擁有各種多樣化的寡醣體對於研究功能性醣體學來說是非常重要的，因不同
醣體在生物體中存在其特定生物活性。為了瞭解這些醣體之生物功能，發展出許
多化學或是化學酵素方法來建立直鏈型或是支鏈型醣體。相較於直鏈型醣體，在
合成支鏈型或是不對稱型醣體上更具有挑戰性。因此，發展簡單、具系統性之複
雜寡醣體合成策略 快速取 這些不對稱醣體，有利於研究、了解他們的生物活性。
本論文第一部分探討細菌來源的四種半乳醣基轉移酶 (GalTs) 及一種岩藻
醣基轉移酶 (FucT) 對於各自修飾過受質之反應活性。我們探討在葡萄醣胺上不
同的N-保護基是否會對這些GalTs及FucT有抑制催化之活性。我們順利地發現
一些特定N-保護基能成功的抑制 GalTs及 FucT之催化活性。藉由篩選出來之特
定N-保護基進行選擇性醣基化反應，我們快速地合成出4120a與5130b兩種支
鏈型人類母乳寡醣 (Branched HMOs)。
在第二部份我們合成具有位置選擇性岩藻醣基化之 VIM-2抗原及其類似物。
雖然已經知道 VIM-2抗原是 E-選擇素的結合配體，但尚未有任何文獻研究在不
同位置岩藻醣基化之 VIM-2抗原類似物與 E-選擇素之間的結合關係。因此，我
們使用控制 N-保護基策略來進行選擇性岩藻醣基化。最後，我們也利用此策略
完成七種 VIM-2抗原類似物的建構。我們透過探討這些醣基轉移酶對於不同受
質之容忍度測試，拓展這些酵素對於位置選擇性醣基化之催化能力。期望透過這
些醣基轉移酶對不同受質有不同催化能力之特性，能更進一步應用於合成更複雜
之 N-聚醣 (N-glycans) 或是人類母乳寡醣。

Accessibility of various kinds of diverse oligosaccharides are essential for the studies of functional glycomics and understanding of their unique biological activities in organisms. In order to realize their biological functions, many chemical and chemoenzymatic methods have been developed to synthesize linear and branched glycans. Compared to linear glycans, it is more challenging to synthesize branched or asymmetric glycans. Hence, developing a facile and systematic method to provide these complicated oligosaccharides will facilitate the progress of understanding the biological activities of asymmetric glycans.
In the first part of this research, we focused on the study of the substrate tolerance of four galactosyltransferases (GalTs) and one fucosyltransferase (FucT) from bacteria. The effect of N-protecting group on glucosamine to inhibit the catalytic activities of GalTs and FucT was investigated. Fortunately, we found that some of N-protecting groups can inhibit the activities of GalTs and FucT. With the help of these specific N-protecting groups, we can easily manipulate site-selective glycosylation to synthesize branched HMOs, 4120a and 5130b.
In the second part, we developed an efficient route to synthesize site-selectivily fucosylated VIM-2 antigen and its analogs. Although VIM-2 antigen is a known ligand of E-selectin, there is not many research about the binding affinity between E-selectin and VIM-2 antigen analogs with various fucosylated site(s). Therefore, we utilized the N-protecting group strategy to investigate the site-selective fucosylaiton. Finally, seven site-selective fucosylation VIM-2 antigen analogs were synthesize. The discovery of acceptor tolerance of these glycosyltransferases expands the scope of their use in site-selective glycosylation. These features can be applied to improve the synthetic efficiency of more complicated N-glycans or HMOs.

中文摘要-------------------------------------------------------------I
Abstract-----------------------------------------------------------II
謝誌---------------------------------------------------------------III
縮寫表-----------------------------------------------------------XVIII
酵素縮寫表---------------------------------------------------------XIX
第一章、緒論---------------------------------------------------------1
1.1. 人類母乳寡醣 (HMOs)---------------------------------------------1
1.1.1. 直鏈型人類母乳寡醣 (Linear HMOs)-------------------------------3
1.1.2. 支鏈型人類母乳寡醣 (Branched HMOs)-----------------------------4
1.2. 不對稱支鏈型寡醣合成策略-----------------------------------------6
1.2.1. 應用於不對稱N-聚醣 (N-glycan) 之合成策略-----------------------6
1.2.2. 應用於不對稱HMOs之合成策略------------------------------------12
1.3. 醣激酶與醣基轉移酶----------------------------------------------16
1.3.1. 醣激酶 (NahK、MtGalK、FKP)-----------------------------------18
1.3.2. 磷酸醣核苷轉移酶 (AtUSP、AGX1)--------------------------------22
1.3.3. 胞苷合成酶 (CMP-Sialic acid synthetase, CSS)-----------------25
1.3.4. 醣基轉移酶 (GTs)---------------------------------------------26
1.4. 研究動機-------------------------------------------------------41
第二章、 醣基轉移酶受質容忍度、不對稱HMOs合成之實驗結果與討論-----------42
2.1. 醣基轉移酶受質容忍度測試應用於不對稱支鏈HMOs合成之策略設計---------42
2.2. 大腸桿菌誘導表現目標蛋白----------------------------------------42
2.2.2. 目標蛋白純化系統---------------------------------------------43
2.2.3. 酵素表達之分析-----------------------------------------------47
2.3. β1,3/4-半乳醣基轉移酶 (β1,3/4-GalTs) 對不同N-修飾葡萄醣胺 (GlcNR) 之受質容忍度測試-----------------------------------------------------57
2.3.1. N-修飾葡萄醣胺受質 (GlcNR) 之合成-----------------------------57
2.3.2. β1,3/4-GalTs受質容忍度測試------------------------------------60
2.4. α1,3/4-岩藻醣基轉移酶 (α1,3/4-FucT) 對不同N-修飾乳醣胺 (LacNR) 之受質容忍度測試--------------------------------------------------------67
2.4.1. N-修飾乳醣胺受質 (LacNR) 之合成-------------------------------67
2.4.2. α1,3/4-FucT受質容忍度測試-------------------------------------71
2.5. 不對稱雙支鏈HMOs合成--------------------------------------------75
2.5.1. 不對稱雙支鏈HMOs合成策略設計----------------------------------75
第三章、VIM-2抗原及其類似物醣體分子庫---------------------------------91
3.1. 選擇素 (Selectin)----------------------------------------------91
3.1.1. E-選擇素 (E-selectin)----------------------------------------93
3.2. VIM-2抗原------------------------------------------------------96
3.3. 化學或酵素方法建構VIM-2抗原-------------------------------------97
3.4. 選擇性岩藻醣基化策略--------------------------------------------99
3.5. 研究動機------------------------------------------------------101
第四章、合成VIM-2抗原及其類似物醣體分子之實驗結果與討論----------------102
4.1. 位置選擇岩藻醣基化 (Site-selective fucosylation) 策略合成VIM-2抗原 ------------------------------------------------------------------102
4.2. 叔丁氧羰基 (Boc) 保護基抑制岩藻醣基化之測試----------------------102
4.2.1. 叔丁氧羰基修飾六醣體之合成------------------------------------102
4.2.2. FucTa對不同六醣體選擇性岩藻醣基化之測試-----------------------106
4.3. 合成VIM-2抗原及其類似物醣體分子---------------------------------107
4.3.1. 合成VIM-2抗原類似物51、56與57--------------------------------108
4.3.2. 合成VIM-2抗原類似物52與54------------------------------------129
第五章、結論與未來展望----------------------------------------------150
第六章、實驗材料與方法----------------------------------------------151
6.1. Materials and Methods----------------------------------------151
6.1.1. General information----------------------------------------151
6.1.2. Equipments used in experiments-----------------------------152
6.1.3. Commercially available enzymes-----------------------------153
6.1.4. Vectors----------------------------------------------------154
6.1.5. E. coli strains--------------------------------------------154
6.2. Enzyme overexpression----------------------------------------155
6.2.1. Overexpression of target enzymes---------------------------155
6.3. Investigation of β1,3/4-GalTs acceptor tolerance-------------158
6.4. Investigation of α1,3/4-FucT acceptor tolerance--------------158
6.5. Synthetic procedures and characterization--------------------159
6.5.1. Synthesis of sugar donor-----------------------------------159
6.5.2. Glycosyltransferase-catalyzed reactions and Purification of Enzymatic reaction products---------------------------------------160
6.5.3. Compound characterization----------------------------------163
第七章、參考文獻----------------------------------------------------213
光譜附錄-------------------------------------------------------------i

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