L’augmentation des activités humaines dans le domaine nucléaire civil comme militaire est à l’origine d’une dissémination effective ou potentielle de radionucléides dans l’environnement. Leur mobilité dépend de plusieurs facteurs tels que le pH, la force ionique, le degré d’oxydation et la présence de ligands organiques. Afin de garantir la sûreté de sites d’entreposage ou de stockage, de développer des procédés de remédiation de sols contaminés, il est nécessaire de disposer de données fondamentales sur les interactions entre actinides et ligands organiques susceptibles d’être présents dans l’environnement, vecteurs de migration ou de piégeage de ces radioéléments. Dans ce contexte, l’étude s’est axée sur les interactions entre Th(IV), U(IV,VI), Cf(III) and Eu(III) et des dérivés hydroxamates, en particulier, la desferrioxamine B, un sidérophore bactérien comportant trois fonctions hydroxamiques. Les constantes de formation des complexes sont déterminées en fonction de l’acidité et de la concentration de ligand à force ionique et température fixées en mettant en jeu plusieurs techniques expérimentales et des concentrations en élément comprises entre 10⁻¹⁰ et 10⁻³ M (extraction liquide-liquide combinée à une détection par spectrométrie γ, électrophorèse capillaire avec détection UV, spectrophotométrie d’absorption UV-visible). L’approche thermodynamique de cette étude est complétée par une étude structurale à l’aide de techniques spectroscopiques telles que la spectroscopie infrarouge à transformée de Fourier et de la spectroscopie d’absorption des rayons X. Ces mesures expérimentales sont alors confrontées à des calculs théoriques (DFT), afin de déterminer l’arrangement structural du métal et les distances interatomiques. / Due to the increasing human activities in the civilian nuclear fields, the actual and potential release of radionuclides into the environment is a matter of concern. The mobility of radio-nuclides depends on several factors such as pH, ionic strength, oxidation state and the presence of organic ligands. In order to guarantee the safety of radioactive waste storage sites and to develop contaminated soil remediation processes, it is necessary to have fundamental data on actinides and natural organic ligands interactions. This study focuses on the interaction between Th(IV), U(IV,VI), Cf(III) and Eu(III) and hydroxamates derivatives, desferrioxamine B, a bacterial siderophore with three hydroxamic functions. The stability constants of complexes are determined as function of acidity and ligand concentration, at fixed ionic strength and temperature, using several techniques and metal concentrations ranging from 10⁻¹⁰ to 10⁻³ M (liquid-liquid extraction coupled with γ-spectrometry, capillary electrophoresis with UV detection, UV-Vis absorption spectrophotometry). The thermodynamic study is supplemented by a structural one using spectroscopic techniques such as Fourier transform infrared spectroscopy and X-ray absorption spectroscopy. Experimental measurements are compared with quantum chemistry calculations (DFT) in order to determine the coordination geometry of the metal ion and the interatomic distances.

Résumé : Grâce à des caractéristiques physiques particulières, le [indice supérieur 64]Cu (T[indice inférieur 1/2]= 12.7 h; β[indice supérieur+], 0.65 MeV [17.8 %]; β[indice supérieur −], 0.58 MeV [38.4 %]) est un candidat idéal pour l’imagerie TEP et la radiothérapie ciblée du cancer. Son utilisation est actuellement limitée par la disponibilité de chélateurs bi-fonctionnels (CBFs) offrant une résistance élevée aux réactions de transmétallation in vivo. Récemment nous avons développés deux nouveaux CBFs cycliques, DOTHA[indice inférieur 2] et NOTHA[indice inférieur 2], portant des ligands hydroxamates pour la complexation au [indice supérieur 64]Cu. Ces CBFs possèdent une cinétique de marquage rapide dans des conditions très douces, une stabilité élevée in vivo et un profil de biodistribution favorable avec une clairance rapide. Nous proposons maintenant d’étendre notre approche à la préparation de CBFs acycliques plus flexibles et compacts afin de moduler les propriétés biologiques et la pharmacocinétique des traceurs peptidiques. Le but de mon projet de maîtrise est de développer une série de chélateurs acycliques dérivés de l'histidine et de l'acide glutamique et fonctionalisés avec des groupements hydroxamates pour identifier un CBF offrant un complexe stable in vivo avec le [indice supérieur 64]Cu(II). Les CBFs ont été préparés en solution pour faciliter l’optimisation de chaque étape réactionnelle. Les groupements chélatants hydroxamates ont été sélectionnés pour leur habilité à former des complexes stables avec différents métaux et ils ont été liés en position N-terminale et sur la chaîne latérale des acides aminés grâce à des réactions de substitution nucléophile. Les groupements para-methoxy-benzyles ont été judicieusement sélectionnés pour la protection des groupements hydroxamates afin de faciliter, au besoin, une déprotection sélective sous des conditions très douces. L’optimisation du marquage a été effectuée avec l’isotope stable du cuivre et ensuite avec le [indice supérieur 64]Cu en faisant varier le contre ion métallique, le pH, la concentration, et la température. Le CBF offrant la plus grande stabilité, soit celui dérivé de l’histidine, a été conjugué à un peptide, le H[indice inférieur 2]N-PEG-[D-Tyr[indice supérieur 6],βAla[indice supérieur 11],Thi[indice supérieur 13],Nle[indice supérieur 14]]bombesin(6-14) (BBN), se liant fortement aux récepteurs de la relâche de la gastrine surexprimés dans les cancers du sein et de la prostate. La stabilité et l’activité spécifique du CBF-histidine et du radiotraceur marqués au [indice supérieur 64]Cu s’est avérée faible in vitro. Il est connu que l’activité antibactérienne de ligands hydroxamates est associée à leur capacité à complexer le fer. En perspective, comme nos chélateurs complexent très fortement le Fe(III), une alternative pour ces composés serait d’évaluer leur capacité à inhiber la croissance et la prolifération des bactéries. || Abstract : Thanks to its particular physical characteristics, [superscript 64]Cu (T[subscript ½= 12.7 h; β[superscript +], 0.65MeV [17.8 %]; β[superscript −], 0.58MeV [38.4 %]) is an ideal candidate for PET imaging and targeted cancer radiotherapy. Currently, its use is limited by the availability of bi-functional chelators (BFCs) which give high resistance to in vivo transmetallation reactions. Recently, we developed two new cyclic BFCs, DOTHA[subscript 2] and NOTHA[subscript 2], bearing hydroxamate pendant arms for the complexation with [superscript 64]Cu. Those BFCs have fast labeling kinetics under very mild conditions, a high in vivo stability and a biodistribution profile which is favorable with a fast clearance. Now, we propose to expand our approach to the preparation of acyclic BFCs, which are more flexible and compact, in order to better modulate biological properties and the pharmacokinetics of the peptidic tracers. The goal of my Master’s degree project is to develop a series of acyclic chelators derived from histidine and glutamic acid and functionalized with hydroxamate pendant arms to identify a BFC that shows highly stable in vivo complexes with [superscript 64]Cu(II). BFCs have been prepared in solution to facilitate the optimization of each reactive step. Hydroxamate chelating groups have been selected for their ability to form stable complexes with different metals and they have been conjugated in N-terminal position and on the lateral chain of amino acids via nucleophilic substitution reactions. Para-methoxy-benzyl groups have been judiciously selected for the protection of the hydroxamate groups to facilitate, if needed, a selective deprotection under mild conditions. The labeling optimization has been performed with a stable copper isotope, and then with [superscript 64]Cu varying the metallic counter-ion, pH, concentration and temperature. The BFC having the highest stability, the one derived from histidine, was conjugated to a peptide, H[subscript 2]N-PEG-[D-Tyr[superscript 6],βAla[superscript 11],Thi[superscript 13],Nle[superscript 14]]bombesin(6-14) (BBN), strongly bounding the gastrin releasing peptide receptor, which is overexpressed in breast and prostate cancers. Both the stability and specific activity of BFC-histidine of the radiotracer labeled with [superscript 64]Cu were low in vitro. It is known that the antibacterial activity of hydroxamate ligands is associated with their ability to complex iron. In perspective, because our hydroxamate ligands strongly complex Fe(III), an alternative for these compounds would be to assess their ability to inhibit the growth and proliferation of bacteria.

Le repositionnement des médicaments est une stratégie visant à trouver de nouvelles indications pour des médicaments existants. Un composé de type thiomorpholine hydroxamate (TMI-1, un inhibiteur d'ADAM-17 utilisé dans le traitement des maladies chroniques inflammatoires) a récemment été repositionné dans le traitement du cancer. Afin d’élucider son mécanisme d'action, une série de dérivés de type d'arysulfonyl thiomorpholine hydroxamate a été synthétisée et évaluée pour déterminer une relation entre structure et activité . Nous avons démontré que l'activité cytotoxique observée dépend du fragment phényl hydroxamate et n'est pas liée à l'inhibition de l'ADAM-17. Grâce à une approche par protéomique inverse, nous avons identifié la protéine membranaire peroxysomale PMP34 / SLC25A17 comme une nouvelle cible de TMI-1. PMP34 est un transporteur transmembranaire de différents cofacteurs nécessaires à la fonction des peroxysomes. Nous démontrons ici que TMI-1 se lie directement à PMP34 et augmente l’interaction entre PMP34 et PEX19, une protéine chaperone impliquée dans l’assemblage et la biologie des peroxysomes, alors que les expériences sur les ARNi ont révélé que PMP34 est essentielle à la viabilité des cellules tumorales. Enfin, le traitement des cellules tumorales par TMI-1 induit une modification de la composition en protéines du peroxysome et augmente le niveau de la protéine PMP70 / ABCD3. Un taux élevé de PMP70 dans le peroxysome augmente la $\beta$-oxydation des acides gras ce qui pourrait entraîner la mort des cellules cancéreuses. En conséquence, PMP34 représente une nouvelle cible intéressante en oncologie. / Drug repositioning is a strategy to find new indications for existing drugs. A thiomorpholine hydroxamate compound (TMI-1, an ADAM-17 inhibitor used for the treatment of chronic inflammatory diseases) has been recently repositioned in cancer. To further elucidate its mechanism of action, a series of arylsulfonyl thiomorpholine hydroxamate derivatives was synthetized and evaluated to determine a structure-activity relationship. We have pinpointed that the observed cytotoxic activity depends on the hydroxamate phenyl moiety and is not related to ADAM-17 inhibition. Using a reverse proteomic approach, we now identify the peroxisomal membrane protein PMP34/SLC25A17 as a new target of TMI-1. PMP34 is a transmembrane transporter of different cofactors required for peroxisome function. We demonstrate here that TMI-1 directly binds to PMP34 and increases the interaction between PMP34 and PEX19, a chaperone protein involved in peroxisome assembly and biology while RNAi experiments revealed that PMP34 is essential for tumor cell viability. Finally, TMI-1 treatment of tumor cells induces modification of peroxisome protein composition and increases the level PMP70/ABCD3 protein. High PMP70 level in peroxisome increases fatty acid $\beta$-oxidation that could lead to cancer cell death. As a result, PMP34 represents a new valuable target in oncology.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21050号 / 薬科博第93号 / 新制||薬科||10(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 竹本 佳司, 教授 二木 史朗 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Iron is essential to the majority of microorganisms; it is an important cofactor in many cellular processes and enzymes. However in an aerobic environment and at biological pH, iron is primarily found as insoluble oxyhydroxides and is unavailable to microorganisms. Many bacteria have the ability to produce siderophores, low molecular weight compounds that have a high affinity for Fe3+. Siderophores are part of a multi-component system that actively transports the iron-siderophore complex into the cytoplasm. Rhizobia are characterized by their ability to form symbiotic relationships with leguminous plants, where they can fix nitrogen for the host plant and the plant provides the bacteria with nutrients. Under iron-limiting conditions, Rhizobia are known to produce siderophores. Rhizobium leguminosarum IARI 917 produces one dihydroxamate-type siderophore. This siderophore has been purified and chemically characterized. Results indicate that this strain is producing schizokinen, which has not been described in a member of the Rhizobia family.

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The groundwater bacterium Pseudomonas fluorescens (CCUG 32456) isolated at a depth of 70 m in the Äspö Hard Rock Laboratory secretes a pyoverdin-mixture with four main components (two pyoverdins and two ferribactins). The dominant influence of the pyoverdins of this mixture could be demonstrated by an absorption spectroscopy study. The comparison of the stability constants of U(VI), Cm(III), and Np(V) species with ligands simulating the functional groups of the pyoverdins results in the following order of complex strength: pyoverdins (PYO) > trihydroxamate (DFO) > catecholates (NAP, 6­HQ) > simple hydroxamates (SHA, BHA). The pyoverdin chromophore functionality shows a large affinity to bind actinides. As a result, pyoverdins are also able to complex and to mobilize elements other than Fe(III) at a considerably high efficiency. It is known that EDTA may form the strongest actinide complexes among the various organic components in nuclear wastes. The stability constants of 1:1 species formed between Cm(III) and U(VI) and pyoverdins are by a factor of 1.05 and 1.3, respectively, larger compared to the corresponding EDTA stability constants. The Np(V)-PYO stability constant is even by a factor of 1.83 greater than the EDTA stability constant. The identified Np(V)-PYO species belong to the strongest Np(V) species with organic material reported so far. All identified species influence the actinide speciation within the biologically relevant pH range. The metal binding properties of microbes are mainly determined by functional groups of their cell wall (LPS: Gram-negative bacteria and PG: Gram-positive bacteria). On the basis of the determined stability constants raw estimates are possible, if actinides prefer to interact with the microbial cell wall components or with the secreted pyoverdin bioligands. By taking pH 5 as an example, U(VI)-PYO interactions are slightly stronger than those observed with LPS and PG. For Cm(III) we found a much stronger affinity to aqueous pyoverdin species than to functional groups of the cell wall compartments. A similar behavior was observed for Np(V). This shows the importance of indirect interaction processes between actinides and bioligands secreted by resident microbes.

Une série de peptides présentant un ou deux motifs hydroxamate a été synthétisée, l'étape clé étant la N-acylation sélective de N-hydroxy-dipeptides terminaux. L'atome d'oxygène du groupe hydroxyle peut servir de point d'ancrage pour une autre chaîne et donne ainsi accès à de nouveaux pseudopeptides. D'une part, l'acylation de N-hydroxy-peptides a été réalisée dans différentes conditions permettant l'introduction de groupements variés, en particulier des acides aminés. Il est possible d'allonger ces N-acyloxy-peptides dans toutes les directions, même si dans certains cas des réarrangements intéressants peuvent avoir lieu. D'autre part, une réaction d'alkylation dans les conditions de Mitsunobu a permis de greffer des groupements allyle et homoallyle sur des N-hydroxy-tripeptides, conduisant notamment à la préparation d'un hexapeptide bis insaturé qui peut être cyclisé via une réaction de métathèse.

As agrarian society developed, the most fertile soils able to sustain the nutritional requirements needed for high crop yield were assigned to farming, while the more penurious soils were left to uphold the forest ecosystems. Some temperate forests are developed on acidic soils considered to be nutrient poor, as much of the inorganic nutrients are entrapped in poorly weatherable soil minerals and not easily accessed by plant roots. In an undisturbed ecosystem, the largest contribution of available nutrients comes from the recycling of organically bound nutrients via the decomposition of dead plant material. If biomass is removed, for instance with a more intensified exploitation of the forest ecosystems including whole tree harvesting, this source of nutrients is consequently decreased. The importance of soil mineral weathering as a source of nutrients, and especially that promoted by soil biota, is thereby emphasized. This thesis addresses biotic parameters associated with mineral weathering. Different aspects of soil solution sampling strategies and analysis of different organic ligands as well as biomarkers for the estimation of fungal biomass were investigated. These chemical parameters were also evaluated as indicators of microbial activity in relation to mineral nutrient availability in soil. With the assumption that the current nutrient status of a soil will affect the microbial interest of certain minerals as sources of inorganic nutrients, a mineral amendment trial was performed in a Swedish boreal forest soil. Overall, the amended soil presented good nutrient status, but with a possible shortage of iron. Due to this, it was hypothesized that the amended mineral with the highest iron content i.e. biotite would cause an elevation of microbial activity in its vicinity when compared to the bulk soil. The level of microbial activity in the vicinity of the amended minerals was evaluated via quantification of organic acids and siderophores, as well as estimation of fungal biomass and enzymatic activity. The highest microbial activity was measured for the O horizon of the investigated podzol, although nothing indicated an elevated association with the amended minerals. In the E horizon, however, elevation in microbial activity was observed in the vicinity of the biotite mineral when compared with bulk soil, although only a few of the investigated parameters differed significantly when evaluated separately.   To enable this study, a highly sensitive analytical method employing liquid chromatography and mass spectrometry was developed to quantify a number of hydroxamate siderophores. On-line pre-concentration enabled detection of these organic ligands in the pico-molar range – a necessity when analyzing natural samples. Furthermore, an analytical method was developed for the estimation of fungal biomass via quantification of chitin-derived glucosamine, which also employed liquid chromatography and tandem mass spectrometry. Unlike currently available methods, the one presented in this thesis did not involve analyte derivatization, which resulted in high sample throughput while simultaneously avoiding complications involved with the additional derivatization procedure. The distribution of a group of organic ligands known as aromatic low molecular mass organic acids was also studied in a boreal forest podzol soil. Different sampling and samples preparation techniques, namely tension-lysimeters, soil centrifugation and liquid-soil extraction, were compared when analyzing soil solution components. Significant differences in analyte amount and species type were found between these sampling techniques. Some of the differences could be accounted for by variation in soil composition at different depths of the investigated podzol, but others could be attributed to structural differences within the studied analyte group. This clearly illustrated the intricacy of sampling and analysis when working with a sample matrix as complex and diverse as soil. As previously, liquid chromatography and mass spectrometry was used to quantify the analytes of interest. A highly sensitive analytical method was developed that was able to detect eleven aromatic low molecular mass organic acids in the nano-molar range. High selectivity was ensured by applying multiple reaction monitoring enabled by collision induced fragmentation of the analytes. / FORE

The groundwater bacterium Pseudomonas fluorescens (CCUG 32456) isolated at a depth of 70 m in the Äspö Hard Rock Laboratory secretes a pyoverdin-mixture with four main components (two pyoverdins and two ferribactins). The dominant influence of the pyoverdins of this mixture could be demonstrated by an absorption spectroscopy study. The comparison of the stability constants of U(VI), Cm(III), and Np(V) species with ligands simulating the functional groups of the pyoverdins results in the following order of complex strength: pyoverdins (PYO) > trihydroxamate (DFO) > catecholates (NAP, 6­HQ) > simple hydroxamates (SHA, BHA). The pyoverdin chromophore functionality shows a large affinity to bind actinides. As a result, pyoverdins are also able to complex and to mobilize elements other than Fe(III) at a considerably high efficiency. It is known that EDTA may form the strongest actinide complexes among the various organic components in nuclear wastes. The stability constants of 1:1 species formed between Cm(III) and U(VI) and pyoverdins are by a factor of 1.05 and 1.3, respectively, larger compared to the corresponding EDTA stability constants. The Np(V)-PYO stability constant is even by a factor of 1.83 greater than the EDTA stability constant. The identified Np(V)-PYO species belong to the strongest Np(V) species with organic material reported so far. All identified species influence the actinide speciation within the biologically relevant pH range. The metal binding properties of microbes are mainly determined by functional groups of their cell wall (LPS: Gram-negative bacteria and PG: Gram-positive bacteria). On the basis of the determined stability constants raw estimates are possible, if actinides prefer to interact with the microbial cell wall components or with the secreted pyoverdin bioligands. By taking pH 5 as an example, U(VI)-PYO interactions are slightly stronger than those observed with LPS and PG. For Cm(III) we found a much stronger affinity to aqueous pyoverdin species than to functional groups of the cell wall compartments. A similar behavior was observed for Np(V). This shows the importance of indirect interaction processes between actinides and bioligands secreted by resident microbes.