α相位固醇/類固醇醣苷之合成研究

Abstract

皂苷為一種廣泛分布於植物、真菌以及低等海洋生物中的的次級代謝物質，依碳骨架結構的不同可分為四環三萜類以及五環三萜類，整體結構主要可分成兩部分:(1)親脂性的苷元 (2)親水性的糖基。而皂苷會因苷元連接醣數的差異，分為單醣鏈皂苷、雙醣鏈皂苷甚至三醣鏈皂苷；皂苷於藥用工業上有著極大的潛力，其可被安全的用於抗癌、抗微生物以及作為生物抑制劑等等，用途可說是相當的廣泛。皂苷的生物合成路徑整體分為三大步驟:(1) 最初的皂苷原碳骨架由氧化鯊烯環化酵素進行之環氧化形成 (2) 碳骨架上特定位置之碳原子與氫氧化酶產生氫氧化作用 (3) 醣基轉移酶於羥基上鍵結上醣基 形成醣苷鍵；轉醣作用中主要會鍵結起疏水性的苷元以及親水性的醣基，此外，皂苷上的醣基對於皂苷本身的生物活性有重要影響。許多研究指出醣化作用對於皂苷的生物活性扮演著相當重要的角色，此外也會對該化合物增進許多的溶解性以及穩定性。而我的研究中，因考慮到許多醣基不易取得或是價格昂貴，故第一步先針對不同的醣基來進行生物合成；接著進一步測試能否與轉醣酶和實驗室現有的四環固醇類成功催化出皂苷，如此一來，不僅可以解決材料上的問題，而且將會有許多種的皂苷可以繼續往後發展及研究。 在此論文中，主要對於比菲德氏嬰兒雙歧桿菌 (Bifidobacterium infantis)中的氮-乙醯六碳糖激酶 (N-acetylhexosamine 1-kinase, NahK)、大腸桿菌(Escherichia coli)中的半乳糖激酶(Galactokinase, Galk)、比菲德氏龍根菌(Bifidobacterium longum)中的尿苷二磷酸醣焦磷酸化酶(UDP-sugar pyrophosphorylase, USP)、多殺型巴斯德桿菌(Pasteurella Multocida)中的無機焦磷酸酶(inorganic pyrophosphatase, PpA)以及幽門螺旋桿菌(Helicobacter pylori)中的HP0421以上五條基因作為研究，其中一條的轉醣酵素基因： 幽門螺桿菌 (Helicobacter pylori) 中的 HP0421 因其特別的α- 轉醣機制，故為主要研究對象。而參考於先前學姊的研究結果，目前主要針對五種四環固醇類作為轉醣酵素的修飾對象。首先利用分子轉殖技術將其基因接合到表現載體上，並利用大腸桿菌 (E.coli) 系統進行蛋白質誘導後，對酵素進行活性分析。初步由薄層層析法確認活性，觀測是否有新分子的產生，接著以高效液相層析法做二次確認，此外兩者分析法皆可近一步做產物的分離。未來將藉由可被催化反應之受質來分析是否可進行更多不同的轉醣，亦或是可經由轉醣酵素形成更多不同組合的多糖鏈皂苷的結構，並以分子模擬的方式，更深入了解酵素催化機制，以期應用於未來之生物活性分析。

Saponins are widespread in our surroundings. They are a group of secondary metabolites expressed in plants, fungi and lower marine animals. Saponins can be divided into tetracyclic triterpenoids and pentacyclic triterpenoids based on their carbon skeletons. The whole structure is classified into two parts: hydrophobic sapogenin and hydrophilic sugar moiety. Saponins are divided into monosaccharide saponins, disaccharides saponins and trisaccharides saponins and have great potential for the pharmaceutical industry, they can be used as anti-cancer and anti-microbe agent as well as biochemical inhibitors. The biosynthesis of saponins are mainly achieved through the following three steps: (1) initial sapogenin backbones are formed by epoxidation of oxidosqualene cyclase; (2) hydroxylation of specific sapogenin carbon; and (3) glycosyltransferase catalyzes saccharides to form glycosidic bonds with sapogenins; glycosylation will bond hydrophobic sapogenin and hydrophilic sugar moieties. Moreover, glycosylation is particularly important for biological activity in several saponins. Furthermore, glycosylation can also improve solubility and stability. In my research, not only does this have an impact on biosynthesis of nucleotide diphosphate sugars, but it also catalyzes different sterols/steroids glycosides. Using this mechanism, development and research of glycosides would be much easier and diversified in the future. In this thesis, the following five genes were successfully cloned and expressed in an E. coli system: NahK (Bifidobacterium infantis), GalK (Escherichia coli), BLUSP (Bifidobacterium longum), PmPpA (Pasteurella Multocida) and HP0421 (Helicobacter pylori). All of these proteins were successfully purified using Ni-NTA chromatography. The glycosyltransferase HP0421 is of most interest due to its unique α-glycosylaion. According to previous studies, we focused on the activities of α-glycosyltransferase on five compounds: trans-androsterone, cis-androsterone, dehydroepiandrosterone, pregnenolone and cholesterol. They were determined and characterized using thin layer chromatography and high performance liquid chromatography.