Etude sur la biosynthèse de Naphtoquinones végétales et bactériennes

Quinones are well dispached in nature, in the animal, vegetal and microbial kindom. Those compounds are synthesized from a small number of simple intermediates. The naphtho-quinone ring can be synthesized from four precusrsors: acetate, toluquinone, p-hydroxy-benzoate and shikimate. The aim of this work is the study of vegetal or bacterial naphthoquinone biosynthesis from shikimic acid (trihydroxy-3,4,5 cyclohexen-1 carboxylic acid), and the search for intermediates in this biosynthetic pathway. At the start of our study no experimental data were available and its development was connected to parallel studie from other teams. Some preliminary results had proposed that shikimic acid was a common precursor of aromatic amino-acid, dihydroxy-3,4-benzoic acide, p-amino-benzoic acid, p-hydroxybenzoic acid and menaquinone from Eschericia coli. Thus at the start of this work we know that shikimic acid is totally incorporated in bacterial menaquinones so that the carboxyle becomes a carbonyle of the quinone. Thus three questions need to be answered: - How does the quinonic ring form: where is the carboxylic carbon? - From which precursor commes the three missing carbons? - Is shikimic acid incorporated in the naphtha-quinone symmetrically or selectively? The first question was solved both by Leistner and Zenk and by Leduc et al. in our laboratory using [1,6-14C] shikimic acid. This method implicates laborious and complex degradation of the formed quinines. We have synthesized [3H-3] shikimic acid specifically labeled in a position allowing a simple and univocal degradation. A partial answer to the second question was obtained by Campbell who showed incorporation of three atoms (2,3 and 4) of glutamate into the quinone ring. From his results and the belief that chorismic acid was a precursor we postulated OSB (ortho-succinoyl-benzoic acid), and could synthesize it and prove its incorporation in the naphtoquinones studied (menaquinone, lawsone and juglone). The work in presented in six chapters: the first describes the synthesis of radioactive precursors. The second describes the methods of incorporation of radioactive precursor into quinones, their isolation and their degradation. The three following chapter report the results of incorporation into menaquinone, vegetal naphthoquinones and vegetal anthraquinones. The six chapter discuss the results and proposes a general scheme for shikimic acid derived quinone biosynthesis. At the end an experimental part and some technical add-on are presented.Les quinones sont des composés largement répandus dans la nature, aussi bien dans le règne végétal, qu'animal ou microbien. Ces molécules sont biosynthétisées à partir d'un petit nombre d'intermédiaires simples. Le noyau des naphtoquinones, en particulier, peut être synthétisé à partir de quatre précurseurs principaux l'acétate, la toluhydroquinone, le p-hydroxybenzoate et le shikimate. L'objet du présent travail est l'étude de la biosynthèse des naphtoquinones végétales ou bactériennes dérivant de l'acide shikimique (acide trihydroxy-3,4,5 cyclohexène-1 carboxylique), et la recherche des intermédiaires de cette voie de biosynthèse. Etant donné, à l'origine, l'absence quasi-totale de résultats expérimentaux dans ce domaine et le développement important qu'il a acquis en même temps que nous commencions notre travail, celui-ci a été constamment lié dans son développement aux données les plus récentes parues dans la littérature. Quelques études antérieures avaient montré que l'acide shikimique devait être le précurseur commun des acides aminés aromatiques et de plusieurs facteurs de croissance aromatiques (acide dihydroxy-2,3 benzoique, dihydroxy-3,4 benzoique, p-aminobenzoique et p-hydroxybenzoique) ainsi que de la ménaquinone de Escherichia coli (16,17). Lorsque nous commençons ce travail, on sait seulement que l'acide shikimique est incorporé in toto dans les ménaquinones bactériennes, de telle sorte que son carboxyle devient un des carbonyles de la quinone. Trois questions se posent alors: - Sur lequel des carbones 2 ou 6 adjacents au carboxyle, le cycle naphtoquinonique se ferme-t-il ? - Quelle est la molécule qui fournit les trois carbones nécessaires à la fermeture du cycle quinonique de la naphtoquinone, et quel est le mécanisme de cette fermeture ? - Le cycle naphtoquinonique ainsi formé est-il incorporé de façon symétrique ou non dans les ménaquinones, c'est-à-dire le carboxyle de l'acide shikimique est-il incorporé indifféremment dans les deux carbonyles quinoniques, ou sélectivement dans l'un des deux ? Pour résoudre la première question, LEISTNER et ZENK d'une part et M. LEDUC dans notre laboratoire d'autre part, ont utilisé l'acide shikimique [14 C-1,6]. Cette méthode nécessite une dégradation laborieuse des quinones formées. Nous avons, quant à nous, synthétisé un acide shikîmique [3H-3] marqué au tritium en une position spécifique, ce qui permet une dégradation simple et univoque. Une réponse partielle à la deuxième question a été donnée par CAMPBELL. Il montrait, en 1969, que le glutamate est capable d'apporter ses carbones 2, 3 et 4 pour la fermeture du cycle naphtoquinonique, mais il ne disposait pas alors des résultats de notre laboratoire lui permettant d'induire correctement le mécanisme de cette biosynthèse. Nous avons pu postuler la formation d'un intermédiaire, I'acide ortho-succinoylbenzoique OBS, dont nous avons pu prouver l'existence par synthèse et incorporation dans les quinones étudiées . L'existence de cet intermédiaire et la possibilité que nous avons eue de réaliser sur l'OSB différents marquages spécifiques nous ont permis de répondre à la troisième question, à savoir, l'incorporation orientée ou non de l'OSB dans les quinones. En même temps, différentes équipes montraient que d'autres quinones existant dans les végétaux telles que la lawsone, la juglone et différentes anthraquinones dérivaient aussi de l'acide shikimique; il nous a semblé intéressant d'appliquer nos techniques à l'étude très voisine de la biosynthèse de ces quinones. Pour la clarté de l'exposé, nous présentons dans le premier chapître les méthodes de synthèse des précurseurs radioactifs, dans le deuxième, les méthodes d'isolement et de dégradation des quinones. Les trois chapîtres suivants rapportent l'incorporation dans les ménaquinones, les naphtoquinones végétales et les anthraquinones végétales. Dans le sixième chapître, nous discutons nos résultats et proposons un schéma général de biosynthèse des quinones dérivant de l'acide shikimique. La partie expérimentale de ce travail est rassemblée après l'exposé théorique

Les inhibiteurs "suicides" des Cytochromes P450 (établissement d'une banque de données, mise au point d'un test de screening et études structures-activité concernant des substrats furaniques du CYP 3A4)

PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

Sulfenic acids as reactive intermediates in xenobiotic metabolism

Sulfenic acid reactive intermediates are formed during the oxidation of cysteine residues of proteins and play key roles in enzyme catalysis, redox homeostasis and regulation of cell signalling. However few data are presently available on the formation and fate of sulfenic acids as reactive intermediates during the metabolism of xenobiotics. This article is a review of the xenobiotic metabolism situations in which the intermediate formation of a sulfenic acid has been reported. Formation of these intermediates has been either proposed on the basis of the isolation of products possibly deriving from sulfenic acids or shown after trapping of the sulfenic acid by specific nucleophiles. This review indicates the different mechanisms by which a sulphur-containing xenobiotic can be metabolized with the intermediate formation of a sulfenic acid. It also indicates the different possible fates of these sulfenic acids that have been reported in the literature. Finally, it discusses the possible implications of the formation of xenobiotic derived sulfenic acid reactive metabolites in pharmacology and toxicology

Engineering of a Water-Soluble Plant Cytochrome P450, CYP73A1, and NMR-Based Orientation of Natural and Alternate Substrates in the Active Site

CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L(–1) yeast culture. This water-soluble enzyme was suitable for (1)H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and (1)H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the (1)H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle

Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis -Dihydroxylation and Epoxidation

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine

Cytochromes P450 Catalyze Both Steps of the Major Pathway of Clopidogrel Bioactivation, whereas Paraoxonase Catalyzes the Formation of a Minor Thiol Metabolite Isomer

The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, clopidogrel, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is a classical cytochrome P450 (P450)-dependent monooxygenation of its thiophene ring leading to 2-oxo-clopidogrel, a thiolactone metabolite. The second step was described as a P450-dependent oxidative opening of the thiolactone ring of 2-oxo-clopidogrel, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding thiol <b>4b</b>. A very recent paper published in <i>Nat. Med.</i> (Bouman et al., (2011) <i>17</i>, 110) reported that the second step of clopidogrel bioactivation was not catalyzed by P450 enzymes but by paraoxonase-1­(PON-1) and that PON-1 was a major determinant of clopidogrel efficacy. The results described in the present article show that there are two metabolic pathways for the opening of the thiolactone ring of 2-oxo-clopidogrel. The major one, that was previously described, results from a P450-dependent redox bioactivation of 2-oxo-clopidogrel and leads to <b>4b cis</b>, two previously reported thiol diastereomers bearing an exocyclic double bond. The second, minor one, results from a hydrolysis of 2-oxo-clopidogrel, which seems to be dependent on PON-1, and leads to an isomer of <b>4b cis, 4b "endo"</b>, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. These results were obtained from a detailed study of the metabolism of 2-oxo-clopidogrel by human liver microsomes and human sera and analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, either as such or after derivatization with 3′-methoxy phenacyl bromide or <i>N</i>-ethyl maleimide (NEM). These results also show that the major bioactive thiol isomer found in the plasma of clopidogrel-treated patients derives from 2-oxo-clopidogrel by the P450-dependent pathway. Finally, chemical experiments on 2-oxo-clopidogrel showed that this thiolactone is in equilibrium with its tautomer having a double bond inside the piperidine ring and that nucleophiles such as CH₃O^– preferentially react on the thioester function of this tautomer. This allowed us to understand why <b>4b cis</b> has to be formed via an oxidative opening of 2-oxo-clopidogrel thiolactone, whereas a hydrolytic opening of this thiolactone ring leads to the "endo" thiol isomer <b>4b "endo"</b>