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Anderson, Crystal Annette. "Reactivity of Re₂(CH₃COO)₂Cl₄·2H₂O with purine DNA dinucleotides." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/603.
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Covalent binding of dinuclear metal carboxylate compounds to purine DNA nucleobases has been shown to be a source of anticancer activity, and has resulted in intense research to understand the coordination of metal complexes to DNA. This investigation focuses on the formation of dirhenium metal:dinucleotide complexes of purine nucleobases. To our knowledge, complexes formed by the reaction of dinuclear rhenium metal compounds with dinucleotides have not been reported in the literature.
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Zhong, Minghong. "N9 Alkylation and Glycosylation of Purines; A Practical Synthesis of 2-Chloro-2'-deoxyadenosine." Diss., CLICK HERE for online access, 2004. http://contentdm.lib.byu.edu/ETD/image/etd433.pdf.
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Barbosa, Sara Isabel Cadinha. "Compostos que interferem no metabolismo dos purina- e pirimidina-nucleótidos: utilização como agentes terapêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5160.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasO conteúdo deste trabalho será desenvolvido em dois temas principais, um referente à utilização de compostos que interferem no metabolismo dos purina- e pirimidinanucleótidos como agentes antineoplásicos e outro referente à sua utilização como agentes antivirais. A síntese dos nucleótidos envolve a construção de ácidos nucleicos e a inserção dos derivados de nucleótidos noutras vias bioquímicas, sendo responsável por inúmeras funções do metabolismo celular. Existem patologias que envolvem enzimas essenciais do metabolismo dos nucleótidos, o que levou à síntese de novos fármacos. As doenças oncológicas continuam a matar milhares de pessoas e um tratamento eficaz e com sucesso tem sido um desafio. O mesmo se passa com algumas infeções virais, nomeadamente infeções provocadas pelo HIV. Para contornar os obstáculos enfrentados na terapia destas doenças têm sido usados análogos de nucleótidos e/ou nucleósidos como agentes terapêuticos. Estes têm o propósito de inibir a síntese de novo dos nucleótidos em determinadas etapas, estando envolvidos na replicação e síntese do RNA e DNA nas células em divisão. Atuam por inibição específica de enzimas no metabolismo dos nucleótidos/nucleósidos ou ainda por incorporação no DNA ou no RNA. This study will be developed into two main subjects; one related to the use of compounds which interfere with the metabolism of purine- and pyrimidine- nucleotides as antineoplastic agents; another related to their use as antiviral agents. The nucleotides’ synthesis involves the construction of nucleic acids and the introduction of the nucleotides’ derivatives into other biochemical pathways and it is responsible for numerous functions of cellular metabolism. There are pathologies involving key enzymes from the nucleotides’ metabolism, which led to the synthesis of new drugs. Cancer is a disease that continues killing thousands of people, an effective and successful treatment has been a challenge. The same happens with some viral infections, mainly infections caused by HIV. To overcome the obstacles faced in the therapy of these diseases it has been used nucleotide and/or nucleoside analogues as therapeutic agents. These agents have the purpose of inhibiting the de novo nucleotide synthesis in certain steps, by being involved in RNA and DNA replication and synthesis in dividing cells. They act by specific enzymes inhibition in nucleotide/nucleoside metabolism and by incorporation into DNA or RNA.
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Frame, Andrew Scott. "The synthesis of some imidazole nucleosides as potential inhibitors of de novo purine nucleotide biosynthesis." Thesis, University of Lincoln, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386384.
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Rayala, Ramanjaneyulu. "Design and Synthesis of Novel Nucleoside Analogues: Oxidative and Reductive Approaches toward Synthesis of 2'-Fluoro Pyrimidine Nucleosides." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2172.
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Fluorinated nucleosides, especially the analogues with fluorine atom(s) in the ribose ring, have been known to exert potent biological activities. The first part of this dissertation was aimed at developing oxidative desulfurization-fluorination and reductive desulfonylation-fluorination methodologies toward the synthesis of 2'-mono and/or 2',2'-difluoro pyrimidine nucleosides from the corresponding 2'-arylthiopyrimidine precursors. Novel oxidative desulfurization-difluorination methodology was developed for the synthesis of α,α-difluorinted esters from the corresponding α-arylthio esters, wherein the arylthio group is present on a secondary internal carbon. For the reductive desulfonylation studies, cyclic voltammetry was utilized to measure the reduction potentials at which the sulfone moiety of substrates can be cleaved.
The 5-bromo pyrimidine nucleosides and 8-bromo purine nucleosides act as crucial intermediates in various synthetic transformations. The second part of the present dissertation was designed to develop a novel bromination methodology using 1,3-dibromo-5,5-dimethylhydantoin (DBH). Various protected and deprotected pyrimidine and purine nucleosides were converted to their respective C5 and C8 brominated counterparts using DBH. The effect of Lewis acids, solvents, and temperature on the efficiency of bromination was studied. Also, N-bromosuccinimide (NBS) or DBH offered a convenient access to 8-bromotoyocamycin and 8-bromosangivamycin.
Third part of this research work focuses on the design and synthesis of 6-N-benzylated derivatives of 7-deazapurine nucleoside antibiotics, such as tubercidin, sangivamycin and toyocamycin. Target molecules were synthesized by two methods. First method involves treatment of 7-deazapurine substrates with benzylbromide followed by dimethylamine-promoted Dimroth rearrangement. The second method employs fluoro-diazotization followed by SNAr displacement of the 6-fluoro group by a benzylamine. The 6-N-benzylated 7-deazapurine nucleosides showed type-specific inhibition of cancer cell proliferation at micromolar concentrations and weak inhibition of human equilibrative nucleoside transport protein (hENT1).
In the fourth part of this dissertation, syntheses of C7 or C8 modified 7-deazapurine nucleosides, which might exhibit fluorescent properties, were undertaken. 8-Azidotoyocamycin was synthesized by treatment of 8-bromotoyocamycin with sodium azide. Strain promoted click chemistry of 8-azidotoyocamycin with cyclooctynes gave the corresponding 8-triazolyl derivatives. Alternatively, 7-benzotriazolyl tubercidin was synthesized by iodine catalyzed CH arylation of tubercidin with benzotriazole.
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Payne, Tiffany Anne. "Analysis of dirhenium carboxylate : purine dinucleotide adducts." Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/629.
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The discovery of cisplatin, cis-[pt(NH3)2Cl2], as an anticancer agent in 1969 by Rosenbert and his colleagues sparked interest in the area of metal complexes as chemotherapeutic agents. Anticancer dimetal complexes such as Re2(O2CCH2CH3)2Br4·2H2O are proposed to prevent replication of cancel cells by coordinating to the purine nucleobases in DNA. To investigate the interaction between dimetal compounds and DNA, dirhenium complexes coordinated to purine dinucleotides were isolated and analyzed. LC/MS, HPLC, 1H NMR, and UV-Visible spectroscopy were used to characterized complexes of Re2(O2CR)2X4·2H2O (R = CH3, CH2CH3; X= Cl, Br) with the purine dinucleotides dApG and dGpG. HPLC, UV-Vis, and 1H NMR are used to investigate the aquation of Re2(O2C2H3)2Cl4μ2H2O which may contribute to its biological activity.
Upon reaction of Re22C2H3)2Cl4μ2H2O with dApG or dGpG, the intact dirhenium:dinucleotide complex is observed by LC/MS after two days. In both of these reactions, dirhenium:GMP complexes are also observes.
1H NMR studies show the appearance of new resonances in the aromatic region that cannot be attributed to starting material or hydrolyzed DNA fragments. These resonances are proposed to result from the formation of dirhenium:dinucleotide complexes. Additionally, MS spectra support the conclusion that a complex between the dinuclear rhenium complex and the purine dinucleotides of dApG and dGpG is formed after two days. A dirhenium:nucleotide product is also formed as a result of the dinucleotide hydrolysis.
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Chen, Xue Bin. "Excretion of purine derivatives by sheep and cattle and its use for the estimation of absorbed microbial protein." Thesis, University of Aberdeen, 1989. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=128449.
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The nucleic acids digested by ruminants are essentially of microbial origin. Absorbed purines are extensively degraded and excreted in urine as allantoin, uric acid, xanthine and hypoxanthine. The measurement of the urinary purine derivatives could be used to estimate the uptake of purines and hence that of microbial protein. 1) Automated methods for measurements of purine derivatives were improved. A HPLC method for determining total adenine and guanine was used to measure the purine contents of mixed rumen bacteria and the digestibility of microbial purines in sheep. 2) Endogenous excretions of purine derivatives by sheep and cattle of different physiological states were measured using animals nourished by intragastric infusions of VFA and casein. The species difference in and the effects of changes in protein supply on the endogenous excretion were studied. 3) A microbial nucleic acid extract was infused into the abomasum of lambs at 6 levels. The subsequent urinary excretion of purine derivatives was examined. The results suggested that exogenous purines were utilised by the sheep. A model is proposed to describe the relationship between purine derivative excretion and purine uptake. 4) Allantoin was infused intravenously to sheep and the changes in plasma concentration, nephric tubular re-absorption and urinary excretion of allantoin were studied. Results showed that plasma allantoin was rapidly removed and a constant proportion of the allantoin entering the blood was excreted in urine of sheep. 5) Secretion of allantoin and uric acid into the gut via saliva was quantified in sheep. The possible decomposition of the allantoin in the rumen by microbes in the rumen fluid and in the rumen wall of sheep was examined in vitro and in vivo. Allantoin infused into the rumen or abomasum of sheep and cattle was not recovered in urine. 6) A model for calculation of microbial protein available to sheep is proposed. It is suggested that a different model should be used for cattle. These models were applied in two feeding experiments with sheep and steers.
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Lemin, David. "Synthèse d'analogues des ligands naturels de récepteurs nicotiniques et purinergiques." Doctoral thesis, Universite Libre de Bruxelles, 2004. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211158.
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Cette thèse s’inscrit dans le cadre de l’étude de la relation structure-activité d’analogues de ligands naturels de récepteurs nicotiniques et purinergiques. Ce travail se divise en deux parties.Dans la première partie de cette thèse, nous avons réalisé la synthèse d’analogues de la 11-homosédinone, alcaloïde isolé de la plante Sedum acre, qui présente une activité agoniste sur différents récepteurs nicotiniques du système nerveux central. Les différents analogues ont été synthétisé par application de la méthoxylation anodique pour introduire succesivement deux substituants en postion 2 et 6 d’un noyau pipéridinique. Les analogues synthétisés se différencient par la nature du noyau aromatique, la présence d’un groupement méthyle sur l’atome d’azote de la pipéridine et l’oxydation du sustituant en position 2. Ce travail a notamment permis de montré l’importance du groupement N-méthyle vis-à-vis de l’activité des analogues. Nous avons également pu mettre en évidence que l’introduction d’un halogène sur le noyau aromatique diminuait l’activité de l’analogue sur le récepteur a7 tout en augmentant l’acitivité sur le récepteur a4b2 et que l’introduction d’un noyau furanique permettait d’augmenter la sélectivité vis-à-vis du récepteur a4b2 tandis que l’introduction sur le noyau aromatique d’un groupement nitro ou méthoxy conduit à une perte totale de l’activité.
Dans la seconde partie de cette thèse, nous avons réalisé la synthèse d’analogues de la dATP, afin d’évaluer leur effet agoniste sur le récepteur P2Y11, impliqué dans différents mécanismes de différentiation cellulaire, dont notamment celui de la maturation des cellules leucémiques HL60 en cellules de type neutrophile. Les analogues synthétisés se différencient de la dATP par la présence d’un groupement méthylène ou dichlorométhylène entre les phosphores b et g de la chaîne polyphosphate, ainsi que par l’estérification de l’alcool en position 3’ du sucre. Ce travail a pu confirmer que les analogues en série 2’-désoxy conduisent à de meilleures activités que ceux de la série 2’-OH. Nous avons également pu montrer que l’estérification de la position 3’ conduit à une diminution de l’activité agoniste, à l’exception du groupement a-naphtoyle qui conduit à une augmentation significative de l’activité sur P2Y11.
Doctorat en sciences, Spécialisation chimie
info:eu-repo/semantics/nonPublished
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Fong, Yuen Ting. "Quantitative structure retention relationships on using high-performance liquid chromatography." HKBU Institutional Repository, 2003. http://repository.hkbu.edu.hk/etd_ra/426.
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Liu, Jiangqiong. "Kinetic Studies of 6-Halopurine Nucleoside in SNAr Reactions; 6-(Azolyl, Alkylthio and Fluoro)-purine Nucleosides as Substrates for Suzuki Reactions." Diss., CLICK HERE for online access, 2007. http://contentdm.lib.byu.edu/ETD/image/etd1821.pdf.
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Humphries, Mark Jonathon. "Synthesis and chemistry of 5-aminoimidazole ribonucleosides." Thesis, Keele University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363753.
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McGuire, Ruth. "Synthesis and studies of modified nucleotides and oligonucleotides." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246868.
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Cormier, James. "The synthesis of nucleoside and silyl nucleotide analogues /." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75458.
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A route to the synthesis of arabino and xylonucleosides is described. This route takes advantage of highly selective ribonucleoside hydroxyl protection procedures which have recently been developed. The route is straightforward and broadly applicable. It may be applied to both purine and pyrimidine nucleosides. Synthesis, deprotection, and characterisation of the target compounds are described. The work is compared to that of others in the field.A novel class of oligonucleotide analogues is described. In this group, the phosphorus atom of the internucleotide link is replaced by silicon. The synthesis of both oligothymidine and oligo-2$ sp prime$-deoxyadenosine nucleotide analogues of this class is described. Various substituents at silicon are employed, and oligonucleotide analogs of up to six units long are synthesised, characterised and deprotected. The circular dichroism spectra of the deprotected hexamers is presented.
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Buckley, Ragan. "The purine world: experimental investigations into the prebiotic synthesis of purine nucleobases and intercalation of homopurine DNA duplexes." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/48971.
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Formamide is a solvent of great interest to prebiotic chemists because it is liquid over a wide range, it is less volatile than either water or HCN, and it possesses a versatile reactivity. When formamide is heated in the presence of minerals or inorganic catalysts, a variety of products including purine nucleobases are generated. Irradiation of formamide reaction solutions with ultraviolet light increases the yield and diversity of products, and eliminates the need for a mineral catalyst. We have also performed formamide reactions in the presence of pyrite, a mineral which is likely to have been available on the primordial Earth, under a variety of atmospheric conditions. Our results indicate the greatest yield and diversity of products result from the combination of a pyrite mineral catalyst, heat, UV irradiation, and a carbon dioxide atmosphere. Purine nucleobases are simple to synthesize in model reactions and they stack well in aqueous solution; it has been hypothesized that the first nucleic acids were composed of only purine bases, and that water-soluble, cationic, aromatic molecules with large stacking surfaces (“”molecular midwives””) may have aided the assembly of the earliest nucleic acid analogs. We have characterized the interactions of various intercalators with a standard DNA duplex as well as with an antiparallel homopurine DNA duplex and have determined that molecules which possess four or more rings and a curved shape interact selectively with all-purine DNA; such molecules can serve as models for putative prebiotic midwives.
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Wei, Shuang. "Modifications du metabolisme des nucleotides en relation avec la differenciation et en reponse a une irradiation dans des cellules tumorales humaines (doctorat : structure et fontionnement des systemes biologiques integres)." Paris 11, 1998. http://www.theses.fr/1998PA114846.
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Juby, Carl D. "Synthesis of acyclonucleotides with potential antiviral activity." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66199.
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Barnes, Colin Lloyd. "Studies in the synthesis of novel antisense oligo nucleotides." Thesis, Institute of Cancer Research (University Of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264958.
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Pavey, John B. J. "The synthesis and properties of 2'-C-functionalised nucleotides." Thesis, University of Liverpool, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367007.
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Honcharenko, Dmytro. "Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides : Design, Synthesis and Properties." Doctoral thesis, Uppsala universitet, Bioorganisk kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8887.
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This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system. The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence. Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.
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Tusa, Girolamo. "Synthesis and biological activity of conformationally constrained nucleosides and nucleotides." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ34032.pdf.
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Turkewitsch, Petra. "The synthesis of fluorescent chemosensors responsive tocAMP and other nucleotides." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0018/NQ44611.pdf.
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Narukulla, Raman. "Synthesis and characterisation of base-modified nucleosides, nucleotides and DNA." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430561.
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Ashe, Kathryn. "Studies towards the prebiotic synthesis of nucleotides and amino acids." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10060452/.
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Understanding how life on Earth might have started is the major goal of prebiotic chemistry. The universal nature of extant life, with integrated informational, catalytic and compartment-forming systems, suggests a common chemical origin. Comprehensive studies of the feasible routes towards the biomolecules necessary for life will refine our knowledge of the prebiotic chemistry that lead to life. The integration of genetic information and proteins in the central dogma of molecular biology indicates the importance of evaluating the prebiotic synthesis of amino acids and peptides. Following the principles of prebiotic systems chemistry, ideally this should take place under similar conditions established for other prebiotically plausible networks that lead to other key class of metabolite using common reagents. Here we focus on resolving the pH incompatibility of nucleotide (pH < 7) and amino acid synthesis (by Strecker reaction of ammonium cyanide and aldehydes, pH > 9). Introducing DAP - a reagent previously implicated in various areas of prebiotic chemistry - into the Strecker reaction enables the synthesis of N-phosphoroaminonitriles at pH 7, and is highly selective for proteinogenic over non-proteinogenic amino acid precursors. Derivatisation of the products of this phosphoro-Strecker reaction provides a handle for further reaction and possible oligomerisation. Phosphate is used as a universal energy currency in extant life, notably in amino acid activation in protein synthesis, so the formation of a phosphorylated amino acid derivative in this work is particularly interesting. The ubiquity of RNA in modern biochemistry and RNA's dual functionality (genotypic and phenotypic) makes RNA a strong contender for the first biopolymer. We also present an account of our results exploring: the replacement of phosphate with borate in the stereochemical rearrangement of 2',2-anhydropyrimidines and 2',8-anhydropurines; the formation of a glycosidic bond between acetylated ribose derivatives (from the UV irradiation of unwanted α-pyrimidine by-products) and purine nucleobases; and a stereospecific reduction of 5',8-cyclopurines.
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Rodrigues, Elisandra Márcia. "Estudos moleculares das fosforribosil pirofosfato sintetases." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-18062008-085454/.
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Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas.Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.
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Hagos, Asmerom M. "Tricyclic purine analogues as antiparasitic and antiviral agents." Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-03292004-141831/unrestricted/hagos%5Fasmerom%5Fm%5F200312%5Fphd.pdf.
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Turner, David Michael. "Design and synthesis of purine isosteres as inhibitors of Nek2 and CDK2." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/1908.
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Nek2 and CDK2 are serine/threonine protein kinases that are implicated in cell cycle control and cancer. The Nek family of enzymes contains 11 known serine/threonine protein kinases (Nek1-11) and are involved in mitotic cell cycle control. There is evidence that of these 11 kinases, Nek2, Nek6, Nek7 and Nek9 play an important role in the regulation of mitotic events through microtubule control. Particular interest has been placed upon Nek2, which has been shown to have a critical role in mitosis, assisting centrosome disjunction. 6-Ethnyl purine 39, was identified as a submicromolar irreversible inhibitor of Nek2 (IC50 = 150 nM), exhibiting good selectivity over other members of the Nek family. It was believed that this compound formed a triplet of H-bonds with the amino acids of the Nek2 ATP-binding site hinge region via the 2-amino N-H, the purine N3 and the imidazole N9H, allowing an initial noncovalent binding interaction, which facilitates subsequent covalent modification. To validate this binding motif and to act as control compounds, N-methylated purines 52-54 were synthesised, along with the 2-phenoxypurine 55 and the 2-benzylpurine 56. These compounds were all essentially inactive in the Nek2 inhibition assay, which demonstrated that the purine 2-amino N-H group and imidazole N9H were essential for non-covalent binding interactions. 5 To investigate the influence of the purine heterocycle upon Nek2 inhibitory activity and ethynyl group reactivity, heterocyclic derivatives of 39 were synthesised. Deazapurines 87 and 88 were weakly active in the Nek2 inhibition assay (IC50 = 30 μM and 24 μM, respectively), whilst pyrazolopyrimidine 89 and triazolopyrimidine 90 had sub-micromolar activity comparable with the parent purine 39. The 5- formylpyrimidine 101 and triazine 91 were modest inhibitors of Nek2 (IC50 = > 10 μM and 17 μM, respectively). By contrast, pyrimidine 95 (10% inhibition at 100 μM) demonstrated weak Nek2 inhibitory activity. Quantitative 1H NMR (q1H-NMR) kinetic studies were performed to model the reaction of ethynyl-functionalised heterocycles with Cys-22 at the Nek2 ATP-binding site. Compounds 54, 56, and 88-89 were reacted with N-acetylcysteine methyl ester in DMSO-d6 at 24 oC under pseudo first order reaction conditions. The most reactive compounds were triazolopyrimidine 90 and pyrazolopyrimidine 89. The 2- benzylpurine 56 and N-methylanilinopurine 54 had moderate reactivity, comparable with the parent purine 39. The least reactive compound was pyrrolopyrimidine 88, which was approximately 150-fold less reactive than purine 39. 6 CDK2 is a member of the cyclin-dependent kinase (CDK) family of enzymes and a mediator of cell cycle progression. Of the 11 known human CDKs, four (CDK1, CDK2 CDK4 and CDK6) have been directly implicated in cell cycle regulation. Mutation of genes coding for CDKs are common in a variety of cancers, making CDKs attractive chemotherapeutic targets. Purines 249 and 37 were identified as moderate (IC50 = 17 μM) and potent (IC50 = 5 nM) ATP-competitive inhibitors of CDK2, respectively. The focus of this research was to determine whether subtle changes to the core heterocycle would allow retention of CDK-inhibitory activity. CDK2 inhibitors based on the pyrazolopyrimidine and triazolopyrimidine heterocycles (256, 252, 257 and 253) were of comparable potency with the corresponding purines (249 and 37). The most potent compound was triazolopyrimidine 253 with an IC50 value of 3 nM against CDK2. Interestingly, the pyrrolopyrimidine 255 proved only weakly active (IC50 > 100 μM), whereas the functionalised pyrrolopyrimidine 251 had potent CDK2 inhibitory activity (IC50 = 26 nM). Finally, imidazopyridine 254 exhibited activity comparable with purine 249; however, imidazopyridine 250 was found to be approximately 30-fold less potent (IC50 = 140 nM) than the parent 37, for reasons that remain unclear. N.B. diagrams not included in this abstract
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Turkewitsch, Petra. "The synthesis of fluorescent chemosensors responsive to cAMP and other nucleotides /." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37551.
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The overall objective of this study was to develop a fluorescent chemosensor for cAMP by creating recognition sites for cAMP, that contain a fluorescent molecule, in a polymer matrix by molecular imprinting. Such a fluorescent molecularly imprinted polymer may, therefore, serve as both the recognition element and the measuring element for the fluorescent detection of cAMP in aqueous media.Two new fluorescent molecules, known as 4-(p-dimethylaminostyryl)pyridinium salts or dyes, were synthesized as possible fluorescent components of a chemosensor. Upon excitation at 469 run, dye 1, trans-4-( p-N,N-dimethylaminostyryl)-N-vinylbenzyl-pyridinium chloride, displays a dramatic quantum yield enhancement in an emission band centered at ∼600 nm, with concomitant slight red shift of the emission maximum, in the presence of the cyclic nucleotides, cAMP and cGMP, in aqueous solution. Other purine nucleotides (AMP, ADP and ATP) and adenosine induce fluorescence quantum yield enhancements of lesser magnitude than those observed for cyclic nucleotides. The pyrimidine nucleotides, CMP and UMP, have almost no effect on the fluorescence of 1, suggesting a specificity of 1 for purine over pyrimidine analytes. Equilibrium association constants for 1 with the purine analytes, in aqueous solution (pH 7.2) range from 13.9 M --1 for cAMP to 0.15 M--1 for adenine. We conclude that the interaction of 1 with these analytes requires the presence of a purine base, and is enhanced by the presence of ribose and phosphate moieties. Dye 1 and a structurally-similar dye 2, trans-4-(p-N,N-dimethylaminostryl)-N-phenethylpyridinium bromide, also display dramatic fluorescence enhancements in the presence of DNA and proteins, suggesting that they also interact with these biomolecules. The environmentally-sensitive fluorescence of dyes 1 and 2 suggests that such compounds may be useful for developing fluorescent chemosensors for purine nucleotides, especially cAMP, and for the fluorescence detection or staining of DNA and proteins.
To increase the recognition ability of 1 for cAMP, we prepared recognition sites for cAMP that contain fluorescent dye 1 in a polymer matrix by molecular imprinting. This is a novel design for such template-selective sites, since dye 1 forms an integral part of the recognition cavity, thereby serving as both the recognition element and the measuring element for the fluorescence detection of cAMP in aqueous media. The polymer displays a concentration-dependent decrease in fluorescence in the presence of aqueous cAMP, whereas almost no effect is observed in the presence of the structurally-similar molecule, cGMP. An association constant of ∼105 M--1 was calculated for cAMP binding. Such fluorescent molecularly imprinted polymers could serve as a starting point in the development of highly effective synthetic fluorescent sensors for cAMP as well as other important biological molecules.
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Derudas, Marco. "Design, synthesis and biological evaluation of novel bioactive nucleosides and nucleotides." Thesis, Cardiff University, 2009. http://orca.cf.ac.uk/55855/.
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At the present there are 36 approved antiviral drugs in the UK of which half are nucleoside analogues. However, the emergence of drug resistance and of new virus strains necessitates new drugs. In particular in this thesis, different nucleoside analogues were studied as potential antivirals. One of the major issues related to nucleoside analogues is the emergence of resistance due to a lack of bioactivation to the monophosphate form. To overcome this issue, the phosphoramidate ProTide technology can be applied. This strategy allows the delivery of the monophosphate form directly inside the cell. Bicyclic nucleoside analogues are a new class of anti-varicella zoster agents of which Cfl743 is the most potent anti-varicella zoster compounds reported to date. Its 5'-valyl derivative, FV100, is currently in phase II clinical trials. A series of derivatives to increase the activity and to investigate the mechanism of action of this new class of compound are reported. Moreover, attempts to improve the scale up synthesis of FV100 are described. Ribavirin is a broad spectrum antiviral drug. The application of the ProTide approach to this compound was not successful. Enzymatic and molecular modelling studies have been performed in order to understand the lack of activity. Acyclovir and its esters are currently the treatment of choice for herpes simplex and varicella-zoster infections. The application of the ProTide technology gave surprising results. In fact, these compounds have been found to be active against HIV, whilst ACV itself did not show any activity. Moreover, these compounds retained activity versus thymidine kinase deficient strains against which acyclovir lost activity. These striking results prompted us to investigate other different nucleoside analogues, through a virtual screening using reverse transcriptase, guanylate or adenylate kinase and human polymerase y. The selected nucleoside analogues from this study include: ganciclovir, penciclovir and their derivatives. ProTides of these are thus pursued.
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Gould, Jayne H. M. "The synthesis of novel nucleosides and nucleotides as potential antiviral agents." Thesis, University of Reading, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339518.
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Lawrence, Anthony John. "The synthesis and properties of 2'-C-functionalised nucleosides and nucleotides." Thesis, University of Liverpool, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321129.
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Pal, Ayan. "Synthesis Of Nucleoside Analogues: Glycosylation, Rigid Nucleosides And Janus Wedge Derivatives." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2750.
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Thesis advisor: Larry W. McLaughlinThesis advisor: Mary F. Roberts
Nucleic Acids are unique biopolymers capable of encoding and transferring genetic information from one generation to the next for every form of life. This fascinating property has made them the topic of intense research from a variety of aspects. Some researchers try to understand how life might have started. Some try to elucidate how the whole process works. Some try to use the properties of nucleic acids as a tool for various purposes. The continuous effort over more than a century explored a lot about the structures and functions of nucleic acids. There is a lot to be discovered yet. This work began with the design and development of a new class of nucleoside analogue with the goal to study their ability to bind nucleic acids. The ongoing research will establish their application as therapeutics and as biomolecular tools. Along the way significant effort went into preparing these analogues. New methodology was developed to address some of the unanswered synthetic problems of nucleoside chemistry
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Grant, Sharon. "The design and synthesis of novel purine based inhibitors of cyclin-dependent kinases." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310023.
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Caton-Williams, Julianne Marie. "Synthesis and Enzymatic Studies of Selenium Derivatized Nucleosides, Nucleotides and Nucleic Acids." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/chemistry_diss/42.
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Nucleoside 5-triphosphates are the building blocks to synthesis of nucleic acids. Nucleic acids (RNA and DNA) participate in many important biological functions in living systems, including genetic information storage, gene expression, and catalysis. Nucleoside 5- triphosphates have many important therapeutic and diagnostic applications. To understand how these triphosphates are utilized in living systems, numerous synthetic mimics have been prepared and used as active metabolites of certain drugs and molecular probes. Over the years, nucleic acids have been modified at the nucleobase, sugar moiety and phosphate backbone with the aim of understanding their structures and functions. We have site-specifically replaced selected oxygen atoms of nucleosides and nucleotides with selenium atom in order to enzymatically synthesize selenium-derivatized DNAs for obtaining insights into the DNA flexibility, duplex recognition and stability. Although triphosphates have important biological and medicinal significance, they are however, very difficult to synthesize and isolate in high purity and yield. There are many approaches to the synthesis of nucleoside 5-triphosphates, but there is no general strategy that allows simple and direct synthesis of nucleoside triphosphates. To face the challenges, we have developed a new approach in the absence of protecting groups to quickly and efficiently synthesized native deoxynucleoside 5-triphosphates and deoxynucleoside 5-(α- P-seleno)- P-seleno)triphosphates. Syntheses of the triphosphates containing selenium-derivatized nucleobases were also successfully accomplished. After replacing the oxygen atoms at the 4-position of thymidine and uridine, and the 6-position of guanosine, we observed most strikingly, a large bathrochromic shift of over 100 nm, relative to their native counterparts of UV absorbance of 260 nm. Consequently, the synthesized selenium base modified triphosphates are yellow. We also synthesized 2-selenothymidine and 5-methylseleno thymidine 5-triphosphates. We conducted stability study on the colored 4-selenothymidine and used the 5- triphosphate analog (4-SeTTP) as substrate for polymerase recognition. The Klenow polymerase incorporated the 4-SeTTP with efficiency equal to that of the native counterpart. Finally, 4-SeTTP was used to demonstrate UVdamage resistance of selenium-derivatized DNAs and plasmid.
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Graham, François. "Regulation of 5-oxo-ETE synthesis by pyridine nucleotides in aging neutrophils." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116087.
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Neutrophils (polymorphonuclear leukocytes) are short lived granulocytes that playa primordial role in host innate defense against invading pathogens. Freshly isolated neutrophils spontaneously undergo apoptosis when cultured, which is associated with oxidative stress. We found that there is a dramatic shift in the metabolism of the 5-lipoxygenase product 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from its biologically inactive o-oxidation product in freshly isolated neutrophils to the potent granulocyte chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in neutrophils cultured for 24 h. o-oxidation of the chemoattractant leukotriene B4 (LTB4) was also reduced in aging neutrophils incubated with arachidonic acid, resulting in higher levels of LTB4. The reduced o-oxidation activity appeared to be due to a decrease in active LTB4 20-hydroxylase. In contrast, the increased 5-oxo-ETE formation was not associated with an increase in the amount of active 5-hydroxyeicosanoid dehydrogenase, which is required for its formation, but rather with a dramatic increase in its cofactor NADP +. NAD+ levels also increased, but NADPH levels remained unchanged after 24 h. There was also evidence for increased oxidative stress (high GSSG/GSH) in aging neutrophils. The changes in 5-HETE metabolism and pyridine nucleotides in cultured neutrophils could be inhibited by neutrophil survival factors and antioxidants. These results suggest that in severe inflammation, aging neutrophils that have evaded rapid uptake by macrophages may produce increased amounts of the chemoattractants 5-oxo-ETE and LTB4, resulting in delayed resolution of inflammation. Similarly, we found that the NADPH oxidase activator PMA caused a very rapid and dramatic increase in NADP + levels in both freshly isolated and cultured neutrophils, accompanied by a rapid increase in 5-oxo-ETE synthesis and reduced o-oxidation activity. Surprisingly, this was not accompanied by a corresponding decline in NADPH levels, which instead initially increased, but rather by a precipitous reduction in NAD+, which mirrored the increase in NADP+. These results suggest that the phosphorylation of NAD+ by NAD kinase may be very important for providing both NADP+ for 5-oxo-ETE synthesis and NADPH for the respiratory burst.
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Alexander, Katie. "The synthesis, detection and repair of nucleotides containing the 8-nitroguanine modification." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/2006939/.
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There is accumulating evidence that reactive nitrogen species derived from nitric oxide metabolism are involved in cancer as they are able to damage DNA largely through oxidation or nitration of the guanine base. The 8-nitroguanine lesion is increasingly associated with cancers that result from chronic inflammation; however due to its instability, very little is known about this base modification. Consequently this thesis focuses upon establishing methods to detect and quantify the lesion and investigate enzymes potentially involved in repair systems directed against 8-nitroguanine in DNA. The approach outlined in this thesis utilises ribonucleoside analogues of the lesion which sufficiently stabilised the labile glycosidic bond. The 8-nitroguanine nucleosides were prepared prior to incorporation into the oligodeoxynucleotide sequences using the traditional 3’- to 5’-solid-phase phosphoramidite chemistry. A number of oligodeoxynucleotides of varying lengths containing a single modification, and dinucleotides containing two modifications were prepared. A variety of reactions of the 8-nitroguanine base both in nucleosides and oligodeoxynucleotides have been investigated. Studies revealed a different pattern of alkylation for the modified base when compared to results reported in the literature for the natural nucleoside. Thus demonstrating the dramatic effect that nitration has on the intrinsic reactivity of the nucleoside. In view of the susceptibility of nitro group to reaction with thiol nucleophiles, displacement of the nitro group from within nucleosides and oligodeoxynucleotides has been achieved. In particular a fluorescent nucleophile has been developed which stabilises the lesion and could enable direct detection of the levels of 8-nitroguanine in DNA. Using a variety of substrates prepared in this thesis, detection of the 8-nitroguanine base in oligodeoxynucleotides has been investigated using surface enhanced Raman spectroscopy in collaboration with Professor Steven Bell at Queen’s University, Belfast. The unique absorption profile of the 8-nitroguanine derivatives allows for signals exclusively associated with the lesion to be identified using this highly sensitive technique. The synthesis of 8-nitroguanosine triphosphate was investigated using a number of different approaches. Although the initial aim was not successful, the principles for the phosphorylation of a nucleoside have been shown. The problems encountered were attributed to the conformational constraints of the molecule.
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Ebel, Susanne. "The synthesis and study of oligo(deoxy)ribo-nucleotides and their analogues." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/13766.
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Laboratory procedures for the synthesis, workup and purification of oligoribonucleotides using RNA phosphoramidites protected with a) Fpmp- and b) TBDMS- groups on the 2'-hydroxl functions have been developed and employed in the following projects: 1. Modified minimised DNA/RNA ribozymes were synthesised and purified, which contained a) a dT4-loop, b) a hexaethylene glycol- or, c) a tetraethylene glycol-loop closing the catalytic site. Their catalytic activity was confirmed. The cleavage activity of the hexaethylene glycol minizyme was superior to that of the tetraethylene glycol minizyme, but cleaved less efficiently than the dT4-minizyme. 2. Tetra-biotinylated antisense RNA was synthesised and purified using two methods. The method using 2 successive additions of a multiple hydroxyl phosphoramidite followed by 1 addition of a single-addition biotin phosphoramidite, generated biotinylated antisense oligoribonucleotides of superior specificity in biotin/streptavidin based pre-mRNA depletion experiments. 3. The stability of tandem GA mismatches was analysed using thermal denaturation techniques. In DNA tandem GA mismatches of the kind 5' pry GA pu 3' adopted an amino-type basepairing and were very stable, tandem AG mismatches were imino-paired and significantly less stable. The amino-type base pair has a distinctly shifted phosphorus resonance in the 32P-NMR spectrum. Tandem GA mismatches in RNA did pair in the amino-form when flanked by 5' pyr..pu 3' but were highly unstable, as were tandem AG mismatches. Tandem GA mismatches in DNA/RNA hybrid sequences were also unstable. Multiple GA mismatches of the kind d(5' pyr GA pu 3') did not destabilise duplexes compared to T.A. base pairs. d(GA)n sequences formed homo-duplexes with exclusively G.A basepairing.
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Lopalco, Maria. "A new class of enzymatically cleavable nucleotides for DNA sequencing by synthesis." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/15233.
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A new family of nucleotides has been designed, synthesised and evaluated for DNA sequencing by synthesis. Each of the nucleotide analogues had a free 3’-OH group and a fluorophore attached to the base through a small peptide linkage that was designed to stop multiple base additions. Analysis by HPLC and MALDI-TOF proved the modified nucleotides were incorporated into a growing DNA strand by a DNA polymerase and that the peptide linker was cleaved with high efficiency after incubation of the extended primer with a protease. Enzymatically cleavable nucleotides were successfully applied to chip-based sequencing by synthesis cycles. The fluorescent emission revealed the identity of the incorporated nucleotide and the removal of the fluorophore by a protease ensured the detection of the next base in the following cycle. Additionally, a practical microwave-mediated solid-phase protocol has been developed for the synthesis of cyanine dyes spanning the visible and the near infrared spectrum to allow the preparation of a series of fluorescent nucleotides for the design of a four colour DNA sequencing technology.
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Boussiengui-Boussiengui, Gino. "Analysis of the role of relative nucleotide concentrations on the regulation of carbohydrate in higher plants." Thesis, Stellenbosch : Stellenbosch University, 2010. http://hdl.handle.net/10019.1/5473.
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Thesis (PhD (Genetics))--Stellenbosch University, 2010.ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines.
AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie.
The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work
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Serdjukow, Sascha [Verfasser], and Thomas [Akademischer Betreuer] Carell. "Chemical synthesis and enzymatic incorporation of artificial nucleotides / Sascha Serdjukow. Betreuer: Thomas Carell." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1102157228/34.
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Hannant, Jennifer. "Synthesis of pyrrolo-modified nucleotides and their incorporation into DNA via enzymatic extension." Thesis, University of Newcastle Upon Tyne, 2011. http://hdl.handle.net/10443/1287.
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A series of pyrrole-containing derivatives of the 2-deoxy-pyrimidines, thymidine and cytidine have been prepared by Pd-catalysed cross-coupling between N-alkyl-alkynyl functionalized pyrrole (py), 2-(2-thienyl)pyrrole (tp) and 2,5-bis(2-thienyl)pyrrole (tpt) with 5-iodo-2’-deoxyuridine and 5-iodo-2’-deoxycytidine. The length of the alkyl chain linking the nucleoside and pyrrolyl-containing unit was varied from three to five carbon units. The series of nucleosides were characterized by 1H NMR spectroscopy, 13C NMR spectroscopy, ES-MS, UV-vis spectroscopy, cyclic voltammetry and in some cases single-crystal X-ray diffraction. Cyclic voltammetry revealed that all the py-, tp- and tpt-alkynyl derivatives can be electrochemically polymerized to form conductive materials. Conversion of the tp-modified nucleosides into their corresponding nucleotides was performed by phosphorylation at the 5’-hydroxyl site via phosphorus (V) and phosphorus (III) chemistry to yield dTTP-5-tp (dTTP*) and dCTP-5-tp (dCTP*), respectively. The purified nucleotides were fully characterized by 1H NMR, 31P NMR, ES-MS, MPLC and HPLC. The incorporation of dTTP* and dCTP* into DNA via enzymatic extension was explored. Polyacrylamide gel electrophoresis indicated that DNA polymerases, Pfu Pol B exoand Klenow Fragment exo-, can tolerate dTTP* and dCTP* respectively. Gel electrophoresis revealed the successful incorporation of the modified bases into a primer template duplex of up to 37 base pairs. In comparison to standard nucleotides, dTTP* and dCTP* were incorporated at a slower rate. In order to produce functionalized DNA duplexes of microns in length, attempts were made to incorporate the nucleotides into an extending primer sequence via a slippage mechanism. Analysis via agarose gel electrophoresis demonstrated that the polymerases employed in this study could not read the modified DNA as a template as efficient as standard nucleotides but limited extension was observed.
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Fijolek, Artur. "Salvage and de novo synthesis of nucleotides in Trypanosoma brucei and mammalian cells." Doctoral thesis, Umeå : Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1850.
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Ferrari, Valentina. "Synthesis and biological evaluation of novel nucleosides and nucleotides as potential therapeutic agents." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/85091/.
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Nucleoside analogues are an important class of antimetabolites, used both as anticancer and antiviral agents for their resemblance to endogenous nucleosides, and their capacity to inhibit metabolic pathways in which these substrates are involved, leading to therapeutic potential. The need for both new anticancer and antiviral cures is significant, and often caused by the emergence of resistance to the available therapies. In the case of therapies with nucleoside analogues, the delivery of a nucleoside monophosphate prodrug inside the cell has potential to overcome resistance to treatment, and proved successful especially with the application of the ProTide approach. This strategy led to the progression into clinical trials of numerous antiviral and anticancer agents. This work focused on the synthesis of novel ProTide prodrugs to different nucleoside analogues that are involved in clinical trials, such as purine and pyrimidine nucleosides with modifications in the base and sugar regions. This strategy was also applied to nucleoside analogues where in vitro evaluation has never been reported. The synthetic strategies to prepare each nucleoside analogue are also reported. The in vitro evaluation of novel nucleotide prodrugs as anticancer and antiviral agents is described and discussed. Moreover the mechanism of activation of the ProTides is supported by studying their bioactivation to the corresponding monophosphate forms, through enzymatic NMR studies and molecular modeling simulations. Modifications on the scaffold of the two most promising families of ProTides were also performed, leading to the introduction of groups on the nucleobase or alteration of the sugar moiety. Moreover, the phosphosphate group was also modified, with the application of alternative phosphorodiamidate and phosphonoamidate prodrug approaches, with the aim of improving the biological profile. Selected analogues from two families were submitted to numerous preclinical assessments, retaining better potency compared to the parent nucleosides. Moreover these analogues were stable in human plasma, serum and liver microsomes. Further investigations on these potential new drugs are currently ongoing.
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Krull, Matthias. "Synthesis of rare nucleobases and artificial nucleotides for investigation of catalytic enzyme activity." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0005-1287-E.
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Theile, Christopher Stone. "Synthesis of Cyclo, Ring Expanded, and Backbone Extended Nucleosides." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2894.
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Thesis advisor: Larry W. McLaughlinNucleic acids are responsible for maintaining the biological information responsible for the activities of all known living organisms. Research of nucleic acids provides opportunities to help understand, prevent, and cure disease in addition to allowing us to gain a greater appreciation for the wonders of nature. This work presents the synthesis and properties of several modified nucleosides. Chapter 2 presents an improved synthesis of R and S 6,5'-cyclouridine, which are rigidified nucleosides locked in the anti conformation. This work helps to understand the properties of these interesting molecules and will allow scientists to synthesize large quantities of these monomers for future research. Chapter 3 presents the synthesis of novel 6,6'-(S)-cyclo-2'-deoxyuridine. This work is highlighted by a zinc mediated cyclization to form a seven-membered ring; the first published reaction of its kind. The compound itself is a mimic of thymidine that also has the base locked in the anti position. Lastly, Chapter 4 presents work on 6' extended backbone nucleosides. These molecules have the potential to form a new type of helical structure and will help us to gain a greater understanding of the properties and dynamics that contribute to duplex stability in DNA
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Romieu, Anthony. "Synthèse d'oligonucléotides modifiés comportant des lésions radio-induites des bases puriques et pyrimidiques." Université Joseph Fourier (Grenoble), 1999. http://www.theses.fr/1999GRE10140.
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Divers facteurs comme des agents oxydants ou cancerigenes, les rayonnement ultraviolets et ionisants, peuvent engendrer des modifications des bases de l'adn. Afin d'evaluer les consequences biologiques et physico-chimiques de ces dommages, l'obtention de courts fragments d'adn (oligonucleotides), de sequence definie (20 a 50 bases de long) et comportant une ou plusieurs modifications en des sites bien precis est primordiale. La synthese oligonucleotidique est aujourd'hui la methode de choix pour preparer de tels composes modeles. Ce travail est consacre a la preparation de fragments d'adn synthetiques contenant des nucleosides modifies formes lors de la radiolyse ou de la photosensibilisation des acides nucleiques. La premiere partie de cette these concerne la preparation d'un synthon phosphoramidite de la 5-hydroxy-2-desoxycytidine et l'incorporation de ce dernier dans des oligonucleotides de synthese de 14 a 33 bases de long. La deuxieme partie (chapitres iii et iv) concerne la synthese et l'incorporation de lesions radio-induites originales : les cyclonucleosides. Les deux diastereoisomeres (5r)- et (5s)- des 5,8-cyclopurine-2-desoxyribonucleosides ont ete inseres separement dans differents oligonucleotides (3 a 22 bases de long) en utilisant la chimie phosphoramidite classique. L'incorporation de la (5s, 6s)-5,6-cyclo-5,6-dihydrothymidine a egalement ete effectuee. La structure particuliere de ce cyclonucleosides (perte du caractere aromatique de l'heterocycle azote) ainsi que sa faible reactivite (determinee au cours de nos experiences) nous ont contraint au developpement d'une strategie de synthese radicalement differente de celle utilisee pour les 5,8-cyclopurine-2-desoxyribonucleosides. La troisieme partie de ce travail est consacre a la preparation d'un synthon phosphoramidite pour la 4-hydroxy-8-oxo-7,8-dihydro-2-desoxyguanosine. Au cours de l'une des etapes de synthese de ce precurseur, nous avons pu facilement separer les deux diastereoisomeres (4r)- et (4s)- de ce nucleoside modifie. Ils ont ete incorpores separemment dans les fragments d'adn synthetiques; l'epimerisation de la position c-4 n'etant pas observee au cours de la synthese sur support solide et lors de l'etape de deprotection ammoniacale. La derniere partie de ce manuscrit concerne la preparation d'oligonucleotides (2 a 9 bases de long) contenant un nucleoside modifie precurseur du radical 5-(2-desoxyuridilyl)methyle : la 5-(phenylthiomethyl)-2-desoxyuridine. Ces substrats ont ete utilises dans des etudes mecanistiques ayant pour but de preciser la reactivite de cet intermediaire radicalaire. Pour chacun des dommages incorpores une attention toute particuliere a ete portee a l'integrite des oligonucleotides synthetises. L'utilisation de differentes methodes analytiques (spectrometrie de masse et analyses clhp et maldi-tof des digestions enzymatiques) a permis de demontrer la purete des produits obtenus.
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Guo, Ruoling. "Chemical synthesis of isotopically labelled (M+4) purine nucleosides and their incorporation into DNA oligomers." Thesis, Aston University, 2004. http://publications.aston.ac.uk/11023/.
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Development of accurate and sensitive analytical methods to measure the level of biomarkers, such as 8-oxo-guanine or its corresponding nucleoside, 8-oxo-2’-deoxyguanosine, has become imperative in the study of DNA oxidative damage in vivo. Of the most promising techniques, HPLC-MS/MS, has many attractive advantages. Like any method that employs the MS technique, its accuracy depends on the use of multiply, isotopically-labelled internal standards. This project is aimed at making available such internal standards. The first task was to synthesise the multiply, isotopically-labelled bases (M+4) guanine and (M+4) 8-oxo-guanine. Synthetic routes for both (M+4) guanine and (M+4) 8-oxo-guanine were designed and validated using the unlabelled compounds. The reaction conditions were also optimized during the “dry runs”. The amination of the 4-hydroxy-2,6-dichloropyrimidine, appeared to be very sensitive to the purity of the commercial [15]N benzylamine reagent. Having failed, after several attempts, to obtain the pure reagent from commercial suppliers, [15]N benzylamine was successfully synthesised in our laboratory and used in the first synthesis of (M+4) guanine. Although (M+4) bases can be, and indeed have been used as internal standards in the quantitative analysis of oxidative damage, they can not account for the errors that may occur during the early sample preparation stages. Therefore, internal standards in the form of nucleosides and DNA oligomers are more desirable. After evaluating a number of methods, an enzymatic transglycolization technique was adopted for the transfer of the labelled bases to give their corresponding nucleosides. Both (M+4) 2-deoxyguanosine and (M+4) 8-oxo-2’-deoxyguanosine can be purified on micro scale by HPLC. The challenge came from the purification of larger scale (>50 mg) synthesis of nucleosides. A gel filtration method was successfully developed, which resulted in excellent separation of (M+4) 2’-deoxyguanosine from the incubation mixture. The (M+4) 2’-deoxyguanosine was then fully protected in three steps and successfully incorporated, by solid supported synthesis, into a DNA oligomer containing 18 residues. Thus, synthesis of 8-oxo-deoxyguanosine on a bigger scale for its future incorporation into DNA oligomers is now a possibility resulting from this thesis work. We believe that these internal standards can be used to develop procedures that can make the measurement of oxidative DNA damage more accurate and sensitive.
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Yang, Xiaochuan. "Design and Synthesis of Boronic Acid-Modified Nucleotides for Fluorescent Sensing and Cell Imaging." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/chemistry_diss/39.
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With the rapidly increasing interest in the field of glycomics, which is the comprehensive study of the roles carbohydrates play in a living system, urgent need for developing quick and highly selective carbohydrate sensors is growing. The boronic acid group, with its electron-deficient structure (6 valence electrons with an open shell), acts as a Lewis acid with high intrinsic affinity towards Lewis bases such as fluoride, cyanide and hydroxyl groups. Specifically, formation of a 5- or 6- membered ring between the boronic acid moiety and a1,2- or 1,3-diol in aqueous solution has been fully explored as a strategy of carbohydrate sensor design. Along this line, those binders were termed as ¡°boronolectins¡± because of their similar functions as lectins. One challenge in developing boronic acid-based carbohydrate sensors is to enhance the discriminating ability among various carbohydrate analytes with diverse building blocks and complex linkage patterns. One approach is using polypeptide or oligonucleotide as a backbone or scaffold with functionalized boronic acid moiety to create a molecular library, and then selecting binders with favorable properties. The work presented here includes three general research parts: synthesis of a naphthalimide-based boronic acid-conjugated thymidine triphosphate (NB-TTP), fluorescence property studies of NB-TTP incorporated DNA (NB-DNA), and cellular imaging studies using NB-TTP: 1) 4-Amino-1,4-naphthalimide (Nap) was chosen as the fluorophore because of its relatively long excitation and emission wavelengths, and stability. The synthesis of naphthalimide-based boronic acid (NB) followed similar route as previously published work. Tethering of boronic acid moiety and TTP was accomplished through Cu(I)-catalyzed azide-alkyne cyclization (CuAAC), known as click chemistry. The synthesized NB-TTP showed fluorescence enhancements at long wavelength (¦Ëem: 540 nm) upon sugar addition. 2) NB-TTP was incorporated into DNA through Klenow fragment-catalyzed primer extension reactions. Different DNA sequences were designed with varying number and spacing for NB-TTP incorporation. The preliminary study provided certain insight into several factors that affect the fluorescent properties of different NB -DNA. 3) NB-TTP was added into Hela cell culture medium to study its cell imaging properties. With the observation under fluorescent microscope, it was demonstrated that NB-TTP showed good cell membrane permeability and significant accumulation in cell nucleus.
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Hilpert, Hans. "Synthesis of novel fluorocabocyclic nucleosides and nucleotides as potential inhibitors of human immunodeficiency virus." Thesis, University of Exeter, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305860.
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Tsang, Hing Wo. "Design, synthesis and evaluation of nucleotides and nucleotide dimers as potential anti-HIV agents." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261107.
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Fletcher, Madison Hill. "Synthesis of Non-Natural Cyclic Di-Nucleotides for the Investigation of Bacterial Signaling Pathways." Diss., Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/429284.
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ChemistryPh.D.
Humans navigate the world and interact with others through a complex series of communicative tools. We experience both internal and external stimuli, such as pangs of hunger or pain from an injury, and both verbal and nonverbal language. Bacteria also possess the ability to communicate, albeit in more discreet, yet no less complex ways. Bacteria rely on an incredibly diverse signaling system of triggers and responses in order to survive and to thrive. While we perceive language with our eyes and ears, bacteria employ a system of small molecules to relay both intra- and extracellular messages. They utilize this ability, known as quorum sensing to "talk" to their neighbors, express otherwise latent genetic characteristics, and to defend themselves against enemies. It has been suggested that this internal and external activity is linked, however, little is known about their interplay. This family of molecules, the cyclic di-nucleotides, which includes c-di-GMP and c-di-AMP, are critical to regulating bacterial processes such as motility, glucose remediation, and cell wall homeostasis. Their importance has spurred numerous investigations into their mechanism of action. Although found in very low concentrations within cells, they are capable of regulating a multitude of processes due to their ability to adopt variable conformations. To date, analog design by other groups has focused on the modification of the innate phosphate moiety as well as various substitutions or deletions at the 2'-position on the ribofuranose ring. However, these analogs have not been water soluble, limiting them to in vitro investigations only. We propose that by replacing the phosphate linkage entirely we can increase water solubility and have pursued a divergent total synthesis of various cyclic di-nucleotides featuring biomimetic linkages. Herein we address the methods we explored to optimize the synthesis of our three monomers, coupling strategies employed, the novel application of a Staudinger ligation to afford our abasic macrocycles and finally our progress towards implementing a bis-glycosylation strategy to install the desired nucleobase. We are able to efficiently provide large amounts of a di-amino, azide methyl ester, and N,O-substituted furanose monomers in no more than six steps from a common intermediate. These monomers are coupled and cyclized to form our four scaffolds, amide, carbamate, squaramide, and urea. Finally, we have begun to successfully implement our Brønsted acid mediated glycosylation strategy and understand its limitations. It is our goal to develop a general method to afford a diverse array of conformationally unique and water soluble cyclic di-nucleotide analogs with which to probe these essential bacterial signaling pathways.
Temple University--Theses