Le Ga1NAc est présent dans un grand nombre de glycoprotéines, protéoglycanes et glycolipides. Il est le sucre immunodominant des antigènes de groupes sanguins et oncofoetaux qui sont des cibles thérapeutiques. Cependant, la synthèse par voie chimique de ces glycoconjugués à Ga1NAc est longue et coûteuse. In vivo, le Ga1NAc est incorporé par des Ga1NActransférases, enzymes spécifiques qui transfèrent le Ga1NAc de l'UDP-GaINAc sur un substrat accepteur. Mais la synthèse chimio enzymatique des glycanes est freinée par l'obtention difficile de quantités importantes d'UDP-Ga1NAc.<br />Nous avons mis au point un système de synthèse chimio enzymatique de glycoconjugués à Ga1NAc qui utilise 4 enzymes et des substrats simples comme le Ga1NAc, l'UTP et la créatine-P. Ce système permet la synthèse rapide et efficace de glycopeptides et d'oligosaccharides à GaINAc ; il a été utilisé pour glycosyler l'antigène MUC1 et nous avons pu évaluer la réponse immunitaire murine contre la petite glycoprotéine obtenue.<br />Le système de synthèse d'UDP-Ga1NAc peut accepter certains dérivés de Ga1NAc et permettre ainsi la synthèse d'analogues d'UDP-Ga1NAc. Ces molécules sont des sondes appréciables pour étudier l'interaction entre substrats et enzymes par les techniques physicochimiques comme la STDNMR.

Made available in DSpace on 2016-06-02T20:34:49Z (GMT). No. of bitstreams: 1 5686.pdf: 10016184 bytes, checksum: e6a42772f0db7983f4cc9525851f9c5b (MD5) Previous issue date: 2014-01-17 / Universidade Federal de Sao Carlos / O foco principal desse trabalho foi a implementação, otimização e aplicações práticas de métodos de ressonância magnética nuclear (RMN) com o propósito de avaliar as interações entre moléculas de diferentes massas molares, sendo que essas técnicas foram implementadas pela primeira vez no laboratório de RMN do DQ-UFSCar. Existem várias abordagens que podem ser utilizadas com esse propósito, e dentre elas, destacamos: STD NMR, Tr-NOESY, WaterLOGSY, SALMON, INPHARMA, DOSY, SAR. Esses métodos são muito úteis para detectar mudanças de comportamento, a nível molecular, quando adicionamos macromoléculas em um meio contendo somente micromoléculas. O entendimento desse comportamento molecular ajuda a desvendar sistemas complexos de interações moleculares existentes no corpo humano, e que, são muito importantes para o descobrimento de novos medicamentos. O primeiro passo para a implementação das técnicas foi a utilização da proteína de soro bovino (do inglês BSA) e proteína de soro humano (do inglês HSA) como fonte de macromoléculas e micromoléculas orgânicas isoladas da fração etanólica do extrato bruto (1 mg) de Rauia resinous e da fração acetato de etila de Strypnodendron polyphyllum, utilizando cromatografia líquida de alta eficiência, extração por fase sólida e a ressonância magnética nuclear (CLAE-EFS/RMN) para a completa elucidação estrutural quando necessário. As técnicas utilizadas foram: saturation transfer difference (STD), transfer nuclear Overhauser spectroscopy (Tr-NOESY) e STD-TOCSY (total correlation spectroscopy). Essa mesma metodologia além de representar um importante mecanismo para avaliar as interações entre moléculas, também pode ser utilizada para outras matrizes variando tanto as macro quanto as micromoléculas. / The main focus of this work was the implementation, optimization and practical applications of nuclear magnetic resonance (NMR) methods for the purpose of evaluating the interactions between molecules of different masses, and these techniques were implemented for the first time in the laboratory NMR DQ - UFSCAR. There are a number of ligand-based screening approaches available, such that, STD NMR, Tr-NOESY, WaterLOGSY, SALMON, INPHARMA, DOSY, SAR by NMR etc. These methods are sensitive towards the perturbations as results of the macromolecular addition in a medium containing the small molecules. The molecular understanding of this behavior helps to uncover the complex systems of molecular interactions existing in the human body, which are very important for the discovery of new medicines. In the first step while implementing these techniques, Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) as a source of organic macromolecules were used. While, for the small organic molecules, a 1 mg crude extract from the hydroethanolic fraction of the Rauia resinous and ethyl acetate fraction of Strypnodendron polyphyllum was utilized, however, for the complete structural characterization, solid phase extraction following high-pressure liquid chromatography in an integrated fashion and then nuclear magnetic resonance (LC-SPE/NMR) was employed when necessary. On the other hand, the ligand screening techniques used were the saturation transfer difference (STD), Nuclear Overhauser Transfer Effect SpectroscopY (Tr- NOESY), Diffusion-Order Spectroscopy (DOSY) and STD- TOCSY (TOtal Correlation SpectroscopY). More importantly, this methodology also represents an important mechanism to evaluate the interactions between molecules or the first hand detection of the active constituents/inhibitors, which can also be used for other matrices varying both the macro and the small molecules.

The research work reported in the thesis deals with the development of dynamic covalent systems and their applications in evaluation and screening of protein-ligands and enzyme inhibitors, as well as in synthetic methodologies. The thesis is divided into four parts as described below. In part one, synthetic methodologies to access 3-functionalized phthalides and 3-thioisoindolinones using the concept of cascade reactions are demonstrated. Efficient syntheses of the target products are designed and performed in one-pot process under mild reaction conditions.  In part two, phosphine-catalyzed disulfide metathesis for the generation of dynamic carbohydrate system in aqueous solution is demonstrated. In the presence of biological target (Concanavalin A), the optimal dynamic ligand is successfully identified in situ by the 1H STD-NMR spectroscopy. In part three, lipase-catalyzed resolutions of dynamic reversible systems using reversible cyanohydrin and hemithioacetal reactions in one-pot processes are demonstrated. The dynamic systems are generated under thermodynamic control in organic solution and subsequently resolved by lipase-mediated resolution under kinetic control. The resolution processes resulted in the lipase-selected substrates with high structural and stereochemical specificities. In the last part, dynamic fragment-based strategy is presented using β-galactosidase as a model target enzyme. Based on our previous study, the best dynamic inhibitor of β-galactosidase was identified using 1H STD-NMR technique from dynamic hemithioacetal systems. The structure of the dynamic inhibitor is tailored by fragment linking and optimization processes. The designed inhibitor structures are then synthesized and tested for inhibition activities against β-galactosidase. / QC 20110526

Orientador: Ljubica Tasic / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T16:55:17Z (GMT). No. of bitstreams: 1 Prando_Alessandra_D.pdf: 9977861 bytes, checksum: 15d25bce7d95433f95b938e68fdeaac8 (MD5) Previous issue date: 2012 / Resumo: Até o momento, pouco se sabe sobre os mecanismos de virulência da bactéria Xanthomonas axonopodis pv. citri (XAC), agente causador do cancro cítrico. Acredita-se que chaperonas de secreção (CS) estão envolvidas no processo de patogenicidade de XAC primeiramente formando complexos com fatores de virulência e auxiliando no encaminhamento desses para os sistemas de secreção utilizando o ATP como fonte de energia. Neste trabalho foram adquiridos dados de fluorescência de emissão, dicroísmo circular, desenovelamento térmico e de ressonância magnética nuclear de H NMR e de 2D {N,H} HSQC de duas proteínas da XAC, a XAC1990 (FlgN) e XACb0033. Para ambas proteínas foram propostas estruturas 3D usando a análise de footprinting com restrições SASA e rmsd. Para as estruturas propostas foi verificado que os dados de fluorescência corroboram com a estrutura 3D não ocorrendo o mesmo para os dados de CD e NMR que revelaram baixo conteúdo helicoidal além de ausência de estrutura 3 D. A interação da proteína FlgN com a sua proteína parceira FlgK também foi sugerida através das análises de CD e fluorescência. Na segunda parte do trabalho foram estudadas as interações entre a proteína Hsp90 da laranja com diferentes ligantes aplicando a técnica de Saturation Transfer Difference (STD-NMR) e espectroscopia de fluorescência. Estas análises revelaram dados que corroboraram com o modelo proposto e, além disso, indicaram que os hidrogênios H-8 e H-2 da adenina e H-1'da ribose estão localizados no sítio ligante da proteína com os fosfatos orientados para fora. Através da fluorescência foram calculados os valores de Kd e foi verificado que a geldanamicina é um potente inibidor de Hsp90 da laranja / Abstract: So far, the Xanthomonas axonopodis pv. citri (XAC) mechanisms of bacterial virulence is unknown. It is believed that secretion chaperones (CS) are involved in the XAC's virulence process by first forming complexes with virulence factors, and assisting in their presentation to corresponding secretion systems using ATP as a source of energy. Fluorescence emission, circular dichroism, thermal unfolding and nuclear magnetic resonance NMR H and 2D {N,H} HSQC data from two proteins of XAC, XAC1990 (FlgN) and XACb0033 were collected. For both proteins, 3D structures were proposed using the footprinting analysis with RMSD and SASA restrictions. For the proposed structures were verified which the fluorescence data were consistent with the 3D structure. The CD and NMR data revealed low-helical content and absence of 3D structure. The interaction of the protein FlgN with its partner, FlgK, was suggested by CD and fluorescence analysis. In the second part, the interactions between the orange's Hsp90 protein with different ligants using Saturation Transfer Difference (STD-NMR) and fluorescence spectroscopy techniques were studied. These analyzes revealed which the data were consistent with the proposed model and moreover showed that the adenine's hydrogens H-8 and H-2 and ribose's hydrogen H-1'are located in the protein binding site with the phosphate driven out. By fluorescence values were calculed Kd and it was verified that geldanamycin is a potent inhibitor of orange's Hsp90 / Doutorado / Quimica Organica / Doutor em Ciências

This thesis deals with the design, formation and evaluation of dynamic systems constructed by means of sulfur-containing reversible reactions, in organic and aqueous media and under mild conditions. In a first part, the synthesis of thioglycoside derivatives, constituting the biologically relevant starting components of the dynamic systems, is described. In addition, the pD-profile of the mutarotation process in aqueous media for a series of 1-thioaldoses is reported and revealed an astonishing beta-anomeric preference for all the carbohydrate analogs under acidic or neutral conditions. In a second part, the phosphine-catalyzed or -mediated disulfide metathesis for dynamic system generation in organic or aqueous media is presented, respectively. The direct in situ 1H STD-NMR resolution of a dynamic carbohydrate system in the presence of a target protein (Concanavalin A) proved the suitability and compatibility of such disulfide metathesis protocols for the discovery of biologically relevant ligands. In a third part, hemithioacetal formation is demonstrated as a new and efficient reversible reaction for the spontaneous generation of a dynamic system, despite a virtual character of the component associations in basic aqueous media. The direct in situ 1H STD-NMR identification of the best dynamic beta-galactosidase inhibitors from the dynamic HTA system was performed and the results were confirmed by inhibition studies. Thus, the HTA product formed from the reaction between 1-thiogalactopyranose and a pyridine carboxaldehyde derivative provided the best dynamic inhibitor. In a fourth and final part, a dynamic drug design strategy, where the best inhibitors from the aforementioned dynamic HTA system were used as model for the design of non-dynamic (or “static”) beta-galactosidase inhibitors, is depicted. Inhibition studies disclosed potent leads among the set of ligands. / QC 20100621