臺灣博碩士論文加值系統

固相胜肽合成法為R. B. Merrifield於1963年所提出。三十多年的發展之後, 這項技術不但使胜肽合成變為快速且簡便, 更被應用到其它的方面, 例如: 醣蛋白、脂蛋白、多醣類及組合式化學資料庫之合成等方面。
本論文共分為二部份
第一部份 微波照射應用於胜肽合成之研究
微波照射應用於有機反應是一個快速發展的領域。微波照射對於具偶極性及離子導電性的分子會有加熱的效應, 而很多的文獻也證實, 使用微波照射可以加快有機反應的速率。
在我們的研究中, 家庭式的微波爐和胜肽自動合成儀被加以組合, 並配合上紫外光偵測系統的建立。希望將微波照射應用在胜肽合成儀上, 且藉由偵測去保護步驟所切下的Fmoc保護基濃度來決定偶合反應的效率, 以研究微波照射對偶合效率的影響。而在胜肽的合成上, 因為胺基酸側鏈結構上的問題或是所謂的 "Difficult Sequences" , 會面臨到偶合不完全的問題, 而導致產物的產率下降。然而目前仍然沒有一個有效且簡便的方法來克服此問題。在此, 我們將微波照射應用在胜肽合成儀上時, 不只希望能縮短偶合反應所需的時間, 也想要解決偶合不完全的問題。
實驗中選擇了三段不同的胺基酸序列做為研究的對象。實驗結果顯示, 微波照射在不減少偶合的效率的情形下, 可以將偶合反應的時間縮短到5分鐘。而在偶合不完全的問題上, 微波照射對偶合的效率也有明顯的提升。因此, 利用微波照射和固相胜肽合成的結合, 不但可以保有固相胜肽合成法方便且自動化的優點, 而且大幅的減少合成時所需的時間, 同時也可以有效的克服偶合不完全的問題, 提高了胜肽合成的產率, 減少後續純化的問題。
第二部份 固相胜肽合成法應用於醣胜肽合成之研究
哺乳動物的肝細胞表面存在著大量的非唾液酸胎蛋白接受器 (asialoglycoprotein receptor, ASGP-r) , 而且在超過80%體內的ASGP-r分佈在肝胞。ASGP-r對末端含有半乳糖或乙醯胺基半乳糖殘基的受質會有專一性的親合力, 而且親合力的大小和末端半乳糖或乙醯胺基半乳殘基的個數及三度空間排列有關。YEE(GalNAcAH)3是由R. T. Lee及Y. C. Lee在1987年所提出, 經由合成而來的ASGP-r受質。然而在已發表的報導中, 因為溶解度的問題使得YEE(GalNAcAH)3的產率不佳, 或是合成步驟太過複雜, 都是有待克服的問題。
在此, 我們利用N-Fmoc aminohexanol glycoside及Fmoc (a-GalNAcAc3AH) Glu作為合成時的建構單元體, 並配合使用固相合成法。YEE(GalNAcAH)3及螢光標定的YEEE(GalNAcAH)3可以被快速、方便的合成, 而且產率在80%以上。而藉由Fmoc (a-GalNAcAc3AH) Glu作為唯一的建構單元體, 我們設計了YEE(GalNAcAH)3的類似物, YEEE(GalNAcAH)3。同樣的, 使用固相胜合成法, 可以合成出高產率的YEEE(GalNAcAH)3及其螢光標定的衍生物。
以HepG2細胞作為生物的模型, 螢光標定的YEE(GalNAcAH)3或是YEEE(GalNAcAH)3對ASGP-r的結合能力及內吞作用的現象被加以研究。經由螢光顯微鏡及雷射共交螢光顯影技術, 可以清楚的觀察到所合成的螢光標定的YEEE(GalNAcAH)3可以和肝腫瘤細胞表面的非唾液酸胎蛋白接受器結合, 並且藉由內吞作用而進到細胞內部。而一千倍的不含螢光標定受質可以競爭螢光標定受質的結合, 表示它們是和相同的接受器結合。而以不含ASGP-r的CHO細胞來進行結合的研究時, 沒有任何的受質結合被發現, 表示受質對ASGP-r有特異性的結合。
經由實驗的結果顯示, 利用固相胜肽合成法, 配合適當的建構單元體, 我們YEE(GalNAcAH)3或是新開發的YEEE(GalNAcAH)3可以被成功的合成。因此, 合成及應用這二種受質作為攜帶藥物專一性的進入肝細胞, 是一種未來臨床研究上極為有潛力的一種工具。

The solid phase peptide synthesis method was proposed by R. B. Merrfield in 1963. During these years, this technology was not only used to make peptide synthesis faster and more simply, but also applied to many fields, such as glycoprotein、lipoprotein、polysaccharides and combinatorial library, etc.
This thesis is composed of two parts:
Part I The Application of Microwave Irradiation on Peptide Synthesis
Recently the use of microwave irradiation in organic reactions is an area of growing interest. Microwave irradiation provides heating effects on molecules that have dipole or ion conductivity. Many papers have demonstrated that microwave enhanced the rate of organic reactions.
In our experiments, a microwave oven was combined with an automated peptide synthesizer. And UV detector was used to monitor the concentration of released Fmoc in the deprotection step. Using the data obtained from UV detector, the effects of microwave irradiation on coupling efficiency was studied. In peptide synthesis, the problem of incomplete coupling results in the low yields. It is mainly due to the structure or so called "Difficult Sequences". Until now, there is still no efficient and convenient method to solve this problem. Here, we hope that the application of microwave irradiation on the peptide synthesizer can not only shorten the coupling reaction time, but also solve the problem of incomplete coupling.
Three different sequences were studied. The results showed that microwave cut down the coupling reaction time to be 5 minutes while the coupling efficiency was improved. Therefore, when microwave irradiation combines with peptide synthesis, the advantages of convenience and automation in solid peptide synthesis can be maintained. Besides, both efficiency and percentage of yields can be improved.
Part II The Application of Solid Phase Synthesis Method on Glycopeptide Synthesis
There are a large number of asialoglycoprotein receptors (ASGP-r) on the cell membrance of hepatocytes in mammalian species. More than 80% of ASGP-r in the body are distributed on liver cells. ASGP-r recognizes specifically the ligands which have terminal galactose (Gal) or N-acetylgalactose (GalNAc) residues, and the affinity depends on the valency of Gal / GalNAc and the three-dimensional arrangement.
YEE(GalNAcAH)3 reported by R. T. Lee, and Y. C. Lee in 1987 is a synthetic ligand for ASGP-r. However, according to the published synthesis methods, the low yield of YEE(GalNAcAH)3 synthesis was due to its solubility and complicated procedures.
Here, we use N-Fmoc aminohexanol glycoside and Fmoc (a-GalNAcAc3AH) Glu as building blocks in solid phase peptide synthesis. The preparations of YEE(GalNAcAH)3 and fluorescein labeled YEE(GalNAcAH)3 are fast and simple. The yields of product are over 80 %. While only using Fmoc (a-GalNAcAc3AH) Glu as a building block, we design YEEE(GalNAcAH)3, an analogue of YEE(GalNAcAH)3. By solid phase peptide synthesis method, YEEE(GalNAcAH)3 and its fluorescien labeled derivative were also synthesized with high yields.
Using HepG2 cells as bioassay models, the binding abilities of fluorescein labeled YEE(GalNAcAH)3 or YEEE(GalNAcAH)3 to ASGP-r were studied. The endocytosis phenomenon was also investigated. The binding of synthetic fluorescein labaled ligands to the ASGP-r was demonstrated by fluorescent microscope. And then the internalization of ligands mediated by ASGP-r was observed by confocal microscope. Additionally, both the competitive binding studies using ligands with or without fluorescein labeled and specificity studies were presented in current paper.
From the results of above experiments, both YEE(GalNAcAH)3 and YEEE(GalNAcAH)3 were synthesized successfully by solid phase peptide synthesis method with suitable building blocks. Therefore, the synthesis and the utilization of these two ligands as a carrier for drug delivery specifically to liver cells become a novel tool for future clinical studies.

緒論
一、 固相胜肽合成法之簡介 1
二、 固相胜肽合成法之流程 3
第一篇 微波照射應用於胜肽合成之研究
第一章 緒論
一、 微波照射的原理及合成上的應用 6
1. 微波的原理 6
2. 微波照射在合成上的應用 6
3. Microwave Effect 8
二、 Difficult Sequences 9
三、 H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2, H-Lys-Val-Val-Val-
Glu-Arg-Trp-Cys-Thr-Leu-Glu-Val-Ala-NH2及H-Gly-MeLeu-Val-
MeLeu- Ala-OH研究 11
第二章 實驗動機及目的
一、 實驗動機 13
二、 實驗目的 14
第三章 實驗試劑及儀器
一、 實驗試劑 15
二、 實驗儀器 16
三、 藥品溶液的配製 17
第四章 實驗方法
一、 實驗設計 18
二、 胜肽自動合成儀的改裝 20
三、 偶合效率的偵測方法 23
1. 定性分析: Kaiser test 23
2. 定量分析: 紫外光吸收偵測 24
四、 微波照射之條件 25
五、 高效能液相層析 (HPLC) 分析方法 26
六、 胜肽合成法 27
1. 胜肽自動合成儀之合成方法 27
2. 由固相擔體切下胜肽之方法 28
第五章 結果與討論
一、 偶合效率定量分析系統之確定 30
二、 微波照射對溶液溫度的影響 35
三、 微波照射對偶合效率之影響 36
四、 胜肽產物的物性分析 43
第六章 結論 52
第二篇 固相合成法應用於醣胜肽合成之研究
第一章 緒論
一、 Asialoglycoprotein Receptor (ASGP-r) 的簡介 53
二、 Asialoglycoprotein Receptor (ASGP-r) 的生理功能 55
三、 YEE(GalNAcAH)3及YEEE(GalNAcAH)3之研究 57
第二章 實驗動機及目的
一、 實驗動機 59
二、 實驗目的 62
第三章 實驗試劑及儀器
一、 實驗試劑 63
二、 實驗儀器 64
三、 藥品溶液的配製 64
第四章 實驗方法
一、 YEE(GalNAcAH)3及YEEE(GalNAcAH)3之逆合成 65
二、 實驗設計 68
三、 醣胜肽之合成 71
1. 胜肽自動合成儀之合成方法 71
2. 由固相擔體切下醣胜肽之方法 72
四、 高效能液相層析 (HPLC) 分析方法 73
五、 生物鑑定的方法 74
1. 細胞株培養 74
2. 結合能力 (binding) 及內吞作用 (endocytosis) 的研究 74
3. 競爭性實驗 74
4. 對ASGP-r特異性的測試 75
5. 雷射共交螢光顯影 (confocal microscopy) 技術 75
第五章 結果與討論
一、 醣胜肽產物的物性分析 76
二、 生物鑑定 80
1. 對ASGP-r之結合 (binding) 能力測試 80
2. 競爭性實驗 80
3. 對ASGP-r之特異性測試 81
4. 內吞作用 (endocytosis) 81
第六章 結論 85

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