Orientador: José Augusto Rosário Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T13:10:57Z (GMT). No. of bitstreams: 1 Nasario_FabioDomingues_M.pdf: 3028989 bytes, checksum: 95b2af06aacbc6c6c5e2ba6bc63d6acf (MD5) Previous issue date: 2013 / Resumo: A desracemização de alcoóis secundários por estereoinversão utilizando apenas um microrganismo é um processo ambientalmente compatível que se enquadra nos princípios da química verde, e apresenta grande interesse visto a utilização desses compostos enantiomericamente puros na síntese de fármacos e outras moléculas biologicamente ativas. Nesse estudo foram empregadas as leveduras S. cerevisiae e C. albicans CCT 5847/18804, separadamente para realizar a desracemização de alguns alcoóis secundários como 1-feniletanol, mandelato de etila, 3-fenil-3-hidroxipropanoato de etila, 4-fenil-2-hidroxibutirato de etila. Esse processo mostrou-se bastante eficiente para obtenção de alcoóis enantiomericamente enriquecidos, visto que dispensa a utilização de enzimas isoladas e uso de custosos cofatores. Foi possível obter (R)-1-feniletanol 90% rendimento cromatográfico ee 99% com desracemização do rac-1-feniletanol catalisada por C. albicans, e o outro enantiômero (S)-1-feniletanol a partir da redução de acetofenona com S. cerevisiae tipo II com 75% de conversão e 96% ee. A desracemização do 4-fenil-2-hidroxibutirato de etila foi possível com C. albicans e S. cerevisiae com apenas 2 horas de reação obteve-se o (S)-4-fenil-2-hidroxibutirato de etila com ee de 96% e rendimento de 99%. Foi possível obter o (S)-mandelato de etila ee de 96% e 90% de rendimento a partir da desracemização com C. albicans e o outro enantiômero com redução do respectivo ceto-ester com S. cerevisiae. O (S)-3-hidroxi-3-fenilpropionato de etila foi produzido a partir da desracemização de 3-hidroxi-3-fenilpropionato de etila com S cerevisiae tipo II após 2 dias de reação com 96% ee e 75% de rendimento. O processo de desracemização de alcoóis secundários utilizando um único microrganismo mostrou-se eficiente para obtenção de compostos quirais com alta economia de átomos e baixo fator de impacto ambiental para a maioria dos substratos estudados / Abstract: The deracemization of secondary alcohols by stereoinversion using only one microorganism is an environmentally compatible process that fits the principles of green chemistry and presents great interest since the use of these compounds in the synthesis of enantiomerically pure drugs and other biologically active molecules. In this study we employed the yeasts of S. cerevisiae and C. albicans CCT 5847/18804 separately to perform deracemization of some secondary alcohols such as 1-phenylethanol, ethyl mandelate, ethyl 3-hydroxy-3-phenylpropanoate, ethyl 2-hydroxy -4-phenylbutyrate. This process proved to be very efficient for obtaining enantiomerically enriched alcohols, since it eliminates the use of isolated enzymes and expensive cofactors. It was possible to perform a deracemization of 1-phenylethanol and obtain 90% of the (R)-1-phenylethanol in 99% ee by C. albicans, and the other enantiomer (S)-1-phenylethanol from the enatioselective reduction of acetophenone with S. cerevisiae with 75% conversion and 96% ee. The deracemization of ethyl 2-hydroxy -4-phenylbutyrate was possible to perform with C. albicans and S. cerevisiae with only 2 hours of reaction to obtain the (S)- ethyl 2-hydroxy -4-phenylbutyrate (ee of 96% and yield of 100%) , which is impressive, since the production of (S)- ethyl 2-hydroxy -4-phenylbutyrate may be performed in any chemistry laboratory, since S. cerevisiae type II is lyophilized and dispenses use of sterile materials and laminar flow hood. It was also possible to obtain (S)-ethyl mandelate (ee of 96% and yield of 90%) by deracemization of ethyl mandelate with C. albicans and other enantiomer by reduction of the corresponding ketone with S. cerevisiae. (S) ethyl 3-hydroxy-3-phenylpropionate (96% ee and 75% yield) was produced by deracemization of ethyl 3-hydroxy-3-phenylpropanoate with S cerevisiae Type II after 2 days of reaction. The deracemization of secondary alcohols using a single microorganism was effective for obtaining chiral compounds with high atomic efficiency and low environmental factor to most substrates studied / Mestrado / Quimica Organica / Mestre em Química

The use of enzymes as catalysts in organic synthesis constitutes an attractive alternative to conventional chemical catalysis. Enzymes are non-toxic and biodegradable and they can operate under mild reaction conditions. Furthermore, they often display high chemo-, regio- and stereoselectivity, enabling specific reactions with single product outcome. By the use of protein engineering, enzymes can be altered for the specific needs of the researcher. The major part of this thesis describes engineering of lipase A from Candida antarctica (CalA), for improved enantioselectivity in organic synthetic transformations. The first part of the thesis describes a highly combinatorial method for the introduction of mutation sites in an enzyme library. By the simultaneous introduction of nine mutations, we found an enzyme variant with five out of the nine possible mutations. This quintuple variant had an enlarged active site pocket and was enantioselective and active for our model substrate, an ibuprofen ester. This is a bulky substrate for which the wild-type enzyme shows no enantioselectivity and very poor activity. In the second part of the thesis, we continued our approach of combinatorial, focused enzyme libraries. This time we aimed at decreasing the alcohol pocket of CalA, in order to increase the enantioselectivity for small and medium-sized secondary alcohols. The enzyme library was bound on microtiter plates and screened by a transacylation reaction in organic solvent. This library yielded an enzyme variant with high enantioselectivity for the model substrate 1-phenyl ethanol, and high to excellent selectivity for other alcohols tested. Screening in organic solvent is advantageous since a potential hit is more synthetically useful. In the third part of the thesis, we used manipulated beads of controlled porosity glass (EziG™) for enzyme immobilization, and demonstrated the generality of this carrier for several enzyme classes. EziG™ allowed fast enzyme immobilization with simultaneous purification and yielded active biocatalysts in all cases. The last project describes the function of the proposed active site flap in CalA. In our study, we removed this motif. The engineered variant was compared to the wild-type enzyme by testing the amount of interfacial activation and the selectivity for certain alcohols. We showed that the motif is indeed controlling the entrance to the active site and that the flap is not part of the enantioselectivity determining machinery. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Microarrays on gold have been used to develop new methodologies for biocatalysis and proteomic applications. The technology applies the logic of solid phase supported chemistry using self-assembled monolayers (SAMs) on a gold chip. The advantages of this technology are: i) an easy to handle platform, ii) parallel screening (64 reactions at once), iii) microliter scale reactions (1µL per sample), iv the use of mild conditions (buffers and t=37 °C), v) the absence of purification steps (only chip washing is required), vi) quick and accurate analysis by MALDI ToF MS.The metallopeptidase thermolysin was studied in peptide-coupling reactions to profile reactivity and specificity (Chapter 2). Reactivity was further investigated in transpeptidation reactions. Comparing the serine peptidase chymotrypsin with the zinc dependent thermolysin, it was found that transpeptidations proceed via N-transacylation reactions independent of a specific enzymatic catalytic mechanism (Chapter 3). These transacylations may be exploited for modifications of biocompatible and selective surfaces in ‘bottom-up’ bionanofabrication technologies. Selected peptidases with different catalytic mechanism were also arrayed to investigate polymerisation ability of dipeptides (Chapter 4). It was shown that oligomerisation can be obtained under mild conditions and a set of peptides was synthesised. Chapter 5 describes a new chemical methodology by which crude tryptic peptide digests can be trapped on chip and analysed by MALDI ToF MS without further purification steps. This dramatically improves time and cost efficiency.Finally, a new stepwise native chemical ligation methodology is proposed for amino acids, and peptides containing N-terminal cysteine residues (Chapter 6).

Cytochromes P450 (P450s) are a superfamily of heme-thiolate monooxygenases. They catalyse a wide variety of reactions on a vast number of substrates and are of particular interest for biocatalyst development due to their ability to oxidise non-activated C-H bonds. Fusion of a P450 to a suitable redox partner protein produces a catalytically self-sufficient enzyme and removes the need to produce electron transfer proteins separately. The well-studied bacterial protein P450cam (Pseudomonas putida) has been fused to the reductase (RhFRed) from the natural fusion protein P450-RhF (Rhodococcus sp.). The P450cam-RhFRed system catalyses the oxidation of camphor and several non-natural substrates and served as the basis for P450cam re-engineering in this current project, with the aim of expanding the substrate scope towards a more mammalian-like activity. The P450cam active site was partitioned into seven paired amino acids and each pair randomised in turn to generate seven sub-libraries of P450cam variants. These were screened for activity using a specially developed colony screen for detection of the blue pigment indigo. In total 94 new variants were identified and then pooled for secondary screening on a number of new substrates, identifying potentially novel activities within the ‘indigo positive’ population. In a separate ‘chimeragenesis’ approach substrate recognition sites (SRSs) within P450cam were targeted for exchange with equivalent portions from a number of human P450s. The B’ helix and F-G loop regions from CYPs 1A2, 2C8, 2D6 and 3A4 were grafted onto the P450cam structure and several of the B’ helix swaps were produced as soluble proteins. The P450cam-2C8-B’-RhFRed chimera gave a Soret peak at 420 nm in the Fe(II)-CO state although an additional substitution next to the proximal cysteine appeared to restore a P450-like state. SRS-exchange therefore offered some insight into structural modularity in P450s, providing a basis for further biocatalyst development.

Amine transaminases are enzymes that catalyze the mild and selective formation of primary amines, which are useful building blocks for biologically active compounds and natural products. In order to make the production of these kinds of compounds more efficient from both a practical and an environmental point of view, amine transaminases were incorporated into multi-step one-pot reactions. With this kind of methodology there is no need for isolation of intermediates, and thus unnecessary work-up steps can be omitted and formation of waste is prevented. Amine transaminases were successfully combined with other enzymes for multi-step synthesis of valuable products: With ketoreductases all four diastereomers of a 1,3-amino alcohol could be obtained, and the use of a lipase allowed for the synthesis of natural products in the form of capsaicinoids. Amine transaminases were also successfully combined with metal catalysts based on palladium or copper. This methodology allowed for the amination of alcohols and the synthesis of chiral amines such as the pharmaceutical compound Rivastigmine. These examples show that the use of amine transaminases in multi-step one-pot reactions is possible, and hopefully this concept can be further developed and applied to make industrial processes more sustainable and efficient in the future. / <p>QC 20170113</p>

The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity. For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines. Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed. Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed. One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents. / <p>QC 20161017</p>

Orientador: Jose Augusto Rosario Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T19:55:40Z (GMT). No. of bitstreams: 1 Zampieri_LuizArthur_M.pdf: 1219190 bytes, checksum: 50f1c90b246b806bd16bb30df4ecbbaf (MD5) Previous issue date: 2006 / Resumo: Neste trabalho estudou-se a bioxidação do sesquiterpeno valenceno (C15H24), produzindo o bioflavorizante nootkatona (C15H22O), buscando as condições ótimas para obtenção do máximo rendimento. Esta reação foi realizada utilizando dois sistemas enzimáticos distintos: o sistema lacase/mediador (LMS) de Trametes versicolor com mediador HBT (hidroxibenzotriazol) ou TEMPO (tetrametilpiperidin-N-oxil) e o sistema utilizando o complexo enzimático do citocromo P-450 de Chaetomium globosum. Outros microorganismos testados foram Botrytis cinerea, Mortierella isabellina e Mortierella ramaniana. As diversas variáveis (pH, tempo de reação, concentrações de enzima, mediadores e indutores, condições de aeração, entre outras) envolvidas nas respectivas reações foram estudadas através de planejamento fatorial e modelagem de superfície de resposta. A utilização do sistema LMS de Trametes versicolor mostrou ser uma ferramenta viável para obtenção de nootkatona a partir de valenceno, embora tenhamos obtido rendimento inferior (17%, agitador orbital em pequena escala e 15 % em escala preparativa, sob aeração externa) descrito na literatura (25%, sob aeração, escala preparativa), o que torna nosso procedimento pouco viável para utilização em maior escala, apesar disso, os resultados foram condizentes com os obtidos em reações semelhantes descritos na literatura científica, onde o rendimento de sistemas LMS dificilmente ultrapassa os 15%. O sistema enzimático CYP-450 apresentou rendimento inferior ao sistema lacase/mediador e, este último sistema, HBT mostrou ser um mediador mais eficiente que TEMPO. / Abstract: In this work, we study the sesquiterpene valencene bioxidation (C15H24), which produces the nootkatona biological flavor substance (C15H22O), in an attempt to achieve the best conditions for optimum yield. This reaction was carried out using two different enzymatic systems: the Trametes versicolor laccase-mediator system with HBT mediators (hydroxybenzotriazol), or TEMPO (tetramethyl-piperidine-N-oxide), and the system using the Chaetomium globosum cythocrome P-450 enzymatic complex. Other microorganisms were tested, such as Botrytis cinerea, Mortierella isabellina and Mortierella ramaniana. The different variables involved in the respective reactions were studied by means of factorial planning and modeling the response system. The use of the Trametes versicolor LMS system proved to be a viable tool to obtain nootkatone from valencene, although we have obtained an inferior yield (17% with orbital agitator in small scale and 15% in preparatory scale, under external aeration) in comparison with the highest value described in literature (25% under aeration). Thus, our procedure presents little viability for large scale use, although results were in agreement with those obtained in similar reactions described in scientific literature, in which the yield produced by LMS systems rarely exceeds 15%. The CYP-450 enzymatic system presented lower yield in comparison with the laccase-mediator system and in the latter, HBT turned out to be more efficient than TEMPO. / Mestrado / Quimica Organica / Mestre em Química

Dans le domaine de la chimie des parfums, l’optimisation des propriétés biologiques et sensorielles de substances naturelles complexes, via la biocatalyse, présente un fort intérêt. Dans un contexte de chimie durable, ces travaux de recherche sont dédiés aux développements de modifications enzymatiques de composés purs, d’extraits et d’huiles essentielles de l’industrie des arômes et parfums. Une étude bibliographique a ainsi été consacrée à la composition des matières premières naturelles, leurs propriétés et les principales voies de biosynthèse des composés présents dans les substances naturelles complexes, ainsi que quelques éléments de réglementation. Dans un premier volet, selon un procédé de chimie durable, le but a été de rendre des produits naturels plus sains tout en conservant leurs propriétés et leur « naturalité ». La détoxification en atranol et en chloroatranol de l’extrait de mousse de chêne a été effectué menant à des absolues de mousse de chêne modifiées par biocatalyse conservant leur qualité odorante. Dans le cadre d’un projet collaboratif public-privé, des protocoles de suivis de transformations biocatalytiques ont été établis et mis en œuvre. Dans un troisième volet, des méthodologies de chimie durable ont été mises à profit afin de proposer de nouveaux ingrédients grâce à l’utilisation de procédés biotechnologiques procédant selon le principe d’économie circulaire. Le caractère naturel a été conservé et la méthode a permis de réaliser des transformations fines et ciblées pour développer des facettes olfactives intéressantes. / In the field of flavor and fragrance industry, the optimization of natural flavoring essential oils and extracts’ properties by biocatalysis is really interesting. In a context of sustainable chemistry, this research project is dedicated to the development of pure compounds, extracts and essential oils by enzymatic modifications. In this way, a bibliographic study has been carried out on the composition of the natural raw materials, their properties and the main biosynthetic pathways of the compounds present in the natural complex substances and some regulation elements. In the first place, according to a process of sustainable chemistry, the goal is to make healthier natural products while keeping their properties and their "naturalness". The detoxification in atranol and chloroatranol of the oak moss extract was carried out with the oak moss absolute by biocatalysis preserving their olfactory quality. Through an academic /industrial collaboration, protocols for monitoring biocatalytic transformations were established and implemented. In a third part, sustainable chemistry methodologies were used to propose new ingredients through the use of biotechnological processes based on the circular economy principle. The natural character has been preserved and the method allowed targeted transformations to develop interesting olfactory facets.

Les macrocycles représentent une catégorie chimique unique en chimie organique et ils possèdent des applications dans les industries pharmaceutique et agrochimique, en parfumerie, et dans les matériaux. Une propriété importante des macrocycles est la possibilité de démontrer de la chiralité planaire menant à des atropoisomères distincts aux propriétés uniques. Très peu de techniques générales existent pour le contrôle de l’atropoisomérisme au sein des macrocycles, rendant leur synthèse un véritable défi. Nous avons accompli le premier exemple d’une synthèse biocatalytique énantio- et atroposélective de p-cyclophanes planaires chiraux. En utilisant une lipase immobilisée commercialement disponible (CALB) et des matériaux de départ pro-chiraux simples, nous avons été en mesure de générer 23 différentes structures avec des rendements entre 11 et 88 %. Des analyses SFC ont permis l’évaluation de l’énantioenrichissement des différents macrocycles, étant compris entre 96 et >99 % ee. Surtout, les macrocycles planaires chiraux ayant des substituants de type halogène ou borylé peuvent subir de la diversification moléculaire au-delà des limites tolérées par l’enzyme. Notre découverte ouvre la porte à l’utilisation de biocatalyseurs pour le contrôle de l’atropoisomérisme lors de la formation de structures macrocycliques. / Macrocycles represent a unique chemotype in organic chemistry, with applications ranging from pharmaceuticals, agrochemicals, aromachemicals and material science. An important property of macrocycles is the possibility of displaying planar chirality yielding distinct atropisomeric structures with unique properties. Very few generalized techniques capable of controlling atropisomerism in macrocycles exist, rendering their synthesis extremely challenging. We have achieved the first example of enantio- and atroposelective biocatalytic synthesis of planar chiral p-cyclophanes. Employing a commercially available immobilized lipase (CALB) and simple pro-chiral starting materials, we were able to generate 23 different structures with yields ranging from 11 to 88 %. SFC analysis permitted evaluation of the enantioenrichment of the different macrocycles, which ranged from 96 to >99 % ee. Importantly, planar chiral macrocycles having halogen or borylated substituents are capable of molecular diversification outside the boundaries of what may be tolerated by the enzyme. Our discovery paves the way for the use of biocatalysts in the control of atropisomerism during macrocycle formation.

Les galactofuranoconjugués, bien que xénobiotiques chez les mammifères, sont des constituants cruciaux de la paroi cellulaire de nombreux micro-organismes pathogènes. La présence de ces motifs font des galactofuranoconjugués des cibles de choix pour le développement d’outils de lutte ou de diagnostic contre ces micro-organismes et leurs maladies associées. Au cours de ces travaux de thèse, une nouvelle stratégie de synthèse utilisant une α-Larabinofuranosidase, capable de reconnaître un mime du motif D-galactofuranose a été utilisée, la CtAraf51 de Ruminiclostridium thermocellum. Des premiers travaux de mutagénèse ont été entrepris, afin d’améliorer l’affinité de l’enzyme pour le motif non naturel D-Galf, sur trois résidus acides aminés de la poche catalytique, identifiés par des études de modélisation. La création de banques de mutants, le criblage de leur activité et la détermination des paramètres cinétiques nous ont permis d’identifier plusieurs mutants à fort potentiel pour le développement d’une néogalactufuranosidase. Dans le but d’étendre l’activité de l’enzyme à d’autres réactions que l’hydrolyse, l’autocondensation et la transglycosylation, un second axe de recherche a été exploré. La mise au point d’une réaction standard à partir du mutant thioglycoligase et le crible d’accepteurs nucléophiles ont permis d’identifier de nouvelles réactions de thioligation et d’acylation. Enfin les deux axes de recherche ont été mis en commun et ont permis de synthétiser de nouveaux S- et O-galactofuranoconjugués de manière efficace par voie biocatalytique. / Galactofuranoconjugates, while xenobiotic in mammals, are crucial constituents on the cell walls of pathogenic microorganisms. The presence of these patterns, in these microorganisms, makes them molecular targets for the development of tools to fight or prevent the associated diseases. This work describes a new strategy to access galactofuranoconjugates mimetic, using an α-Larabinofuranosidase, the CtAraf51 from Ruminiclostridium Thermocellum. Initial mutagenesis work was undertaken, to improve the affinity of the enzyme for the unnatural DGalf motif. Three amino acid residues in the catalytic pocket were identified by modeling studies and subsequently mutated. The screening of activity of the resulting mutants and the determination of kinetic parameters allowed us to identify several mutants with high potential for the development of a néogalactufuranosidase. A second area of research has been explored with the aim to extend the activity of the enzyme to others reactions than hydrolysis, self-condensation and transglycosylation. The optimization of standard condition from the thioglycoligase mutant and the screening of nucleophilic acceptors has led to the identification of new thioligation and acylation reactions. Finally, the two previous researches were combined in order to synthesize new S- and Ogalactofuranoconjugates in an efficient way.