Rapid and Convenient Oxidative Release of Thiol-Conjugated Forms of Microcystins for Chemical Analysis

Abstract

Microcystins are potent cyclic heptapeptide toxins found in some cyanobacteria, and usually contain an α,β-unsaturated carbonyl group that is readily conjugated to thiol-containing amino acids, peptides, and proteins in vivo and in vitro. Methods for deconjugating these types of adducts have recently been reported, but the reactions are slow or result in derivatized microcystins. Mercaptoethanol derivatives of a range of microcystins were therefore used as model compounds to develop deconjugation procedures in which the dialkyl sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate via β-elimination to afford free microcystins with the α,β-unsaturated carbonyl group intact. These free microcystins can be analyzed by LC/MS to determine the toxin profile of bound microcystins. The method was tested on Cys- and GSH-derivatives of [Dha]MC-LR. In solution, the deconjugation reactions were complete within minutes at pH 10.7 and within a few hours at pH 9.2. Oxidation of sulfides to sulfoxides is easier and more rapid than oxidation to sulfones, allowing the use of milder oxidants and shorter reaction times. Oxidation of any methionine residues present in the microcystins occurs inevitably during these procedures, and interpretation of the microcystin profile obtained by LC/MS analysis needs to take this into account. Oxidation of tryptophan residues and degradation of microcystins by excess oxidant were circumvented by the addition of MeSO as a sacrificial reducing agent. These methods may be useful for other compounds that undergo conjugation via thia-Michael addition, such as acrylamide and deoxynivalenol. Oxidation of sulfides to sulfoxides can occur in vivo and could affect the bioavailability of toxins and drugs conjugated via thia-Michael addition, potentially exacerbating oxidative stress by catalytically converting GSH to its sulfenate via conjugation, oxidation, and elimination to regenerate the free toxin.

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards

“…Indeed, as MC-LF was detectable in the liver samples and the covalent interaction of MC-LF and MC-LR are assumed to be comparable, it is currently undiscernible whether the differences of liver tissue levels stem from differences in kinetics (uptake and excretion of MC congeners), dynamics (covalent binding) or indeed difference in extraction of free and protein bound MC. Recently, methods have been developed that are capable of cleaving the thiol bond formed during conjugation and thus release free MC using basic conditions [39,40,41]. Moreover, the regioselective cleavage of the thiol bond in combination with the application of our IS to future in vivo or in vitro experiments would allow to quantify the amount of free, conjugated and protein-bound single MC congeners or MC congener mixtures.…”

Structural Diversity, Characterization and Toxicology of Microcystins

“…These conjugates are also readily semi-synthesised to produce authentic standards for confirmation of their presence in samples and cultures by LC-MS (e.g., [81,100]) (see Section 5.7). In addition, the identity of S-conjugates at position-7 of MCs can be confirmed through a range of deconjugation reactions [101,110,111].…”

Oxidative Release of Thiol-Conjugated Forms of the Mycotoxin 4-Deoxynivalenol

“…Conversion of the sulfide group in DON-10-thiols to a sulfoxide or sulfone was expected to increase the rate of the base-catalyzed deconjugation reaction (Figure ). For example, the rapid release of thiol-conjugated microcystins after oxidation of the sulfide linkage has recently been shown . The rationale for developing such a protocol is the possibility for release and subsequent analysis of thiol-bound toxins, e.g.…”

“…The Michael addition of thiols to the α,β-unsaturated carbonyl moiety in DON is a reversible reaction. ,, Thus, DON may be released from DON-10-thiols by treatment with a base. This has been shown to occur with other toxins that may undergo Michael addition to thiols, such as the microcystins . The kinetics for the deconjugation reaction of such Michael adducts were relatively slow but were significantly higher after oxidation of the sulfide linkage to a sulfoxide or sulfone …”

“…This has been shown to occur with other toxins that may undergo Michael addition to thiols, such as the microcystins . The kinetics for the deconjugation reaction of such Michael adducts were relatively slow but were significantly higher after oxidation of the sulfide linkage to a sulfoxide or sulfone …”