Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17601号 / 工博第3760号 / 新制||工||1573(附属図書館) / 30367 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 松田 建児, 教授 秋吉 一成 / 学位規則第4条第1項該当

Nanofluidic devices have been of great interest due to their applications in variety of fields, including energy conversion and storage, water desalination, biological and chemical separations, and lab-on-a-chip devices. Although these applications cross the boundaries of many different disciplines, they all share the demand for understanding transport in nanoscale conduits. In this thesis, different elusive aspects of molecule and particle transport through nanofluidic conduits are investigated, including liquid and ion transport in nanochannels, diffusion- and reaction-governed enzyme transport in nanofluidic channels, and finally translocation of nanobeads through nanopores. Liquid or solvent transport through nanoconfinements is an essential yet barely characterized component of any nanofluidic systems. In the first chapter, water transport through single hydrophilic nanochannels with heights down to 7 nm is experimentally investigated using a new measurement technique. This technique has been developed based on the capillary flow and a novel hybrid nanochannel design and is capable of characterizing flow in both single nanoconduits as well as nanoporous media. The presence of a 0.7 nm thick hydration layer on hydrophilic surfaces and its effect on increasing the hydraulic resistance of the nanochannels is verified. Next, ion transport in a new class of nanofluidic rectifiers is theoretically and experimentally investigated. These so called nanofluidic diodes are nanochannels with asymmetric geometries which preferentially allow ion transport in one direction. A nondimensional number as a function of electrolyte concentration, nanochannel dimensions, and surface charge is derived that summarizes the rectification behavior of this system. In the fourth chapter, diffusion- and reaction-governed enzyme transport in nanofluidic channels is studied and the theoretical background necessary for understanding enzymatic activity in nanofluidic channels is presented. A simple analytical expression that describes different reaction kinetics is derived and confirmed against available experimental data of reaction of Trypsin with Poly-L-lysine. Finally, in the last chapter translocation of nanobeads through synthetic nanopores is experimentally investigated using resistive pulse sensing. The emphasis is placed on elucidating the effect of nanobead size on the translocation current and time. The key goals pursued in this study are multiplex detection of different nanobead sizes in a mixture of nanobeads as well as determining the concentration of each component. This problem other than its fundamental significance paves the way for developing new biosensing mechanisms for detection of biomolecules. This thesis further explores the molecule and particle transport in nanoscale conduits and serves for better characterization and development of nanofluidic devices for various applications.

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Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.

Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site-directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3-O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting L41M, N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3-O-GT.

La bioluminescence est un processus complexe dans lequel la réaction chimique d'un substrat, catalysée par une protéine, entraîne l'apparition d'une émission de lumière dans le spectre visible. Dans le cas des lucioles, un insecte émettant dans le domaine du jaune-vert, le substrat se nomme luciférine et la protéine luciférase. Cependant, la taille et la complexité de ce système chimique limite sa compréhension, notamment celle du mécanisme réactionnel.L'apport de la chimie théorique dans ce domaine est essentiel et a prouvé son utilité dans de nombreux cas. L'utilisation de la méthode QM/MM, méthode hybride couplant la mécanique quantique et la mécanique moléculaire permet de modéliser et d'étudier ces systèmes biologiques.Cette thèse se focalise sur deux approches différentes de l'étude de la bioluminescence chez les lucioles. La première consiste à étudier l'effet de certaines modifications chimiques sur la couleur de la bioluminescence. On s'intéresse plus particulièrement à un analogue de la luciférine et à certaines luciférases issues d'autres systèmes bioluminescents. Par cette étude on cherche à rationaliser et à prédire l'effet ainsi que l’impact de ces changements sur l’émission. Le deuxième sujet explore deux étapes du mécanisme réactionnel de la bioluminescence: d'une part, la coordination du dioxygène sur un intermédiaire de la réaction, une étape encore non étudiée et d'autre part la réaction de tautomérisation dans l'état excité et au sein de la protéine entre deux formes émissives possibles de la luciférine / The bioluminescence is a complex process that involves the reaction of a substrate, catalysed by an enzyme that sheds light in the visible spectra. In fireflies, the light emitted has a yellow-green tone thanks to the interaction between the substrate luciferin and the protein luciferase. However the size and the complexity of the system prevent its comprehension especially when dealing with the reaction mechanism.The use of computational chemistry is key to understand and improve the comprehension of the bioluminescence. The hybrid QM/MM method that combines quantum mechanics with molecular mechanics is a great tool to model and study bioluminescent systems.This thesis deals with two different approaches of the bioluminescence in fireflies. The first one is related to the study of chemical modifications that tune the emission colour. We will discuss about one analogue of the luciferin and on new luciferases from others bioluminescent species. The goals of this study are to rationalise and predict both the effect and the impact of these modifications on the emission. The second subject deals with two different steps of the bioluminescent mechanism. The first one discusses the binding of the dioxygen to the bioluminescent intermediate, which was so far unstudied and the second one about the tautomerization in the excited state and in the protein of two possible emissive forms of the luciferin

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.