Isolation of the toxin using cyanobacteria cultured from the original Palm Island strain was achieved by gel filtration of an aqueous extract, followed by reverse-phase HPLC. Structure elucidation was achieved via mass spectrometry (MS) and nuclear magnetic resonance (NMR) experiments, and a structure (later proven slightly incorrect) was proposed (Figure 1).
This almost-correct molecule possesses a tricyclic guanidine group (rings A, B & C), along with a uracil ring (D). The zwitterionic nature of the molecule makes this highly water-soluble, as the presence of charged areas within the molecule creates a dipole effect, suiting the polar solvent. Sensitivity of key signals in the NMR spectrum to small changes in pH suggested that the uracil ring exists in a keto/enol tautomeric relationship, where a hydrogen transfer results in two distinct structures (Figure 2). It was originally proposed that a hydrogen bond between the uracil and guanidine groups in the enol tautomer would make this the dominant form.