Preparation and Characterization of Cysteine Adducts of Deoxynivalenol

Abstract

Conjugation with the biologically relevant thiol glutathione is one of the metabolic pathways for the mycotoxin deoxynivalenol (DON) in wheat. The occurrence of putative DON-cysteine conjugates has also been shown in wheat, likely in part as a result of degradation of the glutathione conjugates. It was reported that thiols react in vitro with DON at two positions: reversibly at C-10 of the α,β-unsaturated ketone and irreversibly at C-13 of the epoxy group. We synthesized pure DON-cysteine adducts and made analytical standards using quantitative NMR experiments. Compounds were characterized using NMR and LC-HRMS/MS and tested in vitro for toxicity. Cysteine conjugates were much less toxic than DON at the same concentration, and LC-HRMS analysis demonstrated that there was no detectable metabolism of the conjugates in human monocytes or human macrophages.

Glutathione-Conjugates of Deoxynivalenol in Naturally Contaminated Grain Are Primarily Linked via the Epoxide Group

“…The type of conjugate present in the extract corresponded to a kinetically favored, and thus relatively fast-forming, isomer (see chromatogram 1 for DON–GSH, –CysGly, and –Cys in Figure 2) from Michael addition at C-10 (Figure 1). It has been shown that this initial product from the reaction of DON with mercaptoethanol, Cys or GSH is replaced (under neutral or mildly basic reaction conditions) by other C-10 conjugates and a C-13 conjugate over time [6,7,8], and the same occurred in the present study during conjugation with GSH, CysGly, Cys (Figure 2), γ-GluCys and N -acetylcysteine (NAC) (Figure S1). …”

“…This finding was verified by HRMS/MS targeting the [M − H] − ions of DON–GSH (Figure 3). We previously reported that MS-fragmentation of the [M − H] − ions of DON–thiol conjugates is well suited to distinguishing between the products from Michael addition to C-10 and addition to the epoxide at C-13 [6,7]. Thus, fragmentation of the C-10 conjugates yielded primarily negatively charged amino acid or peptide fragments, while fragmentation of the C-13 conjugates gave product ions primarily related to the trichothecene moiety [6,7].…”

“…We previously reported that MS-fragmentation of the [M − H] − ions of DON–thiol conjugates is well suited to distinguishing between the products from Michael addition to C-10 and addition to the epoxide at C-13 [6,7]. Thus, fragmentation of the C-10 conjugates yielded primarily negatively charged amino acid or peptide fragments, while fragmentation of the C-13 conjugates gave product ions primarily related to the trichothecene moiety [6,7]. A concentrated (approximately 5:1) oat extract was used to obtain HRMS/MS spectra of the DON–GSH isomers present in the sample, and the resulting HRMS/MS spectrum of the presumed DON-13-GSH conjugate in the oats was essentially identical to the HRMS/MS spectrum of DON-13-GSH in the reference mixture (Figure 3).…”

“…However, as no characterized reference standards were available, the exact structures of these adducts could not be determined. It was recently shown that thiols, including GSH, react with DON at both the C-10 and C-13 positions (Figure 1), giving rise to a range of mono- and di-conjugated products [6,7,8,9]. Because the C-8 ketone of DON is in equilibrium with the cyclic 8,15-hemiketal form, and four 9,10-diastereoisomers can be formed by thiol addition at C-10, ten mono- and eight di-conjugated toxin derivatives can be formed for any given thiol [8].…”

“…Because the C-8 ketone of DON is in equilibrium with the cyclic 8,15-hemiketal form, and four 9,10-diastereoisomers can be formed by thiol addition at C-10, ten mono- and eight di-conjugated toxin derivatives can be formed for any given thiol [8]. As the structures of the major thiol addition products now have been determined, and analytical standards are available [6,7], naturally produced DON–thiol adducts can be identified. Thus, the objective of this study was to use LC-HRMS methods in order to identify naturally occurring DON–thiol adducts.…”