Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T04:53:28Z (GMT). No. of bitstreams: 1 Mantovani_SimoneMoraes_M.pdf: 1495773 bytes, checksum: 493a0576675a8aad3c2879147aa7745f (MD5) Previous issue date: 2007 / Resumo: Nas últimas décadas as reações utilizando biocatalisadores tem sido amplamante aplicadas na síntese orgânica, como componentes chave de muitos processos químicos industriais, levando ao aumento na demanda por novas enzimas. A maneira mais rápida e simples de detectar enzimas é através de metodologias de triagem de alto desempenho (HTS) que permitam identificação rápida da atividade enzimática, como por exemplo, os ensaios utilizando compostos fluorogênicos e cromogênicos. Nesse trabalho nós aplicamos HTS baseado em substratos fluorogênicos para detecção de epóxido-hidrolases e esterases em microrganismos brasileiros. Inicialmente foram selecionados cinco microrganismos com alta atividade epóxido-hidrolase, e 18 pela a presença de esterases. Inspirados nesse principio nós adaptamos a metodolgia conhecida como "Quick E" para a avaliação rápida das enantiosseletividades de epóxido-hidrolases em células íntegras através de medidas das velocidades iniciais de substratos fluorogênicos quirais avaliados separadamente com adição de um competidor. Os ensaios de enantiosseletividade mostraram que os experimentos com competidor apresentaram valores de enatiosseletividade muito próximos dos valores de E determinados via biocatálise convencional. Além disso, alguns microrganismos selecionados por HTS foram testados para reações de biotransformação frente a substratos de interesse sintético, o que permitiu, além da confirmação das atividades enzimáticas e seletividades observadas, detectar a capacidade do microrganismo C. albícans CCT 0776 de desracemizar álcoois secundários por estereoinversão, fornecendo o (S)-1,2-octanodiol com 100 % de rendimento teórico e ee > 99 %, e o (S)-4-fenilmetoxi-1,2-butanodiol com ee 45 % / Abstract: Since the past decades the biocatalysts have been applied in organic chemistry, as key components of many industrial chemical processes, thus increasing the demand for novel enzymes. High-Throughput Screening (HTS), using fluorogenic probes are among the best assays to discovery new enzymes, easily adapted to whole cells format. In this work, have been applied fluorogenic probes to screen epoxide hydrolases and esterases in Brazilian Collection Cultures of microorganisms, which allowed to detect epoxide hydrolases in five microorganisms, and esterases in 18 microrganisms. Additionally, were used chiral probes to implement a Quick E assay, for a fast valuation of epoxide hydrolases enantioselectivity by measuring initial rates of pure enantiomers. Optimization of the methodology revealed that almost true E were obtained by competitive experiments of each enantiomer and a substrate of similar reactivity. The quick E assay was validated by determining conversion and ee using GC/MS and NMR (using mandelic acid derivatives) and is now a new method to determine the enantiomeric ratio for epoxide hidrolases. Finally, the outstanding HTS results were better investigated by conventional catalysis detecting a stereoinversion process performed by C. albicans CCT 0776, which furnished (S)-1 ,2-octanodiol in 100 % theoretical yield and ee of 100%, and (S)-4-fenilmetoxi-1,2-butanodiol in ee of 45% / Mestrado / Quimica Organica / Mestre em Química

A ipalbidina é um alcaloide indolizidínico com propriedades analgésica e antirradicais livres. Este alcaloide possui como fonte natural a Ipomoea alba L., uma espécie de dama-da-noite, sendo isolado das suas sementes na forma de aglicona da ipalbina (ligada à D-glicose). A ipalbidina possui estrutura química relativamente simples, porém sua síntese na forma enantiomericamente pura se apresenta como um desafio sintético. Dentre as inúmeras rotas de síntese da ipalbidina, apenas quatro são enantiosseletivas. Neste trabalho de dissertação é apresentada uma nova estratégia sintética visando a preparação da S-(+)-ipalbidina. A estratégia tem como etapas chaves: uma reação de olefinação (Wittig e Horner-Wadsworth-Emmons); a preparação de uma diazocetona e de α-clorocetonas; e a conversão destas no esqueleto indolizidínico por uma reação de ciclização. Como material de partida para a reação de olefinação foi empregado o S-prolinal protegido (Boc e Cbz). Este aminoaldeído foi empregado como fonte do centro estereogênico e de um dos anéis presentes na estrutura final. A partir das reações de olefinação, foram obtidos compostos carbonílicos α, β-insaturados que, em seguida, sofreram redução da dupla ligação (28-94%). A partir dos produtos hidrogenados, foram preparados ácidos carboxílicos (66-83%) que, em seguida, foram convertidos na diazocetona (32%) e nas α-clorocetonas (34-70% a partir de ilídeos β-ceto sulfoxônios). As α -clorocetonas também foram preparadas a partir de ésteres com bons rendimentos (60-70%). O esqueleto indolizidínico foi obtido a partir da reação de ciclização da diazocetona e das α -clorocetonas, porém os rendimentos não foram satisfatórios e a metodologia deve ser melhorada. Uma α-clorocetona mais simples foi reduzida ao álcool correspondente. A β-cloridrina obtida foi convertida no aminoálcool cíclico (indolizidina monoidroxilada, 16%). A preparação deste aminoálcool cíclico sugere que a metodologia desenvolvida é promissora. A nova estratégia sintética pode levar à síntese da (+)-ipalbidina em seis ou sete etapas (uma rota curta). A funcionalização da α-clorocetona e o melhoramento do método a partir da diazocetona podem também levar à síntese outras indolizidinas. / Ipalbidine is an indolizidine alkaloid with analgesic and antioxidant properties. This alkaloid is isolated from the seeds of Ipomoea alba L., as the aglycone of ipalbine (associated to the D-glucose). The ipalbidine has a relatively simple chemical structure, but its synthesis in the enantiomerically pure form is a synthetic challenge. Although a great number of synthetic routes of ipalbidine have been reported, only four are enantioselective. This dissertation reports new studies toward the synthesis of (+)-ipalbidine. The key steps of the synthetic strategy are: olefination reaction (Wittig or Horner-Wadsworth-Emmons); preparation of a diazoketone and α -chloroketones; and the cyclization reaction from the diazoketone and α -chloroketones. A (Boc and Cbz) protected-S-prolinal was used as the initial reagent. This amino aldehyde was employed as the chiral pool and it provides one the rings necessary to build the indolizidine skeleton. A Wittig reaction (88-94%) and a Horner-Wadsworth-Emmons reaction (60-70%) were evaluated for the first step of the synthesis. α, β-Unsaturated carbonyl compounds were prepared from the olefination reactions and, after that, double bond reduction was carried out under some hydrogenation conditions (28-90%). The carboxylic acids were prepared from the hydrogenated compounds (66-83%). The carboxylic acids were used to prepare the diazoketone (32%) and the α -chloroketones (34-70% from sulfoxonium ylides). The cyclization reaction from the diazoketone and α -chloroketones afforded the indolizidine skeleton, but the yields were not satisfactory. The reaction conditions, in this case, need to have to be improved. The α -chloroketones were also prepared from esters in good yields (60-70%). A simple α -chloroketone was reduced to the corresponding β-chlorohydrin. After the preparation of this β-chlorohydrin, the cyclization afforded a cyclic amino alcohol (a mono hydroxylated indolizidine, in 16% yield). These results suggest a promising methodology. The present strategy on the synthesis of (+)-ipalbidine can provide this alkaloid in six or seven steps (a short route) and can also be applicable to the syntheses of other indolizidine alkaloids.

Orientador: Carlos Roque Duarte Correia / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T19:36:09Z (GMT). No. of bitstreams: 1 Angnes_RicardoAlmir_M.pdf: 5518498 bytes, checksum: 67fa707a73dd9c8251132ea7853f9fd7 (MD5) Previous issue date: 2014 / Resumo: O presente trabalho apresenta a viabilidade da síntese e aplicação de diversas bisoxazolinas quirais com ligantes para arilação de Heck-Matsuda enantiosseletiva. A simetria C2 mostra-se vantajosa para a transferência da informação quiral para o aduto das reações testadas. Além disso, observou-se que o aumento do volume estéreo na série: Me < Bn < i-Pr < i-Bu nos substituintes dos ligantes do tipo 2,2¿-bis(2-oxazolina) possui pouca influência na enantioseletividade, os substituintes t-Bu causaram brusca alteração na reatividade do sistema presumivelmente devido a efeitos estéreos não incluídos no modelo de Chartoon. O potencial da classe de ligantes bis(imidazolinas) quirais foi evidenciado através de um único exemplo, embora tais ligantes ainda encontram pouquíssimas aplicações na literatura. A estrutura absoluta dos adutos descritos previamente no trabalho de Correia foi obtida através de cristalografia de difração de raios-X. Uma metodologia para a formação de estruturas quirais do tipo cis-4-aril-2-ciclopenten-1-ol a partir da dessimetrização do 3-ciclopenten-1-ol por meio de uma reação de Heck-Matsuda enantioseletiva foi brevemente estudada / Abstract: This work shows the feasibility of synthesis and application of several chiral bisoxazoline ligands in enantioselective Heck-Matsuda arylation. The C2 symmetry has shown advantages in the transmission of chiral information to the product of the reaction. Besides, the increment of steric hindrance along the series: Me < Bn < i-Pr < i-Bu on the substituents of 2,2¿-bis(2-oxazoline) type ligands shown little effect to the overall enantioselectivity, however, the use of t-Bu substituints caused great change in the system reactivity presumable through steric effects not included in the Chartoon model. The potential use of chiral bis(imidazoline) ligands is shown through a singles example, those ligands have yet found only a few applications in the literature The absolute structure of the aducts described previously in the work of Correia is obtained by the use of X-ray diffraction experiment. A methodology for synthesis of chiral structures like cis-4-aryl-2-cyclopenten-1-ol through desymmetrization of 3-cyclopent-1-ol by enantioselective Heck-Matsuda reaction has been briefly studied / Mestrado / Quimica Organica / Mestre em Química

Abstract The first part of this monograph discusses the asymmetric, ylide based, reagent controlled epoxidations. Both different chiral ylides and epoxidation processes, stoichiometric and catalytic, are reviewed. In the following part, new chiral sulfide catalysts were discovered as enantioselective catalysts for the Corey-Chaykovsky reaction (epoxidation of aldehydes via sulfonium ylides). Using a crystal structure of an oxazolidine derivative as a starting point, a thiazolidine ligand family was designed, synthesized and finally employed as catalysts in the asymmetric epoxidation of benzaldehyde. The ligands were prepared starting from L-valine, L-tert-leucine, D-penicillamine and L-cysteine. The differently tuned thiazolidine ligands were demonstrated to catalyze the formation of trans-stilbene oxide with varying enantioselectivities. On the basis of these results, a mechanistic rationale for the asymmetric induction was presented. The results heavily demonstrated the importance of ring rigidity as an affecting factor in the enantioselectivity of the tested thiazolidines.

Les cycloisomérisations d’énynes sont des réarrangements de substrats polyinsaturés qui permettent d’accéder à des composées cycliques ou polycycliques en une seule étape. Ces réactions sont catalysées par de nombreux métaux de transition et présentent un grand intérêt synthétique. Cependant, peu de cycloisomérisations énantiosélectives sont décrites dans la littérature. Des platinacycles carbéniques comportant une monophosphine comme ligand chiral ont été développés au laboratoire. Ces complexes ont été utilisés avec succès dans la réaction de cycloisomérisation d’allylamides propargyliques en aza[4.1.0]heptènes ; des excès énantiomériques jusqu’à 97% ont été obtenus. Ce système catalytique a été étendu à la désymétrisation par cycloisomérisation d’allylamides propargyliques comportant deux unités vinyliques (ee jusqu’à 95%). Par la suite, ces catalyseurs ont été appliqués à la cycloisomérisation d’énynes-1,5 comportant un groupement oxygéné en position propargylique. Des excès énantiomériques allant jusqu’à 75% ont été mesurés. / Enyne cycloisomerisations induce the formation of cyclic or polycyclic compounds from unsaturated substrates in a single step. These réactions catalysed by transition metals have a great interest in organic synthesis. However enantioselective versions of thèse reactions remain rare. Carbenic platinacycles with chiral monophosphines as ligands have been developped in our team. These complexes have been successfully used in the cycloisomerisation of allylpropargylamides into azabicyclo[4.1.0]heptenes ; enantiomeric excess up to 97% have been attained. This catalytic system has been extended to the desymetrisation of allylpropargylamides bearing a second vinyl moiety (ee’s up to 95%). These catalysts have been used also in the cycloisomerisation of 1,5-enynes bearing an oxygene fonstion at the propargylic position. Enantiomeric excess up to 75% have been obtained.

8 Abstract Epoxide hydrolases (EHs) demonstrating high degree of enantioselectivity or enantioconvergence are useful biocatalysts for the production of optically active epoxides and vicinal diols, which can serve as chiral building blocks for syntheses of biologically active drugs. EHs can play an important role also in degradations of xenobiotics. Genes encoding EHs Kau2 and Kau8 were expressed in E. coli host strains TOP10 and RE3. Enantioselectivities and regioselectivities of Kau2 and Kau8 in supernatants of desintegrated cells were determined for four substrates: tert-butylglycidyl ether, para-chlorostyrene oxide, para-nitrostyrene oxide, α-methylstyrene oxide. The highest values of enantioselectivity and regioselectivity were achieved with Kau2 and para-nitrostyrene oxide as a substrate. The Kau2 was chosen for further experiments on the basis of these results. Kau2 was overexpressed in the recombinant strain RE3(pSEKau2). We performed two batch cultures and one fed-batch culture in stirred bioreactor. The highest volumetric activity of 4500 U/l was obtained in the case of fed-batch culture. Two phase system consisting of polyethylenglycole 6000 and sodium citrate (pH 7.7) was used for Kau2 purification from the supernatant of desintegrated cells. Purification factor 2.6 +/- 0.3 was achieved and...

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This thesis describes the use of chiral counter-ions for the enantiomeric separation of amines in non-aqueous capillary electrophoresis. The investigations have been concentrated on studies of the influence, of the chiral counter-ion, the solvent, the electrolyte and the analyte, on the enantioselective separation. Modified divalent dipeptides have been introduced in capillary electrophoresis for the separation of amino alcohols and chiral resolution of amines. Association constants for the ion-pair between dipeptide and amino alcohol could be utilized for development of separation systems with higher amino alcohol selectivity. Chiral discrimination (ion-pair formation) between the dipeptides and amines are preferably generated in non-aqueous background electrolytes (BGEs). The amount of triethylamine in the BGE determined the dipeptide charge and a divalent dipeptide promoted higher enantioselectivity than a monovalent dipeptide. An N-terminal-end blocking group and glutamic acid at the C-terminal-end of the dipeptide was important for chiral separation of the amines. Chemometric and univariate methods have been employed for evaluation of suitable solvent compositions in the BGE. An experimental design including a single solvent as well as binary, ternary and quaternary mixtures of polar organic solvents, showed that optimal enantioresolution was obtained with an ethanol:methanol 80:20 mixture in the BGE.  Furthermore, water was found to have an adverse influence on enantioselectivity and no enantioresolution was obtained with BGEs containing more than 30 % water. An alkali metal hydroxide added to the BGE affected the chiral separation by competing ion-pair formation with the selector. The electroosmosis was reduced in order of decreasing alkali metal ion solvated radius and became anodic using K, Rb or Cs in ethanolic BGEs. The correlation between the amino alcohol structure and the enantioselectivity was investigated using chemometrics. The obtained models showed that enantioselectivity for the amino alcohols was promoted by e.g. degree of substitution and substituent size on the nitrogen.

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1). A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.    The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104. Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering. For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.

<p>Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.</p>