Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Orientador: Hélia Harumi Sato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-25T13:22:26Z (GMT). No. of bitstreams: 1 Florencio_Moises_M.pdf: 812754 bytes, checksum: c44bcd00e211d80d4501ad8f58305353 (MD5) Previous issue date: 2014 / Mestrado / Engenharia de Alimentos / Mestre em Engenharia de Alimentos

Rhizobium aspartate transcarbamoylase (ATCase; EC 2.1.3.2) was previously believed to be similar to the Pseudomonas ATCase which has been studied extensively. To facilitate the study of the Rhizobium ATCase a pyrimidine-requiring mutant of R. meliloti was isolated and used in the purification of the enzyme.

In the first part of this work, a series of site-specific artificial metallonucleases (AMNs) was developed conjugating clamped-phenanthroline (Clip-Phen) copper complexes to triplex- forming oligonucleotides (TFOs). Several synthetic routes were explored for the synthesis of the TFO-AMNs hybrids, all sharing a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction as the key step. As a consequence, building blocks for enzymatic or chemical synthesis of oligonucleotides (ONs) containing clickable groups, or already conjugated to the Clip-Phen ligand via CuAAC, were prepared. Two new alkynyl-linked nucleoside-5'-O-triphosphates (dNTPs) were designed and developed in order to obtain an efficient polymerase incorporation of clickable alkynyl-tethers into ONs and, at the same time, enhance the efficiency of CuAAC reactions on modified DNA. The relative 3'-O- phosphormaidites were also prepared in order to insert the same alkynyl-linkers into ONs via solid-phase synthesis. The AMN was linked at the 5'- or 3'-ends or in the middle of the TFO stretch, using diverse likers. The hybridization of all the synthesized TFOs with a target DNA duplex was studied. Finally, an extensive study of cleavage efficiency and specificity of the TFO-AMN conjugates towards the target DNA was performed, exploring the...

In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA....

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.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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...

Aspartate transcarbamoylase (ATCase) is encoded by the pyrBI genes in E. coli. Expression of these genes is reduced four-fold by attenuation when grown on uracil. Using plasmid, pRO1727. the pyrB and the pyrBI genes from E. coli were cloned into a P. putida pyrB auxotroph. A recombinant pyrB gene was recovered that encoded a functional hybrid ATCase with a molecular weight of 470 kDa.