細胞表面硫酸乙醯肝素寡醣分子庫的合成研究

Synthesis of Cell Surface Heparan Sulfate Oligosaccharide Library

陳智庚(Zhi-Geng Chen)

指導教授 ： 廖文峯 ； 洪上程

國立清華大學/理學院/化學系/博士(2013年)

摘要

硫酸乙醯肝素（heparan sulfate，HS），是屬於葡胺巨醣（glycosaminoglycan，GAG）的生物分子，在許多重要的生理機制中，扮演著重要的角色，例如：病毒感染、血液凝固、人體的發炎反應、促進細胞附著、細胞生長調控或腫瘤轉移。硫酸乙醯肝素是具有負電荷性質的多醣體，主要的結構是由D式葡萄胺醣（GlcN）衍生物α-1,4方式鏈結D式葡萄醣酸（GlcA）或是L式艾杜醣酸（IdoA）。然而，因為其結構組成的多變，以至於許多重要多醣體的真實結構仍未被了解，而透過化學合成方法，不僅可確認其真實結構，且可大量製備出所需的醣體，為了能了解硫酸乙醯肝素與蛋白質之間的作用關係，在此計畫，透過化學合成的方式，合成出不同長度的寡醣體分子庫，希望藉由寡醣分子庫能更加了解，硫酸乙醯肝素在各種不同蛋白質之中所扮演的角色。
首先，將合成硫酸乙醯肝素結構中的最小重複單元雙醣體分子庫，其合成策略以還原端為GlcA或IdoA雙醣體（GlcN-GlcA/IdoA）為共同中間體並結合不相互干擾的保護基，去合成所有48種可能結構的雙醣分子庫，本論文將介紹其中12種雙醣的詳細合成步驟，完成48種雙醣分子庫之後，初步進行雙醣體與蛋白質（FGF-1）的生物活性測試，發現4個具有最佳生物活性的雙醣體，並透過X-ray解出雙醣體與蛋白質複合體的結構，藉此探討兩者之間的作用關係。
在完成雙醣體分子庫的研究工作之後，選擇六醣體分子庫為下一個目標分子，然而在硫酸乙醯肝素寡醣體的合成中，最重要的是控制葡萄胺醣與醣酸鏈結的立體位向為1,4-順式（1,4-cis），因此利用本實驗室已開發的葡萄胺醣單醣單元，藉此控制得到單一位向的雙醣體或多醣體，此外，為了提高分子庫的合成效率，決定發展新式的一鍋化醣鏈結反應搭配不相互干擾的保護基，最大的特點在於不需製備各種不同的建構單元，即能完成所有六醣體的合成，其策略為，利用兩種硫苷雙醣衍生物（GlcN-GlcA/IdoA）為中間體，透過一鍋化醣鏈結反應，可以快速且高效率的合成出所有的六醣體骨架，並完成其中4個最終六醣產物。

關鍵字

硫酸乙醯肝素 ； 葡胺巨醣 ； 一鍋化醣鏈結 ； 醣共軛分子

並列摘要

Heparan sulfate (HS) is a member of the glycosaminoglycan (GAG) family and plays significant roles in a diverse set of biological processes, including viral infection, blood coagulation, inflammatory response, cell adhesion, cell growth regulation, and tumor metastasis. HS are consisting of linear polyanionic polysaccharides characterized by a repeating disaccharide unit of D-glucosamine (GlcN) attached to uronic acid [either L-iduronic (IdoA) or D-glucuronic acid (GlcA)]. However, unraveling the HS code has been extremely challenging due to the enormous structural variations. Chemical synthesis is considered as one of the most practical way to obtain the pure and structurally verified oligosaccharides at a considerable scale. To understand HS interaction with protein in detail, we investigated the possibility of assembling a library of HS oligosaccharides
Here we employed a divergent strategy to afford a HS repeating disaccharide unit of all 48 theoretical possibilities from just two orthogonally protected disaccharide (GlcN-GlcA/IdoA) precursors. This dissertation describes the detail synthetic steps to achieve 12 disaccharides. With the full disaccharide library in hand, affinity screening with fibroblast growth factor-1 (FGF-1) revealed that four of the synthetic sugars bind to FGF-1. The molecular details of the interaction were further clarified through X-ray analysis of the sugar–protein co-crystals.
After successfully studying the disaccharide library, we choose the hexasaccharide library as our next target. A major challenge in HS oligosaccharide synthesis is stereoselective α 1,4-cis-glycosydic linkage between glucosamine and the uronic acid. Therefore, we choose a new glucosamine building block carrying the orthogonal and stero-directing protecting groups for the formation of cis-1,4-linked

product. On the other hand, in order to achieve the rapid construction of the hexasaccharide library, we developed a new one-pot glycosylation strategy. In addition, from just two orthogonally protected disaccharides all of the required thioglycoside disaccharide modules for library preparation could be generated in a divergent manner, which greatly simplified the building block preparation. This allowed rapid assembly of HS hexasaccharides with systematically varied and precisely controlled backbone structures in a combinatorial fashion. This dissertation summerizes the detail synthetic steps to achieve 4 hexasaccharides.

並列關鍵字

heparan sulfate ； glycosaminoglycan ； one-pot glycosylation ； glycoconjugates

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