Made available in DSpace on 2014-06-11T19:29:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-23Bitstream added on 2014-06-13T19:17:31Z : No. of bitstreams: 1 abe_ro_me_araiq_parcial.pdf: 339743 bytes, checksum: 929e061c798f66e865d33678e4663158 (MD5) Bitstreams deleted on 2015-05-28T14:25:09Z: abe_ro_me_araiq_parcial.pdf,. Added 1 bitstream(s) on 2015-05-28T14:26:13Z : No. of bitstreams: 1 000718858.pdf: 1542164 bytes, checksum: c8fda320ab5ad6c0583c426574dc6e22 (MD5) / Este trabalho teve como principal objetivo a obtenção de substâncias bioativas produzidas pelo fungo endofítico C80, pertencente à família Phaeosphaeriaceae, isolado da alga marinha Bostrychia radicans. O crescimento do endófito em escala ampliada foi realizado em meio sólido de arroz, modo estático e temperatura ambiente por 28 dias. O extrato em AcOEt produzido a partir deste cultivo foi fracionado através de técnicas cromatográficas, levando ao isolamento de seis substâncias: 7-hidroxi-3-(1-hidroxietil)-5-metoxi-3,4-dimetilisobenzofuran-1(3H)-ona (1), 3,7-dihidroxi-5-metoxi-3,4-dimetilisobenzofuran-1(3H)-ona (2), 7-hidroxi-3-(1,2-dihidroxietil)-5-metoxi-3,4-dimetilisobenzofuran-1(3H)-ona (3 e 4), 4,8-dihidroxi-6-metoxi-4,5-dimetil-3-metilenoisocroman-1-ona (5) e 8-hidroxi-6-metoxi-4,5-dimetil-3-metilenoisocroman-1-ona (6). Estas substâncias foram identificadas por análises espectroscópicas e espectrométricas como sendo policetídeos aromáticos, e então encaminhadas para ensaio citotóxico. Visando a mudança na produção metabólica do fungo, assim como na atividade biológica dos extratos brutos, foram realizados estudos da influência da composição do meio de cultura e do tempo de cultivo. Para a variação de composição, foram utilizados meios comerciais (CBD, Malte, Nutriente e arroz) e meios comerciais modificados com nitrato de amônio ou água do mar. Nenhuma alteração foi observada no perfil cromatográfico e nem na atividade citotóxica dos extratos. A variação do tempo de cultivo foi feita em meio sólido de arroz, com extrações em 10, 20, 30 e 40 dias de cultivo. Neste intervalo de tempo monitorado, a proporção dos metabólitos mudou consideravelmente, uma vez que os compostos majoritários no extrato de 10 dias estavam praticamente ausentes em 40 dias / The aim of this study was to obtain bioactive compounds produced by the endophytic fungus C80, belonging to the family Phaeosphaeriaceae, isolated from the marine algae Bostrychia radicans. This endophyte was cultivated in rice solid medium (large scale) in static mode at room temperature for 28 days. After addition of ethyl acetate, broth and mycelia were separated by filtration and the solvent was removed under vacuum to furnish the crude extract. The ethyl acetate extract was fractionated by using different chromatographic techniques resulting in the isolation of six compounds: 7-hydroxy-3-(1-hydroxyethyl)-5-methoxy-3,4-dimethyl-isobenzofuran-1(3H)-one (1), 3,7-dyhydroxy-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (2), 7-hydroxy-3-(1,2-dihydroxyethyl)-5-methoxy-3,4-dimethylisobenzo-furan-1(3H)-one (3 e 4), 4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methyleneiso-chroman-1-one(5) e 8-hydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochroman-1-one (6), which were identified as aromatic polyketides by using spectroscopic and spectrometric analysis and submitted to cytotoxicity assay. In addition, C80 was cultivated in different culture media at different periods of time to evaluate its chemical diversity and metabolites production. The cultures were undertaken using PDB, Malt, Nutrient and Rice media with or without the addition of ammonium nitrate or sea water. These procedures didn’t change the metabolic production and cytotoxicity. The role of time was undertaken only for rice media at 10, 20, 30 and 40 days of incubation. In this time the proportion of the metabolites has changed considerably, since the major compounds in the extract of 10 days, were practically absent in 40 days

CNPq / O Capim Annoni-2 (Eragrostis plana Nees) é uma poaceae que apresenta potencial alelopático conforme observado em trabalhos anteriores. Neste estudo os extratos, obtidos com solventes em ordem crescente de polaridade (éter de petróleo, acetato de etila e metanol), das folhas de Capim Annoni-2, coletadas no verão e inverno, foram analisados e comparados, levando-se em conta composição e/ou concentração de metabólitos produzidos, utilizando-se técnicas de Infravermelho e Cromatografia Líquida de Alta Eficiência, sendo que, os dados de Infravermelho foram comparados estatisticamente por PCA, em seguida realizou-se o fracionamento dos extratos de acetato de etila verão e inverno, as substâncias isoladas foram identificadas através de métodos espectrométricos e espectroscópicos: RMN de 1H e 13C, experimentos de HSQC e HMBC e CG-EM. Verificou-se que os extratos apresentam riqueza em termos de provável variedade de compostos químicos, além disso, observa-se que os solventes utilizados para a extração resultam, estatisticamente, em maiores diferenças, em termos de composição química, do que a estação do ano em que as folhas foram coletadas. Foram identificadas duas substâncias resultantes do fracionamento do extrato de acetato de etila verão, um triacilglicerol e um rotenóide. A presença de um rotenóide nas folhas de Capim Annoni-2, pode justificar a ação alelopática observada nesta espécie em outros trabalhos. / The Annoni-2 grass (Eragrostis plana Nees) is a poaceae that presents allelophatic potential as observed in other research. In this study, the extracts were obtained from leaves collected during summer and winter. The extraction process was performed using solvents in a increasing order of polarity (petroleum ether, etyl acetate and methanol) and the extracts were analyzed and compared regarding the composition and/or concentration of metabolites produced. The techniques utilized were high performance liquid chromatography and infrared spectroscopy, and, the data obtained from the infrared analisys was compared using PCA, then the fractionation of summer and winter ethyl acetate extracts was carried out and the isolated compounds were identified by spectrometric and spectroscopic methods: NMR 1H and 13C, experiments of HSQC and HMBC and GC-MS. The results show the extracts are rich when it comes to probable variety of chemical compounds. It is also observed that, statistically, the solvents used in the extraction process influence the diversity of chemical compounds more than the season in which the leaves were collected. Triacylglycerol and rotenoid were identified from the fractionation summer ethyl acetate extract, and the presence of a rotenoid in the leaves of Annoni-2 grass, can justify the allelopathic action observed in this species in other works.

Cis-diol-containing metabolites have attracted increasing attention in recent years. These metabolites widely exist in the body fluids and tissues. They play important roles in the structure, function and metabolic activity of cells. Some of them are related to cell proliferation and metabolic processes. And they have been used to denote a state of disease as potential biomarkers. Several methods have been developed for the analysis of cis-diol-containing metabolites. However, these methods faced a challenge to separate and detect isomers of these compounds, particularly for compounds with low abundance and high polarity. Therefore, novel methods were necessary to improve the separation and detection sensitivity of this kind of metabolites. With this aim, chemical derivatization methods were developed for cis-diol-containing metabolite detection by using liquid chromatography-mass spectrometry in this project. These methods were optimized and validated to achieve the optimal reaction conditions. And they were applied to study real-world biological systems, including the changes of modified nucleosides in hepatocellular carcinoma (HCC) nude mice and toxic effects of bisphenol A (BPA) exposure. Firstly, the derivatization reaction of cis-diol compounds with acetone were optimized. Factors that affected reaction efficiency were investigated by reacting guanosine (G) with acetone. The optimal reaction conditions were validated by detecting four acetonides of urinary nucleosides by using LC-MS/MS. The results showed that the approach had good linearity, accuracy and precision. The recoveries were ranged from 92.9% to 103.5%. It indicated that the assay was reproducible. The robust method should be potentially useful for the analysis of modified nucleosides and other cis-diol-containing metabolites in biological samples. The validated derivatization method was applied to determine urinary nucleosides by LC-MS. This method not only improved the retention of nucleosides on reversed-phase column, but also reduced the matrix effect from urine samples and enhanced detection sensitivity of mass spectrometry. Isotope labeling method with acetone-d6 and multivariate statistical analysis enabled the positive identification of 56 nucleosides, including 52 modified nucleosides. The obtained results indicated that the derivatization method was practical, fast and effective for the identification of urinary nucleosides. It was successfully applied to study the changes of urinary nucleosides in nude mice bearing HCC. Some significantly changed nucleosides were identified as potential biomarkers. Subsequently, this approach was modified by employing parallel reaction monitoring (PRM) method which was based on high resolution MS to detect urinary nucleosides in rats exposed to BPA. Comparing to the data acquired by triple quadrupole MS with neutral loss scanning, higher specificity and sensitivity were achieved by using PRM scanning mode. Therefore, more nucleosides were identified by using the method in urine samples (from 56 up to 66). The changes of the detected nucleosides were studied in the rats exposed to BPA. Various trends of modified nucleosides were observed with different dose BPA exposure. Specifically, the high-dose exposure group was the most strongly affected. The biomarker of RNA oxidation, 8-hydroxyguanosine (8-oxoG), showed significant change in this group. It proved that BPA exposure could induce RNA damage when the dose of BPA was beyond a certain amount. Except for nucleosides, other cis-diol-containing metabolites, such as carbohydrates, were also studied by using the derivatization method. Acetone and acetone-d6 were applied to label the cis-diol metabolites. Based on the chemical isotope labeling, cis-diol metabolites were easily recognized from urine samples. Influence of BPA exposure on these metabolites was investigated by comparing different doses of BPA administration on rats. Analytes showed noticeable difference were highlighted. Pathway analysis indicated that galactose metabolism, nucleoside and its analogues metabolism were disturbed. The derivatization method was extended to quantify nucleotides in plasma samples. According to the specific physical-chemical properties of nucleotides, the method was improved to fit the requirement of analysis by using 1,1-Dimethoxycyclohexane (DMCH) as derivatization agent and formic acid (FA) as catalyst. Tip micro-columns packed with TiO2 were used for selective adsorption of nucleotides in the plasma. Then in-situ derivatization were carried out to change the polarity of targeted compounds. LC-MS analysis of the derivatization products were employed without using ion-pairing reagents. This method exhibited a high selectivity for the extraction of nucleotides. After derivatization, retention of nucleotides on reversed-phase C18 column was improved. Complete separation of nucleotides with the same base was achieved. The peak shape was symmetrical and the tailing was eliminated by using high pH mobile phase. The method settled the problems of nucleotide detection, which were poor retention, trailing, in-source fragmentation and contamination of ion-pairing reagents. The quantitative method was successfully applied to determine the content of nucleotides in plasma samples of rats exposed to BPA. It was simple and fast, as well as good selectivity and stability. It could be extended to detection of other phosphorylated metabolites with similar structure. To our best knowledge, it was the first time to employ derivatization methods to detect cis-diol-containing metabolites. The methods decreased the matrix effects of complex biological samples, and also decreased the polarity of cis-diol-containing metabolites. The changes of properties not only improved the chromatographic separation, but also enhanced the MS intensities. The methods overcame the problems of cis-diol-containing metabolite detection on reversed-phase column. They were successfully applied to study the changes of cis-diol-containing metabolites of HCC and toxic effects of BPA exposure. The method might be extended to determine other cis-diol-containing metabolites in urine samples as well as in cells, tissues and plasma samples. It might be valuable for the understanding of the roles of cis-diol-containing metabolites in in cell metabolism.

Orientador: Marcia Nasser Lopes / Banca: Jairo Kenupp Bastos / Banca: André Luiz Meleiro Porto / Resumo: Este trabalho teve como principal objetivo a obtenção de substâncias bioativas produzidas pelo fungo endofítico C80, pertencente à família Phaeosphaeriaceae, isolado da alga marinha Bostrychia radicans. O crescimento do endófito em escala ampliada foi realizado em meio sólido de arroz, modo estático e temperatura ambiente por 28 dias. O extrato em AcOEt produzido a partir deste cultivo foi fracionado através de técnicas cromatográficas, levando ao isolamento de seis substâncias: 7-hidroxi-3-(1-hidroxietil)-5-metoxi-3,4-dimetilisobenzofuran-1(3H)-ona (1), 3,7-dihidroxi-5-metoxi-3,4-dimetilisobenzofuran-1(3H)-ona (2), 7-hidroxi-3-(1,2-dihidroxietil)-5-metoxi-3,4-dimetilisobenzofuran-1(3H)-ona (3 e 4), 4,8-dihidroxi-6-metoxi-4,5-dimetil-3-metilenoisocroman-1-ona (5) e 8-hidroxi-6-metoxi-4,5-dimetil-3-metilenoisocroman-1-ona (6). Estas substâncias foram identificadas por análises espectroscópicas e espectrométricas como sendo policetídeos aromáticos, e então encaminhadas para ensaio citotóxico. Visando a mudança na produção metabólica do fungo, assim como na atividade biológica dos extratos brutos, foram realizados estudos da influência da composição do meio de cultura e do tempo de cultivo. Para a variação de composição, foram utilizados meios comerciais (CBD, Malte, Nutriente e arroz) e meios comerciais modificados com nitrato de amônio ou água do mar. Nenhuma alteração foi observada no perfil cromatográfico e nem na atividade citotóxica dos extratos. A variação do tempo de cultivo foi feita em meio sólido de arroz, com extrações em 10, 20, 30 e 40 dias de cultivo. Neste intervalo de tempo monitorado, a proporção dos metabólitos mudou consideravelmente, uma vez que os compostos majoritários no extrato de 10 dias estavam praticamente ausentes em 40 dias / Abstract: The aim of this study was to obtain bioactive compounds produced by the endophytic fungus C80, belonging to the family Phaeosphaeriaceae, isolated from the marine algae Bostrychia radicans. This endophyte was cultivated in rice solid medium (large scale) in static mode at room temperature for 28 days. After addition of ethyl acetate, broth and mycelia were separated by filtration and the solvent was removed under vacuum to furnish the crude extract. The ethyl acetate extract was fractionated by using different chromatographic techniques resulting in the isolation of six compounds: 7-hydroxy-3-(1-hydroxyethyl)-5-methoxy-3,4-dimethyl-isobenzofuran-1(3H)-one (1), 3,7-dyhydroxy-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (2), 7-hydroxy-3-(1,2-dihydroxyethyl)-5-methoxy-3,4-dimethylisobenzo-furan-1(3H)-one (3 e 4), 4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methyleneiso-chroman-1-one(5) e 8-hydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochroman-1-one (6), which were identified as aromatic polyketides by using spectroscopic and spectrometric analysis and submitted to cytotoxicity assay. In addition, C80 was cultivated in different culture media at different periods of time to evaluate its chemical diversity and metabolites production. The cultures were undertaken using PDB, Malt, Nutrient and Rice media with or without the addition of ammonium nitrate or sea water. These procedures didn't change the metabolic production and cytotoxicity. The role of time was undertaken only for rice media at 10, 20, 30 and 40 days of incubation. In this time the proportion of the metabolites has changed considerably, since the major compounds in the extract of 10 days, were practically absent in 40 days / Mestre

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Objetivou-se com o presente estudo avaliar a biometria, parÃmetros sanguÃneos e hormonais de cordeiros Santa InÃs. Foram utilizados 30 cordeiros com peso vivo mÃdio inicial de 13,0  1,49 kg e aproximadamente 60 dias de idade para estimativa daglicose, colesterol, &#946;- hidroxibutirato, N- ureico, proteÃnas totais, albumina, globulina, cloreto, cÃlcio, fÃsforo, magnÃsio e os hormÃnios leptina, insulina e tiroxina (T4). Utilizou-se o delineamento inteiramente casualizado em esquema fatorial 3 x 3 x 2, que consistia detrÃs nÃveis de restriÃÃo alimentar (RA) (controle, 30% e 60%), trÃs perÃodos de coleta correspondente à idade dos animais (14, 18 e 23 semanas de idade) e duas classes sexuais (castrados e nÃo castrados). O sangue foi coletado por punÃÃo da veia jugular nos trÃs perÃodos de coleta. Para as medidas biomÃtricas utilizou-se o delineamento inteiramente casualizado em esquema fatorial 3 x 2, levando-se em consideraÃÃo os nÃveis de restriÃÃo e as classes sexuais. As medidas foram realizadas quinzenalmente durante todo perÃodo experimental.O hormÃnio Tiroxina nÃo foi influenciado pela idade, dieta ou classe sexual. Jà a leptina e a insulina aumentaram com a idade dos animais (P<0,01). As concentraÃÃes de glicose diminuÃram de acordo com o aumento do nÃvel de RA (P<0,001). As concentraÃÃes de BHB foram maiores à medida que se aumentou a restriÃÃo alimentar (P<0,001). O N-ureico foi influenciado pela idade e dieta experimental (P< 0,001) e houve interaÃÃo idade X classe sexual (P<0,01). As concentraÃÃes de colesterol total e proteÃnas totais nÃo foram influenciadas pelos tratamentos experimentais (P<0,05), entretanto a albumina e globulina foram influenciadas pela idade (P<0,001; P<0,01). As concentraÃÃes plasmÃticas de cloretos, cÃlcio, magnÃsio e fÃsforo variaram significativamente em funÃÃo da idade. Os animais submetidos à RA cresceram em ritmo mais lento, sendo que, a influencia negativa foi mais acentuada no R60 (60% de RA). No R30 (30% de RA), o ritmo de crescimento diminuÃdo foi mais perceptÃvel com 100 dias de idade no peso corporal (PC), com 130 dias no escore de condiÃÃo corporal (ECC) e perÃmetro torÃcico (PT), com 145 dias na altura de garupa (AG) e com 160 dias no comprimento de garupa (CG) e largura de peito (LP). A anÃlise da correlaÃÃo de Pearson mostra que as medidas biomÃtricas sÃo co-dependentes do nÃvel de alimentaÃÃo do animal, pois as correlaÃÃes foram altas e significativas nos animais controle (r= + 0,61 a 0,95). Conclui-se que os parÃmetros metabÃlicos foram influenciados mais pela idade do que pela dieta e que a restriÃÃo alimentar afeta o perfil metabÃlico e hormonal, em especial o &#946;-hidroxibutirato e a insulina. O escore de condiÃÃo corporal, peso corporal e largura de peito foram os parÃmetros mais sensivelmente influenciados pela RA. / The present study aimed to evaluate biometric measurements and blood and hormonal parameters of Santa Ines lambs. Thirty lambs with an average live weight of 13.0  1.49 kg and 60 days of age were used to estimate glucose, cholesterol, &#946;-hydroxybutyrate, urea N, total protein, albumin, globulin, chloride, calcium, phosphorus, magnesium, and leptin, insulin, and thyroxine (T4) hormones. Lambs were assigned to a randomized complete design in a 3 à 2 factorial arrangement consisting of three levels of food restriction (FR) (control, 30%, and 60%), three collection periods corresponding the age of the animals (14, 18, and 23 weeks of age), and two sex categories (castrated and uncastrated). Blood was collected by jugular venipuncture in the three collection periods. For biometric measurements, a randomized complete design in a 3 à 2 factorial arrangement was adopted, taking into account restriction levels and sex categories. Measurements were taken every two weeks throughout the experimental period. The hormone thyroxine was not influenced by age, diet, or sex category. Leptin and insulin increased with the animal age (P <0.01). Glucose concentrations decreased as the FR level were increased (P <0.001). &#946;-hydroxybutyrate concentrations increased as the food restriction was increased (P <0.001). Urea nitrogen was influenced by age and experimental diet (P <0.001), and there was an age à sex category interaction effect (P <0.01). Concentrations of total cholesterol and total proteins were not influenced by experimental treatments (P <0.05), but albumin and globulin were influenced by age (P <0.001, P <0.01). Plasma concentrations of chloride, calcium, magnesium, and phosphorus varied significantly according to age. Animals subjected to FR grew at a slower rate, and the negative influence was more pronounced at 60% FR. At 30% FR, the growth rate decline was more noticeable at 100 days of age for body weight (BW); at 130 days for body condition score (BCS) and chest girth (CG); at 145 days for rump height (RH); and at 160 days for rump length (RL) and breast width (BW). Pearson's correlation analysis showed biometric measurements are co-dependent on the feeding level because correlations were high and significant in control animals (r = 0.61 to 0.95). It is concluded that the metabolic parameters were more influenced by age than by diet, and food restriction affects the metabolic and hormonal profiles, in particular &#946;-hydroxybutyrate and insulin. Body condition score, body weight, and breast width were the most significantly affected by FR parameters.

>Magister Scientiae - MSc / The discovery of novel secondary metabolites has declined significantly in recent years whereas there is a rise in the number of multi-drug resistant pathogens and other types of diseases. The decline in natural product discovery was due to high rediscovery of already known compounds and the costs in developing natural products. As a result pharmaceutical companies lost interest in investing in natural product discovery. However, there is a renewed interest in marine sponge associated microorganisms as a rich and untapped source of secondary metabolites. The objective of this study was to design a matrix to investigate the extent to which the One Strain-Many Compounds (OSMAC) approach applies to a collection of marine sponge isolates harvested from two South African marine sponge samples. Terminal restriction fragment length polymorphisms (T-RFLP) analysis was used to investigate and ascertain the two marine sponges which hosted the highest microbial diversities to be used for further culture-dependent studies. The culture-dependent studies, using 33 media which included liquid enrichment, heat treatments and antibiotic treatments, resulted in 400 sponge isolates from the two marine sponges Isodictya compressa and Higginsia bidentifera. Using antibacterial overlay assays, 31 dereplicated isolates showed antibacterial activity. Bioactivities were also exhibited against E. coli 1699 which is genetically engineered for resistance against 52 antibiotics which implies that some of the bioactive compounds could be novel. The 16S rRNA gene sequences revealed that the microbial phyla isolated from the marine sponges belonged to Actinobacteria, Firmicutes and Proteobacteria (Alphaproteobacteria and Gammaproteobacteria).Thirty isolates were selected for an OSMAC-based matrix study, 17 of which showed noantibacterial activities in preliminary screening. The application of the OSMAC approach using co-culture and 36 culture conditions resulted in 6 isolates showing antibacterial activities, three of which did not show activities in preliminary screening. One of these, a Bacillus pumilus isolated from I. compressa displayed antibacterial activity against 5 indicator strains whereas in preliminary screening it had not shown activity. The results show that marine sponges can host novel microbial species which may produce novel bioactive compounds. The results also confirm that traditional methods employing a single culture condition restricts the expression of some biosynthetic pathways of microorganisms and as a result many metabolites have yet to be identified.

The effects of skin burn injury on the levels of oxidized and reduced glutthione, malondialdehyde, and on the activities of glutathione peroxidase, glutathione S-transferase, and glutathione reductase were determined in liver and lung of rabbit models, 24-h post-burn.

The endogenous catecholamines and their metabolites play an integral role in establishing the presence or absence of a suspected sympatho-adrenal tumor. Highly elevated metabolites excreted in the urine are indicative of a tumor. For this reason numerous analytical methods has been developed to accurately quantify the levels of these compounds. However, current methods usually make use of conventional HPLC methods. Although effective, these methods require tedious sample preparation and are usually plagued by interferences. It was the aim of this work to develop a gas chromatographic – mass spectrometric (GC-MS) method that allow for the simultaneous analysis of the endogenous catecholamines, their basic and acidic metabolites using a single extraction procedure (which is easy to use and not tedious) with minimal derivatization steps. Furthermore, to develop GC-MS methods which do not require tedious sample preparation and yet be sensitive and accurate and allow for rapid analysis in the clinical pathology laboratory. Four different gas chromatographic - mass spectrometric methods were developed for the analysis of the catecholamines and their metabolites and are discussed in detail. / Dissertation (MSc)--University of Pretoria, 2009. / Chemical Pathology / unrestricted

Enzymatic halogenation occurs during the biosynthesis of more than 4,000 natural products. The presence of fluorinated natural products is much less common, with only 13 reported to date. The bacterium Streptomyces cattleya is known to biosynthesise two fluorinated secondary metabolites, fluoroacetate and 4-fluorothreonine. The precursor to these secondary metabolites is known to be fluoroacetaldehyde. It had previously been shown that a fluorination enzyme mediates a reaction between S-adenosyl-L-methionine (SAM) and F to generate 5'-fluoro-5'-deoxyadenosine (5'-FDA). This is the first committed step on the biosynthetic pathway. The pathway between 5' -FDA and fluoroacetaldehyde had not been investigated in detail prior to the work carried out in this thesis. A purine nucleoside phosphorylase has been partially purified from cell-free extracts which catalyses the phosphorolytic cleavage of 5'-FDA to 5-fluoro-5-deoxY-D-ribose-1- phosphate (5-FDRP). Substrate specificity shows a profile which shares a close similarity to bacterial 5'-methylthioadenosine phosphorylases (MTAP's). The identification of a gene cluster encoding enzymes responsible for fluorometabolite biosynthesis shows the PNP gene located adjacent to the fluorinase gene, reinforcing the involvement of this enzyme in the fluorometabolite pathway. It is shown that 5-FDRP is converted to 5-fluoro-5-deoxy-D-ribulose-1-phosphate (5- FDRibP) via an isomerase activity. The enzyme responsible for this transformation has been partially purified from cell free extracts (CFE's). Another metabolite was identified as 5-fluoro-5-deoxY-D-xylulose-l-phosphate (5-FDXyuP), a diastereoisomer of 5-FDRibP, which appears to be an adventitious product in CFE's of S. cattleya. Two DHAP dependent aldolases have been identified, one of which is a putative L- fuculose-1-phosphate aldolase which catalyses conversion of 5-FDRibP to fluoroacetaldehyde. The other, an L-fructose 1,6-bisphosphate aldolase has been purified to homogeneity and catalyses an aldol reaction between DHAP and fluoroacetaldehyde to generate 5-FDXyuP. This enzyme is most probably one of primary metabolism.

Toxicities of sulfur-based drugs have been attributed to formation of highly reactive sulfur oxo-acids and depletion of glutathione by the formation of reactive metabolites. Metabolic activation of these sulfur centers to conceivably toxic reactive metabolites (RMs) that can covalently modify proteins is considered the initial step in drug-induced toxicity. Despite considerable effort and research, detection and characterization of these RMs during drug development and therapy remains a challenge. Methimazole (MMI) and 6-propyl-2-thiouracil (PTU) are two commonly used antithyroid, sulfur-based drugs. Though effective, these drugs are associated with idiosyncratic toxicity. PTU has acquired a black box warning and physicians are calling for its withdrawal. RMs resulting from bioactivation of these drugs have been implicated in the aforementioned adverse reactions. Unfortunately, isolating and detecting RMs using traditional analytical techniques has not been successful due to their high reactivity and short life span, typically less than a minute. Current approaches in drug metabolism studies use microsomal incubations to generate RMs, which are then trapped using nucleophiles. Antithyroid drugs, however, are known to deactivate enzymes involved in their oxidation. Moreover, due to the complex nature of biological matrices and low abundance of possible toxic conjugates, this technique results in poor selectivity and sensitivity. This study developed and optimized an analytical method based on coupling electrochemical redox reactions and mass spectrometry to generate, detect and identify RMs from antithyroid drugs. The metabolites were also compared to those that were generated using chemical oxidants and biological microsomes. Mimicry of enzymatic oxidation of the antithyroid drugs was carried out by electrochemically oxidizing them using a coulometric cell coupled on-line to electrospray ionization mass spectrometry (EC/ESI-MS). Oxidation of MMI and subsequent trapping with nucleophile resulted in formation of adducts with N-acetylcysteine, revealing reactive metabolites. The most-postulated metabolite, sulfenic acid, had never been isolated or detected until now, using electrochemistry on-line with electrospray ionization. The results showed that bioactivation of MMI proceeds predominantly through the S-oxide and not through formation of thiyl radicals. These same trapping experiments were also conducted with PTU, but no conjugates were detected. The lack of conjugates from PTU does not preclude formation of RMs, but asserts radical pathway might be dominant in EC oxidation. A double mixing stopped flow was used to investigate the kinetics and mechanism of reaction of the MMI and the biologically relevant hypochlorous acid (HOCl), a product of oxidation of chloride (Cl-) ions by myeloperoxidase. The products from the chemical oxidations were compared to the electrochemically generated metabolites, some differences were apparent. Human liver microsomes (HLM) were also used, to investigate oxidation of PTU. Oxidation of PTU, resulted in the supposedly toxic S-oxide, but this has never been isolated, save for speculation. A comparison of metabolites that were found with HLM to those generated electrochemically showed some degree of similarity. These results show that in vitro techniques such as chemical oxidations and electrochemistry coupled to mass spectrometry can be used to mimic oxidative metabolism and subsequent high throughput screening of reactive metabolites.