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研究生:曾文秀
研究生(外文):Tseng, Wen-Hsiu
論文名稱:裝訂脯胺酸寡肽之合成與其結構穩定性
論文名稱(外文):The Synthesis and Structure Stability of Stapled Polyproline Peptides
指導教授:王聖凱
指導教授(外文):Wang, Sheng-Kai
口試委員:洪嘉呈梁碧惠
口試委員(外文):Horng, Jia-CherngLiang, Pi-Hui
口試日期:2018-07-27
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:149
中文關鍵詞:脯胺酸寡肽裝訂胜肽脯胺酸多肽螺旋圓二色性銅催化疊氮-炔類[3+2]環化加成GROMACS
外文關鍵詞:PolyprolineStapled PeptidePolyproline Helix IICircular DischroismCuAACGROMACS
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在生醫領域中,已發展出數種胜肽藥物,或是以胜肽作為骨架,形成藥物載體。但在生物體內,胜肽易受到酵素辨認,而失去活性,目前發展出裝訂胜肽(stapled peptide)的技術來提升生物體內胜肽的壽命。我們嘗試於脯胺酸多肽螺旋II (Polyproline Helix II, PP II)結構上進行裝訂,利用控制裝訂鏈(Linker)的長度,與改變裝訂位置,觀察此種設計對PP II結構影響。經由交叉測試後,得知i, i+3裝訂會比i, i+2與i, i+1裝訂容易成功。和寡脯胺酸多肽相比,在正丙醇溶液中,可以發現不同長度,位置的裝訂方式會影響結構。除了透過CD光譜鑑定結構外,也使用GROMACS動力學軟體,來模擬研究中合成出來的數種裝訂胜肽。
In recent years, many drugs and carriers based on peptides has been developed. However, drug design based on peptide has a critical disadvantage that they may be degraded by enzymes in organism. For this issue, peptide stapling is one practical solution to extend the lifespan of peptides. While polyproline peptides have unique structure for various applications, we “stapled” polyproline peptide systematically to investigate the effects on polyproline helix II (PP II) structure by change linker length and the locations of stapled residues. From circular dischroism (CD) analysis, we found that stapling at i, i+3 residues of polyproline peptides stabilizing PP II structure better than other locations at i, i+1 and i, i+2. Compared to normal polyproline, the PP II structure stapled polyproline is also affected by the staple length in n-propanol. In addition to CD spectrum, we use GROMACS software to simulate the molecular dynamics of stapled peptides in water.
摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 ix
式目錄 x
縮寫對照表 xi
第一章、 緒論 1
1.1 前言 1
1.2 蛋白質二級結構 2
1.2.1 α螺旋(α Helix) 3
1.2.2 β摺板(β Sheet) 5
1.2.3 310螺旋(310 Helix) 6
1.2.4 π螺旋(π Helix) 7
1.2.5 膠原蛋白螺旋(Collagen Helix) 8
1.3 脯胺酸多肽螺旋(Polyproline Helix) 8
1.3.1 脯胺酸多肽螺旋結構與特性 8
1.3.2 脯胺酸多肽的應用 10
1.4 疊氮-炔類[3+2]環化加成 12
1.4.1 CuAAC反應機構 12
1.4.2 CuAAC的特性 14
1.4.3 CuAAC於生物上的應用 14
1.5 裝訂胜肽(Stapled Peptide) 16
1.5.1 裝訂胜肽的結構特性 16
1.5.2 裝訂胜肽在生物研究上的應用 16
1.6 圓二色性 (Circular Dischroism, CD) 18
1.6.1 圓二色性的原理 18
1.6.2 以圓二色性判斷蛋白質二級結構 18
1.7 分子動力模擬 20
1.7.1 GROMACS 20
1.7.2 SHAKE演算法 21
1.7.3 LINCS 演算法 21
1.7.4 GROMACS分析程式 21
1.8 研究動機 22
1.9 實驗設計 22
第二章、 結果與討論 24
2.1 實驗設計 24
2.2 固相多肽合成之構築單元合成 25
2.3 裝訂鏈合成 26
2.4 固相胜肽合成法之寡脯胺酸多肽合成 28
2.4.1 SPPS流程 28
2.4.2 模板胜肽合成 30
2.4.3 多重裝訂模板胜肽合成 32
2.4.4 裝訂選擇性之模板胜肽合成 33
2.5 裝訂胜肽合成 35
2.5.1 裝訂胜肽裝訂測試 35
2.5.2 不同長度裝訂鏈之裝訂胜肽合成 38
2.5.3 不同位向之裝訂胜肽合成 41
2.5.4 未反應之炔基測試 51
2.5.5 多重裝訂胜肽合成 61
2.5.6 裝訂選擇性測試 64
2.6 裝訂胜肽結構鑑定 70
2.6.1 CD光譜鑑定 70
2.6.2 綜合討論 76
2.7 分子動力模擬 76
2.7.1 與模擬初始結構之RMSD 77
2.7.2 模擬過程中之Potential Energy 83
2.8 結論 89
第三章、 實驗方法與材料 91
3.1 Synthetic procedures and characterization 92
3.1.1 Building blocks 92
3.1.2 Linkers 97
3.1.3 Peptide synthesis 101
3.1.4 Singular stapling 110
3.1.5 Multiple stapling 126
3.1.6 Hydrogenation of stapled peptides 127
3.2 Molecule dynamic simulation 129
第四章、 參考文獻 131
附錄 136
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