臺灣博碩士論文加值系統

唾液酸（Sialic acid）通常位於醣複合體（Glycoconjugate）的非還原端（Non-reducing end），例如位於細胞表面的醣蛋白（Glycoprotein）或醣脂質（Glycolipid）上；唾液酸路易士X（Sialyl Lewis X）或磺基化唾液酸路易士X（Sulfated sialyl Lewis X）已知為選擇素（Selectin）辨識所需要的四醣抗原決定位（Epitope），唾液酸6-O-磺基路易士X為表現在人體周圍淋巴結（Human peripheral lymph nodes）的高內皮小靜脈細胞表面（High Endothelial Venules；HEV），或人類皮膚歸巢輔助記憶T細胞（Skin-homing human helper memory T-cells）表面，它已知可與L-選擇素辨識而結合。然而，先前研究發現唾液酸的環化，也就是說唾液酸的五號N-乙醯胺基去乙醯化，而與一號的羰酸基結合成內醯胺（Lactam），使得相關的醣分子失去了與L-選擇素的結合力，而這樣的結合作用也曾被報導參與淋巴細胞的歸巢作用。
除此之外，有許多研究指出，負責修飾N-乙醯葡萄糖胺三號碳及六號碳上羥基，分別受到岩藻糖轉移酶或磺基化轉移酶的修飾，對於L-選擇素的辨識非常重要，而且它們調控著淋巴細胞的運輸（Lymphocyte trafficking）；也就是說，這樣高度專一性的結合力是透過辨識不同結構之醣體來調控著下游的生物途徑（Biological processes）。為了進一步研究是什麼因素影響著醣體中唾液酸的環化作用，本論文中首先著重於合成一系列環化唾液酸6-O-磺基路易士X以及其衍生物。由於這些醣體分子過去透過化學全合成的方法製備，往往產生的低產率及繁瑣的反應步驟，我們發展以化學結合酵素的合成方法來簡化及加速目標醣體的製程，獲得令人滿意的產率。
在鑑定醣體分子結構後，我們分別建立了核磁共振光譜以及逆向液相層析儀的分析方法，測量各種醣體的環化速率，由實驗可以觀察到磺基化修飾可增加2.30倍，而岩藻糖化修飾則減緩反應速率至0.78倍，若同時修飾磺基與岩藻糖則為1.51倍，因此推測此兩種修飾對環化反應有著拮抗作用，而當由第二型醣鏈（1,4鍵結）置換為第一型醣鏈（1,3鍵結）後反應速率也隨之下降，為了更深入了解這些修飾是如何影響反應速率，分子模擬運算（Molecular Modeling）能提供一些線索，由電腦分析結果得知，磺基基團會與唾液酸支鏈上九號位置的羥基有交互作用，而使整個醣體分子偏好形成C構型，增加羰酸基暴露在外圍環境，後續實驗也證明活化羰酸基為醯胺鍵生成反應（Amide bond formation）的重要步驟，因此能加快環化的速率，由實驗結果得知官能基的修飾對於醣體在液態環境下會形成不同的立體結構，而這樣的改變是否為影響醣體在生物系統裡所執行的任務仍有待進一步實驗的觀察。

Sialic acids are often located in the non-reducing end of glycoproteins and glycolipids, on both the cell surface and intracellular membranes. Sialyl Lewis X or its sulfated derivatives are the essential tetrasaccharide epitopes for recognition by selectin glycoproteins. Sialyl 6-sulfo Lewis X was reported as an L-selectin ligand that is displayed on the surface of high endothelial venules (HEV) in human peripheral lymph nodes as well as skin-homing human helper memory T-cells. Interestingly, the selectin-binding activity of sialyl 6-sulfo Lewis X is abrogated due to the cyclization of its sialic acid moiety.
Serveral studies mediated that the O3 fucosylation and O6 sulfation on GlcNAc of sialyl LacNAc (Sia2-3Gal1-4GlcNAc) are crucial modifications for L-selectin binding to mediate the lymphocyte trafficking. That is to say, specific glycan structures may act as a marker to mediate this highly specific binding and the subsequent biological processes. To further investigate what factors (the presence of sulfate, L-fucose, etc) play a role in affecting the lactam formation, sialyl 6-sulfo Lewis X and its analogues needed to be synthesized in advance.
Since many reaction steps, low total yields and tedious protection/deprotection steps are major obstacles for the chemical synthesis of cyclic sialyl 6-sulfo Lewis X, chemoenzymatic synthesis is an atractive approach to obtain the desire glycans in higher yields. The total yields of 6 target glycans, (compounds 1-6), were greatly improved in this work. Lactonization and molecular dynamic simulations were carried out to discuss the cyclization of sialic acid. The results indicated that rate of lactamization was accelerated in the presence of O6 sulfate owing to the formation of a C-shaped conformation.

口試委員會審定書 #
誌謝 i
縮寫對照表 ii
中文摘要 iii
ABSTRACT v
CONTENTS vi
LIST OF FIGURES viii
LIST OF TABLES xiii
Chapter 1 緒論 1
1.1 含有唾液酸的醣分子：結構及功能 1
1.2 唾液酸6-O-磺基路易士X的生合成 4
1.3 O-磺基化唾液酸路易士X具有重要的生理意義 7
1.4 利用酵素特性製備寡糖 9
1.5 以一鍋化（One-pot）的系統來合成核苷酸醣以及目標醣體 11
1.6 發展以一鍋化系統合成具有後醣化修飾的醣體 15
1.7 唾液酸轉移酶 17
1.8 酵素合成唾液酸6-O-磺基路易士X 19
1.9 化學合成環化唾液酸6-O-磺基路易士X 23
1.10 研究動機 26
Chapter 2 結果 28
2.1 製備環化唾液酸6-O-磺基路易士X衍生物 28
2.1.1 以酵素催化的方式製備雙醣7以及雙醣8 30
2.1.2 測試Neisseria gonorrheae的2,3-唾液酸轉移酶對非磺基化及6-O-磺基化基質的專一性 34
2.1.3 以酵素催化的方式製備N-三氟乙醯基修飾的唾液酸三醣9、10、15、16以及唾液酸四醣15、16 38
2.1.4 以化學合成的方式製備環化衍生物1至6 42
2.2 鑑定環化衍生物及建立環化速率測定的方法 45
2.2.1 鑑定環化衍生物1至6 47
2.2.2 以NMR方法偵測環化反應 50
2.2.3 以HPLC測定各環化衍生物之反應速率 51
2.3 透過內酯化反應及分子模擬來探討影響環化反應的決定性因子 55
2.3.1 唾液酸羰酸基的內酯化促進環化衍生物的生成 56
2.3.2 由分子模擬推測醣體的修飾對於環化作用的影響 60
Chapter 3 討論 64
3.1 以化學生物合成策略來製備醣複合體 64
3.2 以化學酵素法合成具有生物重要性之唾液酸苷 65
3.3 以唾液酸內酯化來看磺基化對於唾液酸醯胺化的影響 66
3.4 岩藻糖影響唾液酸環化的原因 67
3.5 自然界中自發性分子內醯胺鍵生成的案例 70
Chapter 4 實驗步驟 74
參考文獻 101
Appendix: 1H/ 13C NMR Spectra 124

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