Konstanz, Univ., Diss., 2007. / Ersch. auch im Verl. Hartung-Gorre, Konstanz. ISBN 3-86628-176-5.

Le but de ce travail de thèse était de rechercher de nouvelles molécules inhibitrices de la SOD dans des extraits de substances naturelles. Cette enzyme majeure du stress oxydant étant impliquée dans de nombreux mécanismes de défense des cellules cancéreuses contre l'apoptose représente une voie thérapeutique d'avenir. Nous en avons recherché dans les champignons endophytes de plantes péruviennes, ces micro-organismes produisant de nombreux métabolites de défense des plantes hôtes. Ce travail de thèse a d'abord porté sur l'isolement, la culture, l'identification, l'extraction et la caractérisation des souches de champignons endophytes. Les analyses statistiques effectuées sur ces extraits avec les résultats obtenus en HPLC ont confirmé les problèmes de variabilités qualitative et quantitative pouvant être rencontrés au cours de la culture des endophytes et décrites dans la littérature. La seconde partie expérimentale a porté sur la recherche de furocoumarines dans les extraits obtenus, en raison de leur potentiel effet inhibiteur sur la SOD. Nous avons dérépliqué ces composés dans les extraits d'endophytes au cours de deux stratégies de spectrométrie de masse réalisées en mode d'ionisation négatif. La première approche, à l'aide d'un QTOF, a abouti à l'identification de deux furocoumarines, la 5-Methyl-4H-furo[2,3-b][1]benzopyran-4-one et la déhydropachyrrhizone. La seconde, à l'aide d'un OrbiTrap, a vu l'identification de quatre autres furocoumarines : l'Ochrocarpine A, la Moellendorffiline, l'Anisolactone et l'Anhydrorutarétine .Afin de compléter cette approche métabolomique et identifier les molécules inhibitrices de la SOD, nous avons tenté de mettre au point un test d'activité de cette enzyme, rapide, peu coûteux et réalisable en routine. Nous avons sélectionné le test au pyrogallol, mais celui-ci n'a pas démontré les qualités recherchées tant au niveau de la sensibilité que de la reproductibilité. Nous orientons à présent les recherches vers des approches différentes, la recherche directe d'adduits sur la SOD par LC-MS. / Superoxide dismutase is one of the major proteins controlling the oxidizing stress and cellular homeostasis. It is involved in numerous cancer cells proliferation processes. This protein is considered as major anti-cancer target for the development of new anti-cancer drugs. The goal of this work, was to research and identify an inhibitor of the SOD in endophytic fungi from Peruvian plants. These micro-organisms are known to produce numerous metabolites for host plants protection. During the preparation of endophytic extracts and their characterization by HPLC and statistical analyzes, we have pointed out a high quantitative and qualitative variability of the chemical content of endophytic extracts inside a same strain. Considering the literature we have focused our work on the identification of furocoumarins because of their potential inhibitory effect on the SOD. For this purpose two mass spectrometry strategies using negative ionization mode were carried out. With the QTOF mass spectrometer we have identified Methyl-4H-furo [2,3-b] [1] benzopyran-4-one and the déhydropachyrrhizone. With the OrbiTrap, the Ochrocarpine A, Moellendorffiline the Anisolactone and the Anhydrorutarétine were identified. Biological evaluation of the different extracts was performed using pyrogallol test. This investigation did not allow us to identify an inhibitor of the SOD. In the future we may consider seeking SOD inhibitors by looking at the formation SOD-chemical compound adducts using an LC-MS investigation.

Neste trabalho, foram fabricados biossensores amperométricos a partir do uso da polifenol oxidase (PFO), obtida do abacate como fonte enzimática, imobilizada em filmes de polipirrol (PPI) e que foram eletrossintetizados em meio de três diferentes eletrólitos de suporte (NaCl e NaClO4 e NaDDS). O método de imobilização enzimática foi o da adsorção, sendo a solução de enzima adicionada à solução com o pirrol e o eletrólito durante o processo de eletropolimerização. Os filmes de PPI/PFO foram caracterizados por técnicas eletroquímicas, principalmente por voltametria cíclica. A detecção de compostos fenólicos (catecol e pirogalol) foi realizada pela técnica de cronoamperometria após se variar a concentração do analito. A morfologia dos filmes foi estudada por microscopia de força atômica (AFM), sendo observado que a presença da enzima no filme polimérico assim como o uso de diferentes eletrólitos de suporte levou a diferenças na superfície dos filmes. Além disto, verificou-se que o biossensor construído a partir do uso do NaCl, apresentava uma resposta mais eficiente, ou seja, ele foi capaz de detectar catecol e pirogalol em um menor limite de detecção. / In this study amperometric biosensors were manufactured from the use of polyphenol oxidase (PPO) obtained from avocado as a source of enzyme immobilized in polypyrrole (PPY) films that were electrosynthesized with three different support electrolytes (NaCl, NaClO4 and NaDDS). The method of enzyme immobilization was the adsorption. The PPO was added in the solution containing pyrrole and electrolyte during electropolymerization. The PPY/PPO films were characterized by electrochemical techniques mainly by cyclic voltammetry. Detection of phenolic compounds (catechol and pyrogallol) was performed by the technique of chronoamperometry after varying the concentration of the analyte. The morphology of the films was studied by the atomic force microscopy (AFM) and observed that the presence of the enzyme in the polymer film and the use of different electrolytes support led to differences in the surface of films. However it was found that the biosensor constructed from the use of NaCl showed more efficient response and it was able to detect catechol and pyrogallol in a lower limit of detection.

Neste trabalho, foram fabricados biossensores amperométricos a partir do uso da polifenol oxidase (PFO), obtida do abacate como fonte enzimática, imobilizada em filmes de polipirrol (PPI) e que foram eletrossintetizados em meio de três diferentes eletrólitos de suporte (NaCl e NaClO4 e NaDDS). O método de imobilização enzimática foi o da adsorção, sendo a solução de enzima adicionada à solução com o pirrol e o eletrólito durante o processo de eletropolimerização. Os filmes de PPI/PFO foram caracterizados por técnicas eletroquímicas, principalmente por voltametria cíclica. A detecção de compostos fenólicos (catecol e pirogalol) foi realizada pela técnica de cronoamperometria após se variar a concentração do analito. A morfologia dos filmes foi estudada por microscopia de força atômica (AFM), sendo observado que a presença da enzima no filme polimérico assim como o uso de diferentes eletrólitos de suporte levou a diferenças na superfície dos filmes. Além disto, verificou-se que o biossensor construído a partir do uso do NaCl, apresentava uma resposta mais eficiente, ou seja, ele foi capaz de detectar catecol e pirogalol em um menor limite de detecção. / In this study amperometric biosensors were manufactured from the use of polyphenol oxidase (PPO) obtained from avocado as a source of enzyme immobilized in polypyrrole (PPY) films that were electrosynthesized with three different support electrolytes (NaCl, NaClO4 and NaDDS). The method of enzyme immobilization was the adsorption. The PPO was added in the solution containing pyrrole and electrolyte during electropolymerization. The PPY/PPO films were characterized by electrochemical techniques mainly by cyclic voltammetry. Detection of phenolic compounds (catechol and pyrogallol) was performed by the technique of chronoamperometry after varying the concentration of the analyte. The morphology of the films was studied by the atomic force microscopy (AFM) and observed that the presence of the enzyme in the polymer film and the use of different electrolytes support led to differences in the surface of films. However it was found that the biosensor constructed from the use of NaCl showed more efficient response and it was able to detect catechol and pyrogallol in a lower limit of detection.

Coffee contains many antioxidants including purpurogallin, which is a hydrophobic phenolic antioxidant that is difficult to measure with reported methods. A method combining solid-phase extraction and liquid chromatography–mass spectrometry was developed to detect and quantify purpurogallin in brewed beverages, including coffee. For beverage preparation, water extraction was adopted for improved correlation with moka pot brewing. Purpurogallin was detected in all commercial coffee samples, and its content in ground coffees ranged from 455-630 ng/g dry weight. Purpurogallin was only detected in two English breakfast tea samples (335-360 ng/g dry weight) and was not detected in any cocoa sample. Antioxidant activity, total phenolic content, and phenolic profile of coffees with different degrees of roasting were determined and analyzed. The developed methodology was then further improved, and coffees with different roasting degrees were analyzed for their antioxidant activity, total phenolic content, and phenolic profile. The antioxidant activity ranged from 63.9-92.0 mg Trolox equivalents per gram of coffee (dry weight), and the total phenolic content ranged from 36.0-57.7 mg gallic acid equivalents per gram of coffee (dry weight). However, the total phenolic content was not correlated with the roasting degree (p > 0.05). When the roasting degree increases, chlorogenic acid decreases drastically, but shikimic acid, caffeic acid, gallic acid, pyrogallol, and purpurogallin increase correspondingly. The results suggest that purpurogallin is a common antioxidant in roasted coffees, and an increase in roasting degree will not only lead to dramatic breakdown of chlorogenic acid, but also promote significant formation of other phenolic compounds that can provide antioxidant activity.

Rhenium complexes coordinated with suitable organic ligand gain increased interest in radiopharmaceutical medicine. Besides suitable radiological properties given by a metal ion itself, detailed knowledge of overall chemical properties of formed complexes, namely their exact structures, chemical stabilities and possible degradation pathways are essential pre-requisites for their clinical application. Selected rhenium complexes with pyrogallol 2,3-dihydroxynaphthalene and catechol as strongly bound ligands were prepared by a reaction of tetrabutylammonium tetrachlorooxorhenate with twofold molar excess of ligand in presence of various amount of triethylamine. The structures of formed complexes and their consequent reaction products were estimated by means of mass spectrometry with electrospray ionization. The kinetics of reactions in course of complex formation and consequent decomposition were primarily followed by UV- Vis absorption spectra measurement, complemented by single or continuous electrospray mass spectrometry analyses. Optimized HPLC and CZE procedures were utilized for monitoring of the composition of reaction mixtures in course of Re complex formation and for purity check of prepared complexes. Semi-preparative HPLC mode was succesfully utilized for isolation and final purification of...

This thesis is focused on the study of boron coordination compounds, specifically boron pyrogallol and 2,3-dihydroxynaphthalene ligand complexes. In the introductory part of the work are discussed the properties of boron compounds, their preparation, the possibility of separation by capillary electrophoresis and methods of structural characterization. In the first part of the thesis, the methods of preparation of complexes are discussed and for each complex the optimal yield response is chosen. The resulting products were studied by low resolution mass spectrometry. In the second part of the thesis, structures of complexes, including their modeling in the Gaussian program, were described more precisely. Furthermore, the complexes were characterized by high resolution mass spectrometry. The complexes were also studied by 11 B, 1 H and 1 H COSY NMR and IR spectroscopy. The third part was devoted to the separation of these substances from the mixture after the reaction. CE-UV and CE-MS techniques were used for this purpose. To convert the results, the separation method was developed in ammonium formate buffers that are compatible with both instruments. The equilibration between complexes and ligands was also monitored by CE-UV. A special chapter is the study of pyrogallol autooxidation by UV-VIS and...