Preparation and characterization of the conjugated Fusarium mycotoxins zearalenone-4O-β-D-glucopyranoside, α-zearalenol-4O-β-D-glucopyranoside and β-zearalenol-4O-β-D-glucopyranoside by MS/MS and 2D-NMR
Résumé
Glucosides of several Fusarium mycotoxins occur in naturally infected cereals and may contribute to an increased content to the total mycotoxin load of food and feed. Here we present results on a fermentation procedure to produce zearalenone-4O-β-D-glucopyranoside from zearalenone using an engineered Saccharomyces cerevisiae strain, expressing the Arabidopsis thaliana UDP-glucosyltransferase UGT73C6. About 24 mg of zearalenone-4O-β-D-glucopyranoside were obtained from 50 mg zearalenone and further purified. 10 mg of the glucoside were reduced with sodium borohydride, yielding 4.1 mg α-zearalenol-4O-β-D-glucopyranoside and 4.5 mg β-zearalenol-4O-β-D-glucopyranoside at purities higher than 99%. To confirm the identities of the three produced glucosides, MS and MS/MS spectra were acquired using negative electrospray ionization. Besides the deprotonated ions at m/z 479 or 481 respectively in full scan mode, fragments, adducts and dimers were recorded and assigned. MS/MS spectra of the glucosylated substances yielded the deprotonated ions of the mycotoxins zearalenone, α-zearalenol, β-zearalenol and their fragments respectively. Unambiguous structural assignment of the three substances was achieved using 2D-NMR methods. This way, the glucose attachment to position C-4, the β-configuration of the sugar unit and the stereochemical assignment of the zearalenol hydroxyl group at C-6´ were proven.
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