Several hepatotoxic peptides have been isolated from cyanobacteria. Microcystis aeruginosa and M. viridis produce at least nine microcystins as shown in Fig. 1. We previously established an effective isolation and analysis method for these peptides. During separation of these toxins using our method we found that the purified microcystins include consistently 15-20% of minor components (Fig. 2). In this paper we describe isolation and structure determination of the minor components that microcystins LR and RR accompany. The naturally occurring cell samples were treated according to our isolation method. The three minor components, the "small peak" from the RR fraction, and the "small" and "unknown" peaks from the LR fraction could be successfully isolated as shown in Fig. 3. The physicochemical and toxic properties of the five components thus obtained are briefly summarized in Table 1. Both small peaks of microcystins LR and RR possess the same molecular weights and amino acid compositions as the Large peaks. However, while the large peaks of microcystins LR and RR showed the same toxicity as the reported values, no toxicity was found for the small peaks of both toxins up to 1.2mg/kg. Additionally, differences were recognized in the UV and NMR spectra between the large and small peaks. Therefore, the ^1H- and ^<13>C-NMR spectra were carefully examined using COSY, HMQC and HMBC techniques (Table 2). Finally, application of ROESY could make clear that the small peaks are geometrical isomers at C-7 of Adda moiety of the parent microcystins (Figs. 4 and 5).
