction byproducts. The main approach was to study the different reaction conditions, by varying the reaction time, monomer type, solvent used (if applicable) and the type of technique for removal of byproducts, aiming at maximizing polymer molar mass. The condition that provided the material with higher molecular weight was the solution step-growth polymerization, using a mixture cyclohexane:benzene as solvents. Aiming to thoroughly investigate the optimum condition obtained, and to establish standards for comparison with other systems, it was studied, in this condition, parameters such as reaction time, effect of monomer carbon chain length , leaving group, polymers solubility of and dilution of the reaction system. The materials were characterized by gel permeation chromatography (SEC), thermogravimetry (TG), differential scanning calorimetry (DSC), infrared spectroscopy, X-ray diffraction and 1H and 13C Nuclear Magnetic Resonance (NMR). Through this work it was proved that, in spite of a slow reaction kinetics, the enzymatic polymerization of this hindered secondary diol is possible, providing polyester with molecular weight similar to those obtained by chemical catalysis. All results obtained in this work are unprecedented with respect to the enzymatic polymerization of hindered secondary diols, more specifically of Isosorbide.