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Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,β-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,β-unsaturated carbonyl groups and can be expected to behave in a similar manner.

Okadaic acid analogues are well known as protein phosphatase inhibitors and occur naturally in marine shellfish feeding on dinoflagellates of the genus Dinophysis, leading to diarrhetic shellfish poisoning of shellfish consumers. Knowledge of the correct structures for these toxins is important in understanding their toxicology, biochemistry, and biosynthesis. We have performed extensive NMR analyses on okadaic acid (1), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) obtained from natural sources. Consequently, we were able to unambiguously deduce the stereochemistries at C-35 for DTX-1 and DTX-2 based on analysis of NMR coupling constants and NOE interactions. Our results revealed that DTX-2 (3) has a stereochemistry opposite to that of DTX-1 (2) at C-35. Molecular modeling of the docking of 1-3 with protein phosphatase-1 and protein phosphatase 2A (PP2A) suggested that the reduced affinity of DTX-2 for PP2A may be due to the newly defined stereochemistry at the 35-methyl group. The implications of these findings for biosynthesis and toxicology are discussed.