GAA-7為Higuchi研究團隊於1993年從海星Linckia laevigata 體內萃取出的新型神經節苷脂，並利用大鼠腎上腺之嗜鉻細胞瘤作為媒介測試其神經再生活性，研究結果證實GAA-7比神經節苷脂GM1具有更高的神經再生活性，可望開發成為中樞神經系統藥物。在2015年，Kiso教授團隊完成了GAA-7的全合成，然而其過程繁瑣、耗時。
本論文成功地利用酵素結合化學方法，合成出14種GAA-7衍生物。首先以化學方法在唾液酸的八號位置修飾甲氧基、五號位置修飾羥乙醯基，接著利用LgtA, CstI, Psp26ST此三種酵素完成五醣體的建構，並藉由2D NMR光譜分析鑑定出α-2,6唾液酸的鍵結位置在末端的GalNAc。然而，在以八號位置修飾為甲氧基的唾液酸進行α-2,3-唾液酸化反應時，卻得到較低的產率。儘管嘗試將受體改為較接近天然受體的GalNH2，卻仍然無法有效提升產率，並衍生出α-2,3及α-2,6的選擇性問題。
未來，將測試各個衍生物的神經再生活性，並且研究其活性與結構的關係。

It was found that echinodermatousgangliosides (EGs) show neuritogenic activity towards neuron-like rat adrenal pheocromocytoma (PC12) cells in the presence of the nerve growth factor (NGF). GAA-7 is one of echinodermatousgangliosides which show high neuritogenic activity. Till now, GAA-7 was only synthesized by chemical methods that needed several steps and time comsuming.
We synthesized 14 GAA-7 derivatives with different terminal sialic acid derivatives by chemoenzymatic method. First, the modified sialic acid donor is synthesized by chemical method. Then, the synthesis of pentasaccharide can be achieved by 3 transferases (LgtA, Cst-I and Psp26ST). The α-2,6-linkage to GalNAc has been identified by 2D NMR spectrum (COSY, HSQC and HMBC). However, we failed in α-2,3-sialylation for Neu5Gc8OMe donor. Even though we change the acceptor to the GalNH2 which is more similar to the nature substrate of CstI, the reaction yield didn't increase much and the selectivity problem occurred because of unexpected α-2,6-sialylation.
In the future, structure‐activity relationship study of GAA-7 can be achieved by the evaluation of the neuritogenic activity of the synthesized derivatives.

摘要 I
Abstract II
謝誌 III
目錄 V
圖目錄 VIII
流程圖目錄 XI
表目錄 XII
縮寫表 XIII
第一章 緒論 1
1-1 酵素對醣體合成的重要性 1
1-2 唾液酸 3
1-3 神經節苷脂 5
1-3-1 PC12細胞 7
1-3-2 棘皮動物之神經節苷脂 7
1-3-3 神經節苷脂GAA-7 9
1-4 神經節苷脂GAA-7之合成研究 11
1-5 醣激酶及醣基轉移酶 13
1-5-1 醣激酶 (NahK) 14
1-5-2 磷酸醣核苷轉移酶 (GlmU、CSS) 15
1-5-3醣基轉移酶 (LgtA) 16
1-5-4 唾液酸轉移酶 (CstI、PspST) 17
1-6 研究動機 21
第二章 結果與討論 23
2-1 GAA-7衍生物之逆合成分析 23
2-2 唾液酸的修飾 24
2-2-1合成八號位置為甲氧基之唾液酸 24
2-2-2合成Neu5Gc 30
2-3 以大腸桿菌誘導表現目標蛋白 31
2-3-1勝任細胞 31
2-3-2目標酵素純化與表現分析 32
2-3-3酵素表達分析 36
2-4 三醣體之合成 43
2-5 階段性一鍋化建構α-2,3-唾液酸 43
2-6 階段性一鍋化建構α-2,6-唾液酸 47
2-7 以NMR鑑定α-2,6-唾液酸鍵結位置 51
2-8 結論 59
第三章 實驗部分 60
4-1一般實驗方法 (試劑、溶劑與實驗儀器) 60
4-2 實驗步驟及光譜資料 61
參考文獻 96

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