膠原蛋白是哺乳動物中含量最豐富的蛋白質，現今已廣泛應用於生醫材料上，為增加其應用性，許多研究者將不同的官能機修飾在膠原蛋白上，或是利用非共價作用力幫助膠原蛋白自組裝成更大的分子來探討其潛在應用，膠原蛋白的特色之一由個別的聚脯胺酸第二型結構 ( PPII ) 所形成之三股螺旋。另一方面，醣類在生理上的應用，生物體內的機制，扮演著重要的角色。
本研究的主題為以膠原蛋白為基底，將一個經過1.2,3,4,6-penta-O-acetyl-β-D-galactopyranoside修飾的羥脯胺酸，以固相胜肽合成法置入膠原蛋白胜肽中，合成一系列醣化修飾的膠原蛋白胜肽，探討醣化對膠原蛋白三股螺旋熱穩定性與其自組裝結構之影響，也利用了分子模擬探討其結構變化。
經由 CD 測量發現，修飾半乳糖後所形成的胜肽鏈，不論修飾在哪一個位置，其所形成之三股螺旋熱穩定度皆會下降。而分子模擬結果發現，其半乳糖之間的距離過大，並不會有作用力的形成。然而醣化後的膠原蛋白胜肽會加速自組裝形成大型結構。再者，由 TEM 的結果發現，有接上半乳糖的胜肽鏈，其自組裝所形成的纖維寬度都較沒修半乳糖的胜肽鏈來的大，因此可知半乳糖修飾有助於膠原蛋白胜肽鏈的自組裝。

Collagen, the most abundant protein in mammalian tissues, has been widely used in biomedical materials. Many researchers have been performing various modifications on collagen to increase its applicability. Different non-covalent interactions were also applied to promote the self-assembly of collagen into higher order structure. Collagen is a right-handed triple helix, and each helix is a left-handed polyproline type II structure containing many (X-Y-Gly)n repeats. On the other hand, carbohydrates are a necessity to organism and play an important role in biological functions.
In this work, we modified (2S,4R)-hydroxyproline with 1.2,3,4,6-penta-O-acetyl-β-D-galactopyranoside and synthesized a series of collagen-related peptides containing this modified amino acid to study the consequences of glycosylation on collagen stability and self-assembly.
CD measurements indicate that glycosylation will destabilize the collagen triple helices. However, TEM shows that the modification promotes the self-assembly of collagen-related peptides into higher-order structures, suggesting that glycosylation could enhance the interactions between collagen-related peptides to speed their assembly.

中文摘要……………………………………………………………………………..Ⅰ
Abstract…………………………………………………………………………….. Ⅱ
第 1 章 緒論 1
1-1 蛋白質結構簡介 1
1-2 膠原蛋白 2
1-2-1 膠原蛋白結構 3
1-2-2 脯胺酸與羥脯胺酸 (Hyp)穩定膠原蛋白的作用 5
1-3 半乳糖 8
1-3-1 單醣介紹 9
1-3-2 豐富醣修飾的Hyp 蛋白( hydroxyproline-rich glycoproteins , HRGPs) 12
1-4 固相胜肽合成法 ( solid phase peptide synthesis, SPPS ) 15
1-4-1 酯化反應 ( esterification ) / 醯胺化反應 ( amidation ) 19
1-4-2 去保護 ( deprotection ) 19
1-4-3 活化 ( activation ) 20
1-4-4 耦合 ( coupling ) 20
1-4-5 切除 21
1-5 圓二色光譜 21
1-6 穿透式電子顯微鏡 ( Transmission Electron Microscope ) 26
1-6-1原理 28
1-6-2 成像機制 30
1-7 Discovery Studio ( DS ) 31
1-8 研究動機 32
第 2 章 實驗部分 33
2-1 儀器 33
2-2使用藥品 34
2-3實驗步驟流程 37
2-4合成 Fmoc-Pro-Hyp-OH 39
2-4-1 Boc-(2S, 4R)-hydroxyproline (1) 之合成 39
2-4-2 Boc-Hyp-Gly-OBn (2)之合成 40
2-4-3 Fmoc-Pro-Hyp-Gly-OBn(3)之合成 40
2-4-4 Fmoc-Pro-Hyp-Gly-OH (4)之合成 41
2-5 合成 4-O-[(2,3,4,6-Tetra-O-acetyl)-β-D-galactopyranosyl]-Nα- fluoren-9-yl-methoxycarbonyl-(2S,4R)-4-hydroxyproline (6) 42
2-5-1 Fmoc-Hyp-OH (5) 之合成 43
2-5-2 4-O-[(2,3,4,6-Tetra-O-acetyl)-β-D-galactopyranosyl]-Nα- fluoren-9-yl-methoxycarbonyl-(2S,4R)-4-hydroxyproline (6) 之合成 43
2-6 4-O-[(2,3,4,6-Tetra-O-acetyl)-β-D-galactopyranosyl]-Nα- fluoren-9-yl-methoxycarbonyl-(2S,4R)-4-hydroxyproline (6) 與膠原蛋白系列胜肽之合成 44
2-7 將胜肽鏈上的乙醯氧基 (acetoxy group) 轉化為羥基(hydroxyl group) 48
2-8 CD 光譜量測與資料處理 49
2-8-1 Far-UV CD 光譜 (Wavelength scans) 49
2-8-2 變溫 CD 光譜量測 (Thermal denaturation) 49
2-8-3變溫實驗資料處理 50
2-8-4 測量摺疊速率 ( Refolding kinetics) 52
2-8-5 動力學實驗資料處理 52
2-9 Discovery studio 4.0 計算Energy minimization 53
2-10穿透式電子顯微鏡量測 54
第 3 章 實驗結果與討論 55
3-1 N-端 Hyp 修飾之胜肽鏈探討 55
3-2 C端Hyp修飾之胜肽鏈探討 57
3-3 中間 Hyp 修飾之胜肽鏈探討 59
3-4 取代X位置胜肽鏈之探討 61
3-5 胜肽摺疊速率比較 65
3-6利用穿透式電子顯微鏡觀察膠原蛋白與半乳糖系列胜肽的聚集結構形貌 67
3-7 Discovery studio 4.0 模擬半乳糖膠原蛋白胜肽 75
3-7-1 N 端 Hyp 修飾模擬胜肽鏈之探討 75
3-7-2 C端Hyp修飾模擬胜肽鏈之探討 76
3-7-3 中間 Hyp 修飾模擬胜肽鏈之探討 77
3-7-4 X位置 Hyp 修飾模擬胜肽鏈之探討 78
第 4 章 結論 80
參考文獻 (References) 81
附件 84

1. Bella, J., Brodsky, B., and Berman, H. M. (1995) Hydration structure of a collagen peptide, Structure 3, 893-906.
2. Internet : 124.xingshuo.net/siwameinv/ogkukuu.html ( accessible on 06.20)
3. Griffon, N. B., V.; Dieryck, W.; Domoulin, A.; Paginer, J.; Poyart, C.;Marden, M. C. (1998) tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates, Protein Sci. 7, 673-680.
4. Prockop, D. J. K., K. I. (1995) Collagens: molecular biology, diseases, and potentials for therapy, Annu. Rev. Biochem. 64, 403-434.
5. Prockop, D. J. (1998) What holds us together? Why do some of us fall apart? What can we do about it?, Matrix Biol. 16, 519-528.
6. prockop, D. J. (1999) Hopkins Memorial Medal lecture. Pleasant surprises en route from the biochemistry of collagen to attempts at gene therapy, Biochem. Soc. Trans, 15-31.
7. Rich, A. C., F. H. (1961) The molecular structure of collagen, J. Mol. Biol. 3, 483-506.
8. Bella, J., Eaton, M.; Brodsky, B., and Berman, H. M. (1994) Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution, Science 266, 75-81.
9. Fraser, R. D. B. M., T. P. (1973) Conformation in fibrous proteins, Academic Press, London.
10. Ramachandran, G. N. (1967) Structure of collagen at the molecular level. In: Treatise on collagen, Academic Press, London.
11. Hinderaker, M. P. R., R. T. (2003) An electronic effect on protein structure, Protein Sci. 12, 1188-1194.
12. Brodsky, B. R., J. A. M. (1997) The collagen triple-helix structure, Matrix Biol. 15, 545-554.
13. Primalov, P. L. (1982) Stability of proteins: Proteins which do not present a single cooperative system, Protein Chem. 35, 1-104.
14. Rita, B. V., G.; Luigi, V.; Adriana, Z. (2004) Imino acid and collagen triple helix stability: Characterization of collagen-like polypeptides containing Hyp-Hyp-Gly sequence repeats, J. Am. Chem. Soc. 126, 11402-11403.
15. Maton, A. J., H.; Charles, W. M.; Susan, J.; Maryanna, Q. W.; David, L.; Jill D. W. . (1993) Human Biology and Health, Prentice Hall, 52-59.
16. Lamport, D. T. A. (1967) Hydroxyproline-O-glycosidic linkage of the plant cell wall glycoprotein extensin, Nature 216, 1322-1324.
17. Sommer-Knudsen, J. B., A.; Clarke, A. E., 483–497. (1998) Hydroxyproline-rich plant glycoproteins, Phytochemistry 47, 483-497.
18. Jose´, M. P. E., P. . (1993) New Phytol. 125, 259-282.
19. Neil, W. O. J., S.; Erika, L.; Frank, S. (2010) Contiguous O-galactosylation of 4(R)-hydroxyproline residue forms very stable polyproline ll helices, J. Am. Chem. Soc. 132, 5036-5042.
20. Neil W. Owens, C. B., Joe D. O’Neil, Kirk Marat, and Frank Schweizer. (2007) Effects of glycosylation of (2S,4R)-4-hydroxyproline on the conformation, kinetics, and thermodynamics of prolyl amide isomerization, J. Am. Chem. Soc., 11670-11671.
21. Chan, W. C. W., P. D. (2000) Fmoc solid phase peptide synthesis: a practical approach, Oxford University Press : New York, xxiv, 346.
22. 2015), I. C. c. a. o.
23. Berova, N. N., K.; Woody, R. (2000) Circular dichromism:Principles and applications, Wiley, Hoboken.
24. Internet :www.twwiki.com/圓二色性 ( accessible on 05, 2014).
25. 陳建淼/洪連輝編輯. (2009) 穿透式電子顯微鏡 (Transmission Electron Microscopy：TEM), 國立彰化師範大學物理學系.
26. Ackerman, M. S. B., M.; Shenoy, N.; Beck, K.; Ramshaw, J. A. M.; Brodsky, B. (1999) Sequence dependence of the folding of collagen-like peptides: single amino acid affect the rate of triple-helix nucleation, J. Biol. Chem. 274, 7668-7673.
27. Bretscher, L. E. J., C. L.; Taylor, K. M.; DeRider, M. L.; Raind, R. T. (2011) Conformational stability of collagen relies on a stereoelectronic effect, J. Am. Chem. Soc. 123, 777-778.
28. Leonard, D. W. M., K. M. (1997) Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma, Biophys. J. 72, 1382-1387.
29. Michael A. B.; Anil, K. P., ; Agata, A. B.; Neal J. Z. (2014) OGlcNAcylation and phosphorylation have opposing structural effects in tau: phosphothreonine induces particular conformational order, J. Am. Chem. Soc. 136, 3803-3816.
30. 陳佳青. (2009) Cation-π 作用力對膠原蛋白穩定性及自組裝影響之探討, (碩士學位論文), 清華大學化學研究所.
31. Brookhaven Instruction Manual for 90 plus.
32. Chen, Y.-S., Chen, C.-C., and Horng, J.-C. (2011) Thermodynamic and kinetic consequences of substituting glycine at different positions in a Pro-Hyp-Gly repeat collagen model peptide, Biopolymers (Pept. Sci.) 96, 60-68.
33. Kar, K. A., P.; Bryan, M. A.; Persikov, A. V.; Mohs, A.; Wang, Y. H.; Brodsky, B. Self-association of collagen triple helix peptides into higher order structures, J. Biol. Chem. 2006, 33283-33290.
34. 張峻銘. (2014) 膠原蛋白模擬胜肽對貝它類澱粉蛋白聚集影響的探討, (碩士學位論文), 國立清華大學化學系.
35. Roman S. E.;Helma, W. (2010) Functionalizable collagen model peptides, J. Am. Chem. Soc. 132, 13957-13959.