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The isolation and structural determination of new marine ladder-frame polyethers, brevisulcatic acids-1 (1) and -4 (2) are reported. Brevisulcatic acids were isolated from the dinoflagellate Karenia brevisulcata, which was identified as the causative species of a major red tide event in New Zealand in 1998. The ether ring composition and a β-hydroxy, γ-methylene valeric acid side chain of 1 and 2 are common, but 2 has a γ-lactone as the 5-membered A-ring while 1 is the seco acid analogue. Compound 2 has structural and bioactivity similarities to brevetoxin A.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

Preparative HPLC purification of a side-fraction obtained during purification of 44,55-dihydroxyyessotoxin (6) afforded fractions containing previously unidentified yessotoxin analogues. Careful analysis of these fractions by HPLC-UV, LC-MS3, and NMR spectroscopy, revealed the identities of some of these analogues as 45-hydroxy-46,47-dinoryessotoxin (1), 44-oxo-45,46,47-trinoryessotoxin (2) and 9-methyl-42,43,44,45,46,47,55-heptanor-38-en-41-oxoyessotoxin (5). Numerous other analogues were present but could only be characterized by HPLC-UV and LC-MS3 due to their low abundance. The HPLC-UV and LC-MS3 data confirm the presence of large numbers of yessotoxin analogues, some of which may be oxidative degradation products, in extracts of Protoceratium reticulatum. Compound-1 is the first 46,47-dinoryessotoxin to be identified.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC-MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC-MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

Two analogs of yessotoxin were isolated from extracts of a culture of Protoceratium reticulatum. The structures of the analogs were identified as trihydroxylated amides of 41a-homoyessotoxin (1) and 9-methyl-41a-homoyessotoxin (2) by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. Structures were further confirmed by micro-scale chemical conversions combined with LC-MS3 analyses. No toxic effects were recorded in mice injected intraperitoneally with 2 at a dose of 5000 microg/kg.