| 摘要: | 銅綠微囊藻(Microcystis aeruginosa)被認為是自然界當中具有豐富胜肽類的生物資源的一種，一直以來，本實驗室僅專注於單離藻株的培養和其微囊藻毒之提純與結構解析而對於其他二次代謝物接觸較少。本實驗接續了余(2013)在回收非毒性M.TY-1萃取液(已提取其中之Microcystin-LR)中提純micropeptin TY1042、TY1046之研究，進行藻細胞的大量培養、溶劑萃取、配比分離、速分管柱層析及高效液相層析分離，共純化出五個化合物，經由比對前人所分離化合物的層析與核磁共振1D NMR圖譜，確認其中為兩個微囊藻毒Microcystin-LR與[Asp3] Microcystin-LR及三個micropeptin類化合物Micropeptin TY 1046、Micropeptin TY1042、Micropeptin TY992。其中Micropeptin TY992為新的化合物，除與已知化合物比對波譜資料外，也透過2D NMR: HSQC, HMBC, 1H-1H COSY等核磁共振波譜實驗解析其結構。本研究亦修正余(2013)論文中錯誤的micropeptin結構解析進一步的與曹等(unpublish data)的合作，針對micropeptin的組成胺基酸採用高效液相層析的手性分離並分析其酸水解產物的Marfey反應產物鑑定各胺基酸手性碳的立體構型。三個micropeptin依分子量大小差異鑑定其僅在一個胺基酸位置上的取代差異，依序為白胺酸Leu, 酪胺酸Tyr, 及對位羥基環己烯基丙胺酸HcAla的取代。;Microcystis aeruginosa, a cyanbacteria, has been known rich in polypeptides nature products. In the past, our lab in NTU has been working on cultures of the toxic colones of this species and the structure identification of toxins isolated from these clones, and comparably less attentions on other natural products. This study continued the research of Yu, 2013 in recovering the bioactive micropeptin TY1042、TY1046 from the nontoxic fractions of M.TY-1, clone for Microcystin-LR production. Through the mass culture of this species in enriched medium, solvent extractions of the harvested cell masses, solvent partitions, fractionations of a flash column chromatography, and subsequent purifications of a high performance column chromatography (HPLC), we isolated five components. Four of them were identified by the comparisons of chramtographic data, 1D Nuclear Magnetic Resornance (NMR) spectra of previous studies as microcystin-LR, 3-demethylmicrocystin-LR, micropeptin TY1046, micropeptin TY1042 and the rest, micropeptin TY992, a new compound ever published. Structures of these compounds were further confirmed by 2D NMR, including HSQC, HMBC, and 1H-1H COSY where the advanced experiments were needed. We made some corrections on the micropeptin structures determined by Yu, 2013 and we also determined the stereochemistry of the chiral carbons of micropeptins by HPLC analysis of the chiral products from Marfey’s reaction of the mcropeptin hdrolysates through the collaboration with Tsao, 2015. These three micropeptins, namely TY992, TY1042, and TY1046 conatin different L-amino acid, leucine, tyrosine, p-hydroxy cyclohexenyl alanine (HcAla) respectively at the 5 position of their polypeptide chain. |
