Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...

Les méthodes de synthèse d’hétérocycles fonctionnalisés suscitent un vif intérêt de la part de l’industrie pharmaceutique et sont d’une grande importance pour la synthèse de produits naturels et le développement des nano-sciences. La chimie organométallique a révolutionné les approches de fonctionnalisation d’hétérocycles et actuellement le développement de méthodes alternatives aux couplages croisés basées sur la fonctionnalisation directe de la liaison C-H catalysées par un métal de transition, qui sont plus attractives en terme d’économie d’atomes et de chimioselectivité, est fortement étudié. Toutefois une réelle valorisation de ces techniques repose essentiellement sur un accroissement de la diversité et des techniques de contrôle du site de la substitution. Ce travail s’inscrit dans ce programme de développement en série oxazolique dont l’une des principales difficultés réside dans la compétitivité des positions 2 et 5 du noyau oxazole. Le projet a été centré en particulier sur l’étude de la fonctionnalisation directe régiosélective de l’oxazole-4-carboxylate d’éthyle qui a été sélectionné en raison de sa haute valeur ajoutée en synthèse totale, la modularité chimique apportée par la fonction ester et une plus grande discrimination électronique et environnementale des positions 2 et 5 par rapport à l’oxazole nu. La première partie du travail a été axée sur l’établissement d’un nouveau procédé d’hétéroarylation pallado-catalysé régiosélectif en position 2 de l’oxazole-4-carboxylate d’éthyle avec une large gamme d’(hétéro)aromatiques iodés, bromés et chlorés, basé sur l’emploi spécifique de deux ligands, le Cy-JohnPhos et la tri-o-tolylphosphine dans deux solvants, le dioxane et le toluène. Dans le cadre d’une collaboration avec le Dr Doucet de l’Université de Rennes portant sur le développement de systèmes catalytiques plus "éco-compatibles", une étude préliminaire de remplacement du toluène par le diéthylcarbonate a également été réalisée avec succès. Une procédure étendue de vinylation, de benzylation et de méthylation directes régiosélectives en position 2 de l’oxazole-4-carboxylate d’éthyle a également été développée. Les méthodologies de fonctionnalisation directes de l’oxazole-4-carboxylate d’éthyle ont ensuite été mises à profit pour accéder aux oxazoles 2-mono- et 2,5-difonctionnalisés. Trois nouvelles synthèses totales de la balsoxine, de la texaline et de l’annulonine ont ainsi pu être proposées. De plus, dans le contexte actuel du développement de nouveaux fluorophores pour la chimie de détection, un panel d’analogues DPO-4-carboxylates et POPOP-4-dicarboxylates a été obtenu. Trois analogues possédant un déplacement de Stokes amélioré tout en conservant un très bon rendement quantique ont été identifiés. La dernière partie de ce travail a été consacrée à un examen détaillé de trois paramètres du processus d’arylation directe pallado-catalysé de l’oxazole-4-carboxylate d’éthyle, la demande électronique, la force basique mesurée par incorporation de deuterium, et l’influence de la base sur la réussite du couplage. Il a permis de dégager deux modes d’activation principaux, la déprotonation/métallation ortho-dirigée (ODM) et la métallation/déprotonation concertée (CMD), dont le site d’action dépend essentiellement de la nature du solvant. / Development of versatile synthetic routes towards functionalized heterocycles is of main importance for pharmaceutical, total synthesis and materials. The organometallic chemistry has revolutionized the access to functionalized heterocycles and the development of more practical, chimioselective and atom-economy transition metal-catalyzed direct C-H functionalization methodology is currently actively examined as alternatives to the experimentally restrictive and highly used cross-coupling reactions. However, a possible valorisation of such synthetic approach in several fields of chemistry dramatically depends on the development of novel methods for controlling the site and the nature of the substitution. The project is concerned with the challenging oxazole serie for which the 2 and 5 positions are highly competitive, and has been first focused on the study of the direct C-H bond heteroarylation of the selected ethyl oxazole-4-carboxylate which is a ready available and highly valuable in total synthesis scaffold. Moreover interestingly this model exhibits a more electronically and environmental difference between the 2 and 5 competitive sites coupling than the naked oxazole. Thus, the first part of the work lead to the development of a novel regioselective direct C-H bond heteroarylation of ethyl oxazole-4-carboxylate with broad iodo-, bromo- and chloroheteroaromatics using specifically both Cy-JohnPhos and tri-otolylphosphine ligands in dioxane and toluene as solvents. Moreover, the use of the more biodegradable diethylcarbonate as an alternative to toluene as solvent was successfully accomplished through a partnership with Dr Doucet of Université de Rennes concerned with the development of green catalysts. An extended protocol for regioselective direct C-H vinylation, benzylation and methylation of ethyl oxazole-4-carboxylate protocol was also proposed. The previous developed direct C-H heteroarylation methodologies in oxazole-4-carboxylate serie were then first applied to the preparation of 2-mono- and 2,5-difunctionalized oxazoles. Three novel total syntheses of balsoxin, texaline and annulonine, were achieved. In a current context of the designing of original fluorophores for the field of molecular interactions detection, a library of DPO-4-carboxylates and POPOP-4-dicarboxylates was prepared. Interestingly, three novel analogs exhibit a much more better Stokes-shift than DPO and POPOP references with a high-preserved quantum yield. The last part of this work was devoted to the examination of three parameters of the palladium-catalyzed direct C-H arylation in oxazole-4-carboxylate serie, the electronic demand, the basic strength measured by deuterium incorporation and its influence on the success of the direct coupling, allowed us to highlight two main activation pathways, the ortho-directed deprotonation métallation (ODM) and the concerted metallation deprotonation (CMD), that act differently and at different sites depending mainly on the nature of the solvent.

The membrane potential is one of the most important parameters of the living cell. It can be measured using carbocyanine fluorescent probes. In this thesis we examined parameters of several dyes of this family. For further experiments three of them were chosen - diOC3(3), diIC1(3) a diIC2(5) as a supplement to diSC3(3) and diSC3(5), which represent standard probes used at biophysical department of Institut of Physics. We compared the rates of their accumulation in S. cerevisiae cells to determine if they were MDR pumps' substrates. The other goal of this work was to decide whether the results obtained using different probes are equivalent and to determine if the presence of a probe affects the spectral characteristics of another. For this purpose we have chosen diSC3(3) and diSC3(5). With those dyes we examined the influence of the acidification on membrane potencial of the yeast S. cerevisiae. We showed that the information on depolarization obtained using both probes were matching very well.

α-Synuclein (AS) is a small intrinsically disordered protein expressed in neurons and abundantly present in synapses where it is involved in regulation of synaptic vesicle-mediated protein trafficking. Misfolding of AS into amyloid fibrils is a key process in progression of Parkinson's disease (PD), the second most common neurodegenerative disorder which has no cure to date. Inhibition of AS aggregation and blocking of cell-to-cell spreading of AS fibrils is a promising strategy for PD treatment. However, rational design of inhibitors of this type remains complicated due to the lack of thorough knowledge about the mechanisms of aggregation. Therefore, the aim of this thesis was to gain deeper knowledge about AS aggregation and to apply it for developing inhibitors of AS fibrillization. In my work on the mechanisms of AS aggregation, I first determined that the concentration of AS that enables the fibril growth is an order of magnitude lower than the concentration of AS required for initial fibril formation from monomers. I explored fibril disaggregation at AS concentrations below its Kd value, and characterized AS aggregation at low micromolar concentrations. I then investigated how different modifications of AS C-terminus (namely, extensions of various sizes and charges) affect fibril growth and...

Homocysteine is a natural occurring aminothiol. It is an intermediate product in the metabolism of methionine. Methionine is an essential amino acid required for protein synthesis. Metabolic irregularities disrupt homocysteine levels in plasma. Elevated homocysteine levels are directly linked to folate and cobalamin (vitamin B12) deficiencies, and are an independent risk factor for cardiovascular diseases. High homocysteine levels have also been associated with Alzheimer's, osteoporosis, renal failure, cancer, birth defects and pregnancy complications. The association of elevated homocysteine levels with cardiovascular disease and other diseases has generated great interest in the detection of homocysteine. An optical method for the detection of homocysteine has been developed using fluorescein mono- and dialdehydes. Selectivity for homocysteine was achieved based on the characteristic differences between 5- and 6-membered ring heterocyclic amines formed upon the reaction with fluorescein mono- and dialdehydes. 6-membered ring homocysteine-derived thiazinane-4-carboxylic acids were found to be more basic than 5-membered cysteine-derived thiazolidine-4-carboxylic acids. Fluorescence enhancement in response to homocysteine was thus attained by tuning pH and excitation wavelengths. Furthermore, the design and synthesis of a more sensitive fluorophore, fluorescein tri aldehyde has been accomplished based on the aforementioned findings to enable the detection of homocysteine at physiological levels. Calculations of Mulliken charges revealed that the formation of thiazinanes results in modulation of the electron density on the fluorophore leading to higher fluorescence.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20569号 / 医科博第79号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 江藤 浩之, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The development of new multiphoton absorbing materials has attracted the attention of researchers for the last two decades. The advantages that multiphoton absorbing materials offer, versus their one-photon absorbing counterparts, rely on the nature of the nonlinearity of the absorption process, where two photons are absorbed simultaneously offering increased 3D resolution, deeper penetration, and less photobleaching and photodamage as a result of a more confined excitation. The applications of efficient two-photon absorbing materials have been extensively expanding into the fields of photodynamic therapy, microscopy, and optical data storage. One of the fields where an increased interest in multiphoton absorbing materials has been most evident is in bioimaging, in particular, when different cellular processes and organelles need to be studied by fluorescence microscopy. The goal of this research was to develop efficient two-photon absorption (2PA) compounds to be used in fluorescence bioimaging, meaning that such compounds need to posses good optical properties, such as high fluorescence quantum yield, 2PA cross section, and photostability. In the first chapter of this dissertation, we describe the synthesis and structural characterization of a new series of fluorescent donor–acceptor and acceptor-acceptor molecules based on the fluorenyl ring system that incorporated functionalities such as alkynes and thiophene rings, through efficient Pd-catalyzed Sonogashira and Stille coupling reactions, in order to increase the length of the conjugation in our systems. These new molecules proved to have high two-photon absorption (2PA), and the effect of these functionalities on their 2PA cross section values was evaluated. Finally, their use in two-photon fluorescence microscopy (2PFM) imaging was demonstrated. iii One of the limitations of the compounds described in Chapter 1 was their poor water solubility; this issue was addressed in Chapter 2. The use of micelles in drug delivery has been shown to be an area of increasing interest over the last decade. In the bioimaging field, it is key to have dye molecules with a high degree of water solubility to enable cells to uptake the dye. By enclosing a hydrophobic dye in Pluronic® F-127 micelles, we developed a system that facilitates the use of 2PA molecules (typically hydrophobic) in biological systems for nonlinear biophotonic applications, specifically to image the lysosomes. Furthermore, we report in this chapter the efficient microwave-assisted synthesis of the dye used in this study. In addition, linear photophysical and photochemical parameters, two-photon absorption (2PA), and superfluorescence properties of the dye studied in Chapter 2, were investigated in Chapter 3. The steady-state absorption, fluorescence, and excitation anisotropy spectra of this dye were measured in several organic solvents and aqueous media. In Chapter 4, we describe the preparation and the use of an efficient and novel twophoton absorbing fluorescent probe conjugated to an antibody that confers selectivity towards the vascular endothelial growth factor receptor 2 (VEGFR-2) in porcine aortic endothelial cells that express this receptor (PAE-KDR). It is known that this receptor is overexpressed in certain cancer processes. Thus, targeting of this receptor will be useful to image the tumor vasculature. It was observed that when the dye was incubated with cells that do not express the receptor, no effective binding between the bioconjugate and the cells took place, resulting in very poor, nonspecific fluorescence images by both one and two-photon excitation. On the other hand, when the dye was incubated with cells that expressed VEGFR-2, efficient imaging of the cells was obtained, even at very low concentrations (0.4 μM). Moreover, incubation of the bioconjugate iv with tissue facilitated successful imaging of vasculature in mouse embryonic tissue

Probing orientations of fluorescent molecules embedded in or attached to cell membranes has a great potential to reveal information on membrane structure and processes occurring in living cells. In this thesis, we first describe one- and two-photon linear dichroism measurements on a fluorescent probe embedded in a phospholipid membrane with a well- defined lipid composition. On the basis of experimental data, we determine the distribution of the angle between the one-photon transition dipole moment of the probe and the membrane normal. At the same time, we perform molecular dynamics simulations of the fluorescent probe and quantum calculations of its one-photon and two-photon absorption properties. By comparing the orientational distribution gained from experiments with that predicted by simulations, we test the ability of linear dichroism measurements to report on the orientation of a fluorescent molecule in a lipid membrane. We also examine the applicability of molecular simulations as a basis for the interpretation of experimental data.

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-disubstituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak-to-moderately binding to DNA, and symmetrical 1,4-disubstituted compounds were shown to be non-toxic (IC50 = 100 :M) with nonsymmetrical analogues generating IC50 values in the 1-100 :M range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133⁺/ α₂β₁ hi stem cell fraction and upregulated in CD133⁻/ α₂β₁ lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells.