(S)-piperidine-2-carboxylic acid (Pip) 為六員環的脯胺酸衍生物，其被認為與罕見疾病哌啶酸血症 (hyperpipecolic acidemia) 相關，該病症的特徵在於會增加高脯胺酸在血液中的濃度，導致神經疾病和肝腫大。本次研究第一部分即探討將 Pip 置換在脯胺酸 (Pro) 位置對於不同結構胜肽穩定度的影響，我們將 Pip 置換在 polyproline、HP7 及 Trp cage 胜肽上，以圓二色光譜儀 (Circular dichroism；CD) 來觀察其 Far-UV 光譜與熱熔化曲線的改變。結果發現 Pip 的置換對於 polyproline II (PPII) 結構較無影響，但會造成 polyproline I (PPI) 結構穩定度下降。在 HP7 胜肽置換 Pip 的實驗中，發現 Pip 不會對於β-harpin 結構產生太大的改變。最後將 Pip 置換在 Trp cage 上四個 Pro 的位置上，得到 Pip 的置換會造成 Trp cage 胜肽結構相當不穩定，判斷是因為 Pip 的六員環立體障礙過大而導致 α-helix 結構的崩解，也對於不同位置所造成的影響有所討論。
　　我們也利用理論計算試圖佐證我們在 polyproline 實驗中得到的結果，使用 Gaussian 軟體，以 DFT 方法進行結構最佳化，Ac-P2(Pip)P2-NH2 分子模型進行計算。計算所得到的結果為 Pip 主要的構形 boat up 會類似於 Pro 的 exo 構形。在 PPI 結構中四種構形的 Pip 都與 P5 PPI 有很大的差異，可能就是造成我們在 CD 光譜中 PPI 結構較不穩定的主要原因。
　　第二部分我們以 Prion octarepeat 片段來作為醯基轉化催化劑，發現將 Ac-XaaHGGGWGQP-NH2 和 Ac-GQXaaHGGGWGQ-NH2 中的 Xaa 位置置換成 Asp 對於催化醯基轉化有明顯地效果。而 Ac-GQDHGGGWFQ-NH2 (GQD) 的催化效果大於 Ac-DHGGGWGQP-NH2 (DP)，判斷是前者 Asp 位置位在胜肽鏈的內側較不易晃動，使得 Asp 和 His 間的距離較為固定，有比較強的作用造成催化效果較佳。

　　(S)-piperidine-2-carboxylic acid (Pip) is a six-membered ring of proline derivative. Pip has been reported to be associated with hyperpipecolic acidemia－one of the rare disorders, which an increase in pipecolic acid levels in the blood leads to neuropathy and hepatomegaly. In the fist part of this work, we prepared various peptides with Pip incorporated, including polyproline, HP7, and Trp cage. Circular dichroism spectroscopy (CD) was used to characterize their conformation. We found that the incorporation of Pip did not affect polyproline II structure but destabilized polyproline I conformation. When Pip was incorporated into HP7, it seemed to fit well in a β-harpin and did not significantly change the structure. However, the incorporation of Pip into Trp cage dramatically destabilized the α-helical structure, in particular replacing Pro12 to Pip destroyed the structure.
　　We also used a computational approach to study the effect of Pip on polyproline conformation. For each model compound and system, hybrid density functional theory (DFT) calculations, as implemented in Gaussian 09, were carried out to learn the energy difference between different conformations. We used Ac-P2(Pip)P2-NH2 as a model system for the computation. Pip prefers a boat up conformation similar to the Cϒ-exo pucker of Pro, and can also form the conformation of boat down, chair up, and chair down. The calculations indicate that all the conformers of Pip are not appropriate to a PPI helix, which may explain why Pip destabilizes PPI conformation as observed by the experiments.
　　In the second part, we chose Prion octarepeat fragments to study their potential catalytic activity on acyl transfer reaction. The results indicate that it has a high catalytic activity when the Xaa position in Ac-XaaHGGGWGQP-NH2 (GQX) or Ac-GQXaaHGGGWGQ-NH2 (XP) is Asp, and Ac-GQDHGGGWGQ-NH2 (GQD) is more efficient than Ac-GQDHGGGWGQ-NH2 (DP). In GQD, the Asp is at the middle of the peptide, making Asp interact with His more strongly, which might explain why GQD is more effective than DP on catalyzing the reaction.

目錄
謝誌 I
摘要 II
Abstract III
目錄 V
圖目錄 VIII
表目錄 X
第一章 緒論 1
1.1 蛋白質簡介 1
1.2 聚脯胺酸 (polyproline) 介紹 3
1.3 HP7 介紹 7
1.4 色胺酸包覆胜肽 (Trp cage) 介紹 9
1.5 醯基轉化 (Acyl transfer) 原理 11
1.6 Prion Protein 介紹 12
1.7 固相胜肽合成法 (Solid Phase Peptide Synthesis, SPPS) 簡介 15
1.7.1 將胺基酸接上樹脂 19
1.7.2 去保護 (Deprotection) 20
1.7.3 活化 (Activation) 21
1.7.4 耦合 (Coupling) 22
1.7.5 切除 (Cleavage) 23
1.8 圓二色光譜儀 (Circular dichroism spectroscopy, CD) 介紹 23
1.9 研究動機 30
第二章 實驗部分 31
2.1 實驗儀器 31
2.2 實驗藥品 32
2.3 化合物之合成與鑑定 34
2.3.1 Fmoc-Tyr(t-Bu)-resin 之合成 34
2.3.2 4-Nitrophenyl 2-methoxyacetate 之合成 35
2.5 光譜測量 39
2.5.1 UV 光譜測量濃度 39
2.5.2 CD光譜測量 40
2.5.3 醯基轉化實驗測量 41
2.6 光譜數據分析 42
2.6.1 CD 變溫實驗之數據處理 42
2.6.2 催化實驗之數據處理 43
第三章 計算研究方法及步驟 45
3.1 研究系統及計算方法 45
3.2 研究步驟 46
第四章 實驗結果與討論 48
第一部分 高脯胺酸對於胜肽結構的影響 48
4.1 高脯胺酸的構形 48
4.2 polyproline 胜肽 CD 測量結果討論 52
4.2.1 水溶液中的Far-UV CD 光譜 52
4.2.2 n-propanol 溶液中的Far-UV CD 光譜 53
4.2.3 n-propanol 溶液中的變溫實驗 54
4.3 聚脯胺酸置換高脯胺酸後的結構 57
4.4 高脯胺酸的 n→π* 作用力 63
4.5 HP7-Pip 胜肽 CD 測量結果討論 66
4.5.1 Far-UV CD 光譜 67
4.5.2 Near-UV CD 光譜 68
4.5.3 變溫實驗 69
4.5.4 HP7-Pip CD 光譜結果之探討 70
4.6 Trp-cage-Pip 胜肽 CD 測量結果討論 72
4.6.1 Far-UV CD 光譜 72
4.6.2 變溫實驗 73
4.6.3 Trp cage-Pip CD 光譜結果之探討 74
第二部分 利用Prion octarepeat 片段作為醯基轉化催化劑之探討 77
4.7 比較不同 Prion octarepeat 片段對催化醯基轉化反應的效率 77
第五章 結論 82
第六章 參考文獻 84
第七章 附錄 89

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