CiNii Fulltext PDF
In our ongoing search for biologically active metabolites from cyanobacteria, we have found that Oscillatoria agardhii (NIES-204, 205) are a rich source of novel protease inhibitors. Here we mainly report the isolation and structure elucidation of agardhipeptins A (1) and B (2), and aeruginosin 205-A (3), though we succeeded in the isolation of several types of protease inhibitors from O. agardhii. O. agardhii (NIES-204, 205) were obtained from the NIES-collection (Microbial Culture Collection, the National Institute for Environmental Studies, Japan) and cultured in 10L glass bottles containing CB medium. The 80% MeOH extracts of lyophilized cyanobacteria were subjected to solvent partitions, ODS column chromatography followed by ODS HPLC to yield 1-3. Molecular formulae of 1 and 2 were determined as C_<43>H_<51>N_<11O>_7 and C_<57>H_<69>N_<110>_8, respectively, by HRFAB-MS and NMR data. Amino acid analyses of the hydrolyzates of 1 and 2 gave Pro, His, Leu, Trp, and Gly for 1, and Ala, Val, Leu, Pro, and Trp for 2. Interpretation of HMBC and NOESY spectra revealed the structure of 1 and 2. Absolute stereochemistry of amino acid residues in 1 and 2 was determined to be L-form by HPLC analysis of the acid hydrolyzates derivatized with Marfey's reagent. Agardhipeptin A (1) inhibited plasmin with an IC_<50> of 65μg/mL but agardhipeptin B (2) had no plasmin inhibitory activity. Molecular formula of 3 was decided to be C_<34>H_<53>N_<6O>_12ClS from FAB-MS and NMR data. A detailed analysis of the 2D NMR data, including ^1H-^1H COSY, HMQC, HMBC, NOESY and HOHAHA revealed the presence of Pla sulfate (phenyllactic acid 2-O-sulfate), Hleu (3-hydroxyleucine), Ccoi (2-carboxy-6-chlorooctahydroindole), Agma (agmatine), and xylopyranose. The structure of 3 was elucidated by the interpretation of HMBC and NOESY correlations. Absolute stereochemistry of Pla sulfate and xylopyranose was determined to be L- and D-form by HPLC analysis of menthyl ester of acid hydrolyzate and GC analysis of trifluoroacetyl isopropyl derivative, respectively. The absolute configuration of Ccoi and Hleu remains to be defined. Aeruginosin 205-A (3) inhibited trypsin and thrombin with the IC_<50> of 0.07 and 1.5μg/ml, respectively.