Structure elucidation of leadbeaterin-1, an ichthyotoxin associated with fish-killing blooms of Chrysochromulina leadbeateri in Northern Norwegian fjords.

In May-June 2019 the haptophyte Chrysochromulina leadbeateri caused the most damaging fish-killing bloom in northern Europe to date. This study reports the isolation and structural characterization of a new sterolysin, leadbeaterin-1 (1) from C. leadbeateri, based on LC-HRMS/MS and NMR analysis. Although structurally related to karlotoxins, 1 is distinct in having fewer double bonds, the presence of chlorination in the polyol side chain, and in being present in two diastereomeric forms (1a and 1b) in an approximately 1:1 ratio, making it a unique addition to the sterolysin class of compounds. The close structural similarity of 1 from a haptophye, and karlotoxin-2 from a dinoflagellate, raises interesting questions regarding the ancestry of the genes for sterolysin biosynthesis. Quantitative NMR of 1 established the first genuinely quantitative sterolysin standard, enabling precise toxin measurements. Leadbeaterin-1 was highly cytotoxic to the RTgill-W1 (EC50 0.4 ± 0.1 µg/mL) and ASG-10 (EC50 0.5 ± 0.1 µg/mL) gill cell lines using the Alamar Blue assay. Unexpectedly, a high proportion of leadbeaterin-5 (5), identified as a glycolate ester of 1, was detected in freshly extracted cultures. This ester analogue was unstable to hydrolysis, apparently converting to leadbeaterin-1 during harvesting and extraction. The quantitative standard allowed LC-HRMS analysis of the cellular quotas of 1, 5, and other leadbeaterins in C. leadbeateri isolates UIO 035, UIO 393, and UIO 394, revealing minor variations in toxin accumulation.