Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 170-179). Available also in a digital version from Dissertation Abstracts.

Nucleic acids are ubiquitous in nature and modified nucleosides are present in a wide range of anti-viral, anti-cancer drugs and antibiotics. Although a variety of naturally occurring nucleoside analogues exist, few include modifications to the ribose or deoxyribose ring. Intriguingly, the uridyl peptide antibiotics (UPAs), such as pacidamycin, contain a biosynthetically unique 3'-deoxyuridine which resembles synthetic anti-retrovirals. Elucidation of the biosynthesis of this structuraly unique nucleoside motif suggests a degree of substrate flexibility, making it a highly attractive prospect for biosynthetic approaches to nucleoside modification. In order to fully exploit the biotransformative potential, a detailed mechanistic understanding of the individual enzymes involved in the biosynthesis of the nucleoside moiety, and especially the enzyme employed at the installation of the 3'-deoxy modification, is required. Chapter 1, the introduction the thesis, discusses the importance of nucleosides for Chemistry and Biology. The section describes the biosynthesis of the nucleoside antibiotics and reviews the recent advances relating to the synthesis and biosynthesis of 3'-deoxy-nucleosides. The Chapter proceeds to describes the biosynthesis of deoxy-sugars, deoxy-nucleosides and nucleotides, reviewing the most common dehydratase mechanisms in addition to examining unusual dehydratases involved in carbohydrate metabolism. Chapter 2, the study of Pac13, the uridine-5'-aldehyde dehydratase of the pacidamyicin nucleoside cluster, is reported. Through detailed functional, structural and kinetic analysis of the wild-type enzyme as well a series of mutants, Chapter 2 provides insight into the mechanism emplyed by this unusual enzyme. Chapter 3 describes the structural and functional analysis of Pac11, the flavin-dependent oxidoreductase of the nucleoside biosynthetic cluster, while Chapter 4 revolves around Pac5, the PLP-dependent aminotransferase. In Chapter 5, the chemical synthesis of fluorinated nucleosides, as probes for exploring the enzymes' mechanism is investigated. Chapter 7 reports the experimental procedures for the research described in this document. The work described in this thesis broadens the understanding of the biosynthesis of deoxy-nucleosides and constitutes the first structural and mechanistic study of the biosynthesis of the biosynthesis of the valuable yet, synthetically challenging 3'-deoxy nucleosides.

The aim of this thesis was to study the steric influence of the base-modified nucleoside triphosphates (NTPs) on the enzymatic incorporation into RNA, as well as to study their inhibitory effect on different viral RNA polymerases in vitro. Their parent nucleosides and prodrug derivatives were also prepared and their antiviral activity evaluated. In the first part of the thesis, NTPs bearing groups varying in size from small methyl and ethynyl substituents via medium-size phenyl and benzofuryl groups, up to large dibenzofuran ring were prepared. Aromatic substituents were installed via Suzuki coupling on iodinated triphosphates or, in the case of modified guanosines, by the phosphorylation of modified nucleosides. Methyl and ethynyl NTPs were prepared via Pd-catalyzed coupling with AlMe3 and Sonogashira coupling, respectively, followed by the phosphorylation of modified nucleoside. To examine their incorporation into RNA by T7 RNA polymerase, templates coding for 35mer RNA containing one, three or seven modifications were designed. Modified pyrimidine triphosphates worked well for all the sequences, while the biggest dibenzofuryl group was not accepted in the difficult sequence with seven modifications. In the case of AR TPs dibenzofuryl modification did not incorporate at all, while other...

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Structural analyses of DNA oligonucleotides indicate the presence of bound water molecules in the major and minor grooves of DNA. These water molecules participate in DNA charge transfer by their reaction with guanosine radical cation to form 7,8-dihydro-8-oxo-guanine (8-oxoG), which when treated with a base leads to DNA strand cleavage. We probed the reaction of guanosine radical cation with water with series of alkyl substituted cytidines and thymidines by incorporating the modified nucleosides into anthraquinone linked DNA duplexes and irradiating them with UV light at 350 nm. The incorporation of these hydrophobic substituents disrupt the DNA spine of hydration, and we have observed that these modifications in the major and minor groove do not effect the trapping or long distance hopping of radical cations in DNA. The second part of the work reported herein examines the role of charged nucleosides in long range charge transfer in duplex DNA. DNA methylation is a naturally occurring process mediated by enzymes responsible for such functions in biological systems. Hypermethylation of DNA can also occur as a result of environmental alkylating agents leading to mutation of the affected cells. Methylation of the ring nitrogen of a purine base can introduce a positive charge in the ring resulting in the cleavage of the glycosidic bond of the nucleoside. To understand the role of a charged nucleoside on charge transfer in DNA, we designed and synthesized cationic nucleoside mimics, which were incorporated into anthraquinone-linked DNA strands and irradiated at 350 nm. The presence of the cationic bases on the duplexes inhibits the migrating hole from hopping along the DNA strand, and induces a prominent local structural distortion of the DNA as a result of the charged nucleobase.

This master thesis is dedicated to the preparation of fluorinated derivatives of carbocyclic nucleosides, that may serve as flaviviral replication inhibitors. Preparation of both monofluorinated as well as gem-difluorinated analogs of ribo and 2'-deoxyribonucleoside was attempted. While a suitable and reliable route for the preparation of monofluorinated compounds way found, synthesis of gem-difluorinated turned out to be rather challenging. Although most of the presented work dealt with compounds bearing adenine as a nucleobase, the universal applicability of the developed procedures, demonstrated on the preparation of a guanosine-type molecule, suggests that after slight optimization larger series of this type of compounds could be prepared.

In this study, high-performance liquid chromatography (HPLC) was employed to detect and quantify pyrimidine salvage enzymes by monitoring the disappearance of substrates or formation of products.

La squalénisation est une méthode de vectorisation sous forme nanoparticulaire qui consiste àcoupler de manière covalente un dérivé du squalène à des principes actifs hydrophiles tels que lesanalogues nucléosidiques. Les conjugués amphiphiles obtenus sont capables de s’auto-organiserspontanément dans l’eau en nanoparticules d’une centaine de nanomètres de diamètre etpossèdent des activités anticancéreuses ou antivirales remarquables. Notre objectif était d’étendrecette stratégie à différentes classes d’antitumoraux, comme les antimétabolites, les antimitotiqueset les agents alkylants. Différents dérivés du squalène ont ainsi été synthétisés puis couplés à cesprincipes actifs pour former les bioconjugués squalénisés correspondants. Il a été montré que cesprodrogues étaient capables de s’auto-assembler en nanoparticules spontanément en milieuaqueux, que le principe actif soit hydrophile ou hydrophobe. Les suspensions nanoparticulaires deces prodrogues se sont montrées actives in vitro sur différentes lignées cellulaires cancéreuseshumaines et murines et in vivo chez la Souris sur des modèles de cancer. La squalénisation a donc étéétendue à diverses familles de composés anticancéreux confirmant qu’il s’agit d’une méthodegénérale de vectorisation pourvue d’un fort potentiel thérapeutique. / Squalenoylation is a strategy of vectorization consisting in coupling squalene derivatives tohydrophylic drugs as nucleoside analogues. The amphiphilic conjugates obtained are able to selfassembleinto nanoparticles with a diameter of 100 nm in water. In addition those nanoparticleshave shown impressive anticancer and antiviral activities. Our objective was to extend this strategyto different anticancer drugs as antimetabolites, antimitotics and alkylating agents. Differentsqualenoyl derivatives have been synthesised and then coupled to drugs to furnish thecorresponding squalenoylated biconjugates. It has been shown that those prodrugs were able toself-assemble into nanoparticles in water. Nanoparticles of the bioconjugates are active in vitro ondifferent human cancer cell lines and in vivo in Mice on different cancer models. Squalenoylation hasfinally been extended to numerous anticancer drugs, proving that this strategy is a general methodof vectorization with a high therapeutic potential.

The presence and synthesis of transfer RNA (tRNA) is highly conserved in all organisms and a lot of genetic material is dedicated to its synthesis. tRNA contains a large number of modified nucleosides and several diverse functions have been found but much about their function is still unknown. By using a novel frameshifting system to select for tRNA modification mutants, new mutations were isolated and subsequently analyzed. This thesis examines the synthesis and function of a subset of tRNA modifications that have a sulfur (thio) -group as part of the modification. The isc operon encodes for proteins synthesizing iron sulfur centers ([Fe-S]) that are a part of the active site of many key enzymes in the cell and the thiolated nucleosides are dependant on a functional iron sulfur gene (iscS) for their synthesis. By studying thiolated tRNA it is not only possible to learn more about the synthesis of the modifications themselves, but also about the synthesis of [Fe-S] clusters. Based on an analysis of mutations in three of the isc operon genes (iscS, iscU, and iscA), a two-model pathway is proposed for the synthesis of Salmonella enterica Serovar Typhimurium thiolated tRNA modifications. The interactions of IscS with other proteins in the tRNA modification thiolation pathways suggest a more complex sulfur relay than had previously been envisioned. Some of the specificities and the effect of an iscA mutant on the levels of tRNA modifications lead to an examination of the role of IscA in [Fe-S] formation and its importance for tRNA modifications.