在生醫領域中，已發展出數種胜肽藥物，或是以胜肽作為骨架，形成藥物載體。但在生物體內，胜肽易受到酵素辨認，而失去活性，目前發展出裝訂胜肽(stapled peptide)的技術來提升生物體內胜肽的壽命。我們嘗試於脯胺酸多肽螺旋II (Polyproline Helix II, PP II)結構上進行裝訂，利用控制裝訂鏈(Linker)的長度，與改變裝訂位置，觀察此種設計對PP II結構影響。經由交叉測試後，得知i, i+3裝訂會比i, i+2與i, i+1裝訂容易成功。和寡脯胺酸多肽相比，在正丙醇溶液中，可以發現不同長度，位置的裝訂方式會影響結構。除了透過CD光譜鑑定結構外，也使用GROMACS動力學軟體，來模擬研究中合成出來的數種裝訂胜肽。

In recent years, many drugs and carriers based on peptides has been developed. However, drug design based on peptide has a critical disadvantage that they may be degraded by enzymes in organism. For this issue, peptide stapling is one practical solution to extend the lifespan of peptides. While polyproline peptides have unique structure for various applications, we “stapled” polyproline peptide systematically to investigate the effects on polyproline helix II (PP II) structure by change linker length and the locations of stapled residues. From circular dischroism (CD) analysis, we found that stapling at i, i+3 residues of polyproline peptides stabilizing PP II structure better than other locations at i, i+1 and i, i+2. Compared to normal polyproline, the PP II structure stapled polyproline is also affected by the staple length in n-propanol. In addition to CD spectrum, we use GROMACS software to simulate the molecular dynamics of stapled peptides in water.

摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 ix
式目錄 x
縮寫對照表 xi
第一章、 緒論 1
1.1 前言 1
1.2 蛋白質二級結構 2
1.2.1 α螺旋(α Helix) 3
1.2.2 β摺板(β Sheet) 5
1.2.3 310螺旋(310 Helix) 6
1.2.4 π螺旋(π Helix) 7
1.2.5 膠原蛋白螺旋(Collagen Helix) 8
1.3 脯胺酸多肽螺旋(Polyproline Helix) 8
1.3.1 脯胺酸多肽螺旋結構與特性 8
1.3.2 脯胺酸多肽的應用 10
1.4 疊氮-炔類[3+2]環化加成 12
1.4.1 CuAAC反應機構 12
1.4.2 CuAAC的特性 14
1.4.3 CuAAC於生物上的應用 14
1.5 裝訂胜肽(Stapled Peptide) 16
1.5.1 裝訂胜肽的結構特性 16
1.5.2 裝訂胜肽在生物研究上的應用 16
1.6 圓二色性 (Circular Dischroism, CD) 18
1.6.1 圓二色性的原理 18
1.6.2 以圓二色性判斷蛋白質二級結構 18
1.7 分子動力模擬 20
1.7.1 GROMACS 20
1.7.2 SHAKE演算法 21
1.7.3 LINCS 演算法 21
1.7.4 GROMACS分析程式 21
1.8 研究動機 22
1.9 實驗設計 22
第二章、 結果與討論 24
2.1 實驗設計 24
2.2 固相多肽合成之構築單元合成 25
2.3 裝訂鏈合成 26
2.4 固相胜肽合成法之寡脯胺酸多肽合成 28
2.4.1 SPPS流程 28
2.4.2 模板胜肽合成 30
2.4.3 多重裝訂模板胜肽合成 32
2.4.4 裝訂選擇性之模板胜肽合成 33
2.5 裝訂胜肽合成 35
2.5.1 裝訂胜肽裝訂測試 35
2.5.2 不同長度裝訂鏈之裝訂胜肽合成 38
2.5.3 不同位向之裝訂胜肽合成 41
2.5.4 未反應之炔基測試 51
2.5.5 多重裝訂胜肽合成 61
2.5.6 裝訂選擇性測試 64
2.6 裝訂胜肽結構鑑定 70
2.6.1 CD光譜鑑定 70
2.6.2 綜合討論 76
2.7 分子動力模擬 76
2.7.1 與模擬初始結構之RMSD 77
2.7.2 模擬過程中之Potential Energy 83
2.8 結論 89
第三章、 實驗方法與材料 91
3.1 Synthetic procedures and characterization 92
3.1.1 Building blocks 92
3.1.2 Linkers 97
3.1.3 Peptide synthesis 101
3.1.4 Singular stapling 110
3.1.5 Multiple stapling 126
3.1.6 Hydrogenation of stapled peptides 127
3.2 Molecule dynamic simulation 129
第四章、 參考文獻 131
附錄 136

1. Lovell, S. C.; Davis, I. W.; Arendall, W. B.; Bakker, P. I. W. d.; Word, J. M.; Prisant, M. G.; Richardson, J. S.; Richardson, D. C., Structure Validation by Cα Geometry: ϕ,ψ and Cβ Deviation. Proteins 2003, 50 (3), 437-450.
2. Nick Pace, C.; Martin Scholtz, J., A Helix Propensity Scale Based on Experimental Studies of Peptides and Proteins. Biophys. J. 1998, 75 (1), 422-427.
3. Jacobsen, Ø.; Klaveness, J.; Rongved, P., Structural and Pharmacological Effects of Ring-Closing Metathesis in Peptides. Molecules 2010, 15 (9).
4. Hilinski, G. J.; Kim, Y.-W.; Hong, J.; Kutchukian, P. S.; Crenshaw, C. M.; Berkovitch, S. S.; Chang, A.; Ham, S.; Verdine, G. L., Stitched α-Helical Peptides via Bis Ring-Closing Metathesis. J. Am. Chem. Soc. 2014, 136 (35), 12314-12322.
5. Kim, Y.-W.; Kutchukian, P. S.; Verdine, G. L., Introduction of All-Hydrocarbon i,i+3 Staples into α-Helices via Ring-Closing Olefin Metathesis. Org. Lett. 2010, 12 (13), 3046-3049.
6. Wimley, W. C., The Versatile β-Barrel Membrane Protein. Curr. Opin. Struct. Biol. 2003, 13 (4), 404-411.
7. Millhauser, G. L., Views of Helical Peptides: A Proposal for the Position of 310-Helix along the Thermodynamic Folding Pathway. Biochemistry 1995, 34 (12), 3873-3877.
8. Tonlolo, C.; Benedetti, E., The Polypeptide 310-Helix. Trends Biochem. Sci 1991, 16, 350-353.
9. Cooley, R. B.; Arp, D. J.; Karplus, P. A., Evolutionary Origin of a Secondary Structure: π-Helices as Cryptic but Widespread Insertional Variations of α-Helices That Enhance Protein Functionality. J. Mol. Biol. 2010, 404 (2), 232-246.
10. Berisio, R.; Vitagliano, L.; Mazzarella, L.; Zagari, A., Crystal Structure of the Collagen Triple Helix Model [(Pro‐Pro‐Gly)10]3. Protein Sci. 2002, 11 (2), 262-270.
11. Adzhubei, A. A.; Sternberg, M. J. E., Left-Handed Polyproline II Helices Commonly Occur in Globular Proteins. J. Mol. Biol. 1993, 229 (2), 472-493.
12. Rucker, A. L.; Creamer, T. P., Polyproline II Helical Structure in Protein Unfolded States: Lysine Peptides Revisited. Protein. Sci. 2002, 11 (4), 980-985.
13. Wilhelm, P.; Lewandowski, B.; Trapp, N.; Wennemers, H., A Crystal Structure of an Oligoproline PPII-Helix, at Last. J. Am. Chem. Soc. 2014, 136 (45), 15829-15832.
14. Ruggiero, M. T.; Sibik, J.; Orlando, R.; Zeitler, J. A.; Korter, T. M., Measuring the Elasticity of Poly‐L‐Proline Helices with Terahertz Spectroscopy. Angew. Chem. Int. Ed. Engl. 2016, 55 (24), 6877-6881.
15. McCafferty, D. G.; Friesen, D. A.; Danielson, E.; Wall, C. G.; Saderholm, M. J.; Erickson, B. W.; Meyer, T. J., Photochemical Energy Conversion in a Helical Oligoproline Assembly. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (16), 8200.
16. Doose, S.; Neuweiler, H.; Barsch, H.; Sauer, M., Probing Polyproline Structure and Dynamics by Photoinduced Electron Transfer Provides Evidence for Deviations from a Regular Polyproline Type II Helix. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (44), 17400-17405.
17. Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Kumke, M.; Eaton, W. A., Polyproline and the “Spectroscopic Ruler” Revisited with Single-Molecule Fluorescence. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (8), 2754-2759.
18. Kroll, C.; Mansi, R.; Braun, F.; Dobitz, S.; Maecke, H. R.; Wennemers, H., Hybrid Bombesin Analogues: Combining an Agonist and an Antagonist in Defined Distances for Optimized Tumor Targeting. J. Am. Chem. Soc. 2013, 135 (45), 16793-16796.
19. Huisge, R., 1,3-Dipolar Cycloadditions. Proc. Chem. Soc. 1961, 0, 357-396.
20. Meldal, M.; Tornøe, C. W., Cu-Catalyzed Azide−Alkyne Cycloaddition. Chem. Rev. 2008, 108 (8), 2952-3015.
21. Liang, L.; Astruc, D., The Copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) “Click” Reaction and its Applications. An Overview. Coord. Chem. Rev. 2011, 255 (23), 2933-2945.
22. Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V., Copper(I)-Catalyzed Synthesis of Azoles. DFT Study Predicts Unprecedented Reactivity and Intermediates. J. Am. Chem. Soc. 2005, 127 (1), 210-216.
23. Worrell, B. T.; Malik, J. A.; Fokin, V. V., Direct Evidence of a Dinuclear Copper Intermediate in Cu(I)-Catalyzed Azide-Alkyne Cycloadditions. Science 2013, 340 (6131), 457.
24. Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V., Polytriazoles as Copper(I)-Stabilizing Ligands in Catalysis. Org. Lett. 2004, 6 (17), 2853-2855.
25. Li, L.; Zhang, Z., Development and Applications of the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) as a Bioorthogonal Reaction. Molecules 2016, 21 (10).
26. Kolb, H. C.; Sharpless, K. B., The Growing Impact of Click Chemistry on Drug Discovery. Drug Discovery Today 2003, 8 (24), 1128-1137.
27. Beatty, K. E.; Liu, J. C.; Xie, F.; Dieterich, D. C.; Schuman, E. M.; Wang, Q.; Tirrell, D. A., Fluorescence Visualization of Newly Synthesized Proteins in Mammalian Cells. Angew. Chem. Int. Ed. 2006, 45 (44), 7364-7367.
28. Baek, S.; Kutchukian, P. S.; Verdine, G. L.; Huber, R.; Holak, T. A.; Lee, K. W.; Popowicz, G. M., Structure of the Stapled p53 Peptide Bound to Mdm2. J. Am. Chem. Soc. 2012, 134 (1), 103-106.
29. Cromm, P. M.; Schaubach, S.; Spiegel, J.; Fürstner, A.; Grossmann, T. N.; Waldmann, H., Orthogonal Ring-Closing Alkyne and Olefin Metathesis for the Synthesis of Small GTPase-Targeting Bicyclic Peptides. Nat. Commun. 2016, 7, 11300.
30. Greenfield, N. J., Using Circular Dichroism Spectra to Estimate Protein Secondary Structure. Nat. Protocols 2007, 1 (6), 2876-2890.
31. Ronish, E. W.; Krimm, S., The Calculated Circular Dichroism of Polyproline II in the Polarizability Approximation. Biopolymers 1974, 13 (8), 1635-1651.
32. Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R., GROMACS: A Message-Passing Parallel Molecular Dynamics Implementation. Comput. Phys. Commun. 1995, 91 (1), 43-56.
33. Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C., Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23 (3), 327-341.
34. Forester, T. R.; Smith, W., SHAKE, Rattle, and Roll: Efficient Constraint Algorithms for Linked Rigid Bodies. J. Comput. Chem. 1998, 19 (1), 102-111.
35. Tao, P.; Wu, X.; Brooks, B. R., Maintain Rigid Structures in Verlet Based Cartesian Molecular Dynamics Simulations. J. Chem. Phys. 2012, 137 (13), 134110.
36. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M., LINCS: A Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18 (12), 1463-1472.
37. Mihali, V.; Foschi, F.; Penso, M.; Pozzi, G., Chemoselective Synthesis of N‐Protected Alkoxyprolines under Specific Solvation Conditions. Eur. J. Org. Chem. 2014, 2014 (24), 5351-5355.
38. Schwabacher, A. W.; Lane, J. W.; Schiesher, M. W.; Leigh, K. M.; Johnson, C. W., Desymmetrization Reactions:  Efficient Preparation of Unsymmetrically Substituted Linker Molecules. J. Org. Chem. 1998, 63 (5), 1727-1729.
39. Conrow, R. E.; Dean, W. D., Diazidomethane Explosion. Org. Process Res. Dev. 2008, 12 (6), 1285-1286.
40. Bräse, S.; Gil, C.; Knepper, K.; Zimmermann, V., Organic Azides: An Exploding Diversity of a Unique Class of Compounds. Angew. Chem. Int. Ed. 2005, 44 (33), 5188-5240.
41. Northfield, S. E.; Mountford, S. J.; Wielens, J.; Liu, M.; Zhang, L.; Herzog, H.; Holliday, N. D.; Scanlon, M. J.; Parker, M. W.; Chalmers, D. K.; Thompson, P. E., Propargyloxyproline Regio- and Stereoisomers for Click-Conjugation of Peptides: Synthesis and Application in Linear and Cyclic Peptides. Aust. J. Chem. 2015, 68 (9), 1365-1372.
42. Wang, C.; Salmon, L.; Li, Q.; Igartua, M. E.; Moya, S.; Ciganda, R.; Ruiz, J.; Astruc, D., From Mono to Tris-1,2,3-triazole-Stabilized Gold Nanoparticles and Their Compared Catalytic Efficiency in 4-Nitrophenol Reduction. Inorg. Chem. 2016, 55 (13), 6776-6780.
43. Chiba, K.; Asanuma, M.; Ishikawa, M.; Hashimoto, Y.; Dodo, K.; Sodeoka, M.; Yamaguchi, T., Specific Fluorescence Labeling of Target Proteins by Using a Ligand–4-Azidophthalimide Conjugate. Chem. Commun. 2017, 53 (62), 8751-8754.
44. Hemamalini, A.; Mudedla, S. K.; Subramanian, V.; Mohan Das, T., Design, Synthesis and Metal Sensing Studies of Ether-Linked Bis-Triazole Derivatives. New J. Chem. 2015, 39 (5), 3777-3784.
45. Brauch, S.; Henze, M.; Osswald, B.; Naumann, K.; Wessjohann, L. A.; van Berkel, S. S.; Westermann, B., Fast and Efficient MCR-Based Synthesis of Clickable Rhodamine Tags for Protein Profiling. Org. Biomol. Chem. 2012, 10 (5), 958-965.
46. Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E., GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1-2, 19-25.
47. Páll, S.; Abraham, M. J.; Kutzner, C.; Hess, B.; Lindahl, E. In Tackling Exascale Software Challenges in Molecular Dynamics Simulations with GROMACS, Solving Software Challenges for Exascale, Cham, 2015; Markidis, S.; Laure, E., Eds. Springer International Publishing: Cham, 2015; pp 3-27.
48. Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D.; Hess, B.; Lindahl, E., GROMACS 4.5: a High-Throughput and Highly Parallel Open Source Molecular Simulation Toolkit. Bioinformatics 2013, 29 (7), 845-854.
49. Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E., GROMACS 4:  Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory. Comput. 2008, 4 (3), 435-447.
50. Spoel, D. V. D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C., GROMACS: Fast, Flexible, and Free. J. Comput. Chem. 2005, 26 (16), 1701-1718.
51. Lindahl, E.; Hess, B.; van der Spoel, D., GROMACS 3.0: a Package for Molecular Simulation and Trajectory Analysis. J. Mol. Model. 2001, 7 (8), 306-317.
52. Schmid, N.; Eichenberger, A. P.; Choutko, A.; Riniker, S.; Winger, M.; Mark, A. E.; van Gunsteren, W. F., Definition and Testing of the GROMOS Force-Field Versions 54A7 and 54B7. Eur Biophys J 2011, 40 (7), 843.
53. Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Hermans,, Interaction Models for Water in Relation to Protein Hydration. Struct. Bonding 1981, 14, 331.
54. Hockney, R. W., The Potential Calculation and Some Applications. Methods Comput. Phys. 1970, 9, 136-211.
55. Miyamoto, S.; Kollman Peter, A., Settle: An Analytical Version of the SHAKE and RATTLE Algorithm for Rigid Water Models. J. Comput. Chem. 2004, 13 (8), 952-962.
56. Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G., A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103 (19), 8577-8593.
57. Bussi, G.; Donadio, D.; Parrinello, M., Canonical Sampling through Velocity Rescaling. J. Chem. Phys. 2007, 126 (1), 014101.
58. Hentzen, N. B.; Smeenk, L. E. J.; Witek, J.; Riniker, S.; Wennemers, H., Cross-Linked Collagen Triple Helices by Oxime Ligation. J. Am. Chem. Soc. 2017, 139 (36), 12815-12820.
59. Malde, A. K.; Zuo, L.; Breeze, M.; Stroet, M.; Poger, D.; Nair, P. C.; Oostenbrink, C.; Mark, A. E., An Automated Force Field Topology Builder (ATB) and Repository: Version 1.0. J. Chem. Theory. Comput. 2011, 7 (12), 4026-4037.
60. Koziara, K. B.; Stroet, M.; Malde, A. K.; Mark, A. E., Testing and Validation of the Automated Topology Builder (ATB) Version 2.0: Prediction of Hydration Free Enthalpies. J. Comput. Aided Mol. Des. 2014, 28 (3), 221-233.
61. Canzar, S.; El-Kebir, M.; Pool, R.; Elbassioni, K.; Malde, A. K.; Mark, A. E.; Geerke, D. P.; Stougie, L.; Klau, G. W., Charge Group Partitioning in Biomolecular Simulation. J. Comput. Biol. 2013, 20 (3), 188-198.