Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Hung-wen Liu. Vita. Includes bibliographical references. Available also from UMI company.

Thesis (Ph. D.)--University of California, San Diego, 1999. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Abstract not available

The appended sugar residues of many natural products from Actinomyces are important for their biological activities. Many of these unusual sugar biosynthetic gene clusters have been isolated and many glycosyltransferases from various antibiotic-producing organisms have been identified. The increasing knowledge about these sugar biosynthetic pathways opens up the possibility of generating novel bioactive glycosylated compounds through combinatorial biosynthesis. The work described in this dissertation focuses on the investigation of the biosynthetic pathway of a rare nitro-containing sugar, D-kijanose, from an antibiotic, kijanimicin, and the glycosyltransferase helper proteins involved in the glycosylation of macrolide antibiotics. D-Kijanose, especially its nitro group, plays an important role in conferring the biological activities of the parent antibiotics. Cloning and sequencing of the kijanimicin biosynthetic gene cluster have allowed the proposal of the biosynthetic pathway of D-kijanose. The functions of the enzymes encoded by each open-reading frame in the cluster were also assigned based on sequence comparison with known enzymes found in other biosynthetic reactions. In this thesis, the functions of KijB1, a TDP-4-keto-6-deoxy-hexose 2,3-dehydratase, and KijD2, a TDP-hexose C-3 aminotransferase, were verified. The TDP-3-amino-4-keto-2,3,6-trideoxyhexose produced as an intermediate in the early stage of D-kijanose biosynthesis was also identified. In the second part of this dissertation, the in vivo protein-protein interaction between D-desosaminyl glycosyltransferase, DesVII, and its auxiliary protein, DesVIII, was established by yeast two-hybrid assay. The complex formation between these two proteins was also demonstrated by in vitro binding assay. Several strategies were tried to overexpress the D-mycaminosyl glycosyltransferase and its auxiliary protein, TylM2 and TylM3, although none of them were successful. A two-plasmid in vivo glycosylation system was also developed to test the competence of various DesVIII homologues to serve as the helper protein for glycosyltransferase DesVII, MycB and NbmD. In summary, the work in this dissertation has provided important information on the biosynthesis of D-kijanose and also significant insight into the function of the helper proteins of macrolide glycosyltransferases. These results could be useful for future studies of natural product biosynthesis and exploitation of glycodiversification. / text

Thesis (M.S.)--University of Michigan, 1970.

Thesis (M.S.)--University of Michigan, 1970.

Twenty different macrolide and ketolide antibiotics were analyzed by reversed phase high performance liquid chromatography on an ODS-2 cartridge column. Each of these compounds was uniquely separated and purified by varying the flow rate. Retention times of the individual drugs were proportional to the flow rate of the mobile phase. Recovery of antimicrobial activity for most of the drugs was greater than 90% based on a microbiological assay of material recovered from the column. Retention times were related to structural differences between these antimicrobial agents.

My Ph.D. research consists of two components: discovery of novel macrolide antibiotics and methodology development of N-sulfinyl metallodienamines. To tackle bacterial resistance, new antibiotics are desperately needed. My research objective is to address this need by designing, synthesizing, and evaluating novel macrolide antibiotics based on the best-in-class drug candidate, solithromycin. The drug discovery including following three projects: 1) Synthesis of solithromycin analogs wherein the desosamine sugar has been replaced with an acyclic amino alcohol surrogate; 2) In cellulo Click chemistry wherein the bacterial cell serves as the reaction vessel and the ribosome catalyzes the formation of triazole cycloadducts by testing different combinations of azide and alkyne fragments. One of the mechanisms of resistance to macrolide antibiotics is exemplified by methylation of A2058 by the methyltransferase encoded in erm genes. Methylation or dimethylation of A2058 leads to a steric clash with the macrolides and reduces the affinity of the macrolide for the ribosome. Thus, the bacterial resistant can be relieved via disrupting steric clashes between desosamine and A2058 residues. In 2017, I started a new project looking at the scope of the acyclic domino Michael/Mannich reaction to prepare chiral cyclohexenes developed by a previous group member, Dr. Vijay Chatare. This reaction is highly regioselective and stereoselective. Recent research showed that this reaction could be utilized on acroleins, acrylates and unsaturated ketones. Thus, we applied this useful methodology towards the concise total synthesis of (+)-ibogamine. / Chemistry

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Macrolide antibiotics are an important group of prescription drugs; as a consequence of the large and continuous use, they are commonly found in the environment. In the present study, it was developed and optimized a chromatographic method to assess the occurrence of macrolide antibiotics Azithromycin, Clarithromycin, Erythromycin and Roxithromycin in the effluent of the University Hospital of Santa Maria, in two sampling points, by applying high performance liquid chromatography coupled to mass detection with quadrupole ion trap (HPLC-MS/MS_QTrap) and clean-up/pre-concentration by solid phase extraction with the aid of Surface Methodology Response. The concentrations measured during a week in the hospital effluent were 1.32±0.13 and 0.22±0.06 μg L-1 for Azithromycin and Clarithromycin; in the receptor water sream was 1.12±0.20, 0.20±0.05 and 0.01±0.004 μg L-1 for Azithromycin, Clarithromycin and Erythromycin. Roxithromycin was not detected in all effluent samples. After this, it was done the evaluation of the risk quotient of the macrolide antibiotics. The value of the risk quotient for the hospital effluent for Azithromycin and Clarithromycin was 11 (high risk), and for the receptor water stream the risk quotient was 9.3 and 10.0 for Azithromycin and Clarithromycin; for Erythromycin, a quocient risk value of 0.5 (medium risk). For degradation of the antibiotics in aqueous solution, it was used UV-photolysis, by which the influence of pH (3-11) was evaluated. Azithromycin showed low degradation by acid pH; for other pH, as well, for all the other antibiotics, the degradation was above 70% after 60 min of treatment. It was conducted a kinetic study of the degradation process of macrolide antibiotics in different pHs, by which Azithromycin revealed a recalcitrant profile, and Roxithromycin, as the more easily degradable one. For identification of the products formed during the photolysis experiments it was used independent information acquisition and as precursor ions of fragments m/z 116 and 158, characteristic of the macrolide compounds, at three collision energies (30, 45, and 60 V). It was proposed fragmentation routes of the degradation products: 8 products for Azithromycin, 7 for Clarithromycin, 6 of Erythromycin and 8 Roxithromycin. Through the same experiments with independent information acquisition, it was investigated the presence of eventual metabolites in hospital effluent, and three metabolites were found. By applying photolysis to the hospital effluent fortified, at pH 7, it was observed that the degradation occurs above 80% for all compounds after 60 min of irradiation. It was observed the formation of degradation products previously determined by experiments in aqueous solution. It was also found three degradation products for Azithromycin, 2 for Clarithromycin, 1 for Erythromycin and 3 for Roxithromycin. / Os antibióticos macrolídeos são uma importante classe de fármacos preescritos no tratamento das mais variadas infecções, e como consequência se seu grande e continuo uso são comumente encontradas no ambiente. No presente estudo foi desenvolvido e otimizado método de cromatografia líquida de alta eficiência acoplada à detector de massas quadrupolo íon trap (HPLC-MS/MS_QTrap) e de clean-up/pré-concentração por extração em fase sólida com auxílio de Metodologia de Superfície de Resposta para avaliar a ocorrência dos antibióticos macrolídeos Azitromicina, Claritromicina, Eritromicina e Roxitromicina no efluente hospitalar do Hospital Universitário de Santa Maria em dois ponto de amostragem. As concentrações médias durante o ciclo de uma semana de amostragem no efluente do pronto atendimento foram de 1,32±0,13 e 0,22±0,06 g L-1 para Azitromicina e Claritromicina; no corpo recpetor foram de 1,12±0,20; 0,20±0,05 e 0,01±0,004 g L-1 para Azitromicina, Claritromicina e Eritromicina, respectivamente. Roxitromicina não foi detectada. Após foi feita a avaliação do quociente de risco dos antibióticos macrolídeos. O quociente de risco no efluente do pronto atendimento para Azitromicina e Claritromicina foi de 11, risco alto, o qual também foi evidenciado no corpo receptor com quociente de risco de 9,3 e 10 para Azitromicina e Claritromicna, e risco médio para Eritromicina de 0,5. Para degradação dos antibióticos foi utilizado fotólise artificial em solução aquosa, sendo avaliado a influência do pH de 3-11 na degradação destes compostos. Azitromicina apresentou baixa degradação em pH ácido, para os outros pH e demais compostos a degradação foi acima de 70% após 60 min de tratamento. Foi feito um estudo cinético do processo de degradação dos antibióticos macrolídeos em diferentes pH, observando-se que a Azitromicina apresentou um perfil recalcitrante para o processo, e Roxitromicina foi degradada com maior facilidade. Para a identificação dos produtos formados durante os experimentos de fotodegradação foram montados experimentos de informação independente de aquisição utilizando como íons precursores os íons de m/z 116 e 158 característicos dos compostos macrolídeos em três energias de colisão (30, 45 e 60 V). Foram identificadas e propostas rotas de fragmentação para 8 produtos de degradação de Azitromicina, 7 para Claritromicina, 6 para Eritromicina e 8 produtos de degradação de Roxiromicina. Através dos mesmos experimentos de informação independente de aquisição, foi investigada a presença de possíveis metabólitos no efluente hospitalar sendo encontrados 3 metabólitos. Com a aplicação de fotólise ao efluente hospitalar fortificado, em pH 7, observou-se que ocorre degradação acima de 80% para todos os compostos após 60 min de tratamneto. Foi observada a formação de produtos de degradação, que tinham sido previamente determinados em solução aquosa. Foram encontrados 3 produtos de degradação de Azitromicina, 2 para Claritromicina, 1 para Eritromicina e 3 produtos para Roxitromicina.