Association de la spectroscopie de résonance parmagnétique électronique et de la spectrométrie de masse pour l identification de nitroxydes et d espèces diamagnétiques dérivées (application à l étude de mécanismes réactionnels)

L'objectif de ces travaux de thèse porte sur le développement d'une stratégie analytique combinant les techniques de Résonance Paramagnétique Electronique (RPE) et de Spectrométrie de Masse (MS) pour la caractérisation structurale de composés nitroxyde, ainsi que leurs dérivés diamagnétiques. Alors que la RPE permet d'accéder à des informations de façon spécifique et sensible à la fonction nitroxyde, la mise en place d'expériences MS/MS après ionisation électrospray révèle les structures dans leur globalité. L'étude de la réaction d'autoxydation spontanée d'un diénol présente un exemple illustrant l'efficacité de l'association RPE/MS qui a ainsi permis l'identification d'intermédiaires radicalaires de courte durée de vie. Alors que la RPE définit précisément la nature et la position des radicaux piégés, des expériences en MS/MS ont conduit à une caractérisation du nitroxyde formé lors des réactions de spin trapping entre le diénol et une nitrone en présence d'oxygène moléculaire. Dans une seconde étude, la réactivité de divers nucléophiles vis à vis du composé nitroso MNP a également permis de mettre en exergue l'efficacité de la méthode RPE/MS. En particulier, l'addition de ces composés nucléophiles thiols, amines et certains acides carboxyliques et phosphites sur la fonction nitroso dépend fortement des conditions expérimentales. Dans le cas de thiols, le thio-nitroxydes obtenus en présence du MNP peuvent en particulier être envisagés pour des applications dans le domaine du marquage de spin.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Use of X-band EPR to follow the reaction between various nucleophilic compounds and 2-methyl-2-nitrosopropane (MNP)

International audienceContinuous wave X-band EPR spectroscopy was used as an analytical tool to follow the formation of nitroxide radicals after the reaction between various nucleophiles and 2-methyl-2-nitrosopropane (MNP). The addition of amines or of trihalogeno-acetic acids onto the nitroso moiety was found to depend on experimental parameters such as exposure to white light or the nature of the solvent, while no nitroxide adducts were ever detected when alcohols or weak carboxylic acids were used. In contrast to results previously obtained with cysteine, the reaction of ten standard amino acids (Gly, Lys, Arg, His, Trp, Ser, Thr, Tyr, Asp and Glu) on MNP never led to an EPR observable adduct, further confirming the particular reactivity of this nitroso compound towards thiols

Combining EPR and ESI-MS/MS to study the reactivity of alkylthiols and cysteine towards 2-methyl-2-nitrosopropane (MNP)

International audienceAn approach combining EPR spectroscopy and electrospray ionisation mass spectrometry was employed to study the reactivity of two alkylthiols and of cysteine towards 2-methyl-2-narosopropane (MNP) in the presence of H(2)O(2) Depending on the experimental conditions, various nitroxides could be EPR-detected, while their diamagnetic derivatives were structurally characterised after tandem mass spectrometry experiments Sample dilution in methanol before electrospray ionisation was also found to generate methyl-hydroxylamine derivatives and could thus constitute a simple derivatisation process to stabilise hardly MS-detectable radical species Upon white light irradiation, the three thiols tested led to alkoxynitroxides according to an inverted spin trapping process On the other hand, only the reduced form of cysteine was found to react with MNP in the dark, yielding an alkylthionitroxide adduct via a Forrester-Hepburn mechanism Besides a better understanding of the reactivity of thiols towards nitroso compounds, the results obtained underlined the particular behaviour of cysteine, suggesting that the use of a molecule as simple as MNP could allow a nitroxide moiety to be linked to a cysteinyl residu

Ene-reaction between a dienolic compound and 2-methyl-2-nitrosopropane: an EPR-MS study

International audienceThe combination of electron paramagnetic resonance (EPR) and mass spectrometry (MS) was used as an efficient tool to elucidate the mechanism of an ene-reaction between a dienol compound and 2-methyl-2-nitrosopropane. (C) 2010 Elsevier Ltd. All rights reserved

A combined spin trapping/EPR/mass spectrometry approach to study the formation of a cyclic peroxide by dienolic precursor autoxidation

International audienceThe spontaneous addition of air oxygen to a dienolic compound, yielding a cyclic peroxide, was followed by spin trapping (ST) combined with EPR spectroscopy and mass spectrometry (MS). Using two different nitrones, the ST/EPR study allowed the detection of the spin adduct of a radical intermediate, and the radical centre in the addend was identified after similar experiments performed with two different (13)C-labelled analogues of the substrate. The media were also submitted to electrospray ionisation, in both positive and negative modes, for structural characterisation of the spin adducts by tandem mass spectrometry. This allowed the structure of the hydroxylamine derivatives of the nitroxides formed to be identified. Following these results, a mechanism pathway was proposed for this autoxidation

5-Hydroxy-2,2,6,6-tetramethyl-4-(2-methylprop-1-en-yl)cyclohex-4-ene-1,3 -dione, a novel cheletropic trap for nitric oxide EPR detection

International audienceThe title compound behaves as an efficient cheletropic trap for both NO and NO(2) radicals in the presence of oxygen, yielding EPR observable nitroxide and alkoxynitroxide, respectively

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

Exposure to (bi)sulfite (HSO3–) and sulfite (SO32–) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–), peroxymonosulfate (–O3SOO.), and especially the sulfate (SO4. –) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of protein radicals in a mouse model of human lung disease

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

International audienceBackground: Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom ofWalterinnesia aegyptia. Methods: Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionatedWalterinnesia aegyptiasnake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results: Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide fromBungarus candidusvenom. Conclusions: This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue

Heterodimeric Insecticidal Peptide Provides New Insights into the Molecular and Functional Diversity of Ant Venoms

International audienceAnts use venom for predation, defense, and communication; however, the molecular diversity, function, and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modeling, stability studies, and in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain cross-linked by two disulfide bonds in an antiparallel orientation. We chemically synthesized Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blowfly assays (LD50 = 3 nmol/g). Molecular modeling and circular dichroism studies revealed strong α-helical features, indicating its cytotoxic effects could derive from cell membrane pore formation or disruption. The native heterodimer was substantially more stable against proteolytic degradation (t 1/2 = 13 h) than its homodimers or monomers (t 1/2 < 20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterization of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability, a concept that is highly relevant for the design and development of peptide therapeutics, molecular probes, and bioinsecticides