Orientador: Gabriela Alves Macedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T15:45:42Z (GMT). No. of bitstreams: 1 Lopes_DanielleBranta_D.pdf: 3780152 bytes, checksum: ba7b4a54629ff27a24abae14e788d60c (MD5) Previous issue date: 2013 / Resumo: Os aromas despertam grande interesse entre consumidores e indústrias de alimentos, especialmente por estarem diretamente relacionados ao sabor dos alimentos. Dentre os aromas de maior importância industrial destaca-se o grupo das lactonas, presentes em uma grande variedade de produtos naturais e compostos biologicamente ativos. Em virtude do destaque apresentado por essa classe de substâncias, diversos métodos para sua produção têm sido relatados. Como alternativa aos processos químicos tradicionais, propõe-se nesta pesquisa a utilização da biotecnologia de micro-organismos e enzimas para a produção de compostos aromáticos a partir de substratos naturais, como óleos vegetais hidrolisados.A fermentação microbiana é considerada um caminho potencial para a produção de aromas naturais, sendo o grupo formado pelos fungos um dos mais utilizados para esse fim. A biocatálise também é uma alternativa muito vantajosa e capaz de catalisar um grande número de reações estéreo- e regiosseletivas, o que não é alcançado por meio de síntese química clássica. O uso de enzimas isoladas torna-se preferível em relação ao uso de micro-organismos quando existem limitações relacionadas à permeabilidade do substrato na membrana da célula ou quando ocorrem reações secundárias indesejáveis.Considerando-se os argumentos citados, neste trabalho foram estudados dois métodos para a produção de lactonas. O primeiro deles, a biotransformação, foi feita a partir do uso de micro-organismos para a produção dos compostos em questão. O segundo método, a biocatálise, foi realizado através da reação de lactonização com o uso de lipases microbianas brutas isoladas e liofilizadas. Os substratos utilizados no estudo foram os óleos vegetais de mamona e linhaça previamente hidrolisados por lipases fúngicas produzidas por Rhizopus sp. e Geotrichum sp. A produção de lactonas foi alcançada em ambos os métodos. Com o uso da biotransformação, todos os micro-organismos testados (Geotrichum sp., Rhizopus sp., Aspergillus sp. (Linhagens 1068 e 1099) e Fusarium oxysporum) foram capazes de produzir diferentes tipos de lactonas, com destaque para o fungo Fusarium oxysporum, que produziu maior variedade delas (&61543;-decalactona, 2-cumaranona, mevalonolactona e pantolactona), além de alcançar o maior rendimento na produção de &61543;-decalactona (18,93 &956;L L-1), após 48 horas de reação e empregando-se os óleos de mamona e linhaça previamente hidrolisados pela lipase produzida pelo fungo Rhizopus sp.. Através do uso da biocatálise, ótimos resultados também foram alcançados, e a produção de lactonas foi possível ao se utilizar as lipases produzidas pelos fungos Rhizopus sp., Aspergillus sp. (Linhagem 1068) e Fusarium oxysporum, sendo possível a produção de &61543;-undecalactona, &61543;-decalactona e &61543;-dodecalactona, dependendo da lipase empregada e do tempo de reação. A maior produtividade foi obtida na produção de &61543;-undecalactona ao se utilizar a lipase produzida pelo fungo Rhizopus sp. após 24 horas de reação, obtendo-se um rendimento de 21,78 &956;L L-1, também a partir do uso dos óleos de mamona e linhaça hidrolisados previamente pela lipase produzida por Rhizopus sp. As lipases produzidas pelos fungos Geotrichum sp. e Aspergillus sp. (Linhagem 1099), também estudadas neste trabalho, não foram capazes de produzir lactonas / Abstract: Aromas arouse great interest among consumers and food industry, especially because heir direct relationship with food taste. In the midst of the most important industrial flavorings stands lactones group, which are present in a wide variety of natural products and biologically active compounds. Due to the prominence of this class of substances, several methods for the production of lactones have been reported. As an alternative to traditional chemical processes, it is proposed in this research the use of microbial and enzyme biotechnology for the production of aromatic compounds natural substrates, such as hydrolyzed vegetable oils. The microbial fermentation is considered a potential pathway for the production of natural flavor, and fungi group is the most widely used for this purpose. Biocatalysis is also a very useful alternative and is able to catalyze a large number of stereo- and regioive reactions, which is not achieved by classical chemical synthesis. The use of isolated enzymes is preferable over the use of microorganisms when there are limitations concerning the permeability of the substrate in the cell membrane or when undesired side reactions occur. Considering these arguments, two methods were investigated in this work for the production of lactones. The first one, biotransformation, was made the use of microorganisms for the production of these compounds. The second method, biocatalysis, was performed by lactonization reaction using crude microbial lipases that was isolated and lyophilized. Substrates used were the vegetable castor and linseed oils previously hydrolyzed by fungal lipases produced by Rhizopus sp. and Geotrichum sp. Lactones production was achieved in both methods. Using biotransformation, all microorganisms tested (Geotrichum sp., Rhizopus sp., Aspergillus sp. (Strains 1068 and 1099) and Fusarium oxysporum) were able to produce different lactone types, especially using the fungus Fusarium oxysporum, which produced a greater variety (&61543;-decalactone, 2-cumaranone, mevalonolactone and pantolactone), achieving the best performance in y-x decalactone production (18.93 &956;L L-1), after 48 h employing castor and linseed oils hydrolyzed by lipase Rhizopus sp. Through the biocatalysis use, excellent results were also obtained, and the lactones production was possible using lipases produced by fungi Rhizopus sp., Aspergillus sp. (Strain 1068) and Fusarium oxysporum. The production of y-undecalactone, y-decalactone and y-dodecalactone was achieved depending on the lipase employed and the reaction time used. Highest yield was obtained in the &61543;-undecalactone production (21.78 &956;L L-1), using castor and linseed oils hydrolyzed by Rhizopus sp. lipase. Lipases produced by Geotrichum sp. and Aspergillus sp. (Strain 1099) was also studied in this work, but were unable to produce lactones / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

The present work describes the design and synthesis of a series of dimers [(η5 - ring)MCl]2(μ2 -Cl)2, (where (η5 -ring)MCl = (η5 -Me4C5R)Rh(III)Cl or (η5 -Me4C5R)Ir(III)Cl). Iridium and rhodium dimeric complexes were synthesized via a microwave reaction and directly compared through single-crystal X-ray crystallography. Finally, the dimeric complexes were evaluated as potential oxidation catalysts. The modified HCp*R (R = isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, nheptyl, n-octyl, phenyl, benzyl, phenethyl, cyclohexyl, and cyclopentyl) type ligands were synthesized by reaction of 2,3,4,5-tetramethylcyclopent-2-en-1-one with the respective Grignard reagent (RMgX), followed by elimination of water under acidic conditions to produce the tetramethyl(alkyl or aryl)cyclopentadienes in moderate to excellent yields (39 - 98%). Reaction of the HCp*R ligands with [M(COD)](μ2 -Cl)2 (M = Rh, Ir; COD = 1,5-cyclooctadiene) gave the dimeric complexes [Cp*RMCl]2(μ2 -Cl)2 in yields ranging from 16 - 96%. The dimers were characterized by nuclear magnetic resonance (NMR)spectroscopy, single-crystal X-ray diffraction (XRD) (supplemented by powder XRD), high-resolution mass spectrometry (HRMS), and elemental analysis. Complexes studied by XRD were analyzed to understand the bond lengths and bond angles throughout each complex. The dimeric complexes synthesized, will facilitate a complete study on how the R group influences catalytic activity. / Master of Science

No / The naturally occurring bitter principle quassin (1) was converted chemically into the gamma-lactone quassilactone (13) in an attempt to enhance its antiplasmodial activity. The in vitro antiplasmodial activity of 13 against Plasmodium falciparum (K1) (IC(50) = 23 microM) was 40-fold greater than that of 1. However, one of the intermediates, compound 8, the 15beta-hydroxy,16-O-m-chlorobenzoyl analogue of 1, was 506-fold more active than 1 against P. falciparum (IC(50) = 1.8 microM) and only 3-fold less potent than chloroquine. In addition, 8 displayed the best cytotoxic/antiplasmodial ratio (112) of all of the compounds tested. In the course of this work a dimer, neoquassin ether (6), linked at C-16 was also prepared; 6 was found to have weak antiplasmodial activity (IC(50) = 9.7 microM).

Yes / Fused cyclobutanes are found in a range of natural products and formation of these motifs in a straightforward and easy manner represents an interesting synthetic challenge. To this end we investigated an intramolecular variant of the thermal enamine [2 + 2] cyclisation, developing a diastereoselective intramolecular enamine [2 + 2] cyclisation furnishing δ lactone and lactam fused cyclobutenes in good yield and excellent diastereoselectivity. / The work was funded by Yorkshire Cancer Research

L'élaboration de squelettes de pipéridinones et lactones a été accomplie de manière très efficace en alliant processus radicalaires et ioniques. La synthèse d'oximes SEM-protégées par un assemblage multicomposant radicalaire permet, après hydrolyse de la fonction oxime, d'obtenir rapidement un aldéhyde fonctionnalisé. Ce composé est ensuite transformé en lactone ou pipéridinone désirée par un processus ionique.Une approche minimisant le nombre d'étapes élémentaires a été développée autorisant ainsi un accès rapide et convenable à une grande diversité de structures. Ces processus peuvent impliquer jusqu'à cinq composants. / The construction of piperidinone and lactone scaffolds has been performed efficiently, combining radical and ionic processes. The synthesis of SEM-protected oximes by a multicomponent radical reaction enables, after hydrolysis of the oxime functional group, the access to a functionalized aldehyde. This compound is then converted into a lactone or a piperidinone by a ionic process.An approach minimizing the number of steps has been developed, thus allowing a rapid and convenient access to a large diversity of structures. These processes may involve up to five components.

Chez les Vibrio spp., le QS est impliqué dans de nombreuses fonctions comme la colonisation de niche écologiques, les stratégies de survie ou encore la virulence. Cependant, pour la majorité des espèces de Vibrio, la diversité des AHLs produites reste largement sous-estimée et l'étude du QS est encore limitée à quelques espèces modèles ou pathogènes. Toutefois, dans les environnements aquatiques, ces espèces sont minoritaires et les espèces les plus abondantes ne sont que très peu étudiées. Nos résultats ont révélé une importante diversité d'AHLs mais aussi, de façon surprenante, une hétérogénéité dans les phénotypes de production d'AHL au sein d'une même espèce de Vibrio. Pour la première fois, nous avons mis en évidence qu'une même souche de Vibrio pouvait présenter des phénotypes de production d'AHLs différent au cours du temps et une approche statistique a révélé l'implication de certains déterminants biotiques et abiotiques dans ces variations temporelles. Par ailleurs, une approche à micro-échelle a révélé une structuration des populations de Vibrio en unités fonctionnelles constituées de souches phylogénétiquement proches qui partagent des niches écologiques spécifiques et des comportements sociaux. Nos résultats ont mis en évidence que les modalités de communication pouvaient être hétérogènes suggérant l'absence d'un langage commun au sein de ces unités fonctionnelles. En conclusion, ce travail de thèse a permis d'apporter de nouvelles connaissances sur le QS chez les Vibrio dans l'environnement marin, de la souche à la population, et propose une vision intégrée des mécanismes de régulation de la production d'AHLs dans l'environnement. / Quorum sensing is an important mechanism among Vibrio species and is involved in many vital functions such as niche colonization, survival strategies or virulence. However, AHL diversity still largely underestimated for the majority of Vibrio species and the current knowledge on AHL-mediated QS is limited to a few pathogenic or bioluminescent species. Nonetheless, these species are weakly abundant in seawater while dominant species in the environment are poorly studied. Our results revealed a unexpected diversity of AHL molecules but also a quite surprising intra-species diversity of AHL production phenotypes. For the first time, we showed that different isolates of a single genotype switched between different AHL production phenotypes among time and we revealed the potential involvement of abiotic and biotic parameters in these variations. However, it appears that when studied at a microscale, Vibrio populations are showing a functional structuration in ecological units consisting of phylogenetically close strains sharing habitat and social traits. In this context, it was necessary to determine if these different AHL production phenotypes were associated to different micro-habitats in the water column. We did not demonstrate that a common language was spoken within ecological populations. This thesis work provide new insights on AHL-mediated QS among a broader range of species and among Vibrio populations and depicts the potential impact of multiple aspects of marine environments on AHL production.

Les tétrahydropyranes et les pipéridines sont des motifs que l'on retrouve dans de nombreuses molécules naturelles bioactives. L'intérêt pour ces familles de composés ne cesse de croître. En effet le noyau pipéridine est le troisième motif cyclique le plus retrouvé dans les molécules thérapeutiques après les noyaux phényle et pyridine alors que le tétrahydropyrane prend lui la 6ème place. Il apparaît donc opportun de développer des voies d'accès simples et efficaces afin d'obtenir de façon stéréosélective (diastéréo et/ou énantiosélective) ces motifs structuraux. Les travaux de recherche présentés dans ce manuscrit concernent le développement de deux nouvelles méthodologies de la cyclisation d'aza-Prins. La cyclisation d'aza-Prins permet par la réaction entre une amine homoallylique et un aldéhyde en présence d'un acide (Lewis ou Brønsted) la formation de pipéridine. La première a été développée pour pallier à l'absence dans la littérature de méthodologie applicable à tous types d'amines homoallyliques. Cette nouvelle méthodologie repose sur la promotion de la réaction par un effet synergétique entre un acide de Lewis (TiCl4 1 éq.) et un acide de Brønsted (p-TSA 0.1 éq.). Cette méthodologie a permis l'obtention de pipéridine avec de bons rendements et une excellente diastéréosélectivité a été obtenue selon la nature de l'amine homoallylique utilisé. La deuxième méthodologie qui a été développée permet l'aminolyse de pipéridine-lactone. Ces pipéridine-lactones, obtenues par une réaction d'aza-Prins, sont ainsi aminolysées par un acide aminé en présence d'un promoteur puissant LiNTf2. Ces deux méthodologies combinées nous ont permis d'obtenir divers motifs pipéridines. Un certain nombre de ces pipéridines synthétisées seront testées afin de mettre en évidence une éventuelle activité biologique. / Tetrahydropyrans and piperidines are cyclic motifs found in numerous bioactive natural products. Interest in these families of compounds is continuously growing. Indeed, piperidine is the 3rd most common cyclic motif found in therapeutic molecules after phenyl and pyridine structures, while the tetrahydropyran takes the 6th place. Thus the development of simple and effective methodologies to obtain these structures stereoselectively (diastereo and/or enantioselectively) is of great interest. The research presented in this manuscript concerns the development of two new methodologies for aza-Prins cyclization. These reactions involve condensation of homoallylic amines with aldehydes in the presence of a Lewis or Brønsted acid to give piperidine compounds. The first method was promoted by a synergistic combination of a Lewis acid and a Brønsted acid and employs N-alkyl, N-aryl, and unprotected homoallylamines to efficiently form piperidines with good yields and excellent diastereoselectivity, which was controlled by the nature homoallylamine. The second method allows piperidine-lactone aminolysis in presence of aminoacids with the help of a powerful promoter LiNTf2. These two combined methodologies allowed us to obtain a library of piperidine structures, some of which will be tested in order to assess their possible biological activity.

Les poly(hydroxyalcanoate)s (PHAs), et notamment les poly(hydroxybutyrate) (PHB) et poly(malolactonate d’alkyle)s (PMLARs), sont des polyesters biosourcés et biodégradables intéressants pour des applications comme polymères de commodité ou dans le domaine médical. La polymérisation par ouverture de cycle (ROP) de β-lactones est une stratégie très efficace pour la synthèse de PHAs, permettant un contrôle de la masse molaire, de la microstructure et de la fonctionnalité des polymères. Ainsi, il s’agit de moduler ces paramètres pour optimiser les propriétés physico-chimiques des polymères. Cette approche a permis de synthétiser de nouveaux (co)polymères PHAs, présentant des fonctionnalités, des tacticités (atactiques, syndiotactiques) et des topologies (copolymères à blocs, aléatoires et alternés) originales. L’utilisation de différents systèmes catalytiques et amorçants a permis la synthèse de PHAs α-base, ω-crotonate téléchéliques atactiques, mais également des premiers exemples de PMLARs (R = allyle, benzyle et méthyle) syndiotactiques. De la même manière, l’ingénierie catalytique offre la synthèse de copolymères P(BL-b-MLABe) et P(BL-ran-MLABe) par copolymérisation séquentielle et simultanée du couple rac-BL/rac-MLABe. Enfin la copolymérisation simultanée des comonomères (R)-MLAAll/(S)-MLABe en présence de complexe d’yttrium a conduit à la synthèse de copolymères alternés P(MLABe-alt-PMLAAll). Leur modification chimique post-polymérisation ouvre la voie à la synthèse de PHAs portant des fonctions latérales réactives alternées. / Biobased and biodegradable polyesters of the family of poly(hydroxyalkanoate)s (PHAs), and more specifically poly(hydroxybutyrate) (PHB) and poly(alkyl malolactonate)s (PMLARs), display a wide array of valuable uses ranging from commodity plastics to biomedical applications. Ring-opening polymerization (ROP) of β-lactones is an efficient strategy for the synthesis of PHAs with control of the molar mass, the microstructure and the functionalities of polymers. So, it is a question of modulating these parameters to optimize the physicochemical properties of polymers. This approach allowed the synthesis of new (co)polymers PHAs, exhibiting original functionalities, tacticities (atactic, syndiotactic) and topologies (block, random and alternating copolymers). Tunable catalytic/initiating systems afford the synthesis of α-base, ω-crotonate telechelic atactic PHAs and of the first examples of syndiotactic PMLARs (R = allyl, benzyl and methyl). Similarly, catalytic engineering offers the synthesis of P(BL-b-MLABe) and P(BL-ran-MLABe) copolymers from the simultaneous and sequential copolymerization of the comonomers rac-BL/rac-MLABe. Finally, simultaneous copolymerization of the comonomers (R)-MLAAll/(S)-MLABe, in the presence of yttrium complexes, leads to the synthesis of alternating copolymers P(MLABe-alt-PMLAAll). Further post-polymerization modifications of these copolymers foresee a route towards a new class of functionalized alternating PHAs.

Les composés hydrophobes sont connus comme des sources de carbone qui peuvent être utilisées par les levures comme Yarrowia lipolytica pour de multiples applications. Ces composés causent parfois des perturbations aux levures mais sont aussi rapportés comme conférant aux cellules une certaine résistance contre les stress environnementaux. Dans le cadre de cette thèse, nous avons étudié le rôle de l'oléate de méthyle comme source de carbone sur la résistance de la levure Y. lipolytica en réponse au choc d’un composé amphiphile, la -dodécalactone, et au stress thermique. Les résultats obtenus montrent que les cellules ayant poussé sur oléate sont beaucoup plus résistantes au choc lactone ainsi qu’au stress thermique que les cellules ayant poussé sur glucose. L’action de la lactone se trouve au niveau de la membrane où elle cause une fluidification membranaire et une déplétion de stérols qui sont considérés comme la cause de la mort cellulaire. Ce travail met en évidence le rôle des corps lipidiques dans la réponse cellulaire qui se manifeste de différentes manières en réponse à ces stress. Une accumulation des corps lipidiques est importante pour la résistance de la cellule aux stress. Les cellules ayant poussé sur glucose transforment leur stérol libre en esters de stéryle pour former les corps lipidiques en réponse au choc lactone, ce qui augmente leur sensibilité. Tandis que les cellules ayant poussé sur oléate qui ont accumulé des corps lipidiques pendant leur croissance ont tendance à convertir leurs esters de stéryle en stérol libre pour compenser la déplétion de stérol membranaire causée par la lactone ce qui diminue leur sensibilité. L'homéostasie de l'ergostérol, liée à la présence de corps lipidiques, semble donc jouer un rôle clé dans la résistance cellulaire à ces stress. Ce travail relève aussi que la présence de lipides modifie le processus de mort cellulaire programmée de Y. lipolytica en réponse à un stress thermique. / Hydrophobe compounds are known as carbon source which could be used by yeast like Yarrowia lipolytica for multi purposes. These compounds may cause disturbance in yeast but also reported as confer some resistance to cells towards environmental stress. Here, we study the role of methyl oleate as carbon source on resistance of Y. lipolytica in response to stress of an amphiphilic compound, -dodecalactone, and to heat shock. Results show that cells grown in oleate are more resistant to these stresses than cells grown in glucose. This work reveals the role of the lipids bodies in cells response to stress and that cells manifest in different ways in response to these stresses. An accumulation of lipids bodies is required for the resistance of cells towards stress as glucose grown cells transform their free sterol into steryl esters to form the lipids bodies in response to lactone shock which increases their sensibility towards lactone. In the case of oleate grown cells which accumulated the lipids bodies during their growth, these cells have tendency to convert their steryl esters into free sterol in order to compensate sterol depletion causing by lactone shock and decrease their sensibility. Homeostasis of ergosterol, linked with presence of lipids bodies, seems to be the key for cellular resistance to stresses. This work reveals also that the presence of lipids bodies modifies the processes of programmed cells death in response to heat shock.

Les bactéries sont capables de communiquer entre elles afin de coordonner l’expression de certains gènes en fonction de leur densité de population. Ce système de communication utilise de petites molécules appelées autoinducteurs (AIs) comme messagers chimiques et est connu sous le nom de Quorum Sensing (QS). Chez les bactéries pathogènes, les gènes régulés sont, en particulier, ceux codant pour la production des facteurs de virulence et la structuration des biofilms. Ainsi des inhibiteurs du QS chez ces bactéries pourraient être de nouveaux agents anti-bactériens alternatifs aux antibiotiques actuels. Chez les bactéries à Gram-négatif, les AIs sont très majoritairement des acyl-homosérine lactones (AHLs). Utilisant deux approches, la conception rationnelle et le criblage virtuel, nous avons découvert cinq nouvelles familles d’antagonistes des AHLs, ainsi qu’un certain nombre d’agonistes. Nous avons également préparé des analogues d’antagonistes naturels de la famille des bromo-furanones, afin d’établir une étude structure-activité de ce type de composés. / The bacteria are able to communicate with each other for coordinating gene expression depending on their population density. This communication system use small molecules called autoinducers (AIs) as chemical messengers and is referred to as quorum sensing (QS). In pathogenic bacteria, the regulated genes are, in particular, those coding for the production of virulence factors and biofilms formation. Thus, inhibitors of bacterial QS could be used as new anti-bacterial agents providing an alternative to current antibiotics. In Gram-negative bacteria, the main AIs are acyl-homoserine lactones (AHLs). Using two approaches, rational design and virtual screening, we have discovered five new families of AHLs antagonists, and some agonists. We have also prepared analogues of natural bromo-furanones antagonists, in order to establish a structure-activity study of this type of compounds.