天然化學連接法 (native chemical ligation, NCL) 早期被發現可用於含有半胱胺酸 (cysteine, Cys) 序列之胜肽 (peptide) 合成上，本論文利用此反應，將含有硫醇 (thiol) 的光解輔助基團修飾於天門冬胺酸 (aspartic acid, Asp) 之側鏈 (side chain)，以序列為H-AA1-Asp(RSH)-Glu-OH的三肽 (tripeptide) 與C端修飾為硫酯基 (thioester) 之胺基酸Cbz-AA2-SPh進行模型研究 (model study)，透過側鏈輔助以NCL連接。AA2於三肽N端建構四肽 (tetrapeptide)。透過五種AA1 (Gly, Val, Ser, Asp, His) 及六種AA2 (Gly, Ser, Phe, Leu, Pro, His) 經排列組合進行NCL反應性探討，得知三肽的N端若為纈胺酸 (valine, Val)，因立障阻礙而不利於連接反應 (ligation)。最後四肽產物透過溫和的紫外光 (ultraviolet, UV) 照射，移除輔助基得四肽Cbz-AA2-AA1-Asp-Glu-OH，但當天門冬胺酸之N端或C端連接絲胺酸 (serine, Ser) 時，容易於光解 (photocleavage) 時，產生大量副產物。

Native Chemical Ligation (NCL) is a powerful method for peptide synthesis. Originally, NCL occurs between C-terminal thioester and N-terminal cysteine. In this thesis, the thiol containing auxiliaries were assembled at the side-chain of aspartic acid (Asp). The thiol protected Asps were used as a building block to synthesize tripeptides H-AA1-Asp(RSH)-Glu-OH (AA1 = Gly, Val, Ser, Asp, His) which were then reacted with thioester, Cbz-AA2-SPh (AA2 = Gly, Ser, Phe, Leu, Pro, His), by NCL. Photo-irradiation was applied to remove the auxiliaries after ligation to afford tetrapeptides Cbz-AA2-AA1-Asp-Glu-OH. The results showed that the thiol containing auxiliary at Asp can assist the amide bond formation by NCL with moderate to good yields, except that the N-terminal amino acid of tripeptides was valine due to steric hindrance. However, when a serine next by Asp (at either N- or C-terminal), uncharacterized side-product was obtained after photo-irradiation.

目錄 i
圖目錄 iii
表目錄 v
流程目錄 vi
胺基酸總表 vii
縮寫對照表 viii
第一章 緒論 1
1-1 前言 1
1-2 天然化學連接法之起源 1
1-3 天然化學連接法的發展性及限制 2
1-4 不需半胱胺酸之天然化學連接法 4
1-4-1 以自由基方式移除硫醇輔助基 5
1-4-2 光解輔助基團 8
1-4-4 醣輔助連接法 9
1-4-5 側鏈輔助連接法 13
1-5 以固相多肽合成法合成C端為硫酯基之胜肽 15
1-5-1 樹酯之選擇 16
1-5-2 硫醇離去基之選擇 17
1-5-3 硫醇離去基之替代基團 20
1-5-4 硫醇催化劑 21
1-6 以化學動力學控制天然化學連接法 22
1-7 應用於紅血球生成素衍生物之合成 24
第二章 研究動機與構想 26
2-1 於天門冬胺酸側鏈引入光解輔助基團 26
2-2 連續進行兩次醣輔助連接法合成醣胜肽 28
第三章 結果與討論 30
3-1 合成胜肽前驅物 30
3-1-1 光解輔助基團 30
3-1-2 含天門冬胺酸之胜肽骨架 32
3-1-3 結合胜肽骨架及光解輔助基團 34
3-1-4 將胺基酸之羧酸修飾為硫酯基 35
3-2 將側鏈輔助連接法條件最佳化 36
3-2-1 移除光解輔助基團上之甲基 38
3-2-2 增大分子內醯基遷移之環數 39
3-2-3 合成不同胜肽前驅物 41
3-2-4 不同胺基酸之間結合的效率差異 43
3-2-5 反應機構探討 45
3-3 合成醣體前驅物 49
3-3-1 最終以酸去保護獲得醣體前驅物 50
3-3-2 最終以鹼去保護獲得醣體前驅物 54
3-3-3 最終以還原條件去保護獲得醣體前驅物 55
3-3-4 醣體前驅物之結構不穩定 56
3-4 未來展望 57
3-4-1 探討分子內醯基遷移之環數影響 57
3-4-2 改善羧酸被修飾為硫酯基之胜肽水溶性 58
3-4-3 透過固相多肽合成法建構真實例子 59
3-4-4 將光解輔助基團應用於其它胺基酸 60
3-4-5 以光解基團保護醣體不穩定之胺基 61
第四章 實驗部分 63
4-1 一般實驗方法 63
4-1-1 核磁共振光譜 63
4-1-2 質譜 63
4-1-3 薄層色層分析法 63
4-1-4 製備級薄層色層分析法 64
4-1-5 管柱層析法 64
4-1-6 化學藥品 64
4-2 使用高效液相層析法監控反應並純化產物 65
4-2-1 沖提系統 65
4-2-2 儀器與管柱 65
4-2-3 分析及純化條件 65
4-3 實驗步驟及光譜資料 66
參考文獻 123
光譜附錄 129

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