New Nodularins: A General Method for Structure Assignment

A general method has been developed for assigning the structures of nodularin, a potent hepatotoxin, tumor promoter, and protein phosphatase inhibitor, and minor components isolated from a cultured and a bloom sample of the cyanobacterium Nodularia spumigena. It consists of (1) FABMS analysis (determination of molecular weight and molecular formula), (2) ¹H NMR spectroscopy on the parent compound and chiral GC analysis of an acid hydrolyzate (identification and stereochemistry of amino acid components), (3) ozonolysis followed by NaBH₄ reduction (conversion to a linear peptide), and (4) FABMS/CID/MS analyses of the linear peptide and the parent compound (sequence analysis). The method has been employed in assigning structures to three new nodularins (2–4) and can be applied to other cyclic peptides containing α,β-dehydroamino acid unit(s), especially the related microcystins, cyclic heptapeptide hepatotoxins. Two nodularins, [DMAdda³]nodularin (2) and [(6Z)- Adda³]nodularin (3), were obtained from a bloom sample collected from Lake Ellesmere (New Zealand), and [D-Asp¹]nodularin (4) was isolated from cultured cells (strain L-575). The LD50s of 2 and 4 were 150 and 75 μg/kg (ip, mice), respectively, but 3 did not show apparent toxicity at 2.0 mg/kg.