Ce travail décrit l’incorporation de la δ-azaproline dans des pseudopeptides en vue d’analyses conformationnelles et applications biologiques. Le premier chapitre de ce manuscrit est consacré à l’élaboration d’un nouveau substrat de type Suc Ala-X-δazaPro-Phe-ρ-NA (où X est un acide aminé) capable de mesurer l’activité catalytique des PPIases. Ces enzymes ont déjà montré un grand intérêt dans la prévention des rejets de greffes et dans le traitement de la maladie d’Alzheimer car elles sont capables d’isomériser le lien X-Pro de la conformation cis vers la conformation trans. Le second chapitre décrit l’incorporation de la δ-azaproline dans des pseudotripeptides et l’étude de la réactivité des deux atomes d’azote de cet analogue. Il a pu notamment être mis en évidence que l’atome d’azote supplémentaire de la δ azaproline est beaucoup plus réactif que l’atome d’azote natif car l’encombrement stérique généré par le groupement Boc et l’oxydation spontanée de la liaison CHα NHδ représente un frein au bon déroulement du couplage peptidique. Finalement, la dernière partie de ce manuscrit est consacrée à l’analyse conformationnelle des pseudotripeptides synthétisés. Ces analyses ont pour but de déterminer l’influence d’un atome d’azote supplémentaire, en position δ du cycle de la proline, libre ou protégé par un groupement encombré sur la conformation du lien X-ΨPro. Les techniques spectroscopiques comme la RMN et l’IR ont permis de mettre en évidence que l’encombrement stérique généré par un groupement Boc en position δ avec la chaîne latérale de l’acide aminé en amont de cet analogue favorise la conformation cis par formation d’un pseudocycle en C10 / This work describes the incoporation the δ-azaproline in pseudopeptides for conformational analysis and biological applications. The first chapter of this thesis is devoted to the development of a new substrate of the type Suc Ala X δazaPro Phe ρ NA (where X is an amino acid) able to measure the catalytic activity of PPIases. These enzymes have shown great interests in the prevention of transplant rejection and in the treatment of Alzheimer's disease because they can interconvert the cis and trans isomers of X-Pro peptidic bond. The second section describes the incorporation of the δ-azaproline into pseudotripeptides in which the reactivity of both nitrogen atoms of this surrogate was investigated. It could be demonstrated in particular that the supplementary nitrogen atom of azaproline is much more reactive than the native one because of the steric hindrance generated by the Boc protecting group and the spontaneous oxidation of the CHα NHδ bond represent a huge problem in the peptidic coupling.Finally, the last part of this thesis is dedicated to the conformational analysis of pseudotripeptides. These analyzes are designed to determine the influence of a supplementary nitrogen atom, in δ position into proline’s cycle, which can be free or protected by a cluttered group on the conformation of the X-ΨPro bond. Spectroscopic techniques such as NMR and IR enabled to demonstrate that steric hindrance generated by a Boc group on δ position with the side chain of amino acid upstream this surrogate promotes cis conformation by formation of a ten-membered ring

Ce travail décrit la synthèse et l’étude structurale d’oligomères mixtes et de leurs analogues cycliques alternant des résidus acides α aminés et des résidus acides azaaminés. Le premier chapitre concerne la synthèse en solution des 1:1-[α/aza]-oligomères et des 2:1 [α/aza] oligomères obtenus respectivement par oligomérisation par couplage peptidique de l’azadipeptide Boc-azaPhe-Ala-OMe et de l’azatripeptide Boc-Phe-azaPhe-Ala-OMe correctement déprotégés. Pour la première famille en série L, les rendements en dimérisation et en trimérisation sont moyens en raison de la faible nucléophilie du partenaire amine azadipeptidique. Quant aux 2:1 [α/aza]-oligomères de taille variable, ils ont été isolés avec de très bons rendements quelle que soit la nature L et/ou D des résidus acides α aminés de l’azatripeptide. Ce chapitre présente également l’étude conformationnelle de ces oligomères par spectroscopies RMN et IR, par modélisation moléculaire ainsi que dans certains cas par diffraction des RX. L’analyse des 1:1 [α/aza] oligomères a révélé une auto-structuration majoritaire en solution alternant un coude β et un coude γ stabilisés par des liaisons hydrogène intramoléculaires. L’examen des 2:1 [α/aza] oligomères a permis de mettre en évidence, en série homochirale (L), une conformation majoritaire caractérisée par la formation de coudes β stabilisés par un réseau de liaisons hydrogène intramoléculaires. Des repliements, induits par la formation de coudes β et de coudes γ s’imposent en série hétérochirale (DLDL…). Le second chapitre est consacré à la synthèse en solution et à l’étude conformationnelle des analogues cycliques des oligomères présentés dans le premier chapitre. Les 2:1 [α/aza] cyclooligomères sont surtout discutés, leur synthèse par couplage peptidique intramoléculaire étant plus efficace que celle des 1:1-[α/aza]-cyclooligomères. La formation de nanotubes due à l’empilement des 2 :1-[α/aza]-cyclohexamères homochiraux (L) a été mise en évidence à l’état solide par établissement de liaisons hydrogène intermoléculaires puis suggérée en solution. Le pouvoir gélifiant de certains solvants organiques de l’analogue hétérochiral (DLDL) présage également de son auto-structuration en nanotubes discutée uniquement en solution faute de structure à l’état solide / This work describes the synthesis and structural analysis of mixed oligomers and their cyclic analogues containing an alternation of α-acid residues and α-azaamino acids moieties. The first chapter deals with the “in solution” synthesis of 1:1-[α/aza]-oligomers and 2:1 [α/aza] oligomers respectively, obtained by oligomerization of the properly deprotected azadipeptide Boc-Ala-azaPhe-OMe azadipeptide and Boc-Phe-Ala-OMe-azaPhe tripeptide. For the first family series with L aminoacids, yields dimerization and trimerization are moderate due to the low nucleophilicity of the amine azadipeptidic partner. Concerning the 2:1-[α/aza]-oligomers of variable sizes, they were isolated with very good yields whatever the nature L and / or D of the α-aminoacid residues of the azatripeptide. This chapter also presents the conformational study of these oligomers by NMR and IR spectroscopy, molecular modeling and in some cases by X-ray diffraction. Analysis of the 1:1 [α/aza] oligomers reveals mainly an self-assembly in solution with alternation of β- and γ-turns stabilized by intramolecular hydrogen bonds. Examination of the 2:1 [α/aza] oligomers in homochiral series (L) evidenced a main conformation with repetitive β turns. Regarding the heterochiral series (DLDL….), it is an alternation of β- and γ-turns which is observed. The second chapter is devoted to the solution synthesis in solution and conformational study of cyclic analogues oligomers presented in the first chapter. 2: 1-[α/aza]-cyclooligomers are mostly discussed because their synthesis by intramolecular peptide coupling is more effective than the 1:1 [α/aza]-cyclooligomers. The formation of nanotubes due to the stacking of the homochiral (L) 2:1-[α/aza]-cyclohexamers has been shown in the solid state and suggested in solution. Furthermore, in heterochiral series, the gelling properties of some organic solvents suggest a self-assembly in solution

Quaternary ammonium salts are surface active bactericides. A mechanism of their biological activity has been well studied experimentally, and it encompasses two stages. The first stage involves electrostatic interactions of polar functional groups of the salts with oppositely charged functional groups on a bacterial cell surface, and the second stage includes incorporation of their lipophilic groups into a bacterial cell membrane. However, despite numerous experimental studies, computational modelling of this mechanism with the aim to support experimental observations with theoretical conclusions, to the author's knowledge, has not yet been reported. In the current study, linear regression models correlating theoretical descriptors of lipophilicity and electronic properties of mono- and disubstituted imidazolium carboxylates with their biological activity towards Escherichia coli have been developed. These models established that biological activity of these salts is governed by the chemical structures of imidazolium cations, and that the centre of this biological activity is located in the long alkyl side chains of the cations. It was also found that these side chains have an intrinsic electronic potential to form internal C-H- -H-C electrostatic interactions when their lengths reach seven carbon atoms. Additionally, the nature of the C-H- -O-C inter-ionic electrostatic interactions in imidazolium carboxylates has been explored via a topological analysis of these interactions in 1-ethyl-3-methylimidazolium acetate. Thus, it was established that these electrostatic interactions are hydrogen bonds.

La compréhension des mécanismes d’absorption de l’eau, ainsi que l’arrangement moléculaire adopté par le réseau de molécules d’eau lors du confinement à l’échelle nanométrique, est crucial que ce soit pour l’optimisation de plusieurs applications, telles que la production d’énergie propre, la purification et le dessalement de l’eau, ou pour élucider certains processus complexes qui ont lieu dans les systèmes biologiques. Dans le cadre de cette thèse, l’hydratation contrôlée de trois systèmes poreux modèles suivie par spectroscopie infrarouge montre les effets de la nature des surfaces et des limitations stériques qui, en altérant les liaisons hydrogène établies entre les molécules d’eau, déclenche la formation de réseaux atypiques. La brillance de la source de rayonnement synchrotron infrarouge, exploitée par la ligne de lumière AILES au sein du synchrotron SOLEIL, a permis l’étude d’échantillons très absorbants en permettant de mettre en évidence la faible contribution de monocouches de molécules d’eau voire même de chaînes linéaires. L'effet d'une surface hydrophile sur la structure, la densité et la dynamique des molécules d'eau à l'interface a été étudié en mesurant l’absorbance d’une lamelle de Vycor poreux (pores d’environ 5 nm) à l’équilibre avec une pression de vapeur d'eau donnant lieu à la formation d’un réseau d’eau bi-dimensionnel. Les interactions fortes entre l’eau interfaciale et la surface hydrophile provoquent plusieurs transitions structurales du réseau d’eau expliquant ainsi les observations par d’autres techniques. Des similitudes et des différences surprenantes ont été observés entre l’hydratation d’une surface hydrophile et celle d’un système hydrophobe dans des conditions de confinement extrême : les nanotubes de carbone à paroi unique (SWCNTs) ayant un diamètre de quelques Angstrom. Nos mesures ont montré que l’établissement d’un réseau de molécules d’eau unique peut être à l’origine du déplacement rapide des molécules d’eau à l’intérieur des nanotubes de carbone. Enfin, l’étude des mécanismes d’adsorption de la membrane Nafion, membrane modèle dans les piles à combustible à membrane échangeuse de protons (PEMFCs), a permis de clarifier les processus en jeu lors de l’ionisation et la formation d’espèces protonique responsable de la remarquable conductivité ionique propre à cette famille de membranes.Cette méthodologie pourra être étendue à des systèmes plus complexes, tels que les réseaux de molécules d’eau dans les systèmes biologiques. / Understanding the water absorption mechanisms, and the molecular arrangement adopted by the water molecules upon confinement at the nanoscale is crucial both for technological applications, such as clean energy production, purification and desalination of water, and to unveil some of the complex processes occurring in biological systems.In this thesis, the controlled hydration of three porous model systems monitored by infrared spectroscopy shows the effects of surfaces forces and steric limitations that triggers the formation atypical networks by altering the hydrogen bonds established between water molecules. The brightness of the infrared synchrotron radiation source on the AILES beamline at synchrotron SOLEIL allowed to highlight the low contribution coming from water molecules arranged as monolayer or linear chains in highly infrared absorbent matrices. The effect of a hydrophilic surface on structure, density and dynamic of water molecules at interfaces was examined by measuring the absorbance of a porous Vycor slab (pores of about 5 nm) at equilibrium with the water vapor pressure needed to obtain a two-dimensional water network. The strong interfacial interactions between water and the hydrophilic surface cause several structural transitions on the water network elucidating the observations made by other techniques. Surprising similarities and differences are observed between the hydration of a hydrophilic surface and that of a hydrophobic system under extreme confinement: single-wall carbon nanotubes (SWCNTs) having a diameter of several Angstroms. Our measurements have shown that the arrangement of a peculiar water network may be the origin of the enhanced water flux in carbon nanotubes. Finally, the study of the adsorption mechanisms of the Nafion membrane, the benchmark electrolyte in proton exchange membrane fuel cells (PEMFCs), has shed new light on the processes involved in the ionization and the formation of protonic species, accountable for the remarkable ionic conductivity typical of these membranes.This approach may be also extended to more complex systems, such as water molecules networks in biological systems.

The work presented herein focus upon crystal engineering of nutraceutical cocrystals. Cocrystals are considered unique solid dosage form which has many advantages over other traditionally known solid forms. Furthermore, cocrystals have proven to improve stability, solubility and bioavailability of Active Pharmaceutical Ingredient (API) as shown in the case of carbamazepine and other APIs in previous studies. Crystal engineering is commonly used to design new solid forms based on the bases of supramolecular chemistry. In this study, crystal engineering based on intensive Cambridge Structural Database (CSD) analysis used to predict and design new cocrystals of targeted nutraceuticals. Two nutraceuticals were selected for this study; resveratrol and citric acid. The rationale behind selecting resveratrol was to improve its solubility and, accordingly, bioavailability. On the other hand, citric acid is known as a highly soluble and safe nutraceutical, and thus it can be used as a coformer. Five new cocrystals were prepared and characterized using a variety of techniques that include single crystal X-ray diffraction (XRD), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), FT-IR, and thermo-gravimetric analysis (TGA). Most of the reported cocrystals were obtained using different techniques; solvent slow evaporation, mechanichemical approach, slurry, and from melt. Moreover, dissolution test has been performed on resveratrol and two of its cocrystals, using UV-vis spectrophotometer, where the data demonstrate that through cocrystallization with different cocrystal formers, solubility of resveratrol could be greatly modified, and further controlled. The polymorphism phenomenon is encountered, and accordingly addressed, herein where four novel polymorphs were obtained during cocrystallization attempts. Polymorphism has a significant importance in industry, in general, and in pharmaceutical industry, in particular, due to the vast differences in physical properties of polymorphs. Furthermore, the study of polymorphism provides valuable information essential to understand how different crystal forms are attained.

Chloroperoxidase (CPO) is one of the most versatile heme enzyme isolated from the marine fungus, Caldariomyces fumago. Functionally, CPO can catalyze four types of reactions: peroxidation (peroxidase-like), dismutation (catalase-like), halogenation (halogenase-like), and peroxygenation (P450-like). Structurally, CPO has a distal and proximal pockets that can be best described as a hybrid of classical peroxidase and P450s. As a heme-thiolate protein, CPO contains the conserved proximal Pro28-Cys29-Pro30 stretch found in other members of the family. However, the structural and functional roles of these proline residues remain poorly understood. Site-directed mutagenesis was undertaken to generates three CPO mutants, P28A-, P30A-, P28A/P30A-CPO. The replacement of the rigid proline with a more flexible alanine residue, freed up the back bone amide for the formation of additional amide-sulfur hydrogen bond, allowing the investigation of the importance of these residues in CPO catalysis. The three CPO mutants displayed dramatic difference in ligand binding affinity and catalytic activities relative to WT-CPO. Any mutations on the proline resides within the proximal loop eliminated the halogenation and dismutation activities but enhanced the vii epoxidation and peroxidation activities by 4-14 fold. As the binding affinity for cyanide, the CPO mutants displayed significantly higher dissociation constant relative to WT-CPO. Our results revealed that Pro28 and Pro30 play important roles in maintaining the versatility of CPO. As a versatile enzyme, CPO has great application potential in pharmaceutical and chemical industry due to its ability to catalyze the formation of chiral epoxides. Phe103 and Phe186 located on the distal pocket have been proposed to guard the access of substrates to the ferryl oxygen of the heme center. The interactions of these two phenylalanine residues restricted the size of substrates and regulates CPO’s enantioselectivity. F186A- and F103A/F186A-CPO were generated and characterized where the rate of peroxidation and epoxidation were significantly enhanced at the expense of halogenation and dismutation activities. Our results demonstrated that Phe186 played a subtler role relative to Phe103 in terms of substrate specificity and product enantioselectivity of CPO.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20393号 / 工博第4330号 / 新制||工||1671(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 村田 靖次郎, 教授 小澤 文幸, 教授 近藤 輝幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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