Les nanoMOFs – nanoparticles poreuses hybrides ont récemment été introduites dans le domaine de la vectorisation des médicaments afin de combiner les avantages de systèmes purement organiques ou inorganiques. Les nanoMOFs biodégradables et biocompatibles à base de trimesate de fer (MIL-100) ont été étudiées. Une méthode de synthèse «verte» a été développée et validée, ouvrant la voie à la production à grande échelle et à l’utilisation de ces matériaux pour des applications biologiques. Par la suite, les MIL-100 nanoMOFs ont été proposées comme potentiels vecteurs pour l’administration de médicaments hydrophiles antirétroviraux et anti-cancéreux, tels que les analogues nucléosidiques azydothimidines mono et triphosphates ou encore le Topotécan. Finalement, une nouvelle stratégie de modification de surface des nanoMOFs par leur recouvrement avec une couronne à base de dérivés de la β cyclodextrine, a été developpée. / Hybrid porous materials, as Metal Organic Frameworks (MOF) have been recently introduced in the drug delivery field in the attempt to combine advantages of the conventional “purely organic” or “purely inorganic” nanocarriers, such as important loading capability and controlled release.In this work the potential of biodegradable and biocompatible MOF nanoparticles made of iron trimesate (MIL-100 nanoMOF) has been investigated. A “green” synthetic procedure has been developed and validated, opening the way to the scale up synthesis of these materials for biological applications. MIL-100 nanoMOFs have been further applied to the delivery of hydrophilic drugs such as antiretroviral nucleoside analogues mono and triphosphate (azydothimidine mono and triphosphate) and the anticancer drug topotecan. Finally a new method of nanoMOFs surface modification, based on the nanoparticles coating with a β cyclodextrin-based extrenall shell, has been developed

Two classes of C5 azido-modified pyrimidine nucleosides were synthesized and explored as radiosensitizers. The 5-azidomethyl-2'-deoxyuridine (AmdU) was prepared from thymidine and converted to its cytosine counterpart (AmdC). The 5-(1-azidovinyl) modified 2'-deoxyuridine (AvdU) and 2'-deoxycytidine (AvdC) were prepared employing regioselective Ag-catalyzed hydroazidation of 5-ethynyl pyrimidine substrates with TMSN3. AmdU and AmdC were converted to 5'-triphosphates AmdUTP and AmdCTP, and incorporated into DNA-fragments via polymerase-catalyzed reaction during DNA replication and base excision repair. Radiation-mediated prehydrated electrons formed in homogeneous aqueous glassy (7.5 M LiCl) systems in the absence of oxygen at 77 K led to site-specific formation of π-type aminyl radicals (RNH•) from AmdU, AmdC, AvdU, and AvdC. The ESR spectral studies and DFT calculations showed RNH• undergo facile conversion to thermodynamically more stable σ-type iminyl radicals, R=N•. For AmdU, conversion of RNH• to R=N• was bimolecular involving α-azidoalkyl radical as intermediate; however, for AvdU, RNH• tautomerized to R=N•. Our work provides the first evidence for the formation of RNH• attached to C5 position of azidopyrimidine nucleoside and its facile conversion to R=N• under reductive environment. These aminyl and iminyl radicals can generate DNA damage via oxidative pathways. The azido-nucleosides were successfully applied as radiosensitizers in EMT6 cancer cells in both hypoxic and normoxic conditions. To explore the generation and reactivity of 2'‑deoxyguanosin-N2-yl radical (dG(N2-H)•) postulated to generate from guanine moiety towards •OH, 2-azido-2'-deoxyinosine (2-N3dI) was prepared by conversion of 2-amino group in protected dG into 2-azido via diazotization with tert-butyl nitrite followed by displacement with azide and deprotection. The investigation of dG(N2-H)• generated from 2-N3dI and its subsequent reactions using ESR will be discussed. Cycloaddition between 5-ethynylpyrimidine or 8-ethynylpurine nucleosides and TMSN3 in the presence of Ag2CO3, CuI, or CuSO4/sodium ascorbate provided N-unsubstituted 1,2,3-triazol-4-yl analogues of the parental DNA bases (i.e. 5-TrzdU, 5‑TrzdC, 8-TrzdA, and 8-TrzdG). These novel triazolyl nucleosides showed excellent fluorescent properties: 8-TrzdA exhibits the highest quantum yield (ΦF) of 44% while 8‑TrzdG had ΦF of 9%. The 5-TrzdU and 5-TrzdC showed a large Stokes shift of ~110 nm. The application of these fluorescent nucleosides to cell imaging and DNA modifications will also be discussed.

The PhD work "Hydroacylation and C-N coupling Reactions. Mechanistic Studies and Application in the Nucleoside Synthesis" tackle two different objectives, a) developing new methods of synthesis of nucleosides (introduction, and chapters 1 and 2) and b) to carry out a mechanistic study of the intermolecular hydroacylation and hydroiminoacylation reaction with and cationic rhodium complexes (chapter 3). Concerning the synthesis nucleosides, in chapter 1 we have explored new methods of synthesis of 2',3'-dideoxynucleosides and isonucleosides using a palladium or copper catalyzed C-N coupling reaction, aiming to overcome the stereoselectivity problems of the glycosylation reaction. The synthesis of the iodo-vinyl derivatives required as starting materials has been tried by different procedures, all of them unsuccessful. Finally, the coupling reaction has been explored in 1-iodo-glucal derivatives. Palladium catalysts were unsuccessful in coupling with benzimidazol used as model of purinic bases. Copper catalysts provided very low conversions. However, the oxidative addition of 1-iodo-glucal to palladium was proved and it was also observed that the reaction with aniline proceeds. That, suggest that the problem is in the steps involving the benzimidazol.In chapter 2, it has been developed a new method of synthesis of carbocyclic nucleosides using and enantioselective intramolecular hydroacylation reaction as a key step. This reaction leaded to the 3-hydroxymethyl-cyclopentanones in good yields and excellent enantioselectivities. When (S,S)-Me-Duphos was used the 3S-cyclopentanone was obtained, in contrast whether the (R,R)-Me-Duphos was employed the reaction proceed giving the opposite enantiomer. In both cases. The reduction of the ketone can be carried out in a stereoselective way using a hydroxyl-assited reduction with NaBH(OAc)3. Alternatively, the diastereomeric mixture obtained by a direct reduction can be resolved by using a DKR process using a combined enzyme/Ru complex catalytic system. A Mitsunobu reaction has allowed finally to link adenine to the cyclopentane moiety. In the third chapter, the mechanism of both cationic and neutral rhodium catalyst precursors in the hydroiminoacylation of alkenes was studied. The oxidative addition step was studied using both NMR and DFT techniques. Using the neutral complex, this step is a thermodynamically favoured process, as demonstrated by the isolation of the stable complex. Furthermore, DFT calculations showed the existence of an agostic intermediate on the route to the C-H activation product. In the cationic system, the oxidative addition reaction was shown by DFT calculations to be an endothermic process, hence un-favoured. This was in agreement with the NMR experiments, in which an oxidative addition product was only detected in the presence of a chloride source. Furthermore, the transition states involved in both systems were identified using DFT calculations, which proved that the presence of chloride not only stabilize the oxidative addition product but also lower the energy barrier of the overall process.Using the neutral system, it was identified the coupling product still coordinated to rhodium, which is in an enamine tautomeric form. After removal of the coupling product the stable complex [Rh(μ-Cl)(PPh3)2]2 was formed. This species was reported as a precursor for the oxidative addition step, from which the catalytic cycle can start again. However in the cationic system, the system did not yield any stable rhodium species and quickly evolved towards decomposition. / Durante la última década la terapia del SIDA ha experimentado una evolución notable. El conocimiento del modo de actuación y proliferación del virus ha permitido incrementar el número de dianas biológicas para su neutralización. Así, hoy en día se conocen compuestos que inhiben la entrada del virus en la célula, la transcripción del RNA en DNA, la integración del DNA vírico en DNA celular, la producción del envolvente proteico del virus, entre otros. Todo ello, ha permitido la realización de tratamientos dirigidos a diferentes dianas, que han neutralizado la evolución del virus mejorando la calidad de vida de los pacientes.Existen numerosas metodologías diseñadas para obtener los retrovirales mencionados anteriormente, pero en la mayoría de ellas se requieren numerosos pasos de síntesis y además en muchas de ellas se obtienen mezclas de los isómeros α/β. De este modo se pretende diseñar una alternativa sintética general para la preparación de la familia de nucleósidos arriba indicadas y al mismo tiempo que sea una alternativa práctica y eficaz a los métodos descritos hasta el momento.En el capítulo uno la obtención de isonucleosidos y 2',3'-dideoxinucleosidos se abordó utilizando como etapa clave de reacción el acoplamiento C-N entre los derivados de 4-halo-2,3-dihidrofurano y 5-halo-2,3-dihidrofurano con bases púricas y pirimidínicas, la posterior hidrogenación enantioselectiva del doble enlace nos permitiría obtener los mencionados compuestos de una forma sencilla. En el estudio realizado bajas conversiones de los productos de acoplamiento cruzado fueron detectados aunque actualmente se están intentado mejorar los resultados.Referente a la obtención de carbociclonucleosidos abordada en el capítulo 2, se ha llevado cabo una nueva metodología sintética en la que se ha aplicado la reacción de hidroacilación intramolecular enantioselectiva catalizada por rodio. Así pentenales substituidos en posición cuatro han sido convertidos en las ciclopentanonas correspondientes. En función de la quiralidad de la fosfina empleada se han obtenido tanto los enantiomeros R como S con excelentes conversiones y enantioselectividades.Con el fin de incorporar la base nitrogenada en la ciclopentanona la reducción diastereoselectiva se ha llevado a cabo dos procedimientos: a) reducción racémica de la ciclopentanona y posterior resolución cinética dinámica, b) reducción diastereoselectiva utilizando como agente reductor el triacetoxiborohidruro de sodio. En ambos casos se obtuvieron diastereoselectividades excelentes pudiendo así obtener un distereoisomero u otro en función del procedimiento y el enantiomero utilizado como material de partida. La posterior reacción de Mitsunobu sobre el alcohol y la desprotección del grupo protector nos ha permitido obtener el carbociclonucleosido con buenos rendimientos y excelentes esteroselectividades.En el capitulo tres se ha realizado un estudio sobre la reacción de hidroacilación intramolecular de alquenos cataliza por rodio, donde se ha estudiado la diferencia de comportamiento de los sistemas catiónicos y neutros de rodio en la etapa de adición oxidante. Estos estudios se han realizado utilizando técnicas espectroscópicas de resonancia magnética nuclear y cálculos teóricos mediante técnicas DFT. El estudio computacional ha mostrado que en el caso de los sistemas neutros la etapa de adición oxidante es una etapa termodinámicamente favorable hecho que se gratifica con el hecho de que el producto de adición oxidante es estable y aislable. Además se ha encontrado la existencia de un intermedio agóstico en el proceso de activación del enlace C-H. Sin embargo, en los sistemas catiónicos la etapa de adición oxidante resultó ser un proceso endotérmico. Los estados de transición encontrados no solo han demostrado que la presencia de cloruro estabiliza el producto de adición oxidante sino que también disminuye la barrera energética del proceso global. La etapa de inserción del alqueno también fue estudiada para ambos sistemas utilizando estireno como sustrato. En el sistema neutro se detectó una nueva especie de rodio la cual no había sido descrita anteriormente y fue completamente caracterizada mediante RMN multinuclear.En el sistema catiónico se consiguió detectar el hidruro correspondiente al producto de adición oxidante el cual también fue completamente caracterizado por técnicas de RMN. Sin embargo, en el estudio de la inserción del alqueno no se observó la ningún producto que indicase que el mencionado proceso se llevará acabo indicado que la inserción de alqueno es además la etapa lenta del proceso.

Adenosine (ADO) is an endogenous neuroprotectant. Under ischemic conditions ADO levels rise in the brain up to 100-fold. ADO in the brain is dependent on the movement across cell membranes by equilibrative nucleoside transporters (ENT) or produced from membrane bound ecto-5’ nucleotidase (eN). We used transgenic neurons with neuronal specific expression of human ENT1 (hENT1) and eN knockout (CD73 KO) astrocytes. The aim of this research was to determine the role of ENT1 and eN in ADO release from ischemic-like conditions in primary cultured neurons, astrocytes or co-cultures. Neurons primarily release intracellular ADO via ENTs; this effect was blocked by transporter inhibitor, dipyridamole (DPR). Astrocytes primarily convert ADO extracellularly from eN; this effect was with eN inhibitor α, β-methylene ADP (AOPCP). Combined neuron and KO astrocytes produced less ADO, extracellular ADO was inhibited by DPR but not AOPCP. Overall these results suggest that eN is prominent in the formation of ADO but other enzymes or pathways contribute to rising ADO levels in ischemic conditions.

Adenosine (ADO) is an endogenous neuroprotectant. Under ischemic conditions ADO levels rise in the brain up to 100-fold. ADO in the brain is dependent on the movement across cell membranes by equilibrative nucleoside transporters (ENT) or produced from membrane bound ecto-5’ nucleotidase (eN). We used transgenic neurons with neuronal specific expression of human ENT1 (hENT1) and eN knockout (CD73 KO) astrocytes. The aim of this research was to determine the role of ENT1 and eN in ADO release from ischemic-like conditions in primary cultured neurons, astrocytes or co-cultures. Neurons primarily release intracellular ADO via ENTs; this effect was blocked by transporter inhibitor, dipyridamole (DPR). Astrocytes primarily convert ADO extracellularly from eN; this effect was with eN inhibitor α, β-methylene ADP (AOPCP). Combined neuron and KO astrocytes produced less ADO, extracellular ADO was inhibited by DPR but not AOPCP. Overall these results suggest that eN is prominent in the formation of ADO but other enzymes or pathways contribute to rising ADO levels in ischemic conditions.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 128-140).

La découverte de nouvelles substances thérapeutiques nécessite la synthèse de série de molécules soumises au criblage biologique sur une cible donnée. Ce projet de recherche a pour objectif de développer des inhibiteurs de nucléosides monophosphate kinases (NMPK) en se basant sur le concept de chimie dynamique combinatoire in situ. La synthèse de ces molécules a nécessité l’association via des fonctions réactives d’un analogue d’accepteur de phosphate et d’un mime d’ATP donneur de phosphate. La mise au point de ce dernier a fait l’objet de ce travail de thèse et a été orientée vers la pharmacomodulation d’un hétérocycle azoté et soufré: la 2-thiohydantoïne. La synthèse de ce composé a été réalisée par la méthode de Schlack-Kumpf et par celle d’Edman provenant de techniques d’analyses peptidiques. Ces deux voies ont été exploitées pour étudier la réactivité et la fonctionnalisation sélective de cet hétérocycle notamment par des couplages de type Suzuki. La réaction de Vilsmeier-Haack-Arnold a par la suite constitué l’étape clé permettant de transformer un cycle 2-thiohydantoïne en un cycle de type imidazole qui a pu être fonctionnalisé en diverses positions. La synthèse de dérivés 2-thiohydantoïne et imidazole diversement substitués par des groupements utiles, au couplage in situ avec les analogues d’accepteur de phosphate ainsi qu’à l’affinité enzymatique a permis l’accès à une bibliothèque de molécules. Des tests biologiques ont permis d’évaluer leur affinité vis-à-vis de plusieurs NMPK ainsi que leur cytotoxicité sur cellules cancéreuses ; cet ensemble de résultats permettant de trouver les déterminants nécessaires à l’activité biologique. / New therapeutical compounds determination requires the formation of a library of molecules and their screening on specific biological targets. The aim of this project was to design new inhibitors targeting nucléoside monophosphate kinases (NMPK) based on in situ dynamic combinatorial chemistry. These molecules were synthesized by ligation between analogues of phosphate acceptors and donors on which reactive functions were introduced. The topic of this PhD was to develop the ATP mimetics using chemical transformation and pharmacomodulation of a small heterocycle: 2-thiohydantoin. Its synthesis was achieved using the Schlack-Kumpf and the Edman methods initially develop for peptidic analysis. These two pathways have been explored in order to study the reactivities and the selective functionalizations of the heterocycle allowing for example Suzuki cross coupling reactions. Furthermore we used the Vilsmeier-Haack-Arnold reaction as a key step to the formation of a highly substituted imidazole ring directly from a 2-thiohydantoin. The synthesis of 2-thiohydantoin and imidazole derivatives, on which reactive groups for the in situ coupling reactions and the enzymatic affinity have been introduced, leads to a library of molecules. Their affinity toward to ATP donor site of NMPK and their toxicity on cancer cells were evaluated by biological tests.

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08-18Bitstream added on 2014-06-13T20:29:19Z : No. of bitstreams: 1 silva_don_me_sjrp.pdf: 1054782 bytes, checksum: 832047dd271670cc0bc6debdbd5dc8d8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Purina Nucleosídeo Fosforilase (PNP) catalisa a fosforólise de nucleosídeos de purina para suas respectivas bases e açucares (ribose ou desoxirribose) 1-fosfato. A PNP desempenha uma função central no metabolismo das purinas, normalmente operando na via de recuperação do DNA das células. Mais ainda, a PNP cliva ligações glicosídicas com inversão da configuração para produzir a-ribose 1-fosfato. Acredita-se que no organismo do Mycobacterium tuberculosis a PNP desempenha tarefas similares, o que levanta o interesse em desenvolver ciência que dê suporte para o desenvolvimento de drogas baseadas na estrutura desta proteína. A proteína é um homotrímero simétrico com um arranjo triangular das subunidades, similar às PNPs triméricas de mamíferos. Cada monômero consiste de um enovelamento a/ß formado por onze fitas ß circundadas por oito hélices a. O estudo desta PNP visa proporcionar comparações com outras estruturas, na intenção de identificar as bases estruturais de possíveis diferenças ou similaridades funcionais entre esta e outras PNPs, num esforço para desenvolver pesquisa que dê suporte para o desenho de novas drogas mais seletivas e poderosas contra a tuberculose. / The Purine nucleoside phosphorylase (PNP) catalyses the phosphorolysis of purine nucleosides to corresponding bases and ribose 1-phosphate. PNP plays a central role in purine metabolism, normally operating in the purine salvage pathway of cells. Moreover, PNP cleaves glycosidic bond with inversion of configuration to produce á-ribose 1-phosphate. It is believed that in the MtPNP is responsible for the same labor in the Mycobacterium tuberculosis organism, which arouses the interest in developing science for giving support to the development of structure based drugs. The protein is a symmetrical homotrimer with triangular arrangement of the subunits, similar to the trimeric mammalian PNPs. Each monomer consist of a á/â folding formed by eleven â sheet surrounded by eight á helices. The study of this PNP aims the possibility of caring out comparisons with other structures, in order to identify the structural basis of possible differences or functional similarities between this and other PNPs, in an effort to develop research which gives support to the design of more selective and powerful new drugs against tuberculosis.

Orientador: Mário Tyago Murakami / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T16:32:04Z (GMT). No. of bitstreams: 1 Martins_NadiaHelena_D.pdf: 33217097 bytes, checksum: 6cbd9ad5dcfddd06e2357b3c680cd3f6 (MD5) Previous issue date: 2011 / Resumo: A enzima purina nucleosídeo fosforilase hexamérica de Bacillus subtilis (BsPNP233) c uma nucleosídeo fosforilase do tipo 1 , responsável pela catalise reversível da reação de guebra de urn nucleosídeo em base nitrogenada e ribose-1-fosfato na via de salvação de purinas. Essa enzima possui interesses biomédicos e biotecnológicos devido ao uso na terapia gênica em canceres sólidos e na biossíntese de análogos de nucleosídeos...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The hexamcric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) is a nucleoside phosphorylase typc-1 involved in purine salvage pathway by the nucleoside phosphorolysis resulting in purine base and ribose-1-phosphatc. The interest about this enzyme involves gene therapy application in solid cancers treatment and nucleoside analogs biosynthesis...Note: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Les traitements actuels contre le virus de l’hépatite B (VHB) combinent un ou plusieurs analogues de nucléos(t)ides qui inhibent directement la réplication virale en bloquant l’étape de transcription inverse. Ces traitements très efficaces sont pourtant confrontés à l’émergence de virus résistants à ces traitements. Ces résistances sont la conséquence de l’émergence et la sélection de mutants parfois complexes présentant des mutations à la fois dans le gène de la polymérase (pol) et de l’enveloppe virale. Les objectifs principaux de ce doctorat ont été d’étudier la sensibilité des variants résistants du VHB vis-à-vis d’analogues de nucléos(t)ides et de nouveaux composés nonnucléos(t)idiques agissant contre la nucléocapside, mais également de comparer les performances virales de différents mutants afin de comprendre le processus de sélection des mutants qui s’opère chez le patient sous pression thérapeutique. Ces études ont caractérisé la sensibilité de certaines mutations de résistance aux analogues de nucléos(t)ides, de souligner l’importance des modifications de l’enveloppe dues aux mutations de résistance dans le processus d’émergence et de sélection des variants dans la quasi-espèce virale et d’identifier de nouvelles molécules antivirales efficaces permettant, en combinaison avec les analogues de nucléos(t)ide, de diminuer fortement les phénomènes de résistance du VHB. Mieux comprendre les phénomènes de résistance, les procédés d’émergence, de sélection et de transmission des mutants du VHB pour élaborer les meilleures stratégies cliniques de combinaisons thérapeutiques peut réduire considérablement le nombre de personnes touchées par ce virus / Current therapies against the hepatitis B virus (HBV) combine one or more nucleoside analogues that directly inhibit viral replication by blocking reverse transcription step. These treatments are very effective, however, faced with the emergence of viruses resistant to these treatments. These resistances are the result of the emergence and selection of mutants with mutations can be complex in both the polymerase gene (pol) and the viral envelope. The main objectives of this PhD was to study the sensitivity of resistant HBV variants vis-à-vis similar nucleos(t)ides and new compounds non-nucleos(t)idic acting against the nucleocapsid, but also compare the performance of different viral mutants to understand the process of selection of mutants that occurs in patients under therapeutic pressure. These studies have characterized the sensitivity of some resistance mutations to nucleoside analogues, to highlight the importance of the envelope changes due to resistance mutations in the process of emergence and selection of variants in the quasispecies virus and to identify new effective antiviral drugs may allow, in combination with nucleoside analogues, to greatly reduce the phenomenon of HBV resistance. Better understanding the phenomenon of resistance, the processes of emergence, selection and transmission of HBV mutants to develop the best clinical strategies of combination therapy can significantly reduce the number of people affected by this virus