膠原蛋白模擬胜肽與硼烷共軛化合物的製備及雙硫鍵與cation-π作用力對膠原蛋白異源三股螺旋摺疊探討

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研究生：	李育昇 Li, Yu-Sheng
論文名稱：	膠原蛋白模擬胜肽與硼烷共軛化合物的製備及雙硫鍵與cation-π作用力對膠原蛋白異源三股螺旋摺疊探討 Preparation of the collagen-mimetic peptide-borane conjugate and the effects of disulfides and cation-π interactions on the folding of collagen heterotrimers
指導教授：	洪嘉呈 Horng, Jia-Cherng
口試委員:	江昀緯 Chiang, Yun-Wei 許馨云 Hsu, Hsin-Yun
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2017
畢業學年度：	105
語文別：	中文
論文頁數：	63
中文關鍵詞：	硼烷共軛化合物、雙硫鍵、異源三股螺旋
外文關鍵詞：	borane conjugate, disulfides, heterotrimers
相關次數：	點閱：2 下載：0
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膠原蛋白是哺乳動物中含量最多的蛋白質，也是人體內非常重要的蛋白質。膠原蛋白因為具有高度的生物相容性、低免疫性、生物降解性，已被廣泛的運用在生醫材料上。本研究的第一部分中，我們將硼烷分子 (boroncage)修飾到膠原蛋白模擬胜肽(POG)7上；硼烷分子是目前中子捕捉療法的其中一環，當硼原子接受到中子時會放出熱能進而殺死癌細胞，此研究期望利用膠原蛋白模擬胜肽的生物相容性，使此硼烷分子可以進入到Hela癌症細胞內。實驗結果顯示修飾上硼烷分子的(POG)7的熱穩定性僅有些微下降，且在細胞胞吞實驗中經過ICP- MASS的檢測發現細胞中有硼原子的訊號，所以此類膠原蛋白模擬胜肽具有運送硼進入細胞的潛在應用。

　　大多數的膠原蛋白都是由二條 (AAB型)或是三條(ABC型)不同胜肽鏈形成的異源三股螺旋結構，因此研究異源三股螺旋結構更能模擬天然的膠原蛋白。我們實驗室先前的研究中，成功的利用cation-π作用力來促使(POG)6(PKG)3與(POG)6(FOG)3胜肽形成AAB型的異源三股螺旋。在第二部分的研究中，我們在 (POG)6(PKG)3的N端先修飾上三個Gly當spacer，再修飾上一個Cys形成CGGG(POG)6(PKG)3，氧化後可以成為雙硫鍵相接的胜肽鏈，此外在CGGG(POG)6(PKG)3上其中一個POG的Gly置換成Sar，因為置換成Sar會使三股螺旋損失氫鍵使結構不穩定，希望藉由雙硫鍵和cation-π作用力形成更穩定的AAB型異源三股螺旋並探討氫鍵對此結構的貢獻。研究結果發現，CGGG(POG)6(PKG)3與(POG)6(PKG)3的性質相似，且與(POG)6(FOG)3混合後會形成異源三股螺旋結構，並觀察到cation-π作用力，但是在氧化後形成雙硫鍵的CGGG(POG)6(PKG)3與CGGG(POG)5 (POSar) (POG) (PKG)3並不能與(POG)6(FOG)3形成類似AAB型的異源三股螺旋結構，顯示位於N端的雙硫鍵並無助於異源三股螺旋的摺疊，而Sar的置換對三股螺旋造成結構很大的破壞。

　Collagen is the most abundant protein in mammals and is an important protein in human body. Collagen has been widely used in biomedical materials because of its biocompatibility, low immunity, and biodegradability. In the first part of this thesis, we prepared the collagen mimetic peptides (POG)7-boroncage conjugate, and investigated the delivery of boron into cells for the potential application in boron neutron capture therapy (BNCT). The results show that the boroncage moiety only slightly destabilize the collagen triple helix and the peptide-boroncage conjugate can enter cells.

Most of natural collagens are heterotrimers composed of two (AAB) or three (ABC) different peptide chains, and thus studying heterotrimers can better mimic natural collagens. In our early study, we showed that (POG)6(PKG)3 can form heterotrimers with (POG)6(FOG)3 via cation-π interaction. Here we futher added Cys into the N-terminus of (POG)6(PKG)3, CGGG(POG)6(PKG)3, and replaced Gly to Sar, CGGG(POG)5(POSar)(POG) (PKG)3 to study the effects of disulfides and hydrogen bonding on heterotrimers. The results reveal that CGGG(POG)6(PKG)3 is similar to (POG)6(PKG)3 can form AAB-type heterotrimers with (POG)6(FOG)3, and the cation-π interactions could induce the AAB-type heterotrimers. However, neither disulfide-linked CGGG(POG)6(PKG)3 nor disulfide-linked CGGG(POG)5(POSar) (POG)(PKG)3 can form AAB-type heterotrimers with (POG)6(FOG)3. The resulted showed that the disulfide bond at the N-terminus could not facilitate to the folding of collagen heterotrimers and the replacement of Gly to Sar significantly destabilized the triple helix.

中文摘要    I
Abstract    II
謝誌    III
目錄    IV
圖目錄    VII
表目錄    X
第一章 緒論    1
1-1 膠原蛋白    1
1-1-1    膠原蛋白的結構    1
1-1-2 脯胺酸 (Pro)與羥脯胺酸 (Hyp)使膠原蛋白穩定    2
1-1-3 置換鹼基胺基酸與芳香環胺基酸對膠原蛋白之影響    4
1-1-4異源三股螺旋 (heterotrimer)    5
1-2 Cation-π作用力    6
1-2-1鹼基胺基酸與芳香環胺基酸的cation-π作用力    7
1-2-2生物系統上的cation-π作用力    8
1-3 蛋白質胺基酸序列突變    8
1-4硼中子捕獲療法 (BNCT)    9
1-5 固相胜肽合成法 (Solid Phase Peptide Synthesis)37    10
1-5-1 酯化反應(Esterification)/ 醯胺化反應(Amidation)    10
1-5-2 去保護 (Deprotection)    11
1-5-3活化 (Activation)    12
1-5-4耦合 (Coupling)    12
1-5-5切除 (Cleavge)    13
1-5-6固相胜肽合成法流程    13
1-6 圓二色光譜儀 (Circular Dichrosim)38    16
1-7 研究動機    20
第二章 實驗部分    21
2-1 實驗儀器    21
2-2 實驗藥品    22
2-3 實驗步驟流程    24
2-4 合成Fmoc-Pro-Hyp-Gly-OH    25
2-4-1 合成 Boc-Hyp-OH    25
2-4-2 合成Boc-Hyp-Gly-OBn    26
2-4-3 合成 Fmoc-Pro-Hyp-Gly-OBn    26
2-4-4 合成 Fmoc-Pro-Hyp-Gly-OH    27
2-5胜肽之合成    28
2-5-1 模擬膠原蛋白系列之合成    28
2-5-2  N端修飾炔基的膠原蛋白模擬胜肽合成    29
2-5-3 胜肽的切除與純化    29
2-5-4 雙硫鍵膠原蛋白模擬胜肽合成    30
2-5-5 膠原蛋白模擬胜肽與硼烷共軛化合物合成    30
2-6細胞實驗    30
2-6-1 培養細胞之藥品    31
2-6-2 細胞回養與繼代    31
2-6-3 細胞保存    31
2-6-4 細胞胞吞作用測試實驗    32
2-7 圓二色光譜實驗    32
2-7-1 Far-UV光譜 (Wavelength scan)    33
2-7-2 熱變性實驗 (Thermal denaturation)    33
2-7-3 動力學實驗    33
2-8 2D-NMR光譜    34
2-8-1 1H,15N-HSQC光譜    34
2-8-2 1H,1H-TOCSY、1H,1H-NOESY光譜    34
2-9 示差掃描量熱法 (DSC)    34
2-10 圓二色光譜實驗數據之處理    34
2-10-1 變溫實驗數據之處理    34
2-10-2 動力學實驗數據之處理    36
2-11 DSC實驗數據處理    36
2-11-1熱力學實驗數據處理    36
第三章 實驗結果與討論    38
3-1 膠原蛋白模擬胜肽與硼烷共軛化合物實驗    38
3-1-1 Far UV CD光譜    38
3-1-2 變溫實驗測量    39
3-1-3 細胞實驗    41
3-2 雙硫鍵以及胺基酸Sar對膠原蛋白三股螺旋的影響    42
3-2-1 CD光譜    42
3-2-2 1H,15N-HSQC NMR圖譜    48
3-2-3 1H,1H-TOCSY、1H,1H-NOESY NMR光譜    52
3-2-4 DSC分析    54
3-2-5 胜肽摺疊速率比較    56
第四章 結論    58
參考資料    60
附錄    63


                                

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