Introdução: O estudo da ação biológica de compostos bioativos e de nutrientes, a fim de que se possa explicar a relação entre o consumo de alimentos e a redução do risco de doenças, é uma das áreas que tem aplicação direta com a saúde pública. A erva mate (Ilex paraguariensis) é uma planta rica em compostos fenólicos (ácidos clorogênicos), extensivamente metabolizados após a ingestão. O conhecimento detalhado sobre os compostos formados pela metabolização dos mesmos, concentrações e tecidos-alvo é fundamental para o completo esclarecimento sobre os mecanismos de ação envolvidos. Objetivo: Avaliar a biotransformação dos ácidos fenólicos do chá mate in vivo em ratos Wistar. Métodos: Os animais foram eutanasiados 90 min (ensaio piloto) ou 30, 60, 120, 240 e 480 minutos (ensaio principal) após a administração de chá mate ou padrão de ácido 5-cafeoilquínico (5CQA) por gavagem. O grupo Controle recebeu solução salina. No ensaio piloto foram analisados plasma, fígado, rins, músculo, estômago e intestino delgado para identificação dos compostos fenólicos e com base nos resultados definida a dose de 2g de chá mate solúvel/kg de peso do animal para ser usada no ensaio principal, que corresponde a 240 mg de fenólicos totais/kg peso, dose administrada ao grupo Padrão na forma de 5-CQA. Quantificação dos compostos fenólicos foi realizada no plasma, fígado, estômago, intestino grosso e urina dos animais do ensaio principal. As análises foram realizadas por UPLC/DAD-MS, após desenvolvimento e validação das metodologias para extração e análise dos ácidos fenólicos nas amostras. O chá mate foi avaliado quanto ao perfil e teor de compostos fenólicos por UPLC/DADMS. Resultados: As metodologias desenvolvidas para extração e análise dos ácidos fenólicos nas amostras biológicas apresentaram bons níveis de recuperação e precisão. Os limites de detecção e quantificação foram determinados para cada fluido/tecido. No ensaio piloto, foram detectados ácidos clorogênicos intactos em todas as amostras, assim como uma série de metabólitos de fase I e II. No ensaio principal, os ácidos clorogênicos livres foram os principais ácidos fenólicos presentes no estômago e intestino grosso, enquanto no plasma, fígado e urina os compostos mais abundantes eram os metabólitos, em ambos os grupos, em especial o ácido caféico ligado ao ácido glicurônico/grupos sulfato e o ácido 3hidroxifenilpropiônico (livre) no grupo Erva Mate e os ácidos feruloilquínicos (FQAas) e ácido 3-hidrofenilpropiônico no grupo Padrão. Demonstrou-se que a absorção e metabolização dos ácidos clorogênicos começa no estômago, mas a maior parte é absorvida no intestino grosso, especialmente após metabolização por bactérias. Cerca de 4,0 por cento dos compostos ingeridos pelo grupo Erva Mate e 3,3 por cento pelo grupo Padrão (mol/mol) estavam presentes na urina na forma de ácidos clorogênicos e dos metabólitos avaliados, 8 hs após a gavagem. Conclusão: A absorção e metabolização dos ácidos clorogênicos começa no estômago. Houve diferenças no tipo e quantidade dos diferentes compostos formados a partir dos fenólicos do chá mate e do 5-CQA puro, demonstrando que o perfil de ácidos clorogênicos presentes no alimento influencia qualitativamente e quantitativamente os metabólitos formados. Maior ênfase deve ser dada aos metabólitos em estudos que avaliem as propriedades biológicas e mecanismos de ação dos compostos fenólicos da erva mate e outros alimentos fonte / Introduction: Evaluation of biological properties of bioactive compounds and nutrients, aiming to explain the relationship between food consumption and decreased risk of diseases, is a field of study directly related to public health. Yerba maté (Ilex paraguariensis) is a plant rich in phenolic compounds (chlorogenic acids) which are extensively metabolized after ingestion. Detailed knowledge about the metabolites, its concentrations and target tissues is fundamental to clarify the action mechanisms involved in disease prevention. Objective: Evaluating the biotransformation of Yerba maté phenolic acids in vivo in Wistar rats. Methods: Animals were euthanized 90 min (pilot study) or 30, 60, 120, 240 and 480 (main study) after administration of maté tea or 5-caffeoylquinic acid (standard) by gavage. Control group received saline solution. In the pilot study plasma, liver, kidneys, muscle, stomach and small intestine were analyzed for identification of phenolic compounds and the dose of 2 g maté tea/kg body weight was defined for the main study, which corresponds to 240 mg of total phenolic compounds/kg bw, dose administered to the Standard group as 5-CQA. Quantification was performed in plasma, liver, stomach, large intestine and urine in the main study. Analyses were performed using UPLC/DAD-MS, after development and validation of methodologies for extraction of phenolic acids from fluids and tissues. Maté tea phenolic compounds amount and profile were evaluated by UPLC/DAD-MS. Results: Developed methodologies showed good levels of recovery and precision. Limits of quantification (LQ) and detection (LD) were calculated for each biological matrix. In the pilot study, chlorogenic acids and their phase I and II metabolites were detected in all biological matrices. In the main study, the main compounds in gastric large and intestinal tissues were intact chologenic acids, whereas in plasma, liver and urine their metabolites were present in larger quantities, specially caffeic acid, bound to glucuronic acid and/or sulfate groups, and 3-hydroxyphenylpropionic acid in the free form on Yerba Mate group, and 3-hydroxyphenylpropionic acid and feruloylquinic acid on the group that received 5-CQA. It was demonstrated that chlorogenic acids absorption and metabolism begins in stomach, but most of the absorption takes place in the large intestine, especially after microbial metabolization. Approximately 4,0 per cent of compounds ingested by Yerba Mate group and 3,3 per cent by Standard group (mol/mol) were recovered in urine collected up to 8 hs after the gavage, in the form of chlorogenic acids and the evaluated metabolites. Conclusion: The absorption and metabolization of chlorogenic acids begins in the stomach. There were differences in the amount and type of compounds formed from maté tea or pure 5-CQA, showing that the profile of chlorogenic acids on food products may influences qualitatively and quantitatively the metabolites formed on the body. Greater emphasis should be given to metabolites in studies that assess biological properties and mechanisms of action of phenolic compounds from yerba mate and other food source

This dissertation describes efforts to improve the oxidative capacity of n-alkane- induced Beauveria bassiana; a fungus and a versatile whole cell biocatalyst used in the biotransformation of steroids. n-Hexadecane was used as the carbon source during the growth of B. bassiana, presumably to induce the expression of oxidative enzymes, thus enhancing the oxidation of unactivated carbons. Dehydroepiandrosterone (DHEA) is an essential endogenous male-hormone and serves as a metabolic intermediate in the production of more potent androgens. Using DHEA as a substrate also provides the opportunity to study the hydroxylation of an unfunctionalized carbon, an attractive reaction that produces valuable intermediates for chemical synthesis. Results showed that exposing and inducing cells in n-hexadecane improves the synthesis of 11α-hydroxy derivatives. Reactions were carried out with cells grown on n-hexadecane, resulting in 65 ± 6.3 % conversion of DHEA to androstenediol (40.3% mM) and 3β,11∝,17β- trihydroxyandrost-5-ene (22.8% mM), as determined by HPLC, NMR and LCMS analyses. However, experiments with non-induced cells resulted in a poor substrate conversion (17%). To extend use of B. bassiana to pharmaceutical applications, it was necessary to optimize reaction conditions such as biocatalyst preparation, substrate concentration, agitation reaction temperature and pH. Higher substrate conversion, selectivity and yield of desired product were achieved with the reactor arrangement of “Resting Cells”. The apparent rate of reaction fits a Michaelis-Menten kinetic model with a maximum reaction rate of 4.45 mM/day, revealing that the transformation of intermediate androstenediol to desired 3β,11∝,17β-trihydroxyandrost-5-ene is the limiting step in the reaction. Interestingly, when a diluted amount of substrate was used, a higher yield of 11∞-hydroxy steroid was achieved. Also, reactions at 26°C with pH ranges between 6.0 and 7.0, resulted in the highest conversion (70%) and the higher product yield (45.8%). The maximum conversion of DHEA (71%) was achieved in experiments with high biomass loading, and the increment of desired product yield (11∝-hydroxy) was directly proportional to the amount of biomass used. Moreover, a high VMax/KM value was achieved with high biomass yields. Interestingly, the changes in biomass yield did not have a considerable effect on reaction selectivity. The main drawbacks of biocatalysis for production of steroids were addressed and approaches to minimize the drawbacks have been presented. The production of desired product (11∝-DHEA) was significantly improved using cells previously adapted to n-hexadecane.

No description available.

Der Fettsäurestoffwechsel unterliegt vielfältigen Kontrollmechanismen. So wird der Fettsäureabbau über die Induktion und Aktivität spezifischer Enzyme reguliert. Ein zentraler Regulator ist dabei der nukleäre Rezeptor Peroxisomenproliferator-aktivierter-Rezeptor-α (PPARα). PPARα wird durch freie Fettsäuren in der Zelle aktiviert und fördert über die Induktion von Zielgenen den Fettsäuretransport und -abbau sowie die Gluconeogenese und Ketogenese. Der Anstieg an freien Fettsäuren beim Fasten, aber auch im Diabetes aktiviert PPARα. Unabhängig davon wurde in beiden Stoffwechsellagen auch eine erhöhte Expression des nukleären Rezeptors Constitutiver-Androstan-Rezeptor (CAR) und einiger CAR-Zielgene, vorrangig Enzyme des Fremdstoffmetabolismus wie Cytochrom P450 2B (CYP2B), festgestellt. Bei der Adaption an eine Fastensituation scheinen PPARα- und CAR-Signalwege über einen bisher unbekannten Mechanismus miteinander verschaltet zu sein. In der vorliegenden Arbeit sollte der der Verschaltung zugrunde liegende Mechanismus anhand eines Modelsystems, der PPARα-Agonisten-vermittelten Verstärkung der Phenobarbital (PB)-abhängigen Induktion des CAR-Zielgens CYP2B, in vitro untersucht werden. Zudem sollte die physiologische Relevanz einer durch PPARα-Agonisten vermittelten Modulierung der CYP2B-Aktivität in einer Ganztierstudie in vivo belegt werden. Die verwendeten synthetischen PPARα-Agonisten steigerten in primären Hepatozyten der Ratte signifikant die Phenobarbital (PB)-abhängige mRNA- und Protein-Expression sowie die Aktivität von CYP2B. Ohne vorherige PB-Behandlung induzierten PPARα-Agonisten CYP2B nicht. In Gegenwart von PB war die Steigerung der CYP2B-Aktivität durch PPARα-Agonisten dosisabhängig. In einem Luciferase-Reportergenassay wurde gezeigt, dass die Induktion durch PB unter der Kontrolle des CYP2B1-Promotors von einem distalen PBREM (PB-responsive-enhancer-module), an welches CAR binden kann, abhängig war. PPARα-Agonisten steigerten diese PB- und PBREM-abhängige Reportergentranskription und induzierten die CAR-mRNA und CAR-Proteinexpression. Sie aktivierten die Transkription eines Reportergens unter der Kontrolle eines Promotorfragments von bis zu 4,4 kb oberhalb des mutmaßlichen CAR-Transkriptionsstarts. Mit Hilfe von Deletionskonstrukten konnte ein potentielles Peroxisomenproliferator-aktivierter-Rezeptor-responsives Element (PPRE) im CAR-Promotorbereich von -942 bp bis -930 bp identifiziert werden, welches essentiell für die Initiation der Transkription durch PPARα-Agonisten ist. In band shift Experimenten akkumulierte verstärkt Kernprotein mit diesem PPRE. Ein Überschuss an unmarkiertem Wildtyp-CAR-Reportergenvektor, nicht aber an CAR-Reportergenvektor mit PPRE-Deletion, konnte mit dem markierten PPRE um die Bindung von Kernprotein konkurrieren. Nach Chromatin-Immunpräzipitation mit einem PPARα-Antikörper wiederum wurde das betreffende PPRE amplifiziert. Bei in vivo Experimenten an männlichen Ratten resultierte die Behandlung mit PPARα-Agonisten in einer signifikanten Induktion der CAR-mRNA-Expression und signifikant erhöhter PB-abhängiger CYP2B-Aktivität. Die physiologisch Relevanz wurde durch weiterführenden Experimente unterstrichen, in denen gezeigt wurde, dass die Fasten-abhängige Induktion von CAR in PPARα-defizienten Mäusen unterdrückt war. Diese Experimente legen nahe, dass durch PPARα-Agonisten aktiviertes PPARα an das PPRE im CAR-Promotorbereich von -942 bp bis -930 bp bindet und dadurch die CAR-Transkription induziert. Somit kann CAR als PPARα-Zielgen betrachtet werden, was die Schlussfolgerung zulässt, dass die PPARα- und CAR-Signalwege über die direkte Bindung von PPARα an den CAR-Promotor unmittelbar miteinander verknüpft sind. Allerdings ist davon unabhängig eine Aktivierung von CAR, etwa durch PB, für die vermehrte Induktion von CAR-Zielgenen notwendig . Die physiologische Relevanz der PPARα-abhängige CAR-Expression zeigt sich in den Ganztierexperimenten, bei denen die Wirksamkeit der PPARα-Agonisten bestätigt werden konnte. CAR-abhängig induzierte Enzyme sind nicht nur in großem Umfang am Fremdstoffmetabolismus beteiligt, sondern auch am Abbau von Schilddrüsenhormonen und Glucocorticoiden. Sie können damit direkt Einfluss auf den Kohlenhydrat- und Energiestoffwechsel sowie die Regulation der Nahrungsaufnahme nehmen. Über eine PPARα-abhängige Induktion von CAR im Rahmen der Fastenadaption könnten die CAR-Zielgene UDP-Glucuronyltransferase 1A1 und Sulfotransferase N beispielsweise verstärkt Schilddrüsenhormone abbauen und in der Folge den Grundumsatz senken. Der in dieser Arbeit erstmals beschriebene Mechanismus ist dafür von zentraler Bedeutung. / Fatty acid metabolism is tightly regulated. Thus the activity and expression level of specific enzymes involved in fatty acid turnover are controlling fatty acid catabolism. The nuclear receptor peroxisome proliferator activated receptor α (PPARα) acts as the key regulator of these pathways. PPARα is activated by intracellular free fatty acids and promotes the fatty acid transport and break down, as well as gluconeogenesis and ketogenesis, via induction of target genes. An increase in free fatty acids as seen in fasting and diabetes activates PPARα. Under these conditions, an elevated expression of another nuclear receptor, the constitutive androstane receptor (CAR) and its target genes, mainly enzymes catalysing biotransformation such as cytochrome P450 2B (CYP2B1), was also observed. It is therefore likely that as yet unidentified modes of interaction between PPARα and CAR signalling exist. The object of the present work was to discover these underlying mechanisms utilising an in vitro model, the PPARα-agonist induced increase of the phenobarbital (PB)-dependent induction of the CAR target gene CYP2B1. Furthermore, an in vivo study would serve to demonstrate the physiological relevance of a PPARα-agonist induced modulation of the CYP2B activity. The synthetic PPARα agonists under investigation significantly enhanced the PB-dependent mRNA and protein expression as well as activity of CYP2B in primary rat hepatocytes. Without prior treatment with PB, PPARα agonists did not induce CYP2B activity. In the presence of PB, PPARα agonists increased the CYP2B activity dose-dependently. Luciferase reporter gene assays showed that the PB-dependent induction of the CY2B1 promoter relied on a distal PBREM (PB-responsive enhancer module), a well-known CAR binding site. PPARα agonists enhanced this PB- and PBREM-dependent reporter gene transcription and induced the upregulation of CAR mRNA and CAR protein expression. The PPARα agonists also activated the transcription of a reporter gene controlled by up to 4.4 kb upstream of the putative CAR-transcription start site. A potential peroxisome proliferator activated receptor responsive element (PPRE), essential for the initiation of transcription by PPARα agonists, could be identified between -942 bp to -930 bp upstream of the transcription start site using CAR promoter deletion constructs. In subsequent band shift experiments, enhanced nuclear protein accumulation with this specific promoter region was observed. In contrast to unlabelled wild-type CAR reporter gene vector, an excess of unlabelled CAR reporter gene vector with PPRE deletion did not compete with the binding of nuclear protein. Furthermore, this PPRE could be amplified with specific primers after chromatin immunoprecipitation with a PPARα antibody. Treatment of rats with a PPARα agonist resulted in a significant induction of CAR mRNA expression and significantly increased PB-dependent CYP2B activity. A physiological relevance of this newly-discovered mechanism is confirmed by the observation that PPARα-deficient mice, unlike wild-type mice, do not respond to fasting with an increase of CAR mRNA expression. The results of these experiments suggest that activated PPARα binds to the PPRE of the CAR promoter to initiate transcription of the CAR gene. CAR therefore could be regarded as a PPARα target gene, which implicates that PPARα- and CAR-signalling are directly linked through binding of PPARα to the CAR promoter. For subsequent enhanced induction of CAR target genes, activation of CAR, for instance using PB, is required. In vivo studies with PPARα agonists in rats support the relevance of the PPARα-dependent CAR expression. CAR target genes code for enzymes that metabolise not only a wide range of xenobiotics, but also thyroid hormones and glucocorticoids. CAR target genes could therefore directly interfere with carbohydrate and energy metabolism, as well as with food intake. PPARα-dependent induction of CAR upon fasting could lead to an increased expression of the CAR target genes UDP-glucuronyl transferase 1A1 and sulfotransferase N, resulting in an enhanced degradation of thyroid hormones, and decreased resting energy expenditure. The findings of this present study are of primary importance since it is the first time that this mechanism has been described.

Enzymes are attractive catalysts in organic synthesis since they are efficient, selective and environmentally friendly. A large number of enzyme-catalyzed transformations have been described in the literature. If no natural enzyme can carry out a desirable reaction, one possibility is to modify an existing enzyme by protein engineering and thereby obtain a catalyst with the desired properties. In this thesis, the development of enantioselective enzymes and their use in synthetic applications is described.  In the first part of this thesis, enantioselective variants of Candida antarctica lipase A (CALA) towards α-substituted p-nitrophenyl esters were developed by directed evolution. A highly selective variant of CALA towards p-nitrophenyl 2-phenylpropanoate was developed by pairwise randomization of amino acid residues close to the active site. The E value of this variant was 276 compared to 3 for the wild type. An approach where nine residues were altered simultaneously was used to discover another highly enantioselective CALA variant (E = 100) towards an ibuprofen ester. The sterical demands of this substrate made it necessary to vary several residues at the same time in order to reach a variant with improved properties. In the second part of the thesis, a designed variant of Candida antarctica lipase B (CALB) was employed in kinetic resolution (KR) and dynamic kinetic resolution (DKR) of secondary alcohols. The designed CALB variant (W104A) accepts larger substrates compared to the wild type, and by the application of CALB W104A, the scope of these resolutions was extended. First, a DKR of phenylalkanols was developed using CALB W104A. An enzymatic resolution was combined with in situ racemization of the substrate, to yield the products in up to 97% ee. Secondly, the KR of diarylmethanols with CALB W104A was developed. By the use of diarylmethanols with two different aryl groups, highly enantioselective transformations were achieved. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows:<strong>  </strong>Paper 5: Submitted.

Naphthenic acids (NAs) are carboxylic acids found in crude oil and petroleum products. The objectives of the research presented here were to: a) assess the occurrence and fate of NAs in crude oil and refinery wastewater streams; b) evaluate the biotransformation potential and inhibitory effects of NAs under nitrifying, denitrifying and methanogenic/fermentative conditions; c) investigate the factors affecting NA biotransformation under aerobic conditions and the microbes involved; and d) assess the toxicity of individual model NAs using quantitative structure-activity relationships (QSAR) and examine the effect of structure on NA biotransformation potential. NAs are ubiquitous in refinery wastewater streams and the desalter brine was found to be the main source of NAs in refinery wastewater. A commercial NA mixture was not biodegraded under nitrate-reducing or methanogenic/fermentative conditions. NAs were degraded under aerobic conditions by an NA-enriched culture; however, a residual fraction was not degraded under all conditions studied. The results indicated that NAs are not inherently recalcitrant and the residual fraction was due to the individual NA concentrations being below the minimum substrate concentrations at which they are no longer degraded. A fraction of the NA mixture was completely mineralized to carbon dioxide, with the remaining portion biotransformed to more oxidized intermediates. Overall, the results indicated that NAs were degraded under aerobic conditions; however, biological treatment of NA-bearing wastewater will not completely remove NA concentrations and thus, biological treatment must be combined with physical/chemical treatment to achieve complete NA removal.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

The objective of the study presented here was to develop either enzymatic or whole cell mediated green procedures for the syntheses of a-hydroxy ketones. Production of optically active synthons is crucial for the preparation of fine chemicals. Enzymes and whole-cell biocatalysts have proven to be excellent vehicles with their chiral nature for the biotransformations. Under the light of this discussion, firstly benzaldehyde lyase [BAL, (EC 4.1.2.38)] was used in novel C-C bond formation reactions to obtain interesting and biologically important precursors / 2-Hydroxy-1-arylethan-1-ones and functionalized aliphatic acyloin derivatives. All the compounds were obtained with high yields and in the case of aliphatic acyloin derivatives with high enantiomeric excesses (ee&rsquo / s). Another strategy was to use whole cell biocatalysis. A.flavus 200120 was found to be a promising biocatalyst with the ability to catalyze a broad range of reactions / reduction, hydrolysis and deracemization, while another fungus / A. oryzae 5048 was utilized in bioreduction reactions of benzil and its derivatives. Each reaction was investigated, optimized and thus enhanced via medium design. Products were obtained with high yields and ee&rsquo / s. To sum up, in this study novel efficient green procedures were developed to synthesize various ahydroxy ketones with high yield and stereoselectivity. These newly established methods present promising alternatives to classical chemical methodologies.

La structure et la dynamique de la matière organique mobile (lipides), peu soluble (acides humiques) et insoluble (humine et kérogène) ont été étudiés dans différents environnements. Les sols, les tourbes qui représentent la première phase d'accumulation du carbone organique (diagenèse précoce) et les sédiments anciens dans lesquels le carbone organique s'est transformé. L'étude de ces différents milieux pouvant permettre d'appréhender les mécanismes d'accumulation du carbone.<br />L'étude de la structure et de l'évolution de la matière organique des sols et des sédiments a nécessité la mise en œuvre de techniques d'analyse globales comme la spectroscopie infra-rouge ou la RMN 13C (CP-MAS) et plus approfondies comme la pyrolyse off line ou on line (Py-GC/MS). En fonction des informations obtenues, nous avons appliqué aux macromolécules biologiques, des réactions de dégradation plus sélectives par voie chimique ou enzymatique. Le développement ou l'adaptation de ces techniques et réactions de dégradation a donc constitué une large partie de ce travail.<br /> L'étude de la matière organique complexe des sols et des sédiments fait apparaître la présence de chaînes aliphatiques, liées au réseau macromoléculaire par des liaisons éther ou ester, ou formant des ponts dialkyles. L'importance des liaisons de faible énergie a pu être soulignée dans la structure des substances humiques ainsi que la contribution ligneuse. <br />L'accumulation du carbone dans les tourbes et les sédiments s'est déroulée en milieu anoxique (conditions réductrices), cependant il existe des témoins de phénomènes d'oxydation qui ont pu se produire au préalable ou lors d'échanges possibles avec une phase mobile.<br />L'étude des processus de biodégradation du carbone dans les sols montre que les molécules simples, après oxydation, peuvent s'incorporer dans le réseau macromoléculaire par liaisons ester ou éther. Nous avons ainsi montré que l'apport de carbone organique sur des sols cultivés en améliore les qualités.