合成胜肽鏈中保護胺基酸之溶解度參數測定

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姓名

鍾瑩(Ying Zhong) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

合成胜肽鏈中保護胺基酸之溶解度參數測定
(Solubility parameter determination for protected amino acids in synthetic peptides)

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摘要(中)

近年來胜肽(peptide)藥物廣泛地應用在疾病治療方面，因此胜肽的合成與純化扮演相當重要的角色。胜肽合成技術於近數十年間已日趨純熟，其中以固相合成(Solid-phase synthesis)為最大宗，但往往胜肽鏈的增長與結構效應大幅降低了合成的產率。因此，期望能以固相分段合成短片段之胜肽，再於液相中接合，來改善低產率之問題，但由於固相合成過程中有許多胺基酸具有疏水的保護基團，所以我們不僅要考慮接點問題，更要找到合適的溶劑以利接合。故本研究著重於疏水胺基酸與保護胺基酸溶解度參數之求得，並進一步地估算胜肽的溶解度參數，根據溶解度參數理論來尋找合適之溶劑。本研究設計了21條胜肽(Fmoc-Gly-Phe~X~Phe-Gly-OH, ~X~ 位置分別放置20個胺基酸，另外未放置的為控制組)，以逆相層析(Reverse-phase chromatography)之方式求得胜肽之溶解度參數，進一步的計算疏水胺基酸與保護胺基酸的溶解度參數。與Hoy軟體計算結果之比較顯示，實驗所得之極性(polarity，δp)作用力較大，氫鍵作用力較小(hydrogen bonding，δh)，又與文獻上兩液相分配法量測之疏水性成正相關。此外我們也找一條具保護胺基酸之胜肽來計算其溶解度參數、溶劑之選擇和預測其滯留行為，結果顯示確實精準預測胜肽於何種溶劑條件下可被沖堤。這表示導入溶解度參數之概念於層析系統中，不僅可準確預測具有側鏈保護基團的胜肽之滯留行為，亦可有助於我們找到合適的溶劑。

摘要(英)

In the past few decades, with the widely application of the peptide drug in medical treatment, the peptide synthesis and purification play important roles. The technology development of liquid-phase and solid-phase synthesis becomes mature state. However, there exists a problem that the purification yield decreases with the length of peptide chain or conformation effect. Therefore, we propose a new idea to improve the yield. First, use the solid-phase synthesis method to obtain part of the long-chain peptide. And after purifying these fragments, operate the ligation step in liquid phase. In order to find the common solvents for these peptides in the ligation step, we utilize the solubility parameter theory that widely used in polymer-solvent selection to study the hydrophobic and protected amino acids. We use the reverse-phase chromatography to investigate the solubility parameters of the designed 21 model peptides (Fmoc-Gly-Phe-X-Phe-Gly, -X- is replaced by 20 different amino acids), and then do the calculation to obtain the solubility parameters of hydrophobic and protected amino acids. In addition to comparing the results with Hoy software calculated data, the correlation of the experimental partition energy and hydrophobicity from the literature is also to be study. Finally, we take a peptide composed of protected amino acids to estimate its solubility parameters, find the suitable solvents and predict the retention behavior successfully. The result shows that apply the solubility parameter theory in RP-HPLC to study the hydrophobic and protected amino acids can not only do the help in peptide-solvent selection but also prediction the elution conditions.

關鍵字(中)

★ 溶劑選擇
★ 胜肽純化
★ 胜肽合成
★ 溶解度參數

關鍵字(英)

★ solvent selection
★ solubility parameter
★ peptide synthesis
★ peptide purification

論文目次

第一章緒論 1
第二章文獻回顧 2
2.1 胜肽合成 2
2.1.1 胜肽合成之方法 2
2.1.2 胺基酸側鏈之特定保護基團 4
2.2 疏水作用力之簡介及其對於生化分子之重要性 5
2.3 胺基酸疏水性之量測方式 6
2.3.1 胺基酸在水相及油相之溶解度比 6
2.3.2 兩液相分配法 (Aqueous two-phase partition method) 8
2.3.3 表面可接觸表面積之方法 (Accessible surface area method) 13
2.3.4 定點突變方法 (Site-directed mutagenesis) 15
2.3.5 以層析系統量測胺基酸疏水性 (Chromatography method) 16
2.4 溶解度參數 21
2.4.1 溶解度參數之理論及其發展 21
2.4.2 計算溶解度參數之方法 28
2.4.3 影響溶解度參數之因子 29
2.4.3.1 溫度對於溶解度參數之影響 30
2.4.3.2 莫耳體積對於溶解度參數之影響 30
2.4.3.3 濃度對溶解度參數之影響 31
2.4.4 溶解度參數之應用 31
2.4.4.1 高分子聚合物之相溶性預測以及溶劑的選擇 32
2.4.4.2 塗料工業之應用 33
2.4.4.3 生化領域之應用 34
2.4.4.4 其他應用 34
第三章材料與方法 35
3.1 實驗藥品 35
3.1.1 胜肽鏈 35
3.1.2 層析用品及藥品 36
3.2 實驗儀器 37
3.2.1 一般設備 37
3.2.2 高效能液相層析儀 38
3.3 實驗方法 39
3.3.1 高效能液相層析實驗 39
3.3.1.1 移動相配置 39
3.3.1.2 樣品溶液的配置 39
3.3.1.3 高效液相層析(HPLC)系統之操作 39
3.3.2 固定相之溶解度參數量測 43
3.3.3 胜肽之溶解度參數量測 43
第四章結果與討論 44
4.1 層析之滯留行為與溶解度參數之關係 44
4.2 以逆相層析測定固定相之溶解度參數 47
4.3 以逆相層析測定胜肽之溶解度參數 51
4.3.1 胜肽序列之設計 51
4.3.2 胺基酸序列對滯留性之影響 54
4.3.3 胜肽之溶解度參數 56
4.4 疏水性胺基酸與保護胺基酸對胜肽溶解度參數之貢獻 59
4.5 層析實驗與Hoy軟體計算之胺基酸溶解度參數比較 60
4.6 層析實驗之胺基酸溶解度參數與疏水性的比較 62
4.7 胺基酸之溶解度參數於胜肽純化分離的應用 64
4.7.1 估算胜肽之溶解度參數 64
4.7.2 尋找適合胜肽之溶劑以及沖提濃度之預測 66
第五章結論 70
第六章參考文獻 71
第七章附錄 73

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指導教授

阮若區、陳文逸
(Ruoh-Chyu Ruaan、Wen-Yih Chen)

審核日期

2011-7-28

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