Metabolism of fungicidal cyanooximes, cymoxanil and analogues in various strains of Botrytis cinerea.

BACKGROUND: The metabolism of cymoxanil [1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea] and fungicidal cyanooxime analogues was monitored on three phenotypes of Botrytis cinerea Pers. ex Fr. differing in their sensitivity towards cymoxanil. For this purpose, labelled [2-(14)C]cymoxanil was added either to the culture medium of these strains or to its cell-free extract. RESULTS: In the culture medium of the most sensitive strain, four main metabolites were detected. Three were isolated and identified. Cymoxanil was quickly metabolised by at least three concurrent enzymatic pathways: (i) cyclisation leading, after hydrolysis, to ethylparabanic acid, (ii) reduction giving demethoxylated cymoxanil, (iii) hydrolysis followed by reduction and then acetylation leading to N-acetylcyanoglycine. In the cell-free extract of the same strain, only the first and the second of these enzymatic reactions occurred. By comparing the metabolic profile of the most sensitive strain with that of the less sensitive ones, it was shown that the decrease in sensitivity to cymoxanil correlates with a reduced acetylcyanoglycine formation. Among all metabolites, only N-acetylcyanoglycine is active against the most sensitive strain. Moreover, in a culture of this strain, two other fungicidal cyanooximes were also metabolised into this metabolite. CONCLUSION: The formation of N-acetylcyanoglycine may play an important role in the fungitoxicity of cymoxanil and cyanooxime derivatives.

Biodegradation of 2-ethylhexyl nitrate by Mycobacterium austroafricanum IFP 2173.

2-Ethyhexyl nitrate (2-EHN) is a major additive of fuel that is used to increase the cetane number of diesel. Because of its wide use and possible accidental release, 2-EHN is a potential pollutant of the environment. In this study, Mycobacterium austroafricanum IFP 2173 was selected from among several strains as the best 2-EHN degrader. The 2-EHN biodegradation rate was increased in biphasic cultures where the hydrocarbon was dissolved in an inert non-aqueous-phase liquid, suggesting that the transfer of the hydrophobic substrate to the cells was a growth-limiting factor. Carbon balance calculation, as well as organic-carbon measurement, indicated a release of metabolites in the culture medium. Further analysis by gas chromatography revealed that a single metabolite accumulated during growth. This metabolite had a molecular mass of 114 Da as determined by gas chromatography/mass spectrometry and was provisionally identified as 4-ethyldihydrofuran-2(3H)-one by liquid chromatography-tandem mass spectrometry analysis. Identification was confirmed by analysis of the chemically synthesized lactone. Based on these results, a plausible catabolic pathway is proposed whereby 2-EHN is converted to 4-ethyldihydrofuran-2(3H)-one, which cannot be metabolized further by strain IFP 2173. This putative pathway provides an explanation for the low energetic efficiency of 2-EHN degradation and its poor biodegradability.

Characterization of metabolites of fungicidal cymoxanil in a sensitive strain of Botrytis cinerea.

The metabolism of cymoxanil [1-(2-cyano-2-methoxyiminoacetyl)-3-ethyl urea] by a very sensitive strain of Botrytis cinerea toward this fungicide was studied by using [2-(14)C]-cymoxanil. Labeled cymoxanil was added either in a culture of this strain or in its enzymatic extract. The main metabolites, detected in biological samples, were isolated and identified by mass and NMR spectrometry. Their identification allowed us to show that this strain quickly metabolized cymoxanil according to at least three enzymatic pathways: (i) cyclization leading, after hydrolysis, to ethyl parabanic acid, (ii) reduction giving demethoxylated cymoxanil, and (iii) hydrolysis and reduction followed by acetylation leading to N-acetylcyanoglycine. In a cell-free extract of the same strain, only the first and the second enzymatic reactions, quoted above, occurred. Biological tests showed that, among all the metabolites, only N-acetylcyanoglycine is fungitoxic toward this sensitive strain.

Trace analysis of sulfonylurea herbicides and their metabolites in water using a combination of off-line or on-line solid-phase extraction and liquid chromatography-tandem mass spectrometry.

Two alternatives for the rapid simultaneous quantification of six sulfonylurea herbicides and five of their main degradation products in natural water are proposed. For concentration, the compounds were extracted on a polystyrene-divinylbenzene solid phase under pH and elution conditions that suppressed any hydrolysis. The eluates were analysed by liquid chromatography coupled to electrospray tandem mass spectrometry within 20 min. The whole method was validated and shown to give no hydrolysis artefacts. The application of off-line and on-line SPE of sulfonylureas enabled the 0.1 microg L(-1) and 1 ng L(-1) LOQ levels to be reached, respectively. The on-line SPE-LC-MS-MS method allowed the accurate quantitation of all sulfonylureas and three degradation products at 0.1 microg L(-1) or below in natural water, with an average repeatability of 8%.

Glyphosate and AMPA analysis in sewage sludge by LC-ESI-MS/MS after FMOC derivatization on strong anion-exchange resin as solid support.

An innovative analytical method has been developed for the determination of glyphosate and aminomethylphosphonic acid (AMPA), its major metabolite, in sewage sludge. This method involved an alkaline extraction from sludge and purification on a strong anion-exchange resin. While the analytes remained fixed by ionic interactions, an "on-solid support" derivatization by FMOC-Cl was carried out. This versatile approach allowed a 10 min reaction and simple elimination of the excess of reagent. The resulting derivatives remained retained by ionic and hydrophobic interactions with the resin until being eluted by a mixed NaCl water/acetonitrile, 70/30, v/v, solution. After an appropriate dilution and adjustment of the pH, the sample was concentrated on an Oasis HLB solid-phase cartridge. For quality analysis of traces in complex matrixes, LC-ESI-MS/MS in the multiple reaction monitoring positive mode was used fulfilling the European Union requirements (Decision 2002/657/CE). To overcome the matrix effects, stable isotopically labeled standards were added to the sludge extracts as internal standards and were thus derivatized during the procedure in parallel to the analytes. Mean recovery values were 70% +/- 7% for glyphosate and 63% +/- 3% for AMPA. Limits of detection (20 and 30 ppb dw) and limits of quantification (35 and 50 ppb dw) for glyphosate and AMPA, respectively, were sufficient to monitor samples taken from Ile-de-France wastewater treatment plants where contamination currently reached 0.1-3 ppm and 2-35 ppm dw for glyphosate and AMPA, respectively.

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry.

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.

Structural characterization of the major flavonoid glycosides from Arabidopsis thaliana seeds.

Information gains from the seed of the model plant Arabidopsis thaliana (Brassicaceae) have greatly contributed to a better understanding of flavonoid synthesis and may be used for crop improvement. However, exhaustive identification of the flavonoid accumulated in Arabidopsis seed was still lacking. Complementary investigations of seed flavonoids by LC-ESI-MS, LC-ESI-MS-MS, and NMR spectroscopy in Arabidopsis led to full characterization of monoglycosides, namely, quercetin 3-O-alpha-rhamnopyranoside and kaempferol 3-O-alpha-rhamnopyranoside, and diglycosides, namely, quercetin and kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and quercetin and kaempferol 3,7-di-O-alpha-rhamnopyranoside. Interestingly, the tt7 mutant that lacks flavonoid-3'-hydroxylase and consequently accumulates only kaempferol derivatives was shown to contain three additional products, kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-alpha-rhamnopyranoside-7-O-beta-glucopyranoside, and the triglycoside kaempferol 3-O-beta-[alpha-rhamnopyranosyl(1-->2)-glucopyranoside]-7-O-alpha-rhamnopyranoside. Taken together these results allow a scheme for flavonol glycosylation to be proposed.

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana.

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.

Iridoid glucosides from the aerial parts of Globularia alypum L. (Globulariaceae).

From the hydromethanolic extract of the aerial parts of Globularia alypum grown in Morocco, a new chlorinated iridoid glucoside, globularioside has been isolated beside 5 known iridoid glycosides, globularin, globularicisin, globularidin, globularinin and globularimin. This is the first report of a chlorinated iridoid in G. alypum and in the Globulareaceae. Unlike all other known 7-chlorinated iridoid glucosides where the chlorine atom exhibits an alpha configuration, globularioside incorporate the chlorine atom as a 7beta substituent. The structures of the isolated compounds were established on the basis of ESI-MS, MS-MS, 1D and 2D NMR spectral analysis.

TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat.

The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.

Application of positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry to a study of the fragmentation of 6-hydroxyluteolin 7-O-glucoside and 7-O-glucosyl-(1 --> 3)-glucoside.

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the structural characterization of 6-hydroxyluteolin 7-O-glucoside and 7-O-glucosyl-(1 --> 3)-glucoside. In-source fragmentation of both glycosides at an increased potential yielded the protonated and deprotonated aglycone, allowing CID spectra to be obtained. The differentiation between quercetin and 6-hydroxyluteolin aglycones was achieved by product ion analysis of the protonated and deprotonated aglycone (m/z 303 and 301), that showed the characteristic product ions (1,3)A at m/z 151 and 153 for quercetin, and m/z 167 and 169 for 6-hydroxyluteolin, consistent with the trihydroxylated A-ring skeleton. In the negative ion mode both glycosides were shown to undergo collision-induced homolytic and heterolytic cleavages of the O-glycosidic bond producing the aglycone radical-anion [Y0-H]-* and Y0(-) product ions. At lower collision energy, various fragmentations involving the glucose moieties were observed with a relatively higher abundance for the monoglucoside compared to the diglucoside. In the latter case both the inner and the terminal glucose residues were involved in the fragmentations, giving useful information on the 1 --> 3 interglycosidic linkage. CID MS/MS analysis of the sodiated molecules gave complementary information for the structural characterization of the studied compounds. Fragmentation mechanisms are proposed for the observed product ions.

Antioxidant constituents of the aerial parts of Globularia alypum growing in Morocco.

Three new phenolic compounds were isolated from the aerial parts of Globularia alypum. Their structures were determined as 6-hydroxyluteolin 7-O-laminaribioside (1), eriodictyol 7-O-sophoroside (2), and 6'-O-coumaroyl-1'-O-[2-(3,4-dihydroxyphenyl)ethyl]-beta-D-glucopyranoside (3). In addition, three phenylethanoid glycosides (acteoside, isoacteoside, and forsythiaside) and two flavonoid glycosides (6-hydroxyluteolin 7-O-beta-D-glucopyranoside and luteolin 7-O-sophoroside) were also isolated and are reported here for the first time in this plant. The structures of compounds 1-3 were established on the basis of their spectroscopic data analysis. Evaluation of the antioxidative activity, conducted in vitro, showed that the isolated phenylethanoids and flavonoid glycosides possess strong effects of this type.

Profiling isoflavone conjugates in root extracts of lupine species with LC/ESI/MSn systems.

Extracts obtained from roots of three lupine species (Lupinus albus, L. angustifolius, L. luteus) were analysed using LC/UV and LC/ESI/MS(n). The experiments were performed using two mass spectrometric systems, equipped with the triple quadrupole or ion trap analysers. Thirteen to twenty isomeric isoflavone conjugates were identified in roots of the investigated lupine species. These were di- and monoglycosides of genistein and 2'-hydroxygenistein with different patterns of glycosylation, both at oxygen and carbon atoms; some glycosides were acylated with malonic acid. It was not possible to establish the glycosylation sites of the aglycone only on the basis of the registered mass spectra; however, it was possible to differentiate C- and O-glucosides of isoflavones. Only comparison of retention times with those of standard compounds permitted to indicate the correct glycosylation pattern. In the case of diglycosides, the glycosylation pattern (O-diglucoside or O-glucosylglucoside) was distinguishable on the basis of the relative intensities of daughter ions in the mass spectra of protonated molecular ions. It was not possible to elucidate the site of malonylation on the sugar moiety from mass spectra, however, protonated molecules [M + H](+) of isoflavone glucosides with different placement of the malonyl group on the sugar ring were recognized in the extracts. In addition to the isoflavone glycosides, some flavone or flavonol glycosides were identified in the samples on the basis of collision-induced daughter ion spectra of the aglycone ions. A comparison of results obtained with the triple quadrupole and ion trap analysers was done in the course of the investigations.

Diaeretiella rapae limits Myzus persicae populations after applications of deltamethrin in oilseed rape.

In fall, Myzus persicae (Sulzer) (Homoptera: Aphididae) may exhibit population resurgence in winter oilseed rape in France. This resurgence may arise from pyrethroid treatments against Coleoptera (Psylliodes chrysocephala L.) that either kill parasitoids present during treatment or prevent recolonization by off-crop parasitoids. We studied the impact of Diaeretiella rapae (M'Intosh) (Hymenoptera: Braconidae) on populations of M. persicae when parasitoids were introduced on deltamethrin-treated plants at increasing intervals after treatment. Parasitoids were introduced 1, 2, 7, or 14 d posttreatment on individually caged plants infested with established populations of M. persicae. Aphids were counted 7, 14 and 21 d after parasitoid introduction. First, we observed that both the pesticide and the parasitoid reduced aphid population growth and that their effects were additive. Second, there was no mortality of parasitoids exposed to treated leaves in a device with a refuge area, and only 20% of mortality without the refuge area. Furthermore, deltamethrin residues had no effect on the reproduction of D. rapae females. Compared with the known toxicity of deltamethrin to D. rapae on glass, this low mortality may have been due to both the high liposolubility of deltamethrin (leading to a rapid diffusion of residues in the oilseed rape leaf cuticle) and to the existence of a refuge area. This work suggests that D. rapae could limit populations of M. persicae in the fall, even after pyrethroid treatment, because the presence of deltamethrin residues had little impact on the parasitoid.

Synthesis of modified proanthocyanidins: introduction of acyl substituents at C-8 of catechin. Selective synthesis of a C-4-->O-->C-3 ether-linked procyanidin-like dimer.

The regioselective introduction of substituents at C-8 of (+)-catechin is described, leading to the synthesis of several catechin derivatives with various substitution patterns to be used for the further synthesis of modified proanthocyanidins. Thereafter, a new 3-O-4 ether-linked procyanidin-like derivative was synthesized. Its formation was selectively achieved through TiCl(4)-catalyzed condensation of 4-(2-hydroxyethoxy)tetra-O-benzyl catechin with the 8-trifluoroacetyl adduct of tetra-O-benzyl catechin.

Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745.

Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi. The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity. We used purified laccases to transform the chemical and then we separated the metabolites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes. versicolor. We identified 13 oligomers formed from 2,5-xylidine. (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethylcyclohexa-2,5-dienone at 1.25 x 10(-5) M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture. Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer.

Kinetics and mechanism of the degradation of the pesticide metsulfuron methyl induced by excitation of iron(III) aqua complexes in aqueous solutions: steady state and transient absorption spectroscopy studies.

The degradation of the pesticide metsulfuron methyl induced by excitation of iron(III) aqua complexes was studied at 365 nm. The process involved the formation of hydroxyl radicals and was shown to depend on Fe(OH)(2+) concentration. The second order rate constant of the reaction of metsulfuron methyl and hydroxyl radical was evaluated by laser flash photolysis experiments to k = 3.5 x 10(9) mol(-1) L s(-1). Various products were formed. Among them 2-amino-4-methoxy-6-methyl-1,3,5-triazine, 2-(carbomethoxy) benzenesulfonamide and hydroxylated derivatives represent the major photoproducts. Compounds arising from the cleavage of the triazine moiety were also found. After prolonged irradiation, a total disappearance of metsulfuron methyl and a complete mineralization of the solution were obtained.