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

蓝绿藻蛋白酶抑制剂的寻找及生物合成途径研究

• 批准号: 05453172
• 负责人: MURAKAMI Masahiro
• 金额: $ 4.74万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05453172/
• 关键词: Protease; Protease inhibitor; Trypsin; Trypsin inhibitor; Blue-green algae; Microcystis; Cytotoxicity; -; エラスターゼ; キモトリプシン; Nostoc; ペプチド

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.

铜绿微囊藻（NIES-98）获自NIES保藏中心（Microbial Culture Collection，the National Institute for Environmental Studies，Japan）。将冷冻干燥的黄芪的80%甲醇提取物在水和乙醚之间分配。经正丁醇萃取、ODS柱层析、ODS HPLC分离得到了具有胰蛋白酶抑制活性的AERCOSIN 98-A（1）、B（2）、C（3）和AERCOUNAIDINE 98-A（4）、B（5）、C（6），经HRFABMS和NMR分析确定其分子式为C_<29>H_<45>N_6 O_9 ClS，酸水解产物经氨基酸分析得到了一种未知的亚氨基酸--异亮氨酸。~ 1H-~ 1H COSY、HMQC和HMBC测定结果表明，存在Chloro Hpla（3-chloro-4-hydroxyphenyllactic acid）、allo-Ile、Cho sulfate（2-carboxy-6-hydroxyoctahydroindolsulfate）和agmatin。HMBC光谱的解释揭示了1的结构。通过手性GC分析确定allo-Ile的立体化学为D-型。用NOESY光谱确定了其相对立体化学结构。2和3的结构与1的不同之处仅在于Chloro Hpla的苯环被取代。化合<50>物1、2和3对胰蛋白酶的IC分别为0.55、0.6和3.93 mg/mL，经HRFABMS和NMR数据确定化合物5的分子式为C_<34>H_<59>N_9O_<14>S_3。1H-^1HCOSY、HOHAHA、HMQC和HMBC数据显示存在Hphpa三硫酸盐（1-（4-羟基-3-羟甲基）苯基-1-羟基-2-丙胺4 '，3'，1-三-O-硫酸盐）、N-MeArg和NmgArg（N，α-甲基-N，N-香叶基精氨酸）。HMBC和NOESY光谱的解释揭示了5的结构。4和6的结构被确定为5的类似物，具有不同的N-MeArg侧链。这些化合物对P388白血病细胞显示出弱的细胞毒性。

项目成果

M.Murakami: "Aeruginosin 98-A and B,Trypsin Inhibitors from the Blue-Green Alga Microcystis aeruginosa (NIES-98)" Tetrahedron Lett.(in press).

M.Murakami：“铜绿素 98-A 和 B，来自蓝绿藻铜绿微囊藻 (NIES-98) 的胰蛋白酶抑制剂”四面体快报（正在出版）。

Masahiro Murakami: "Aeruginosin 98-A and B,Trypsin Inhibitors from the Blue-Green Alga Microcystis aeruginosa(NIES-98)" Tetrahedron Letters. (in press.).

Masahiro Murakami：“铜绿素 98-A 和 B，来自蓝绿藻铜绿微囊藻 (NIES-98) 的胰蛋白酶抑制剂”四面体信件。