膠原蛋白的結構是由三條左旋第二型聚脯胺酸螺旋結構，沿著一個相同的主軸右旋轉組成，序列中每三個胺基酸有一個 Gly 、高含量的亞胺基酸和氫鍵是促進其穩定性的重要因素。另一方面， 正電荷與芳香環四極矩間的非共價鍵作用力 - cation-π 作用力，也陸續被發現其在蛋白質的 ligand 鍵結等生物辨識上的重要性。因此，我們利用一系列的主客胜□系統去探討 cation-π 作用力對膠原蛋白三股螺旋的影響；我們合成了一系列胜□： Ac-(POG)3-(PYGXOG)-(POG)3-NH2 ，在 X 位置為 Phe、 Tyr 、 Trp 、 Phe(4-F), 、 Phe(F5) 和 Phe(4-Me)； Y 位置為 Lys 、 Arg。 而 X 位置的 Trp 由於其立體障礙的緣故，使其形成 cation-π 作用力不如預期的大。而當 Y 位置為 Arg 的 Tm 值比 Lys 時高，因為 Arg 側鏈形狀的幾何特性，所以比 Lys 易產生 cation-π 作用力，與之前的文獻結論一致。此外，非自然胺基酸取代則顯現出，當芳香環側鏈上有電子基的去活化者 – 氟，會減少膠原蛋白模擬胜□的穩定性，因其降低 π 電子密度而減弱了 cation-π 作用力，證實 cation-π 作用力對於膠原蛋白三股螺旋的穩定性有重要的貢獻。
更進一步，我們設計膠原蛋白模擬胜□序列： Ac-RG(POG)8F-NH2 和 Ac-RG(POG)10F-NH2 ，發現 cation-π 作用力對促進膠原蛋白模擬胜□的自組裝速率的影響。最後，我們亦試著利用 cation-π 作用力去形成異源三聚體膠原蛋白模擬胜□。
以上這些結果顯示出，強健的非共價鍵作用力 – cation-π 作用力，可以被應用在穩定膠原蛋白模擬胜□，及促使膠原蛋白模擬胜□自組裝成高階結構。
Collagen is a right-handed triple helix composed of three left-handed PPⅡ helices. The presence of Gly as every third residue, a high content of imino acids and hydrogen bonding are significant characteristics to improve the stability of collagen. Cation-π interactions are considered as an electrostatic attraction between a positive charge and the quadrupole moment of the aromatic ring and are important in a variety of proteins that bind to ligands or substrates. Here, we used a host-guest peptide system to study the cation-π interactions in the collagen triple helix. A series of Ac-(POG)3-(PYGXOG)-(POG)3-NH2 peptides were synthesized and characterized, where X is Phe, Tyr, Trp, Phe(4-F), Phe(F5), Phe(4-Me), and Y is Lys, Arg. Owing to the steric hindrance, the cation-π interactions formed by Trp are not as stable as what we predicted. The peptides with Arg in the Y position have a higher Tm value than those with Lys at this position consistent with the previous conclusion that Arg is more likely than Lys to be in a cation-π interaction due to its geometric features. Moreover, the unnatural residue substitutions showed that an electron-withdrawing deactivator, fluorine, in the aromatic ring will reduce the thermal stability because it would reduce the π-electron density as well as the cation-π interaction. These results suggest that cation-π interactions significantly contribute to the collagen triple helix stability.
Furthermore, we design another two peptides, Ac-RG(POG)8F-NH2 and Ac-RG(POG)10F-NH2 to promote the rate of self-assembly by cation-π interactions. We have also tried to use cation-π interactions to form the heterotrimeric collagen helices. These results exhibit that the strong and non-covalent force, cation-π interactions, could be applied to stabilize collagen triple helices and assemble them into a higher order structure.

中文摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 xii
Scheme xiii
第一章 緒論 1
1-1 前言 1
1-2 Cation-π作用力 (Cation-πinteractions) 1
1-2-1 芳香環π電流 2
1-2-2 Cation-π作用力與其他作用力 4
1-3 Cation-π作用力在水溶液中的研究 4
1-4 生物系統中的cation-π作用力 7
1-4-1 帶電荷與芳香環側鏈的胺基酸 7
1-4-2 胺基-芳香環作用力 11
1-4-3 乙醯膽鹼 (Acetylcholine) 與相關蛋白質鍵結 13
1-5 膠原蛋白 (Collagen) 15
1-6 膠原蛋白的結構 18
1-6-1 脯胺酸 (Pro) 穩定膠原蛋白的能力 18
1-6-2 氫氧脯胺酸 (Hyp) 穩定膠原蛋白的能力 20
1-6-3 膠原蛋白模擬胜□上的鹼基胺基酸與芳香環胺基酸 23
1-7 膠原蛋白的自組裝 (Self-association or self-assembly) 25
1-8 研究方向 31
第二章 實驗部分 33
2-1 實驗步驟流程 33
2-2 實驗儀器 34
2-3 實驗藥品 36
2-4 Fmoc-Pro-Hyp-Gly-OH tripeptide 之合成 38
2-4-1 Boc-(4R,2S)-hydroxyproline 之合成 39
2-4-2 Boc-Hyp-Gly-OBn 之合成 39
2-4-3 Fmoc-Pro-Hyp-Gly-OBn 之合成 40
2-4-4 Fmoc-Pro-Hyp-Gly-OH 之合成 40
2-5 Fmoc-Phe-Hyp-Gly-OH tripeptide 之合成 41
2-5-1 Fmoc-Phe-Hyp-Gly-OBn 之合成 41
2-5-2 Fmoc-Phe-Hyp-Gly-OH 之合成 42
2-6 固相胜□合成法 (Solid-phase peptide synthesis, SPPS) 42
2-6-1酯化反應 (Esterification) 46
2-6-2 去保護 (Deprotection) 46
2-6-3活化 (Activation) 47
2-6-4耦合 (Coupling) 49
2-6-5 切除 (Cleavage) 49
2-7 固相胜□合成膠原蛋白模擬胜□鏈 50
2-7-1 合成 Ac-(POG)3-(PYGXOG)-(POG)3-NH2 系列膠原蛋白模擬胜□鏈 50
2-7-2 合成 Ac-RG-(POG)n-F-NH2, n= 8, 10、Ac-(FOG)8- NH2、Ac-(PRG)8- NH2 系列膠原蛋白模擬胜□鏈 51
2-8 利用高壓液相層析儀 HPLC 進行純化 52
2-9 圓二色光譜儀 (Circular dichroism spectroscopy, CD) 介紹 54
2-10 CD 光譜量測與資料處理 57
2-10-1 Far-UV CD 光譜 (Wavelength scans) 57
2-10-2 變溫 CD 光譜量測 (Thermal denaturation) 58
2-10-3測量折疊速率 ( Refolding kinetics) 58
2-10-4 變溫實驗資料處理 58
2-10-5 動力學實驗資料處理 61
2-11 觀察膠原蛋白模擬胜□自組裝實驗 61
2-11-1 UV-Vis量測濁度 (turbidity) 62
2-11-2量測動態光散射光譜 62
2-11-3動態光散射 (dynamic light scattering, DLS) 介紹 62
2-11-4 以穿透式電子顯微鏡 (TEM) 鑑定結構 64
第三章 結果與討論 66
3-1 Ac-(POG)3-(PYGXOG)-(POG)3-NH2系列胜□探討 66
3-1-1 CD 光譜探討 66
3-1-2 利用 Swiss-PDB Viewer 和 Accelrys Discovery Studio 計算 cation-π 作用力的距離 81
3-2 Ac-RG-(POG)n-F-NH2 (n= 8,10) 系列胜□自組裝探討 83
3-2-1 CD 光譜探討 83
3-2-2 UV-Vis 光譜探討濁度 (turbidity) 85
3-2-3 觀察穿透式電子顯微鏡 (TEM) 86
3-2-4量測動態光散射光譜 92
3-3 異源三聚合胜□ (Heterotrimeric peptides) 系列探討 94
3-3-1 CD 光譜探討 94
第四章 結論 99
第五章 參考文獻 (References) 100
附錄 105

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