High Resolution LC-MSⁿ Fragmentation Pattern of Palytoxin as Template to Gain New Insights into Ovatoxin-a Structure. The Key Role of Calcium in MS Behavior of Palytoxins

Abstract

Palytoxin is a potent marine toxin and one of the most complex natural compounds ever described. A number of compounds identified as palytoxin congeners (e.g., ovatoxins, mascarenotoxins, ostreocins, etc.) have not been yet structurally elucidated due to lack of pure material in quantities sufficient to an NMR-based structural investigation. In this study, the complex fragmentation pattern of palytoxin in its positive high resolution liquid chromatography tandem mass spectra (HR LC-MS n ) was interpreted. Under the used conditions, the molecule underwent fragmentation at many sites of its backbone, and a large number of diagnostic fragment ions were identified. The natural product itself was used with no need for derivatization. Interestingly, most of the fragments contained calcium in their elemental formula. Evidence for palytoxin tendency to form adduct ions with calcium and other divalent cations in its mass spectra was obtained. Fragmentation pattern of palytoxin was used as template to gain detailed structural information on ovatoxin-a, the main toxin produced by Ostreopsis ovata, (observe correct font) a benthic dinoflagellate that currently represents the major harmful algal bloom threat in the Mediterranean area. Either the regions or the specific sites where ovatoxin-a and palytoxin structurally differ have been identified.

Chemical, molecular, and eco-toxicological investigation of Ostreopsis sp. from Cyprus Island: structural insights into four new ovatoxins by LC-HRMS/MS

“…This results in an atypical fragmentation behavior with regard to cleavage #17. In more detail, cleavage #17, which generally generates clearly detectable A-and B-side fragments in the LC-HRMS 2 spectra [19,23,24], did not occur in OVTX-i. The structural modification could involve oxidation and addition of a C2 unit to the methyl group at C50.…”

“…Since OVTX-i, OVTX-j 1 , OVTX-j 2 , and OVTX-k were contained in the extracts at levels too low to be isolated and studied by NMR, the LC-HRMS n approach developed by Ciminiello et al [19] was applied to investigate their structural features. The fundamentals of this approach are that all palytoxin-like compounds share the same MS 2 behavior, fragmenting at many sites of their backbone (Fig.…”

“…Considering that the B-side fragment due to cleavage #19 matched again that of ovatoxin-a, we could deduce that the oxygen was lacking in the region stretching from C53 to C78. We could not further restrict the region of difference as this part structure does not fragment further, as already observed for other palytoxin-like compounds [19,23,24], likely as a consequence of the formation of a conjugated polyene during the fragmentation process [37].…”

“…Net exponential growth rate (μ t −1 ) was calculated as the slope of a regression line of ln (N) versus time (t −1 ), where N was the [20,21] and ovatoxin-a (OVTX-a) [22,36] as determined by NMR studies. Cleavages #14, #20, #28, and #6+12 occur only in PLTX [19]. The stereochemistry of palytoxin is depicted.…”

“…strains were analyzed by liquid chromatography-high-resolution multiple-stage mass spectrometry (LC-HRMS n ) (n=1, 2). The employed approach [19] uses the fragmentation pattern of a structurally defined palytoxin congener, such as palytoxin [20,21] or ovatoxin-a (OVTX-a) [22] (Fig. 1), as a template for structural characterization of the unknown [23,24], providing tentative structures.…”