A fosfatase Cdc25B atua na progressão do ciclo celular através da ativação de complexos Cdk/Ciclina. Atualmente, nos modelos estruturais propostos do domínio catalítico da Cdc25B não estão incluídos os últimos 16 resíduos da região C-terminal. Este segmento tem importante papel no reconhecimento do substrato proteico e pode estar envolvido na complexação de pequenas moléculas com a Cdc25B. Assim, o principal objetivo desta tese foi avaliar a flexibilidade conformacional do domínio catalítico completo da Cdc25B em solução através de simulações computacionais e por medidas experimentais de ressonância magnética nuclear (RMN). A similaridade entre as estruturas cristalográficas e em solução foi confirmada pela previsão de ângulos diedrais φ/ψ da cadeia principal a partir dos deslocamentos químicos (CS) e pela concordância entre os acoplamentos dipolares residuais (RDC) medidos e calculados a partir da geometria cristalina. Medidas de parâmetros de relaxação de 15N e RDC evidenciaram a presença de desordem conformacional na região C-terminal, em acordo com a ausência de densidade eletrônica desse segmento no experimento de difração de raios-X. Através da comparação entre CS experimentais e previstos de simulações de dinâmica molecular (DM) longas (total de 6µs de duração) foram apontados artefatos de cristalização, possíveis erros nos campos de força usados nas simulações, falhas na composição do sistema simulado e estados conformacionais populados pela Cdc25B em solução distintos da geometria cristalográfica. De maneira geral, os CS previstos a partir das simulações para a flutuação estrutural dos resíduos da região C-terminal desordenada estão em acordo com os valores experimentais, sugerindo que os estados conformacionais deste segmento foram razoavelmente bem amostrados nas simulações. Em particular, verificou-se que o contato tipo cátion-π entre as cadeias laterais dos resíduos 550W do C-terminal desordenado e 482R do núcleo proteico, ausente na estrutura cristalográfica, pode ser importante em solução. A formação desse contato na simulação de DM também está de acordo com medidas experimentais de perturbação de deslocamentos químicos (CSP) entre construções completa e truncada do domínio catalítico da Cdc25B. Assim, através do uso conjunto de simulações computacionais e medidas experimentais foi possível obter uma representação mais completa e realista da flexibilidade conformacional do domínio catalítico da Cdc25B em solução, incluindo a determinação de possíveis contatos intramoleculares entre a região C-terminal desordenada e o núcleo proteico. Essas informações poderão ser usadas na construção de um ensemble conformacional da Cdc25B. / Cdc25B phosphatase acts on the progression of cell cycle through the activation of Cdk/Cyclin complexes. Currently, the proposed structural models of Cdc25B catalytic domain lack the last 16 residues from the C-terminal region. This segment is important for protein substrate recognition and might be involved in small molecule binding to Cdc25B. Thus, the main goal of this thesis was to evaluate the conformational flexibility of the complete catalytic domain from Cdc25B through computer simulations and experimental nuclear magnetic resonance (NMR) measurements. Similarity between crystal and in solution structures was confirmed by the prediction of backbone φ/ψ dihedral angles from chemical shifts (CS) and by the agreement between observed and back-calculated residual dipolar couplings (RDC). 15N relaxation and RDC measurements pointed to the conformational disorder of the C-terminal region, in agreement with the X-ray diffraction experiment where this segment showed no electronic density. Comparison between experimental and predicted CS from long molecular dynamics (MD) simulations (6µs total running time) pointed to the presence of crystallographic artifacts, possible deficiencies in simulation force fields, inaccurate composition of the simulated system and conformational states visited by Cdc25B in solution that were not observed in the crystallographic geometry. Generally, CS predicted from simulations for the structural fluctuation of the disordered C-terminal region were in agreement with experimental values, suggesting that the simulations sampled the conformational states populated by this segment reasonably well. In particular, a cation-π contact not observed in the crystal structure between side chains of residue 550W from the disordered C-terminal tail and residue 482R from the protein core might be important in solution. This contact is also in agreement with experimental chemical shift perturbations (CSP) measured between complete and truncated constructs of Cdc25B catalytic domain. Therefore, the joint use of computer simulations and experimental measurements allowed the achievement of a more complete and realistic representation of the conformational flexibility of the Cdc25B catalytic domain in solution, including intramolecular contacts between the disordered C-terminal region and the protein core. This information might be used to obtain a conformational ensemble of Cdc25B.

A presente tese trata da análise conformacional e estudo das interações eletrônicas de algumas 2-(fenilseleno)-2-(etiltio)acetofenonas (série I), 2-(fenilseleno)-2-(etilsulfinil)- acetofenonas [série IIa para (CRSR/CSSS) e série IIb para (CRSS//CSSR)] e 2-(fenilseleno)-2- (etilsulfonil)acetofenonas (série III) contendo substituintes doadores e atraentes de elétrons, além de hidrogênio na posição para do grupo fenacila. Foi constatada a ocorrência de isomerismo conformacional por espectroscopia no infravermelho em solventes de constante dielétrica crescente (n-C6H14, CCl4, CHCl3, CH2Cl2, CH3CN) em todas as séries estudadas. Os resultados obtidos por infravermelho são concordantes com as estruturas obtidas por otimização estrutural no nível de teoria B3LYP/6-31+G(d,p) com o efeito do solvente simulado pelo método PCM. As interações eletrônicas foram investigadas a partir dos contatos interatômicos e por meio da análise das interações orbitalares (NBO). Foram obtidas as estruturas cristalográficas de alguns compostos selecionados das séries IIa, IIb e III por difração de raio-X. Na série I, o confôrmero mais estável é caracterizado pelas interações orbitalares entre carbonila e enxofre, com o grupo etiltio numa geometria sinclinal em relação à carbonila, enquanto o grupo fenilselenila se aproxima da carbonila devido à repulsão eletrostática ser pequena. O efeito do solvente nas populações relativas dos confôrmeros ao longo da série I é menor que nas demais séries. As interações são significativamente modificadas com o grupo etilsulfinila (séries IIa e IIb): o átomo de enxofre é positivo, de modo que as interações orbitalares πCO→σ*CS e π*CO→σ*CS são mais intensas. Assim, em solventes de baixa polaridade, o grupo etilsulfinila mantém a preferência conformacional sinclinal em relação à carbonila na série IIa enquanto na série IIb esse confôrmero é altamente desestabilizado devido à repulsão eletrostática Oδ-(CO)...Oδ-(SO). O aumento na constante dielétrica do solvente favorece a conformação na qual o grupo etilsulfinila tende a geometria sinperiplanar em relação à carbonila, com os dipolos S=O e C=O apontando na mesma direção. Na série IIb o grupo fenilselenila adota a geometria sinclinal (que favorece as interações orbitalares) enquanto o grupo etilsulfinila tende à geometria sinperiplanar em relação à carbonila nos dois confôrmeros estáveis. A orientação do oxigênio sulfinílico em relação ao oxigênio carbonílico define a estabilidade dos confôrmeros da série IIb: enquanto solventes apolares e grupos doadores de elétrons estabilizam a conformação na qual os dipolos S=O e C=O apontam em direções opostas, solventes polares e grupos atraentes de elétrons estabilizam a conformação na qual os referidos dipolos apontam na mesma direção. Por fim, na série III (que contém o grupo etilsulfonila) a atração eletrostática cruzada entre os grupos carbonila e sulfonila é suficientemente forte para estabilizar uma mesma conformação em todos os solventes. Nessa conformação, o arranjo geométrico desfavorece as interações orbitalares entre heteroátomos e carbonila, reforçando a importância do fator eletrostático para o equilíbrio conformacional nos compostos estudados. Conclui-se que a soma das interações orbitalares envolvendo enxofre ou selênio são praticamente as mesmas para os diferentes confôrmeros de todas as séries estudadas (I-III), sendo o balanço entre as interações eletrostáticas repulsivas e atrativas o fator principal que determina a estabilidade relativa dos confôrmeros em solução. / This thesis reports the conformational analysis and the electronic interactions study of some 2-(phenylseleno)-2-(ethylthio)acetophenones (series I), 2-(phenylseleno)-2-(ethylsulfinyl)- acetophenones [series IIa (CRSR/CSSS) and series IIb (CRSS//CSSR)] and 2-(phenylseleno)-2- (ethylsulfonyl)acetophenones (series III) bearing in the para position of the phenacyl group electron donating, hydrogen and electron attracting substituents. It has been found the occurrence of conformational isomerism through infrared spectroscopy in solvents of increasing dielectric constant (n-C6H14, CCl4, CHCl3, CH2Cl2, CH3CN) in all studied series. The infrared results are consistent with the conformers obtained by structural optimization on the B3LYP/6-31+G(d,p) level of theory along with the solvent effect simulated by the PCM method. The electronic interactions were investigated from interatomic contacts and by orbital interactions analysis (NBO). Structures for selected solids of series IIa, IIb and III were obtained through X-ray diffraction analysis. In the series I, the most stable conformer is characterized by interactions between carbonyl and sulfur orbitals, with the ethylthio group bearing a synclinal geometry relative to the carbonyl group while the phenylselenyl moiety approaches the carbonyl group, due to the reduced electrostatic repulsion. The solvent effect on the relative population of the conformers through the series I is smaller than the other series. The interactions are significantly modified due the ethylsulfinyl group (series IIa and IIb): the sulfur atom is positive, leading to stronger πCO→σ*CS and π*CO→σ*CS orbital interactions. Thus, in solvents of low polarity, the ethylsulfinyl group retains the synclinal geometry relative to the carbonyl group in the serie IIa while in serie IIb this conformer is highly desistibilized due the Oδ-(CO)...Oδ-(SO) electrostatic repulsion. The increase in the dielectric constant of the solvent stabilizes the conformation in which the ethylsulfinyl group adopts a sinperiplanar geometry in relation to the carbonyl group, with the S=O and C=O dipoles pointing towards the same direction in both series. In the series IIb, the phenylselenyl group adopts the synclinal geometry (which favors the orbital interactions) while the ethylsulfinyl group tends to a sinperiplanar geometry relative to the carbonyl group in both conformers. The orientation of the sulfinyl oxygen relative to the carbonyl oxygen defines the stability of the conformers in series IIb: while nonpolar solvents and electron donating substituents stabilize the conformation in which the S=O and C=O dipoles point in opposite directions, on the other hand, polar solvents and attractive electron groups stabilize the conformation in which these dipoles point in the same direction. Finally, in the series III (which contains the ethylsulfonyl group) the crossed electrostatic attraction between the carbonyl and sulfonyl groups is strong enough to stabilize the same conformation in all solvents. In this conformation, the geometrical arrangement disfavors the orbital interactions between heteroatoms and carbonyl group, reinforcing the importance of the electrostatic factor for the conformational equilibrium in the studied compounds. It may be concluded that the sum of the orbital interactions involving sulfur or selenium atoms are practically the same for the different conformers of the studied series (I-III), being the balance of the attractive and repulsive electrostatic interactions the main factor which determine their relative stabilization in solution.

A presente tese trata da síntese e a análise conformacional de algumas N-metóxi-N-metil-amidas α-heterossubstituídas (amidas de Weinreb) Z-C(O)-N(OMe)Me (Z= CH2F (1), CH2OMe (2), CH2OPh (3), CH2Cl (4), CH2Br (5), CH2SEt (6) e Me2CSEt (7). A análise da banda da carbonila no infravermelho apoiada por cálculos teóricos B3LYP/6311++G(3df, 3pd), juntamente com a análise de NBO indicou a existência de um equilíbrio conformacional cis/gauche, isto é, (c) e (g) para (1) e (3), (c1, c2) e (g1, g2) para (2), (c) e (g1, g2) para (4-6) e (g1, g2) para (7). Em fase gasosa a população do confôrmero gauche prevalece ligeiramente sobre a população do confôrmero cis para os compostos (1) e (3); a população de (c1 + c2) prevalece sobre a população de (g1 + g2) para o composto (2) e a soma das populações de (g1 + g2) é mais abundante que a população de (c) para os compostos (4), (5) e (6). Já para o composto (7) a população do confôrmero g1 prevalece sobre a do g2. Nos compostos (4), (5) e (6), em solução de n-hexano, a ocorrência da ressonância de Fermi na região da vibração de estiramento da carbonila (VCO), impede qualquer estimativa das populações relativas dos confôrmeros (c, g1 e g2). Já, no composto (7) constata-se, em n-hexano, somente a presença do confôrmero (g1). Os resultados dos espectros da banda do composto (7), que contém duas metilas ligadas ao carbono α exclui a possibilidade da ocorrência da ressonância de Fermi. Os cálculos SCI-PCM concordam com o efeito do solvente sobre a intensidade relativa dos componentes da banda da carbonila para os compostos (1-3). A análise de NBO mostrou que a interação orbitalar nN→π*CO é o principal fator que estabiliza o confôrmero gauche (g, g1,e g2) em maior extensão do que o confôrmero cis (c, c1 e c2) para os compostos de (1-6). As interações orbitalares nY→π*CO, σC-Y→π*CO, πCO→σ*C-Y e π*CO→σ*C-Y também contribuem para a estabilização do confôrmero gauche (g, g1, g2) em relação ao confôrmero cis (c, c1, c2), porém em menor extensão. A existência de uma piramidalização no átomo de nitrogênio das amidas de Weinreb é responsável pela ocorrência dos contatos curtos entre Yδ-(4)…Oδ-(9) e Yδ-(4)…Nδ-(7) nos confôrmeros gauche (g, g1, g2), que origina uma forte interação coulombica repulsiva, agindo fortemente em oposição à estabilização dos confôrmeros gauche (g, g1, g2) em relação aos confôrmeros cis (c, c1, c2). Portanto, um delicado equilíbrio entre as interações Coulômbicas e as interações orbitalares, pode ser responsável pela estabilização observada nos confôrmeros cis (c, c1, c2) em relação aos confôrmeros gauche (g, g1, g2), tanto em fase gasosa, quanto em solução. Contrariamente ao que seria esperado, a predominância do confôrmero cis (em solução de n-hexano e tetracloreto de carbono) para as N-metóxi-N-metil amidas α-heterossubstituídas (1-3), contendo em α-átomos da primeira fila (flúor e oxigênio) está em direção oposta à preferência do confôrmero gauche para as N-metóxi-N-metil amidas α-heterossubstituídas remanescentes (4-6) contendo em α átomos da segunda e terceira filas (cloro, enxofre, bromo), onde os confôrmeros g1 e g2 são preponderantes ou exclusivos no caso de (7). / This thesis deals with the synthesis and conformational analysis of some α-heterosubstituted N-methoxy-N-methyl-amides (Weinreb amides) Z-C(O)-N(OMe)Me (Z= CH2F (1), CH2OMe (2), CH2OPh (3), CH2Cl (4), CH2Br (5), CH2SEt (6) and Me2CSEt (7). The analysis of the carbonyl band in the IR spectra supported by B3LYP/6 311++G(3df, 3pd calculations along with the NBO analysis indicated the existence of a cis-gauche conformational equilibrium i.e. (c) and (g) for (1) and (3), (c1, c2) and (g1, g2) for (2), (c) and (g1, g2) for (4-6) and (g1, g2) for (7). In the gas phase the g conformer population prevails slightly over the c one for (1) and (3); the (c1 + c2) population prevails over the (g1 + g2) for (2), and the (g1 + g2) conformer population is more abundant than the population (c) for (4), (5) and (6). In n-hexane solution the cis conformer is more abundant for (1-3). The occurrence of Fermi resonance in the VCO region, in n-hexane, precludes the estimative of relative populations of the (c, g1, g2) conformers for (4-6). The SCI-PCM calculations agree with the solvent effect on the VCO band component relative intensities for (1-3). NBO analysis showed that the nN→π*CO orbital interaction is the main factor which stabilizes the gauche (g, g1, g2) conformers for (1-6) into a larger extent relative to the cis (c, c1, c2) ones. The nY→π*CO, σC-Y→π*CO, πCO→σ*C-Y and π*CO→σ*C-Y orbital interactions still contribute, but into a minor extent for the stabilization of the gauche conformers relative to the cis ones. The existence of some pyramidalization at the nitrogen atom of the Weinreb amides (1-6) is responsible for the occurrence of Yδ-(4)…Oδ-(9) and Yδ-(4)…Nδ-(7) short contacts in the gauche (g, g1, g2) conformers, which originates strong repulsive Coulombic interactions, acting in opposition to the large orbital stabilization of the gauche conformer with respect to the cis one. The same effects are responsible for the larger stabilization of the (g1, g2) conformers of (7) which in turn precludes the existence of the c conformer. Therefore, a delicate balance of the Coulombic and orbital interactions seems to be responsible for the observed stabilization of the gauche (g, g1, g2) and cis (c, c1, c2) conformers, both in the gas phase and in the solution for (1-6) and (7). However, the cis conformer predominance, in non polar solvents, for the α-substituted N-methoxy-N-methyl acetamides (1-3), bearing in α first row (fluorine and oxygen) atoms, is in the opposite direction to the gauche conformer preference for the remaining α-substituted N-methoxy-N-methyl acetamides (4-6), bearing in α second and third rows (chlorine, sulfur, bromine) atoms. However the g1 and g2 conformers are the only ones present for (7).

Les Rotavirus appartiennent à la famille des Reoviridae, famille du groupe III des virus à ARN double brin. Identifiés en 1973 par Ruth Bishop, ces virus non enveloppés sont la première cause de diarrhée aiguë sévère du jeune enfant dans le monde. La capside virale icosaédrique est constituée de 3 couches protéiques de structure : la couche externe formée par la glycoprotéine VP7 d’où émergent les spicules de protéine VP4, la couche intermédiaire constituée par la protéine VP6 représentant près de 50 % du poids du virus et enfin, la couche interne appelée core, résultant de l’assemblage des protéines VP2, d’où émergent vers l’intérieur les protéines VP1 et VP3. Cette capside renferme un génome divisé en 11 segments d’ARN bicaténaires. A ces 6 protéines structurales s’ajoutent les protéines non structurales qui interviennent lors de la réplication du virus. Les deux protéines structurales, VP4 et VP7 sont essentielles pour la fixation de la particule triple couche (TLP) aux membranes des cellules hôtes, par interaction aux récepteurs intégrines, elle sont également impliqués dans la déstabilisation des membranes endosomales, indispensable à la libération de la particule double couche (DLP) infectieuse dans le cytoplasme. Actuellement, contrairement au mécanisme d’action de la protéine VP5*, celui de la glycoprotéine VP7 est inconnu. L’objectif de cette thèse, a été de comprendre le mécanisme moléculaire de déstabilisation des membranes par les peptides dérivés de VP7. Dans un premier temps nous avons montré, par des études in silico, l’existence d’un domaine prédit en hélice membranaire bordé de résidus arginine et lysine hautement conservés, situé à l’extrémité C-terminale de la glycoprotéine VP7. Ces résultats ont conduit à la synthèse de quatre peptides avec lesquels des tests de perméabilisation de membranes modèles de larges vésicules unilamellaires (LUVs) ont été menés. Ceux-ci ont permis d’identifier le domaine minimum le plus actif, VP723, parmi les peptides sélectionnés. Dans un second temps nous avons déterminé la structure de ces peptides par RMN, dans des conditions mimant l’environnement hydrophobe de la membrane. Le peptide minimal VP723 s’organise en hélice α-amphipathique, structure souvent impliquée dans la déstabilisation des membranes cellulaires. La comparaison de sa structure obtenue par RMN à celle du domaine correspondant dans la structure cristallographique de la protéine native montre le réarrangement conformationnel de ce segment après maturation par la trypsine. Ces résultats ont été confirmés par deux mutants de synthèse, dont l’un est inactif pour la perméabilisation des membranes modèles. Ces travaux ont été complétés par des expériences de Résonance Plasmonique aux Ondes guidée (PWR). Des études par RMN du solide sont en cours afin de déterminer l’orientation du peptide dans les membranes modèles. En conclusion, nos résultats mettent en évidence l’importance du domaine C-terminal VP723 de la protéine VP7 dans la déstabilisation des membranes, permettant d’assurer la translocation de la particule virale infectieuse (DLP) de l’endosome vers le cytoplasme. Un modèle du mécanisme d’entrée du virus, médié par les peptides dérivés de la maturation par la trypsine de la glycoprotéine VP7 est proposé. / Rotaviruses belong to the Reoviridae family, belonging to the group III of dsRNA viruses. Identified in 1973 by Ruth Bishop, these non-enveloped viruses are the leading cause of severe diarrhea in young children worldwide. The icosahedral capsid is composed of three structural protein layers: the outer one, formed by the glycoprotein VP7, emerges spicules protein VP4, the intermediate one consists of VP6 protein representing nearly 50% of the weight of the virus and finally, the inner one called core, results from the assembly of proteins VP2, emerges towards the inside of proteins VP1 and VP3. The capsid contains a genome divided into 11 segments of dsRNA. To these six structural proteins are added nonstructural proteins involved in virus replication. The two structural proteins, VP4 and VP7, are involved in the interaction of the triple layer particle (TLP) to integrin receptors, necessary for the release of the infectious double layer particle (DLP) into the cytoplasm following the permeabilization of the membrane of the endosome compartments. Currently, unlike the mechanism of action of the protein VP5*, the glycoprotein VP7 remains unknown. The objective of this work was to understand the molecular mechanism involved in the destabilization of membranes by peptides derived from VP7. In a first step, we have shown, by in silico studies, the existence of a helical trans-membrane domain predicted containing a highly conserved arginine and lysine residues, located at the C-terminus of the VP7 glycoprotein. These results led to the synthesis of four peptides with which permeabilizing tests of model membranes were conducted. We have identified the minimum of the most active domain, named VP723, among the selected peptides. In a second step, we determined the structure of these peptides by NMR under conditions mimicking the hydrophobic environment of the membrane. The VP723 peptide is organized like an α-helical amphipathic structure often involved in the destabilization of cell membranes. The comparison of the structure obtained by NMR to that of the corresponding domain in the crystallographic structure of the native protein shows a conformational rearrangement of the segment after trypsin maturation. These results were confirmed by two synthetic mutants, one of which is inactive for the permeabilization of model membranes. These studies were complemented by experiments Plasmon Resonance guided the Waves (PWR). Studies by solid state NMR are in progress to determine the orientation of the peptide in model of membranes. In conclusion, our results highlight the importance of the C-terminal domain of the VP7 protein, named VP723, in the destabilization of membranes, to ensure the translocation of the infectious viral particle (DLP) from the endosome into the cytoplasm compartments. A mechanism of virus entry mediated by peptides derived from trypsin maturation of the VP7 glycoprotein is proposed in this study.

A presente tese trata da análise conformacional e estudo das interações eletrônicas de algumas 2-(fenilseleno)-2-(etiltio)acetofenonas (série I), 2-(fenilseleno)-2-(etilsulfinil)- acetofenonas [série IIa para (CRSR/CSSS) e série IIb para (CRSS//CSSR)] e 2-(fenilseleno)-2- (etilsulfonil)acetofenonas (série III) contendo substituintes doadores e atraentes de elétrons, além de hidrogênio na posição para do grupo fenacila. Foi constatada a ocorrência de isomerismo conformacional por espectroscopia no infravermelho em solventes de constante dielétrica crescente (n-C6H14, CCl4, CHCl3, CH2Cl2, CH3CN) em todas as séries estudadas. Os resultados obtidos por infravermelho são concordantes com as estruturas obtidas por otimização estrutural no nível de teoria B3LYP/6-31+G(d,p) com o efeito do solvente simulado pelo método PCM. As interações eletrônicas foram investigadas a partir dos contatos interatômicos e por meio da análise das interações orbitalares (NBO). Foram obtidas as estruturas cristalográficas de alguns compostos selecionados das séries IIa, IIb e III por difração de raio-X. Na série I, o confôrmero mais estável é caracterizado pelas interações orbitalares entre carbonila e enxofre, com o grupo etiltio numa geometria sinclinal em relação à carbonila, enquanto o grupo fenilselenila se aproxima da carbonila devido à repulsão eletrostática ser pequena. O efeito do solvente nas populações relativas dos confôrmeros ao longo da série I é menor que nas demais séries. As interações são significativamente modificadas com o grupo etilsulfinila (séries IIa e IIb): o átomo de enxofre é positivo, de modo que as interações orbitalares πCO→σ*CS e π*CO→σ*CS são mais intensas. Assim, em solventes de baixa polaridade, o grupo etilsulfinila mantém a preferência conformacional sinclinal em relação à carbonila na série IIa enquanto na série IIb esse confôrmero é altamente desestabilizado devido à repulsão eletrostática Oδ-(CO)...Oδ-(SO). O aumento na constante dielétrica do solvente favorece a conformação na qual o grupo etilsulfinila tende a geometria sinperiplanar em relação à carbonila, com os dipolos S=O e C=O apontando na mesma direção. Na série IIb o grupo fenilselenila adota a geometria sinclinal (que favorece as interações orbitalares) enquanto o grupo etilsulfinila tende à geometria sinperiplanar em relação à carbonila nos dois confôrmeros estáveis. A orientação do oxigênio sulfinílico em relação ao oxigênio carbonílico define a estabilidade dos confôrmeros da série IIb: enquanto solventes apolares e grupos doadores de elétrons estabilizam a conformação na qual os dipolos S=O e C=O apontam em direções opostas, solventes polares e grupos atraentes de elétrons estabilizam a conformação na qual os referidos dipolos apontam na mesma direção. Por fim, na série III (que contém o grupo etilsulfonila) a atração eletrostática cruzada entre os grupos carbonila e sulfonila é suficientemente forte para estabilizar uma mesma conformação em todos os solventes. Nessa conformação, o arranjo geométrico desfavorece as interações orbitalares entre heteroátomos e carbonila, reforçando a importância do fator eletrostático para o equilíbrio conformacional nos compostos estudados. Conclui-se que a soma das interações orbitalares envolvendo enxofre ou selênio são praticamente as mesmas para os diferentes confôrmeros de todas as séries estudadas (I-III), sendo o balanço entre as interações eletrostáticas repulsivas e atrativas o fator principal que determina a estabilidade relativa dos confôrmeros em solução. / This thesis reports the conformational analysis and the electronic interactions study of some 2-(phenylseleno)-2-(ethylthio)acetophenones (series I), 2-(phenylseleno)-2-(ethylsulfinyl)- acetophenones [series IIa (CRSR/CSSS) and series IIb (CRSS//CSSR)] and 2-(phenylseleno)-2- (ethylsulfonyl)acetophenones (series III) bearing in the para position of the phenacyl group electron donating, hydrogen and electron attracting substituents. It has been found the occurrence of conformational isomerism through infrared spectroscopy in solvents of increasing dielectric constant (n-C6H14, CCl4, CHCl3, CH2Cl2, CH3CN) in all studied series. The infrared results are consistent with the conformers obtained by structural optimization on the B3LYP/6-31+G(d,p) level of theory along with the solvent effect simulated by the PCM method. The electronic interactions were investigated from interatomic contacts and by orbital interactions analysis (NBO). Structures for selected solids of series IIa, IIb and III were obtained through X-ray diffraction analysis. In the series I, the most stable conformer is characterized by interactions between carbonyl and sulfur orbitals, with the ethylthio group bearing a synclinal geometry relative to the carbonyl group while the phenylselenyl moiety approaches the carbonyl group, due to the reduced electrostatic repulsion. The solvent effect on the relative population of the conformers through the series I is smaller than the other series. The interactions are significantly modified due the ethylsulfinyl group (series IIa and IIb): the sulfur atom is positive, leading to stronger πCO→σ*CS and π*CO→σ*CS orbital interactions. Thus, in solvents of low polarity, the ethylsulfinyl group retains the synclinal geometry relative to the carbonyl group in the serie IIa while in serie IIb this conformer is highly desistibilized due the Oδ-(CO)...Oδ-(SO) electrostatic repulsion. The increase in the dielectric constant of the solvent stabilizes the conformation in which the ethylsulfinyl group adopts a sinperiplanar geometry in relation to the carbonyl group, with the S=O and C=O dipoles pointing towards the same direction in both series. In the series IIb, the phenylselenyl group adopts the synclinal geometry (which favors the orbital interactions) while the ethylsulfinyl group tends to a sinperiplanar geometry relative to the carbonyl group in both conformers. The orientation of the sulfinyl oxygen relative to the carbonyl oxygen defines the stability of the conformers in series IIb: while nonpolar solvents and electron donating substituents stabilize the conformation in which the S=O and C=O dipoles point in opposite directions, on the other hand, polar solvents and attractive electron groups stabilize the conformation in which these dipoles point in the same direction. Finally, in the series III (which contains the ethylsulfonyl group) the crossed electrostatic attraction between the carbonyl and sulfonyl groups is strong enough to stabilize the same conformation in all solvents. In this conformation, the geometrical arrangement disfavors the orbital interactions between heteroatoms and carbonyl group, reinforcing the importance of the electrostatic factor for the conformational equilibrium in the studied compounds. It may be concluded that the sum of the orbital interactions involving sulfur or selenium atoms are practically the same for the different conformers of the studied series (I-III), being the balance of the attractive and repulsive electrostatic interactions the main factor which determine their relative stabilization in solution.

Les azacycles tels que les morpholines ou les pyrrolidines, sont très répandus dans le domaine de l’organocatalyse et de la chimie médicinale. Cette thèse traitera d’analogues contraints de ces azacycles, qui peuvent moduler de par leurs structures, les propriétés de certains médicaments ou la sélectivité de certaines réactions. Le cas de l’halopéridol, qui est connu pour son activité sur les récepteurs dopaminergiques D2 et D4, est au centre de la première partie de cette thèse, dans laquelle de nouveaux analogues contraints de type 2-oxa-5-azabicyclo[2.2.2]octane ont été développés pour pallier ses problèmes de stabilité métabolique. Dans une seconde partie, la synthèse de deux nouvelles chimères morpholine-proline pontées est rapportée. Leurs structures rigides et conformationnellement verrouillées permettent aux doublets d’électrons non liants sur les atomes d'azote et d'oxygène d’être respectivement orientés dans des directions « Est-Ouest » et « Nord-Est » spatialement différentes. En combinaison avec la présence d'un acide carboxylique, les propriétés électroniques de ces composés peuvent être utiles dans le contexte de la conception peptidomimétique de composés biologiquement pertinents. Des estimations quantitatives de la basicité des atomes d'azote ont été obtenues en utilisant une analyse DFT conceptuelle. Dans la troisième partie de cette thèse, la synthèse de nouvelles pyrrolidines oxabicycliques sera développée. Les cyclopentan[c]pyrroles sont très répandus dans la littérature, et connus pour leurs propriétés analgésiques. Des dérivés fonctionnalisés en positions 4 et 5, synthétisés par Servier, ont notamment présenté de bonnes activités en tant que ligands nicotiniques 7, mais aussi des problèmes d’inhibition de hERG. Afin d’obtenir des composés moins lipophiles et donc de pallier les problèmes d’inhibition hERG, de nouveaux analogues oxygénés de type furo[2,3-c]pyrroles ont été développés par différentes voies de synthèse. Ces nouveaux composés pourront notamment être obtenus sous formes énantiomériquement enrichies, grâce à une étape clé de résolution enzymatique. La proline a été largement utilisée ces dernières années comme organocatalyseur au sein d’importantes transformations asymétriques comme les aldolisations ou les additions de Michael. Cependant, malgré le succès de ce motif, plusieurs de ses dérivés ont été rapportés dans la littérature. C’est notamment le cas des 4,5-méthanoprolines qui furent rapportées pour la première fois par Hanessian en 1997, dont l’efficacité en tant qu’organocatalyseur pour la réaction de Hajos-Parrish ainsi que plusieurs autres types de réaction fut par la suite établie. Les dernières parties de cette thèse viennent compléter ces études. La synthèse de nouvelles 4,5-ethanoprolines a été développée, ainsi que leurs utilisations comme catalyseur lors de réactions de Hajos-Parrish et d’addition catalytique asymétrique de nitroalcanes sur des énones cycliques. Une étude DFT a été effectuée afin d’expliquer l’inversion de sélectivité observée pour ces nouveaux catalyseurs lors des réactions de Hajos-Parrish, le mécanisme de formation des énamines réactives a aussi été investigué. / Azacycles such as morpholines and pyrrolidines, are very widespread in chemistry, especially in the fields of organocatalysis and medicinal chemistry. This thesis will deal with constrained analogues of those azacycles, which, depending on their structures, can modulate the properties of certain drugs or the selectivity of certain reactions. The case of haloperidol, which is known for its activity on the dopamine D2 and D4 receptors, is at the center of the first part of this thesis, in which new constrained analogs of the 2-oxa-5-azabicyclo type [2.2.2] octane have been developed to overcome its metabolic stability problems. In a second part, the synthesis of two new bridged morpholine-proline chimeras are reported. Their rigid structures allow the lone pairs on the nitrogen and oxygen atoms to be oriented in spatially different "East-West" and "North-East" directions, respectively. In combination with the presence of a carboxylic acid, the electronic properties of these compounds could be useful in the context of the design of biologically relevant peptidomimetics. Quantitative estimations of the basicity of the nitrogen atoms were obtained using DFT analysis. In the third part of this thesis, the synthesis of new oxabicyclic pyrrolidines is described. Cyclopentan[c]pyrroles are widely encountered and known for their analgesic properties. The Servier laboratories have synthesized derivatives with substituents at positions 4 and 5, exhibiting good activities as 7 nicotinic ligands, but problems of hERG inhibition. In order to obtain less lipophilic compounds and therefore overcome the problems of hERG inhibition, new oxygenated analogs of the furo[2,3-c]pyrrole type have been developed by different synthetic routes. These new compounds were obtained in enantiomerically enriched forms, using enzymatic resolution. Proline has been widely used in recent years as an organocatalyst in asymmetric transformations such as aldolizations or Michael additions The success of this motif, has inspired synthesis of derivatives, such as 4,5-methanoprolines, which were first reported by Hanessian in 1997, and shown to be effective as organocatalysts in the Hajos-Parrish and other reactions. The last parts of this thesis develop further these studies by the synthesis of new 4,5-ethanoprolines which act as a catalysts in the Hajos-Parrish reaction and the asymmetric catalytic addition of nitroalkanes to cyclic enones. A DFT study was carried out to explain the reversal of selectivity observed for the new catalysts in Hajos-Parrish reaction and to investigate formation of a reactive enamine in the mechanism.

MSc (Chemistry) / Department of Chemistry

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

L’objectif de cette thèse est d’analyser et prédire les conformations d’un système moléculaire en combinant la théorie des graphes et la chimie computationnelle.Dans le cadre des simulations de dynamique moléculaire, une molécule peut avoir une ou plusieurs conformations au cours du temps. Dans les trajectoires de simulation de dynamique moléculaire, on peut avoir des trajectoires n’explorant qu’une seule conformation ou des trajectoires explorant plusieurs conformations, donc plusieurs transitions entre conformations sont observées. L’exploration de ces conformations dépend du temps de la simulation et de l'énergie (température) fixée dans le système. Pour avoir une bonne exploration des conformations d’un système moléculaire, il faut générer et analyser plusieurs trajectoires à différentes énergies. Notre objectif est de proposer un algorithme universel qui permet d’analyser la dynamique conformationnelle de ces trajectoires d’une façon rapide et automatique. Les trajectoires fournissent les positions cartésiennes des atomes du système moléculaire à des intervalles de temps réguliers. Chaque intervalle contenant un ensemble de positions est appelé image. L’algorithme utilise des règles de géométrie (distances, angles, etc.) sur les positions pour trouver les liaisons (liaisons covalentes, liaisons hydrogène et interactions électrostatiques) créées entre les atomes, permettant par la suite d’obtenir le graphe mixte qui modélise une conformation. Nous ne considérons un changement conformationnel que s’il y a un changement dans les liaisons calculées à partir des positions données. L’algorithme permet de donner l’ensemble des conformations explorées sur une ou plusieurs trajectoires, la durée d’exploration de chaque conformation, ainsi que le graphe de transitions qui contient tous les changements conformationnels observés.Les conformations se caractérisent par une énergie appelée énergie potentielle. Cette énergie est représentée par une courbe appelée surface d’énergie potentielle. En chimie théorique et computationnelle, certains s’intéressent à trouver des points particuliers sur cette surface. Il s'agit des minima qui représentent les conformations les plus stables et des maxima ou états de transition qui représentent les points de passage d'une conformation à une autre. En effet, d'une part, la conformation la plus stable est celle de plus basse énergie. D'autres part, pour aller d’une conformation à une autre il faut une énergie supplémentaire, le point maximum représente l'état de transition. Les méthodes développées pour calculer ces points nécessitent une connaissance de l’énergie potentielle ce qui est coûteux en temps et en calculs. Notre objectif est de proposer une méthode alternative en utilisant des mesures ah doc basées sur des propriétés des graphes qu’on a utilisées dans le premier algorithme et sans faire appel à la géométrie ni aux calculs moléculaires. Ces mesures permettent de générer des conformations avec un classement énergétique ainsi de définir le coût énergétique de chaque transition permise. Les conformations possibles avec les transitions représentent respectivement les sommets et les arcs de ce qu’on appelle le “graphe des possibles”. Les hypothèses utilisées dans le modèle proposé est que seules les liaisons hydrogène peuvent changer entre les conformations et que le nombre de liaisons hydrogène présentes dans le système permet de déterminer son coût énergétique.L’algorithme d'analyser des trajectoires a été testé sur trois types de systèmes moléculaires en phase gazeuse de taille et de complexité croissantes. Bien que la complexité théorique de l’algorithme est exponentielle (tests d’isomorphisme) les résultats ont montré que l’algorithme est rapide (quelques secondes). De plus, cet algorithme peut être facilement adapté et appliqué à d’autres systèmes. Pour la prédiction conformationnelle, le modèle proposé a été testé sur des peptides isolés. / This PhD is part of transdisciplinary works, combining graph theory and computational chemistry.In molecular dynamics simulations, a molecular system can adopt different conformations over time. Along a trajectory, one conformation or more can thus be explored. This depends on the simulation time and energy within the system. To get a good exploration of the molecular conformations, one must generate and analyse several trajectories (this can amount to thousands of trajectories). Our objective is to propose an automatic method that provides rapid and efficient analysis of the conformational dynamics explored over these trajectories. The trajectories of interest here are in cartesian coordinates of the atoms that constitute the molecular system, recorded at regular time intervals (time-steps). Each interval containing a set of positions is called a snapshot. At each snapshot, our developed algorithm uses geometric rules (distances, angles, etc.) to compute bonds (covalent bonds, hydrogen bonds and any other kind of intermolecular criterium) formed between atoms in order to get the mixed graph modelling one given conformation. Within our current definitions, a conformational change is characterized by either a change in the hydrogen bonds or in the covalent bonds. One choice or the other depends on the underlying physics and chemistry of interest. The proposed algorithm provides all conformations explored along one or several trajectories, the period of time for the existence of each one of these conformations, and also provides the graph of transitions that shows all conformational changes that have been observed during the trajectories. A user-friendly interface has been developed, that can de distributed freely.Our proposed algorithm for analysing the trajectories of molecular dynamics simulations has been tested on three kinds of gas phase molecular systems (peptides, ionic clusters). This model can be easily adapted and applied to any other molecular systems as well as to condensed matter systems, with little effort. Although the theoretical complexity of the algorithm is exponential (isomorphism tests), results have shown that the algorithm is rapid.We have also worked on computationally low cost graph methods that can be applied in order to pre-characterize specific conformations/points on a potential energy surface (it describes the energy of a system in terms of positions of the atoms). These points are the minima on the surface, representing the most stable conformations of a molecular system, and the maxima on that surface, representing transition states between two conformers. Our developed methods and algorithms aim at getting these specific points, without the prerequisite knowledge/calculation of the potential energy surface by quantum chemistry methods (or even by classical representations). By avoiding an explicit calculation of the potential energy surface by quantum chemistry methods, one saves computational time and effort. We have proposed an alternative method using ad doc measures based on properties of the graphs (already used in the first part of the PhD), without any knowledge of energy and/or molecular calculations. These measures allow getting the possible conformations with a realistic energy classification, as well as transition states, at very low computational cost. The algorithm has been tested on gas phase peptides.

Les protéines d’échafaudage de la famille Elmo forment des complexes avec les facteurs d’échange de nucléotides de la guanine (GEF) de la famille des protéines Dock. Globalement, le complexe Elmo/Dock est caractérisé par une régulation conformationnelle, où au niveau basal il se retrouve dans un état fermé, en raison de la présence de sites de contact bloquant la liaison des interacteurs d’Elmo et la fonction GEF de Dock qui permet l’activation de Rac1. Une fois dans un état activé, le complexe adoptera une conformation ouverte et les sites de liaison d’Elmo seront disponibles. Par exemple, il a été démontré que la liaison des GTPases RhoG ou Arl4A au domaine RBD d’Elmo induit le recrutement du complexe à la membrane cellulaire. Le domaine DHR-2 étant alors accessible, celui-ci assurera l’activation spécifique de la GTPase Rac1 afin d’induire, entre autres, le remodelage du cytosquelette d’actine. Divers processus cellulaires seront alors d clench s, tels que la migration cellulaire, la phagocytose et la fusion des cellules musculaires (nommées myoblastes). Dans le cadre de cette thèse, nous avons étudié par l’entremise de deux objectifs l’importance de la régulation conformationnelle d’Elmo. Pour le premier objectif, nous avons étudié la régulation conformationnelle d’Elmo durant la myogenèse. La formation de fibres multi-nuclées est fondamentale pour l’établissement du muscle squelettique. Durant ce processus, la fusion des myoblastes est une étape clé pour permettre le développement et la régénération musculaire. Afin d’étudier les fonctions d’Elmo in vivo dans ce contexte, nous avons généré une série de modèles de souris. Tout d’abord, via la génération de souris double knock-out pour Elmo1 et Elmo2 (Elmo1KOElmo2cKO), nous avons démontré la fonction essentielle d’Elmo durant la fusion des myoblastes embryonnaires. En effet, uniquement des myofibres mononuclées sont observées suite à l’inactivation génétique d’Elmo1 et Elmo2. Par la suite, nous avons également généré des lignées de souris knock-in, où des mutations ont été introduites dans des domaines spécifiques d’Elmo2 afin d’induire sa conformation ouverte (Elmo2EID) ou fermée (Elmo2RBD). Nous avons ainsi démontré qu’en présence d’Elmo2EID, la capacité de fusion est augmentée et des fibres musculaires plus larges sont développées. De plus, la régénération musculaire est plus efficace chez ces souris. À l’opposé, lorsqu’Elmo2 a perdu l’activité de son domaine RBD (Elmo1KOElmo2RBD), des fibres musculaires plus étroites sont retrouvées chez les jeunes souris adultes ainsi qu’une régénération du muscle moins efficace. Finalement, nous avons démontré que l’augmentation de l’activité de la voie Elmo2-Dock1-Rac1, directement via le contrôle de la régulation conformationnelle d’Elmo2, améliore les phénotypes dystrophiques retrouvés chez les souris DysferlinKO, un modèle récapitulant la dystrophie musculaire des ceintures de type 2B (LGMD2B). Ainsi, pour la première fois, nous avons établi la possibilité d’exploiter la fusion des myoblastes en tant que thérapie régénérative pour des maladies musculaires. Pour le deuxième objectif, nous avons étudié Elmo3 dans un cas clinique dans le cadre d’une collaboration internationale. Plus précisément, des mutations bi-alléliques dans le gène Elmo3 ont été identifiées chez un jeune patient atteint d’une déficience intellectuelle. En effectuant des études biochimiques et fonctionnelles, nous avons démontré que ces mutations ont un impact sur l’activation de Rac1, sans toutefois influencer l’interaction entre les protéines du complexe Elmo3/Dock1. Cette étude apporte les premières évidences de fonctions biologiques pour Elmo3. Pour conclure, nos études ont permis de souligner l’importance d’un control approprié de la régulation conformationnelle d’Elmo. En effet, en manipulant cette régulation dans un modèle de muscle squelettique, via l’introduction d’une mutation maintenant la protéine dans une conformation ouverte, cela a permis un effet positif sur la fusion des myoblastes et sur la régénération musculaire, menant à l’amélioration des phénotypes de dystrophie musculaire.   l’opposé, la présence chez l’humain de mutations dans Elmo peut également affecter l’activation de Rac1 par Dock1, contribuant ainsi à une déficience intellectuelle chez le porteur. / The scaffold proteins Elmo forms a complex with guanine nucleotide exchange factors (GEFs) of the Dock family. The Elmo/Dock complex is characterized with a conformational regulation and at the basal level, the complex is found in a closed state, owing to the presence of contact sites blocking the binding of Elmo interactors and the GEF activity of Dock for the activation of Rac1. In their activated state, the complex adopts an open conformation and Elmo binding sites will be available. For example, binding of the GTPases RhoG or Arl4A to the RBD domain of Elmo has been shown to induce the recruitment of the complex at the cell membrane. Likewise, the DHR- 2 domain of Dock being available, the GTPase Rac1 will then be specifically activated by Dock and thus induce the remodeling of the actin cytoskeleton. Various cellular processes will then be triggered, such as cell migration, phagocytosis and muscle cell (named myoblast) fusion. In this thesis, we have emphasized the importance of the conformational regulation of Elmo by achieving two objectives. For the first objective, we studied the conformational regulation of Elmo during myogenesis, i.e. during the establishment of skeletal muscle. The formation of multinucleated myofibers is fundamental for skeletal muscle. During this process, myoblast fusion is a key step to allow the development as well as the regeneration of the muscle. In order to study Elmo in this in vivo context, we generated a series of mouse models. First, through the generation of double knockout mice for Elmo1 and Elmo2 (Elmo1KOElmo2cKO), we demonstrated the essential function of Elmo during embryonic myoblast fusion. Indeed, only mononucleated myofibers are observed following the genetic inactivation of Elmo1 and Elmo2. Subsequently, we also generated knockin mouse lines, where mutations were introduced in specific domains of Elmo to induce its opened (Elmo2EID) or closed (Elmo2RBD) conformation. Thus, we have demonstrated that when Elmo2EID is expressed, the fusion capability is increased and the myofibres are larger. Moreover, muscle regeneration is more efficient in these mice. At the opposite, when Elmo2 has lost its RBD activity (Elmo1KOElmo2RBD), smaller myofibers are observed as well as a less efficient muscle regeneration. Finally, we demonstrated that increasing the Elmo-Dock1-Rac1 pathway activity, directly through the control of the conformational regulation of Elmo, leads to the improvement of the dystrophic phenotypes found in DysferlinKO mice, a mouse model of the limb-girdle muscular dystrophy type 2B (LGMD2B). Thus, for the first time, we have established the possibility of exploiting myoblast fusion as a regenerative therapy for muscle diseases. For the second objective, we studied Elmo3 in a clinical case, as part of an international collaboration. More specifically, biallelic mutations in Elmo3 gene have been identified in a young patient with intellectual disability. Through biochemical and functional studies, we have shown that the mutations have an impact on the activation of Rac1, without however affecting the interaction between the proteins of the Elmo3/Dock1 complex. This study provides the first evidence of biological functions for Elmo3. In conclusion, our study has emphasis the relevance of the proper control of the conformational regulation of Elmo. In fact, by manipulating this regulation in a skeletal muscle model, through the introduction of a specific mutation promoting the open conformation of Elmo, it promotes myoblast fusion and induce a more efficient muscle regeneration, thus improving the dystrophic phenotypes. In contrast, the presence of human mutations in Elmo can also affect the activation of Rac1 by Dock1, hence contributing to intellectual disability in the carrier.