寡醣是自然界中含量最豐富的生物寡聚合物，在許多生物和病理過程中發揮重要的作用。Ganglio-系列醣體是具有多唾液酸的鞘醣脂，能夠與特定唾液酸免疫球蛋白產生親合作用力進而調節生物的免疫功能。Globo-系列醣體主要為中性的鞘醣脂，與許多的疾病及腫瘤的發展有密切的關聯，具有作為抗癌疫苗與藥物的潛力。然而一些結構較為複雜的鞘醣脂，包括 GP1c、Globo A 與Globo B，在生物體內的含量稀少、取得不易，使我們難以對其生物功能與機制做更進一步的研究，因此在本篇論文中藉由實驗室建立的酵素系統有效率的合成出 c-系列神經節苷脂和 Globo A 與 Globo B 等天然鞘醣脂，以及利用 UDP-GalNH2 作為醣核苷予體合成 Forssman antigen 等醣體。
此外，人類母乳寡醣 (HMOs) 是母乳的第三大成分，能夠預防細菌感染、培養健康的腸道菌群和調節免疫反應等，對嬰幼兒的健康有許多益處。然而不對稱支鏈型 HMOs 結構複雜且種類繁多，因此開發快速、有效率合成這類醣體的方法有助於了解醣體中不同結構的生物活性與特性。在本篇論文中，為了克服合成複雜支鏈聚醣的困難，利用兩個正交的胺保護基 N3 和 Boc 作為抑制半乳醣基轉移酶活性的基團，首先以酵素方法建構一端含有 N3 修飾的不對稱四醣核心，再藉由改變酵素催化選擇性合成出多種以 LNO 和 iLNO 為骨架的不對稱雙支鏈和三支鏈 HMOs，最後比較不同醣體結構與醣基轉移酶催化效率的關係，了解酵素對此類複雜醣體結構的催化特性。

Oligosaccharides are the most abundant biopolymers in nature and play essential roles in many important biological and pathological processes. Gangliosides, sialic acid-containing glycosphingolipids (GSLs), can interact with specific sialic acid-binding Ig-like lectins (Siglecs) to regulate the immune function of organisms. Globo-series glycan, mainly neutral GSLs, are associated with the development of various diseases and tumors and have the potential to be developed as anticancer vaccines and drugs. However, some structurally complex GSLs, including GP1c, Globo A, and Globo B, are scarce and difficult to be obtained from natural sorces. Therefore, in this thesis, chemoenzymatic methods were developed to synthesize these oligosaccharides to facilitate future investigation of their characteristics and functions.
Furthermore, human milk oligosaccharides (HMOs), third major components of breast milk, can provide significant beneficial effects to health by preventing infection, nurturing healthy gut microiota, and maintaining immune homeostasis. However, complex structures of asymmetrically branched HMOs make them difficult to be synthesized. Thus, it is important to develop efficient methods for synthesizing these glycans to understand their biological functions. In this thesis, to overcome the challenge in the synthesis of complex branched glycans, two orthogonal amino protecting groups, N3 and Boc, were applied to inhibit the activities of galactosyltransferases and fucosyltransferases. With these efficient site-selective chemoenzymatic strategies, we accessed a series of asymmetric, multiantennary LNO-based and iLNO-based HMOs. Finally, the relationships between different glycan structures and glycosyltransferase catalytic efficiency were compared to understand the catalytic characteristics of enzymes towards such complex glycan structures.

摘要 I
Abstract II
謝誌 III
目錄 V
圖目錄 IX
表目錄 XII
流程目錄 XIV
縮寫表 XVIII
酵素全名與來源表 XXI
單醣中英文名稱與代表符號 XXIII
第一章、緒論 1
1.1 醣體與腫瘤相關醣體抗原 1
1.2 醣神經鞘脂質 3
1.2.1 神經醯胺的重要性 5
1.2.2 唾液酸 7
1.2.3 唾液酸免疫球蛋白凝集素 8
1.3 Ganglio-系列抗原 11
1.3.1 c-系列神經節苷脂 13
1.3.2 以酵素方法合成神經節苷脂文獻回顧 14
1.3.3 GP1c合成文獻回顧 17
1.4 Globo-系列抗原 19
1.4.1 合成 Globo-系列醣體文獻回顧 22
1.4.2 Forssman 與 para-Forssman 抗原 26
1.4.3 Globo A 與 Globo B 29
1.5 人類母乳寡醣 31
1.5.1 支鏈型不對稱母乳寡醣 32
1.5.2 不對稱合成策略之建構 35
1.6 醣激酶與轉移酶 37
1.6.1 醣激酶 (GalK、NahK、FKP) 38
1.6.2 磷酸醣核苷轉移酶 (AtUSP、AGX-1、CSS) 41
1.6.3 唾液酸轉移酶 (Cst-I、Cst-II、Psp2,6ST、Pd2,6ST) 43
1.6.4 N-乙醯半乳醣胺轉移酶 (BgtA、CgtA) 46
1.6.5 半乳醣轉移酶 (CgtB、BvαGalT、h13GTB、LgtB、HP0826、CvGalT) 47
1.6.6 N-乙醯半乳醣胺轉移酶/半乳醣轉移酶 (LgtD) 51
1.6.7 岩藻醣轉移酶 (FutC、FucTa) 52
1.6.8 N-乙醯葡萄醣胺轉移酶 (HP1105、GCNT2) 53
1.6.9 唾液酸水解酶 (SpNanA) 55
1.7 研究動機與目標 56
第二章、實驗結果與討論 57
2.1 以大腸桿菌誘導表現目標蛋白 57
2.1.1 勝任細胞 57
2.1.2 目標蛋白純化系統 58
2.1.3 酵素表達分析 60
2.2 以哺乳類細胞誘導目標蛋白 69
2.2.1 GCNT2 (β-1,6-GlcNAcT) 表達分析 70
2.3 酵素系統合成 c-系列神經節苷酯醣體 71
2.3.1 GM3 醣體合成 71
2.3.2 GT3 醣體合成 72
2.3.3 GT2 醣體合成 73
2.3.4 GT1c 醣體合成 74
2.3.5 GQ1c 醣體合成 75
2.3.6 GP1c 醣體合成 76
2.4 化學酵素系統合成 Forssman 與 para-Forssman 抗原 80
2.4.1 Forssman 與 para-Forssman 抗原合成路徑探討 80
2.4.2 Gb4NH2 醣體合成 82
2.4.3 para-Forssman antigen 醣體合成 83
2.4.4 Forssman antigen 醣體合成 83
2.5 化學酵素系統合成 globo-系列醣神經鞘脂質 84
2.5.1 Gb4-Sph 醣體合成 85
2.5.2 Gb5-Sph 醣體合成 86
2.5.3 Globo H-Sph 醣體合成 87
2.5.4 Globo A 與 GloboB 醣體合成路徑探討 88
2.5.5 Globo A-Sph 醣體合成 89
2.5.6 Globo B-Sph 醣體合成 90
2.5.7 Globo A-Cer 與 GloboB-Cer 合成 91
2.6 化學酵素方法合成不對稱人類母乳寡醣 92
2.6.1 不對稱四醣核心 (27) 建構 93
2.6.2 疊氮基 (N3) 修飾六醣中間體 (29) 合成 95
2.6.3 iLNO 醣體合成 97
2.6.4 DF-iLNO V 醣體合成 98
2.6.5 不對稱雙支鏈醣體 34 合成 99
2.6.6 DF-LNO I 醣體合成 101
2.6.7 TF-LNO 醣體合成 103
2.6.8 DF-iLNO VI 醣體合成 104
2.6.9 LNO 醣體合成 109
2.6.10 不對稱雙支鏈醣體 52 合成 113
2.6.11 不對稱雙支鏈醣體 54 合成 114
2.6.12 F-LNO 醣體合成 115
2.6.13 DF-LNO II 與 DF-iLNO III 醣體合成 116
2.6.14 DF-iLNO II 醣體合成 117
2.6.15 LNnD 醣體合成 117
2.6.16 F-LNnD I 醣體合成 119
2.6.17 醣體結構與醣基轉移酶催化效率分析 122
第三章、結論與未來展望 127
第四章、實驗材料與方法 130
4.1 Materials 130
4.1.1 General information 130
4.1.2 Instruments 131
4.1.3 Bacterial strains 132
4.1.4 Vectors 132
4.2 Overexpression and purification of target enzymes 133
4.2.1 General procedure of E. coli expression system 133
4.2.2 General procedure of purification of target enzymes 134
4.2.3 General procedure of Expi293™ expression system 135
4.3 Synthetic procedures and characterization 137
4.3.1 Synthesis of UDP- Galactosamine 137
4.3.2 General enzymatic synthetic procedure of HMOs 137
4.3.3 General purification procedure 138
4.3.4 HPLC purification procedure 139
4.3.5 The synthesis and characterization of compounds 139
第五章、參考文獻 195
附錄目錄 208

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