Studies on search of protease inhibitors in blue-green algae and biosynthetic pathways

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Microcystis aeruginosa (NIES-98) was obtained from the NIES-collection (Microbial Culture Collection, the National Institute for Environmental Studies, Japan) . The 80% methanol extract of freeze-dried alga was partitioned between water and diethyl ether. The aqueous layr that showed potent trypsin inhibitory activity was further extracted with n-butanol and subjected to ODS column chromatography, followed by ODS HPLC to yield aeruginosin 98-A (1), B (2) and C (3) and aeruginoguanidine 98-A(4) , B (5) and C (6).Molecular formula of 1 was decides as C_<29>H_<45>N_6O_9ClS from HRFABMS and NMR data.Amino acid analysis of the acid hydrolyzate gave allo-Ile and an unknown imino acid. ^1H-^1H COSY,HMQC and HMBC date showed the presence of Choloro Hpla (3-chloro-4-hydroxyphenyllactic acid), allo-Ile, Choi sulfate (2-carboxy-6-hydroxyoctahydroindole sulfate) andagmatin. Interpretation of HMBC spectra revealed the structure of 1. The stereochemistryof allo-Ile was determined to be D-form by the chiral GC analysis. The relative stereochemistry of Choi sulfare was elucidated by NOESY spectra. The structures of 2 and 3 only differ from 1 in the substitution of the benzene ring of Chloro Hpla. The IC_<50> of 1,2 and 3 against trypsin was 0.55,0.6 and 3.93 mg/mL,respectively.Molecular formula of 5 was decided as C_<34>H_<59>N_9O_<14>S_3 from HRFABMS and NMR data. ^1H-^1HCOSY,HOHAHA,HMQC and HMBC data showed the presence of Hphpa trisulfate (1- (4-hydroxy-3-hydroxymethyl) phenyl-1-hydroxy-2-propylamine 4', 3', 1-tri-O-sulfate) , N-MeArg and NmgArg (N^a-methyl-N^W-geranylarginine). Interpretation of HMBC and NOESY spectra revealed the structure of 5. The structures of 4 and 6 were elucidated asanalogs of 5, having the different side chains of N-MeArg. These compounds showed weak cytotoxicity against P388 leukemia cells.