Origins of Oxygen Atoms in a Marine Ladder-Frame Polyether: Evidence of Monooxygenation by 18O-Labeling and Using Tandem Mass Spectrometry

Yessotoxin is a ladder-frame polyether produced by the dinoflagellate Protoceratium reticulatum. Previous labeling experiments using C-13-acetate established the unique assembly of the carbon chain from intact and cleaved acetate units. The origins of ether and hydroxy oxygens in the molecule, which would yield further information regarding the assembly of the ladder-frame structure, have yet to be established. In this study, we describe the incorporation of O-18 in one experiment where the dinoflagellate was cultured under O-18(2) atmosphere and in a second where the culture media was supplemented with [O-18(2)]acetate. Labeled yessotoxin obtained from these experiments was subjected to collision-induced dissociation tandem mass spectrometry to determine the positions of O-18-incorporation pattern in the molecule. Detailed analyses of product ions from the fragmentation processes led to the identification of O-18-labeled positions and the incorporation ratios. The data revealed that the ether oxygens were labeled from O-18(2) and the hydroxy oxygen on C32 was derived from [O-18(2)]acetate. These results support a proposed biosynthetic mechanism of marine ladder-frame polyethers that a polyene precursor was oxidized by a monooxygenase after acetate condensation.