This thesis describes approaches towards the synthesis of 20-deoxybryostatins and 20,20-difluorobryostatins. Towards the 20-deoxybryostatins, a route previously developed within the group was followed with a protecting group change designed to prevent problems encountered with a late-stage deprotection. To this end, (R)-pantolactone was transformed through to dimethyl (4S,6R,8R)-[4-(para-methoxybenzyloxy)-10-hydroxy-6,8-O-isopropylidine-3,3-dimethyl-2-one] phosphonate which was subsequently subjected to a Horner-Wadworth-Emmons condensation with (5R,E)-6-(4-methoxybenzyloxy)-5-triethylsilyloxy-3-(2’-triisopropylsilyloxyethylidene)hexanal. The reactions of the resulting enone through to the advanced intermediate allyl [(4R,6R)-6-((S)-2’-(4-methoxybenzyl)oxy-5-{(2’’S,6’’R)-6’’-(4-methoxybenzyloxymethyl)-4’’-[2-triisopropylsilyloxyeth-(Z)-ylidene]-tetrahydropyran-2’’-yl}-3,3-dimethyl-4’-oxopentyl)-2,2-dimethyl-1,3-dioxan-4-yl]-acetate are described, and this compound corresponds to a C1-C16 northern bryostatin fragment. A synthetic route to 20,20-difluorobryostatins was begun by the synthesis of three 3,3-difluoro-2-hydroxytetrahydro-4H-pyrans which started from an indium-mediated coupling of 3-bromo-3,3-difluoro-1-propene with an aldehyde. A strategy was employed which culminated in the preparation of 3,3-difluoro-2-hydroxy-2-(3-methylbut-1-en-3-yl)-4-(E)-methoxycarbonylmethylenetetrahydro-4H-pyran which corresponds to a C16-C23 20,20-difluorobryostatin southern fragment. Of particular interest is the selectivity observed in the reaction of α,α-difluoroketones with a stabilised Wittig reagent. The indium-mediated coupling was extended to include the first example of the coupling of a 2-alkyl substituted bromide.

Per- and polyfluoroalkyl substances (PFAS) are synthetic compounds that have beenaround since 1940. They can be used in a variety of products such as fire-fighting foam, food packaging and cosmetic products. Many PFAS have potential toxic effects on both humans and animals which poses a problem due to their wide distribution and persistency. Another problem concerns the remediation of PFAS, where the substances ends up circulating between the different disposal types (landfills, wastewater treatment and incineration). Active methods are therefore needed to remove or retain the contaminants. Some examples of these remediation technologies can be biomaterials, organoclays and more advanced methods such as activated carbon and ion exchange. The more advanced being suited for remediation of drinking water. In this study, the sorption efficiency of two pine bark fractions has been studied and also compared to the efficiency of activated carbon. The evaluation was done for both target analysis (PFAS-11) and non-specific analysis of extractable organofluorine (EOF) compounds in contaminated water from a hazardous waste management facility. The two pine bark fractions indicated similar sorption efficiencies, for both the PFAS-11 and the EOF compounds. The sorption efficiency ranged from 10% up to 75% for perfluorinated sulfonic acids (PFSA) and from below zero up to 40% for perfluorinated carboxylic acids (PFCA). A general pattern can be seen, the sorption efficiency increases with increasing length of the PFAS chain. The activated carbon had a higher sorption efficiency, where the majority of PFAS had an average sorption of 100%. In comparison, the PFAS-11 and total EOF analysis displayed high concentrations of unidentified EOF compounds. These compounds indicated a negative sorption, which might imply that non-targeted PFAS or other fluorinated organic compounds desorb from the bark. Therefore, the pine bark might be suitable as a rough remediation of long-chain PFAS (≥8C), but further studies on the sorption of unidentified fluorinated organic compounds are of interest.

Per- and polyfluoroalkyl substances (PFAS) are synthetic compounds that have beenaround since 1940. They can be used in a variety of products such as fire-fighting foam,food packaging and cosmetic products. Many PFAS have potential toxic effects on bothhumans and animals which poses a problem due to their wide distribution and persistency.Another problem concerns the remediation of PFAS, where the substances ends upcirculating between the different disposal types (landfills, wastewater treatment andincineration). Active methods are therefore needed to remove or retain the contaminants.Some examples of these remediation technologies can be biomaterials, organoclays andmore advanced methods such as activated carbon and ion exchange. The more advancedbeing suited for remediation of drinking water. In this study, the sorption efficiency oftwo pine bark fractions has been studied and also compared to the efficiency of activatedcarbon. The evaluation was done for both target analysis (PFAS-11) and non-specificanalysis of extractable organofluorine (EOF) compounds in contaminated water from ahazardous waste management facility. The two pine bark fractions indicated similarsorption efficiencies, for both the PFAS-11 and the EOF compounds. The sorptionefficiency ranged from 10% up to 75% for perfluorinated sulfonic acids (PFSA) and frombelow zero up to 40% for perfluorinated carboxylic acids (PFCA). A general pattern canbe seen, the sorption efficiency increases with increasing length of the PFAS chain. Theactivated carbon had a higher sorption efficiency, where the majority of PFAS had anaverage sorption of 100%. In comparison, the PFAS-11 and total EOF analysis displayedhigh concentrations of unidentified EOF compounds. These compounds indicated anegative sorption, which might imply that non-targeted PFAS or other fluorinated organic compounds desorb from the bark. Therefore, the pine bark might be suitable as a roughremediation of long-chain PFAS (≥8C), but further studies on the sorption of unidentifiedfluorinated organic compounds are of interest.Keywords:

Chapter 1 gives an overview of the fluorine chemistry field, from its early developments to recent applications in medicinal chemistry. The development of asymmetric electrophilic or nucleophilic installation of fluorine in organic molecules is highlighten. Chapter 2 of this thesis discusses the enantioselective synthesis of α-fluoroamides. The study is applied to the synthesis of fluoroenantiomers of the bioactive molecule capsaicin and short-chain analogues. The biological activity of these compounds is assayed with the TRPV1 receptor. Results show that enantioselective α-fluoroamides (R)-97, (R)-99 and (S)-99 can generate differentiated biological responses, from TRPV1 agonists to TRPV1 antagonists. Chapter 3 focuses on the optimisation and development of 2-(aminomethyl)piperidine (R)-251 dihydrochloride. The development of 2-(aminomethyl)piperidine (R)-251 as its ditetrafluoroborate salt proved to offer excellent reactivity and solubility for the preparation of derivatives. This tetrafluoroborate salt was used to improve the syntheses of organocatalysts 2,2,2-trifluoro-N-(piperidin-2-ylmethyl)acetamide 363 and 4-methyl-N-(piperidin-2-ylmethyl)benzenesulfonamide 364.The catalytic properties of these latter two molecules for asymmetric Mannich reaction is demonstrated. Both (R)-363 and (R)-364 show up to 86% ee, in a typical 20 mol% loading, but loading of (R)-363 as low as 5 mol% still induces the catalysis.

Per- and polyfluoroalkyl substances (PFASs) are persistent organic pollutants used in industrial applications and are globally distributed in the environment. A group of PFASs that are difficult to measure with today’s method are perfluoroalkyl acid precursors (PFAA precursors) that, when degraded, serves as indirect sources of PFAAs. This study has optimized a previously developed method for quantification of PFAA precursors in soil; through total oxidizable precursor assay (TOP assay) under alkaline conditions, to be applicable on sewage sludge. To achieve and maintain an alkaline environment during the entire oxidative treatment, several parameters were tested: concentrations of NaOH, persulfate and sample; additional clean-up with graphitized non-porous carbon and reaction time. Solid phase extraction-weak anion exchange (SPE-WAX) was used for clean-up and separation of analytes, and LC-MS/MS was used for quantification. The optimal conditions with the highest levels of PFAAs detected was obtained with 1.33 M NaOH, 60 mM persulfate, 3.57 g/L sludge with a reaction time of 6 hours. The use of graphitized non-porous carbon reduced matrix effects on oxidative conversion resulting in a higher pH as well as a higher degree of oxidation, but with some analyte loss.

Per- and polyfluoroalkyl substances (PFAS) have been manufactured and used in differentapplications for several decades, including food packaging materials. During the last 20 yearsthese compounds have been acknowledged as hazardous for humans and the environment, anddifferent regulations on PFAS have been established on both national and international levels.Companies started to phase-out long-chain PFAS, including both PFOA and PFOS, around 20years ago. Since PFAS are persistent, this cause concerns both for our health and theenvironment, as well as possible PFAS contamination in new products due to the recycling ofmaterials. The aim of this study was to find an effective method to extract PFAS from differentfood packaging materials; analyze the samples for their extractable organofluorine (EOF)content using combustion ion chromatography; as well as analyze targeted PFAS in the samplesusing ultra-high performance liquid chromatography tandem mass spectrometry. The findingsof this study suggest that none of the selected samples had EOF contents above the Danishindicator value of 20 mg/kg dw TOF set to determine whether PFAS has been intentionallyadded to a material, and that only two samples exceeded the limit of detection for EOF. Atakeaway bowl made out of 100% sugarcane contained the highest EOF content while the outerpackaging of a cereal box contained the second highest EOF. Both PFOA and PFOS, alongwith other long-chain PFAS were detected in a majority of the samples. The lowest total PFASconcentrations when analyzing for targeted PFAS was detected in the sugarcane take awaybowl. The highest total PFAS concentration was detected in an egg carton, followed by theouter packaging of a cereal box (same as above) and the outer box of a waffle mix. The targetedPFAS was detected in almost all samples, with PFNA and 6:2 diPAP being the most frequentlydetected PFAS. PFCAs, PFSAs, FTSAs, FOSAAs and PAPs were detected in a majority of thesamples. The highest concentrations were measured for diSAmPAP in a majority of thesamples. Mass balance calculations of the sugarcane bowl showed that the targeted PFAS onlyaccounted for 0.04% of the extractable organofluorine content. In conclusion, none of thesamples displayed EOF contents higher than the Danish indicator value, suggesting that noneof the samples were intentionally treated with PFAS. However, targeted PFAS analysis of thesesamples showed that they still contain PFAS, that could be further recycled along with therecycling of paper and board food packaging materials. Considering the persistence of PFASand that these compounds can remain in the recycling chain, with the risk of also being releasedinto the environment, it is of importance that PFAS is not introduced in any of the stages in apaper or board containers life cycle.

This thesis is divided into three main results chapters that reflect the path my research took. In the first results chapter, the first organocatalyst for the carbonyl-ene reaction was discovered and found to give high conversion using 1,3-bis(3,5-bis(trifluoromethyl)phenyl)thiourea. Various carbonyl and alkene precursors were examined in the ene reaction in both catalysed and uncatalysed reactions. It was found that ene reactions using fluoral and ethyl trifluoropyruvate give higher rates of reaction when compared to other carbonyl compounds. A novel enantiopure thiourea was synthesised and the ene reaction was catalysed enantioselectively to 33% e.e. In an attempt to catalyse the reaction to a further extent a new thiourea bonded to a P(=S)R2 group was developed. However, the intramolecular hydrogen bonding of this catalyst was thought to be so strong that this it did not catalyse the reaction. The synthesis of a chiral phosphoric acid was achieved but this was an unsuccessful catalyst in the ene reaction. Two component achiral thiourea and chiral acids were also examined in the ene and Mannich-type reaction. The new easily synthesised thiourea for this reaction has an interesting intermolecular hydrogen bonding coordination in the solid state. Asymmetric fluorination of ketoesters using palladium is a dynamic kinetic resolution. In the 2nd chapter cationic palladium complexes were synthesised and used to determine the optimum parameters for bidentate ligands in this reaction. Four carbon chain phosphines were found to give the highest conversion for this reaction among those ligands tested such as 1,4-bisdiphenylphosphinobutane (bite angle 99º). A new bis-phosphinous amide chiral ligand was developed with a bite angle of 96.7º. The dichloropalladium complex of this phosphine was isolated and structurally characterised. The use of the palladium complex in asymmetric fluorination was attempted however this was found to be unsuccessful. Mechanistic studies reveal that the formation of the desired cationic catalyst did not occur under conditions shown to work well for other palladium phosphine complexes. The ligand was investigated further in hydrogenation reactions. The phosphinous amide was protected as its borane and was used in the rhodium catalysed hydrogenation of alkenes to give high conversion and up to 93% e.e. The borane protected phosphinous amide was also found to catalyse the hydrogenation of acetophenone using copper complexes with up to 84% e.e for the hydrogenation of acetophenone, although conversion was quite low.

This thesis is focussed on the biosynthesis of three toxins: fluoroacetate, 4-fluoro-L-theronine and β-ethynyl-L-serine which are biosynthesised by the soil bacteria Streptomyces cattleya. The two fluorinated metabolites originate from a common biosynthetic pathway and the thesis describes studies carried out on an aldose-ketose isomerase enzyme of the pathway. The biosynthetic origin of β-ethynyl-L-serine is not known. A total synthesis of this acetylenic amino acid is descibed along with the development of a new analytical method for identifying the metabolite and for future isotope-labelling based biosynthetic studies. Chapter 1 presents the background of this research. It is focussed on the biosynthesis of fluoroacetate and 4-fluoro-L-threonine by S. cattleya and it also introduces alkyne-containing natural products and their biosynthesis. Chapter 2 describes the work carried out on crystallisation of the aldose-ketose isomerase of the fluorometabolite pathway in S. cattleya. Crystals of the isomerase were obtained and they were diffracted by X-ray, however a structure could not be solved. Chapter 3 contains site-directed mutagenesis studies of the isomerase from S. cattleya. Chapter 4 describes an enantioselective total synthesis of β-ethynyl-L-serine. A robust analytical technique based on derivatisation using 'Click' chemistry and LC-MS was developed for the detection of this amino acid directly from the fermentation broth. Chapter 5 details the experimental procedures for compounds synthesised in this thesis and the biological procedures for gene cloning and protein purification.

Made available in DSpace on 2014-06-11T19:31:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-05-16Bitstream added on 2014-06-13T20:22:04Z : No. of bitstreams: 1 zuanaze_rcc_dr_botfmvz.pdf: 2365698 bytes, checksum: 5ed70ccdd25955ad8f5b2e015a21dec7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / O monofluoroacetato de sodio (MFAS) ou composto 1080 e um rodenticida que foi amplamente utilizado para o controle de roedores e predadores domesticos, apos sua descoberta em 1945. Este potente rodenticida age bloqueando o ciclo de Krebs por acao do seu metabolito toxico, o fluorocitrato, e como consequencia, inibe a resposta celular e a producao de ATP. O MFAS foi proibido por lei no Brasil e em diversos paises, mas seu uso indiscriminado continua causando diversos casos de intoxicacoes potencialmente fatais no homem e animais domesticos, principalmente em caes e gatos, representadas por alteracoes neurologicas e cardiacas. Pretendeu-se com este estudo caracterizar o perfil hematologico e bioquimico, as alteracoes histopatologicas e toxicologicas de gatos intoxicados experimentalmente com MFAS, com o objetivo de determinar metodos diagnosticos eficazes. Para tanto, foram utilizadas amostras de sangue de 16 gatos intoxicados experimentalmente com 0,45mg/kg de MFAS, por via oral. Estas amostras foram colhidas por puncao jugular e analisadas quanto ao perfil hematologico, bioquimica serica e analises toxicologicas em cromatografia liquida de alta eficiencia (CLAE), para deteccao e quantificacao do MFAS no soro dos animais. Avaliaram-se tambem as lesoes macro e microscopicas dos animais intoxicados que vieram a obito. Observaram-se leucopenia e trombocitopenia transitorias; hiperglicemia, aumento das enzimas musculares creatinoquinase (CK) e creatinoquinase fracao cardiaca (CK-MB); observaram-se tambem hipocalemia, hipofosfatemia e hipomagnesemia. Os achados macroscopicos e histopatologicos demonstraram lesoes caracteristicas de processos isquemicos e as analises toxicologicas demonstraram um metodo diagnostico simples e eficiente... / Sodium monofluoroacetate (SMFAC) or 1080 compound is a potent rodenticide, largely used since 1945 for rodent and domestic pest control. The toxic effects of SMFAC are caused by fluorocitrato, a toxic metabolite, which has a competitive action with aconitase enzyme, leading to citrate accumulation and interferes in energy production by Krebs cycle blockade. In Brazil, although prohibited by law, there is illegal use, keep causing intoxication in children and domestic animals, specially dogs and cats. The most common intoxication clinical signs are from the cardiac and neurological alterations. In the present study, 16 domestic cats were intoxicated with oral doses of monofluoroacetate (0.45mg/kg). The hematologic and biochemical profiles, and histophatological and blood serum toxicological analysis by high performance liquid chromatography (HPLC) were made to looking for a efficient diagnosis methods. The hematologic profile showed transitory leucopenia and trombocitopenia; the biochemical profile presented hiperglycemia, increase of creatinoquinase enzyme (CK) and creatinoquinase cardiac fraction (CK-MB), hypokalemia and hypophosfatemia were observed. The macrocospic and histopathological findings showed lesions characteristic of degenerative and ischemic processes. The toxicological analysis was shown to be a simple and efficient diagnostic method. SMFAC was detected in 75% of the serum samples analysed, and it was verified an average concentration of 0.32 ìg/mL in them; 4.81% of the serum samples did not show metabolized SMFAC six hours after the induced intoxication of the animals used in this study.

Orientador: Michiko Sakate / Banca: Noeme Sousa Rocha / Banca: Manoel Lima de Menezes / Banca: Mara Regina Stipp Balarin / Banca: Silvana Lima Gorniak / Resumo: O monofluoroacetato de sodio (MFAS) ou composto 1080 e um rodenticida que foi amplamente utilizado para o controle de roedores e predadores domesticos, apos sua descoberta em 1945. Este potente rodenticida age bloqueando o ciclo de Krebs por acao do seu metabolito toxico, o fluorocitrato, e como consequencia, inibe a resposta celular e a producao de ATP. O MFAS foi proibido por lei no Brasil e em diversos paises, mas seu uso indiscriminado continua causando diversos casos de intoxicacoes potencialmente fatais no homem e animais domesticos, principalmente em caes e gatos, representadas por alteracoes neurologicas e cardiacas. Pretendeu-se com este estudo caracterizar o perfil hematologico e bioquimico, as alteracoes histopatologicas e toxicologicas de gatos intoxicados experimentalmente com MFAS, com o objetivo de determinar metodos diagnosticos eficazes. Para tanto, foram utilizadas amostras de sangue de 16 gatos intoxicados experimentalmente com 0,45mg/kg de MFAS, por via oral. Estas amostras foram colhidas por puncao jugular e analisadas quanto ao perfil hematologico, bioquimica serica e analises toxicologicas em cromatografia liquida de alta eficiencia (CLAE), para deteccao e quantificacao do MFAS no soro dos animais. Avaliaram-se tambem as lesoes macro e microscopicas dos animais intoxicados que vieram a obito. Observaram-se leucopenia e trombocitopenia transitorias; hiperglicemia, aumento das enzimas musculares creatinoquinase (CK) e creatinoquinase fracao cardiaca (CK-MB); observaram-se tambem hipocalemia, hipofosfatemia e hipomagnesemia. Os achados macroscopicos e histopatologicos demonstraram lesoes caracteristicas de processos isquemicos e as analises toxicologicas demonstraram um metodo diagnostico simples e eficiente...(Resumo completo, clicar acesso eletrõnico abaixo) / Abstract: Sodium monofluoroacetate (SMFAC) or 1080 compound is a potent rodenticide, largely used since 1945 for rodent and domestic pest control. The toxic effects of SMFAC are caused by fluorocitrato, a toxic metabolite, which has a competitive action with aconitase enzyme, leading to citrate accumulation and interferes in energy production by Krebs cycle blockade. In Brazil, although prohibited by law, there is illegal use, keep causing intoxication in children and domestic animals, specially dogs and cats. The most common intoxication clinical signs are from the cardiac and neurological alterations. In the present study, 16 domestic cats were intoxicated with oral doses of monofluoroacetate (0.45mg/kg). The hematologic and biochemical profiles, and histophatological and blood serum toxicological analysis by high performance liquid chromatography (HPLC) were made to looking for a efficient diagnosis methods. The hematologic profile showed transitory leucopenia and trombocitopenia; the biochemical profile presented hiperglycemia, increase of creatinoquinase enzyme (CK) and creatinoquinase cardiac fraction (CK-MB), hypokalemia and hypophosfatemia were observed. The macrocospic and histopathological findings showed lesions characteristic of degenerative and ischemic processes. The toxicological analysis was shown to be a simple and efficient diagnostic method. SMFAC was detected in 75% of the serum samples analysed, and it was verified an average concentration of 0.32 ìg/mL in them; 4.81% of the serum samples did not show metabolized SMFAC six hours after the induced intoxication of the animals used in this study. / Doutor