Globo-系列醣體及 sialyl Lewis a (sLea) 分別與乳癌及結腸癌有著極大的關聯，在正常細胞上卻鮮少觀察到它們的存在，因此這些醣體具有發展成抗癌疫苗的潛力。除此之外，常表現於神經細胞表面的 Ganglio-系列醣體亦相當重要，其疏水端的神經醯胺會嵌入細胞膜，親水端的醣體則與唾液酸免疫球蛋白有特定的作用力。因此神經節苷脂的取得有助於了解生物體內醣體與蛋白質之間的親和力
本篇論文利用多種酵素，並以醣核苷再生系統及連續性酵素催化系統合成一系列醣體。其中在神經節苷脂 GD1a 及 GM1 的α2,6-唾液酸化遇到選擇性問題，無法專一將唾液酸鍵結於 Gal 或是 GalNAc，而 GM1b 及 GA1 的α2,6-唾液酸化則可以有特定的產物；在 sialyl Lewis a 的部分，FucTIII 無法專一地將岩藻糖鍵結於內部 GlcNAc，導致生成鍵結於還原端 Glc 的異構物；而在 Globo-series 的部分，Globo H 醣體可以透過酵素法催化合成得到，最後再將含有馬來醯亞胺的連接物利用醯胺鍵的方式連結至醣體 (Globo H-maleimide)。

Globo-series glycans and sialyl Lewis a (sLea) are strongly associated with malignant diseases such as breast or colon cancers while they are rarely detectable in normal cells. Therefore, these glycans have good potential to be developed as anticancer vaccine. Furthermore, ganglio-series glycans are also vital since they are widely expressed on vertebrate cells and nerve cells. Ceramide of gangliosides are anchored to the plasma membrane while glycans extend into the extracellular space through sugar-specific interactions with glycan binding proteins such as Siglecs (sialic-acid-binding Ig-like lectins). Thus, obtainning gangliosides is necessary to elucidate the physiological mechanism of glycan-protein interaction.
In this thesis, multiple enzymes were applied on the enzymatic synthesis of glycans in regeneration system and sequential one-pot system. The challenge in synthesis of gangliosides, GT1aα and GD1α, was selectively sialylation at C6-OH in either Gal or GalNAc. As for sLea, the same problem we are facing is the selective fucosylation in either inner GlcNAc or Glc. For further application, Globo H with maleimde linker can be chemoenzymatically synthesized and conjugated with carrier protein for developing anticancer vaccine.

摘要 I
Abstract II
謝誌 III
目錄 V
圖目錄 XII
表目錄 XVII
流程目錄 XVIII
縮寫表 XX
酵素中英文名稱對照表 XXII
單醣中英文名稱及代表符號 XXIV
第一章、緒論 1
1.1 腫瘤相關醣體抗原 1
1.1.1 Ganglio-series 3
1.1.1.1 神經相關神經節苷脂 5
1.1.1.2 癌症相關神經節苷脂 5
1.1.1.3 gangliosides 合成文獻回顧 6
1.1.2 Globo-series 9
1.1.2.1 癌症相關醣體 10
1.1.2.2 globo-series 醣體合成文獻回顧 13
1.1.3 Lacto-series 18
1.1.3.1 母乳寡醣 18
1.1.3.2 癌症相關醣體 19
1.1.3.3 sLea 合成文獻回顧 21
1.2 唾液酸 22
1.3 唾液酸免疫球蛋白凝集素 24
1.3.1 唾液酸免疫球蛋白凝集素-4 (Siglec-4) 26
1.4 醣激酶與轉移酶 28
1.4.1 醣激酶 29
1.4.1.1 半乳糖激酶 (MtGalK) 29
1.4.1.2 N-乙醯己糖激酶 (NahK) 30
1.4.1.3 岩藻糖激酶 (FKP) 31
1.4.2 磷酸醣核苷酸轉移酶 31
1.4.2.1 尿苷磷酸糖焦磷酸酶 (AtUSP) 32
1.4.2.2 磷酸-N-乙醯葡萄糖胺尿苷轉移酶 (GlmU) 33
1.4.2.3 尿苷二磷酸-N-乙醯半乳糖焦磷酸酶 (AGX1) 34
1.4.2.4 胞苷單磷酸唾液酸合成酶 (CSS) 35
1.4.3 醣基轉移酶 35
1.4.3.1 β-1,3-N-乙醯半乳糖胺轉移酶 / β-1,3-半乳糖轉移酶 (LgtD) 35
1.4.3.2 β-1,3-半乳糖轉移酶 (CgtB) 36
1.4.3.3 β-1,3-半乳糖轉移酶 (WbgO) 37
1.4.3.4 β-1,3-N-乙醯葡萄糖胺轉移酶 (HP1105) 38
1.4.3.5 β-1,4-N-乙醯半乳糖胺轉移酶 (CgtA) 39
1.4.3.6 β-1,4-半乳糖胺轉移酶 (NmGalT) 40
1.4.3.7 α-1,4-半乳糖轉移酶 (LgtC) 42
1.4.3.8 α-1,2-岩藻糖轉移酶 (FutC) 43
1.4.3.9 α-1,3-岩藻糖轉移酶 / α-1,4-岩藻糖轉移酶 (FucTIII) 43
1.4.4 唾液酸轉移酶 44
1.4.4.1 α-2,3-唾液酸轉移酶 (CstI) 44
1.4.4.2 α-2,6-唾液酸轉移酶 (Pd2,6ST) 45
1.4.4.3 α-2,6-唾液酸轉移酶 (Psp2,6ST) 47
1.5 研究目標與動機 48
第二章、實驗結果與討論 49
2.1 以大腸桿菌誘導表現目標蛋白 49
2.1.1 勝任細胞 49
2.1.2 目標酵素純化與表現分析 50
2.1.2.1 IMPACTTM 系統 50
2.1.2.2 IMAC 系統 51
2.1.2.3 麥芽糖結合蛋白 (maltose binfing protein) 純化系統 52
2.1.2.4 穀胱甘肽 S-轉移酶 (GST) 純化系統 53
2.1.3 酵素表達分析 54
2.1.3.1 GalK 55
2.1.3.2 NahK 56
2.1.3.3 FKP 57
2.1.3.4 AtUSP 58
2.1.3.5 GlmU 59
2.1.3.6 LgtD 60
2.1.3.7 CgtB 61
2.1.3.8 WbgO 62
2.1.3.9 HP1105 63
2.1.3.10 CgtA 64
2.1.3.11 NmGalT 65
2.1.3.12 LgtC 66
2.1.3.13 FutC 67
2.1.3.14 FucTIII 68
2.1.3.15 CSS 69
2.1.3.16 CstI 70
2.1.3.17 Pd2,6ST 71
2.1.3.18 Psp2,6ST 72
2.1.3.19 CMK 73
2.1.3.20 IP 74
2.2 酵素系統合成ganglio-series 75
2.2.1 asialo-series 75
2.2.1.1 GA2 醣體合成 75
2.2.1.2 GA1 醣體合成 77
2.2.1.3 GM1b 醣體合成 80
2.2.1.4 GM1α 醣體合成 80
2.2.1.5 GD1α醣體合成 86
2.2.2 a-series 93
2.2.2.1 GD1a 醣體合成 93
2.2.1.2 GD1aα 醣體合成 93
2.2.1.3 GT1aα 醣體合成 99
2.3 酵素系統合成 Globo H 104
2.3.1 Gb3 醣體合成 104
2.3.2 Gb4 醣體合成 105
2.3.3 Gb5 醣體合成 106
2.3.4 GbH 醣體合成 107
2.3.5 GbH-maleimide 合成 108
2.4 酵素系統合成 sialyl Lewis a (sLea) 109
2.4.1 LNTri II 醣體合成 109
2.4.2 LNT 醣體合成 109
2.4.3 sialyl Lewis a (sLea) 醣體合成 110
2.5 結論 114
第三章、未來展望 116
第四章、實驗材料與方法 117
4.1 Materials 117
4.1.1 General 117
4.1.2 Machines 118
4.1.3 Enzymes 119
4.1.4 Vectors 119
4.2 General procedure of overexpression and purification of target enzymes 121
4.2.1 General procedure of enzyme overexpression 121
4.2.2 Purification of target enzymes 122
4.2.2.1 IMPACTTM system 122
4.2.2.2 IMAC system 123
4.2.2.3 MBP system 124
4.2.2.4 GST system 125
4.3 Synthetic procedures and characterization 126
第五章、文獻參考 180
附錄目錄 196

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