為了研究蛋白質的結構與功能，科學家可以透過化學全合成的方式得到具有特定後轉譯修飾之蛋白質。其中最為廣泛使用的是天然化學連接法。由於蛋白質中半胱胺酸的含量非常稀少，因此其應用性受到侷限。為此，我們開發了新的策略，進而突破胜肽序列需含有Cys的限制。本篇論文合成了硫酯胜肽以及具有硫醇修飾之光敏感輔助基團胜肽，在完成NCL後藉由光照移除輔助基團，不需另外加入化學試劑。在研究中發現在賴胺酸的例子中，16元環之反應過渡態不能完成分子內醯基轉移，而19、22、25元環可以順利完成NCL以得到醯胺鍵鍵結之產物。
為了證明本策略的可行性，本篇研究嘗試合成大鼠抗微生物肽部分序列。首先利用Fmoc-SPPS合成硫酯胜肽rCRAMP (7-11) 以及光敏感輔助基團之胜肽rCRAMP (12-18)，通過NCL將此兩段胜肽進行連接後，照光移除輔助基得到rCRAMP (7-18)。未來將以此為基礎，繼續完成大鼠抗微生物肽之合成。

Total chemical synthesis provides a unique approach for the access to post-translationally modified protein for structural and functional studies. One of these methods, Native chemical ligation (NCL), is widely used. Because of the rare amount of cysteine in proteins, NCL has been limited. Therefore, we develop a method which allows for the synthesis of protein from cysteine-free peptide. We synthesize a thioester peptide and a photocleavable auxiliary peptide, which is modified with a thiol on the auxiliary. Based on this strategy, we can use photo-irradiation instead of using extra chemical reagents to remove the auxiliary and get an unmodified peptide. In our study, we found that transition state of ligation reaction with 16 membered ring can’t facilitates intramolecular acyl transfer. However, the transition state with 19, 22 and 25 membered ring can proceed NCL smoothly to complete the amide bond formation.
To illustrate the usefulness of our strategy, we attempted the synthesis of native polypeptide rCRAMP, a rat homologue of the human cathelicidin LL-37, which is a kind of antimicrobial peptide for the innate immune system and playing an important role in the brain. We synthesized rCRAMP (7-11) thioester and rCRAMP (12-18) auxiliary peptide via Fmoc-SPPS. And these two peptide fragment can proceed NCL successfully to complete rCRAMP (7-18). In the future, we will continue the assembly of N-terminal auxiliary peptide and C-terminal thioester peptide toward the synthesis of rCRAMP.

Abstract I
摘要 II
謝誌 III
目錄 IV
圖目錄 VIII
流程圖目錄 XII
表目錄 XIV
縮寫表 XV
胺基酸總表 XIX
第一章 緒論 1
1-1 蛋白質的化學合成 1
1-1-1 固相多肽合成法 2
1-1-2 選擇性化學連接法 3
1-2 天然化學連接法 7
1-2-1 天然化學連接法之起源 8
1-2-2 天然化學連接法之發展與限制 8
1-3 不含半胱胺酸之天然化學連接法 10
1-3-1 利用自由基方式移除硫醇輔助基 10
1-3-2 利用輔助基團之連接法 13
1-3-3 側鏈輔助連接法 16
1-4 以固相多肽合成法合成C端為硫酯基之胜肽 19
1-4-1 合成硫酯胜肽之文獻回顧 20
1-4-2 近年之發展 21
1-5 利用天然化學連接法組裝胜肽片段的策略 26
1-5-1 從N端合成至C端 27
1-5-2 從C端合成至N端 29
1-6 利用微波輔助固相多肽合成法 30
1-7 研究動機 31
第二章 結果與討論 33
2-1 合成胜肽前驅物 33
2-1-1 胜肽前驅物之逆合成分析 33
2-1-2 光敏感輔助基團之合成 33
2-1-3 合成具光敏感輔助基團之胺基酸建構單元 34
2-1-4 微波條件之測試 37
2-1-5 合成具光敏感輔助基團之胜肽 38
2-1-6 硫酯二肽之合成 49
2-2 利用胜肽前驅物進行側鏈輔助連接法 51
2-2-1 胜肽前驅物15、16之側鏈輔助連接法 52
2-2-2 增大分子內醯基遷移之環數 57
2-2-3 胜肽前驅物20之側鏈輔助連接法 60
2-2-4 結論 63
2-3 合成真實胜肽：rCRAMP 65
2-3-1 rCRAMP之逆合成分析 66
2-3-2 合成具有光敏感輔助基之rCRAMP（12-18）胜肽片段 66
2-3-3 合成rCRAMP硫酯胜肽片段 68
2-3-4 rCRAMP(7-18)之側鏈輔助連接法 75
第三章 未來展望 79
第四章 實驗部分 80
4-1 Reagents and Solvents 80
4-2 Spectra Notes 81
4-3 High-performance liquid chromatography (HPLC) 82
4-3-1 Analytical HPLC 82
4-3-2 Semi-preparative HPLC purification 82
4-4 Microwave-assisted Solid Phase Peptide Synthesis 83
4-4-1 General manual Fmoc-SPPS 83
4-4-2 Monitoring of solid phase reactions 85
4-4-3 Synthesis of thioester peptide by Fmoc-SPPS 85
4-5 UV-irradiation 86
4-6 Synthetic procedures and characterization 87
第五章 參考文獻與資料 115
光譜附錄 122

1. Bondalapati, S.; Jbara, M.; Brik, A., Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins. Nat. Chem. 2016, 8, 407.
2. Merrifield, R. B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149-2154.
3. Dawson, P. E.; Kent, S. B. H., Synthesis of Native Proteins by Chemical Ligation. Annu. Rev. Biochem 2000, 69, 923-960.
4. Kent, S. B. H., Total chemical synthesis of proteins. Chem. Soc. Rev. 2009, 38, 338-351.
5. Raibaut, L.; Ollivier, N.; Melnyk, O., Sequential native peptide ligation strategies for total chemical protein synthesis. Chem. Soc. Rev. 2012, 41, 7001-7015.
6. Harmand, T. J. R.; Murar, C. E.; Bode, J. W., New chemistries for chemoselective peptide ligations and the total synthesis of proteins. Curr. Opin. Chem. Biol 2014, 22, 115-121.
7. Hackenberger, C. P.; Schwarzer, D., Chemoselective ligation and modification strategies for peptides and proteins. Angew. Chem. Int. Ed. 2008, 47, 10030-10074.
8. Nilsson, B. L.; Kiessling, L. L.; Raines, R. T., Staudinger ligation: a peptide from a thioester and azide. Org. Lett. 2000, 2, 1939-1941.
9. Saxon, E.; Armstrong, J. I.; Bertozzi, C. R., A “traceless” Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2000, 2, 2141-2143.
10. Nilsson, B. L.; Kiessling, L. L.; Raines, R. T., High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide. Org. Lett. 2001, 3, 9-12.
11. Köhn, M.; Breinbauer, R., The Staudinger ligation—a gift to chemical biology. Angew. Chem. Int. Ed. 2004, 43, 3106-3116.
12. Zhang, Y.; Xu, C.; Lam, H. Y.; Lee, C. L.; Li, X., Protein chemical synthesis by serine and threonine ligation. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 6657-6662.
13. Bode, J. W.; Fox, R. M.; Baucom, K. D., Chemoselective amide ligations by decarboxylative condensations of N‐alkylhydroxylamines and α‐ketoacids. Angew. Chem. Int. Ed. 2006, 45, 1248-1252.
14. Medina, S. I.; Wu, J.; Bode, J. W., Nitrone protecting groups for enantiopure N-hydroxyamino acids: synthesis of N-terminal peptide hydroxylamines for chemoselective ligations. Org. Biomol. Chem. 2010, 8, 3405-3417.
15. Patil, M., A revised mechanism for the α-ketoacid hydroxylamine amide forming ligations. Org. Biomol. Chem. 2017, 15, 416-425.
16. Pattabiraman, V. R.; Ogunkoya, A. O.; Bode, J. W., Chemical protein synthesis by chemoselective α-ketoacid–hydroxylamine (KAHA) ligations with 5‐oxaproline. Angew. Chem. Int. Ed. 2012, 51, 5114-5118.
17. Li, Y. M.; Huang, Y. C.; Liu, L., KAHA Ligation at Serine. ChemBioChem 2016, 17, 28-30.
18. Pusterla, I.; Bode, J. W., An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations. Nat. Chem. 2015, 7, 668.
19. Conibear, A. C.; Watson, E. E.; Payne, R. J.; Becker, C. F. W., Native chemical ligation in protein synthesis and semi-synthesis. Chem. Soc. Rev. 2018, 47, 9046-9068.
20. Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H., Über Peptidsynthesen. 8. Mitteilung Bildung von S‐haltigen Peptiden durch intramolekulare Wanderung von Aminoacylresten. Justus Liebigs Ann. Chem. 1953, 583, 129-149.
21. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S., Synthesis of proteins by native chemical ligation. Science 1994, 266, 776-779.
22. Johnson, E. C.; Kent, S. B., Insights into the mechanism and catalysis of the native chemical ligation reaction. J. Am. Chem. Soc. 2006, 128, 6640-6646.
23. Bang, D.; Kent, S. B., A one‐pot total synthesis of crambin. Angew. Chem. Int. Ed. 2004, 43, 2534-2538.
24. Johnson, E. C.; Durek, T.; Kent, S. B., Total chemical synthesis, folding, and assay of a small protein on a water‐compatible solid support. Angew. Chem. Int. Ed. 2006, 45, 3283-3287.
25. Torbeev, V. Y.; Kent, S. B., Convergent chemical synthesis and crystal structure of a 203 amino acid “covalent dimer” HIV‐1 protease enzyme molecule. Angew. Chem. Int. Ed. 2007, 46, 1667-1670.
26. McCaldon, P.; Argos, P., Oligopeptide biases in protein sequences and their use in predicting protein coding regions in nucleotide sequences. Proteins: Struct., Funct., Bioinf. 1988, 4, 99-122.
27. Yan, L. Z.; Dawson, P. E., Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 2001, 123, 526-533.
28. Wan, Q.; Danishefsky, S. J., Free‐radical‐based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. 2007, 46, 9248-9252.
29. Rohde, H.; Seitz, O., Invited reviewligation—Desulfurization: A powerful combination in the synthesis of peptides and glycopeptides. J. Pept. Sci. 2010, 94, 551-559.
30. Wong, C. T.; Tung, C. L.; Li, X., Synthetic cysteine surrogates used in native chemical ligation. Mol. BioSyst. 2013, 9, 826-833.
31. Malins, L. R.; Payne, R. J., Synthetic amino acids for applications in peptide ligation–desulfurization chemistry. Aust. J. Chem. 2015, 68, 521-537.
32. Crich, D.; Banerjee, A., Native chemical ligation at phenylalanine. J. Am. Chem. Soc. 2007, 129, 10064-10065.
33. Chen, J.; Wan, Q.; Yuan, Y.; Zhu, J.; Danishefsky, S. J., Native chemical ligation at valine: a contribution to peptide and glycopeptide synthesis. Angew. Chem. Int. Ed. 2008, 47, 8521-8524.
34. Haase, C.; Rohde, H.; Seitz, O., Native chemical ligation at valine. Angew. Chem. Int. Ed. 2008, 47, 6807-6810.
35. Tan, Z.; Shang, S.; Danishefsky, S. J., Insights into the finer issues of native chemical ligation: an approach to cascade ligations. Angew. Chem. Int. Ed. 2010, 49, 9500-9503.
36. Siman, P.; Karthikeyan, S. V.; Brik, A., Native chemical ligation at glutamine. Org. Lett. 2012, 14, 1520-1523.
37. Chen, J.; Wang, P.; Zhu, J.; Wan, Q.; Danishefsky, S. J., A program for ligation at threonine sites: application to the controlled total synthesis of glycopeptides. Tetrahedron 2010, 66, 2277-2283.
38. Shang, S.; Tan, Z.; Dong, S.; Danishefsky, S. J., An advance in proline ligation. J. Am. Chem. Soc. 2011, 133, 10784-10786.
39. Yang, R.; Pasunooti, K. K.; Li, F.; Liu, X.-W.; Liu, C.-F., Dual native chemical ligation at lysine. J. Am. Chem. Soc. 2009, 131, 13592-13593.
40. Ajish Kumar, K.; Haj‐Yahya, M.; Olschewski, D.; Lashuel, H. A.; Brik, A., Highly efficient and chemoselective peptide ubiquitylation. Angew. Chem. Int. Ed. 2009, 48, 8090-8094.
41. Marinzi, C.; Offer, J.; Longhi, R.; Dawson, P. E., An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications. Bioorg. Med. Chem. 2004, 12, 2749-2757.
42. Botti, P.; Carrasco, M. R.; Kent, S. B., Native chemical ligation using removable Nα-(1-phenyl-2-mercaptoethyl) auxiliaries. Tetrahedron Lett. 2001, 42, 1831-1833.
43. Offer, J.; Boddy, C.; Dawson, P. E., Extending synthetic access to proteins with a removable acyl transfer auxiliary. J. Am. Chem. Soc. 2002, 124, 4642-4646.
44. Loibl, S. F.; Harpaz, Z.; Seitz, O., A type of auxiliary for native chemical peptide ligation beyond cysteine and glycine junctions. Angew. Chem. Int. Ed. 2015, 54, 15055-15059.
45. Kawakami, T.; Aimoto, S., A photoremovable ligation auxiliary for use in polypeptide synthesis. Tetrahedron Lett. 2003, 44, 6059-6061.
46. Johnson, T.; Quibell, M.; Sheppard, R. C., N, O‐bisFmoc derivatives of N‐(2‐hydroxy‐4‐methoxybenzyl)‐amino acids: useful intermediates in peptide synthesis. J. Pept. Sci. 1995, 1, 11-25.
47. Nadler, C.; Nadler, A.; Hansen, C.; Diederichsen, U., A photocleavable auxiliary for extended native chemical ligation. Eur. J. Org. Chem. 2015, 2015, 3095-3102.
48. Brik, A.; Ficht, S.; Yang, Y.-Y.; Wong, C.-H., Sugar-Assisted Ligation of N-Linked Glycopeptides with Broad Sequence Tolerance at the Ligation Junction. J. Am. Chem. Soc. 2006, 128, 15026-15033.
49. Lutsky, M.-Y.; Nepomniaschiy, N.; Brik, A., Peptide ligation via side-chain auxiliary. Chem. Commun. 2008, 1229-1231.
50. Kumar, K. A.; Harpaz, Z.; Haj-Yahya, M.; Brik, A., Side-chain assisted ligation in protein synthesis. Bioorg. Med. Chem. Lett. 2009, 19, 3870-3874.
51. Spasser, L.; Ajish Kumar, K.; Brik, A., Scope and limitation of side‐chain assisted ligation. J. Pept. Sci. 2011, 17, 252-255.
52. Li, X.; Kawakami, T.; Aimoto, S., Direct preparation of peptide thioesters using an Fmoc solid-phase method. Tetrahedron Lett. 1998, 39, 8669-8672.
53. Clippingdale, A. B.; Barrow, C. J.; Wade, J. D., Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation. J. Pept. Sci. 2000, 6, 225-234.
54. Raz, R.; Rademann, J. r., Fmoc-based synthesis of peptide thioesters for native chemical ligation employing a tert-butyl thiol linker. Org. Lett. 2011, 13, 1606-1609.
55. Kenner, G.; McDermott, J.; Sheppard, R., The safety catch principle in solid phase peptide synthesis. J. Chem. Soc. D 1971, 636-637.
56. von Eggelkraut-Gottanka, R.; Klose, A.; Beck-Sickinger, A. G.; Beyermann, M., Peptide αthioester formation using standard Fmoc-chemistry. Tetrahedron Lett. 2003, 44, 3551-3554.
57. Flemer Jr, S., Efficient method of circumventing insolubility problems with fully protected peptide carboxylates via in situ direct thioesterification reactions. J. Pept. Sci. 2009, 15, 693-696.
58. Nagalingam, A. C.; Radford, S. E.; Warriner, S. L., Avoidance of epimerization in the synthesis of peptide thioesters using Fmoc protection. Synlett 2007, 2007, 2517-2520.
59. Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H., Native chemical ligation through in situ O to S acyl shift. Org. Lett. 2004, 6, 4861-4864.
60. Kawakami, T.; Sumida, M.; Vorherr, T.; Aimoto, S., Peptide thioester preparation based on an NS acyl shift reaction mediated by a thiol ligation auxiliary. Tetrahedron Lett. 2005, 46, 8805-8807.
61. Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O., Bis (2-sulfanylethyl) amino native peptide ligation. Org. Lett. 2010, 12, 5238-5241.
62. Hou, W.; Zhang, X.; Li, F.; Liu, C.-F., Peptidyl N, N-bis (2-mercaptoethyl)-amides as thioester precursors for native chemical ligation. Org. Lett. 2010, 13, 386-389.
63. Blanco‐Canosa, J. B.; Dawson, P. E., An efficient Fmoc‐SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem. Int. Ed. 2008, 47, 6851-6855.
64. Mahto, S. K.; Howard, C. J.; Shimko, J. C.; Ottesen, J. J., A reversible protection strategy to improve Fmoc‐SPPS of peptide thioesters by the N‐acylurea approach. ChemBioChem 2011, 12, 2488-2494.
65. Blanco-Canosa, J. B.; Nardone, B.; Albericio, F.; Dawson, P. E., Chemical protein synthesis using a second-generation N-acylurea linker for the preparation of peptide-thioester precursors. J. Am. Chem. Soc. 2015, 137, 7197-7209.
66. Fang, G. M.; Li, Y. M.; Shen, F.; Huang, Y. C.; Li, J. B.; Lin, Y.; Cui, H. K.; Liu, L., Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 2011, 50, 7645-7649.
67. Selvaraj, A.; Chen, H.-T.; Ya-Ting Huang, A.; Kao, C.-L., Expedient on-resin modification of a peptide C-terminus through a benzotriazole linker. Chem. Sci. 2018, 9, 345-349.
68. Wang, J. X.; Fang, G. M.; He, Y.; Qu, D. L.; Yu, M.; Hong, Z. Y.; Liu, L., Peptide o‐aminoanilides as crypto‐thioesters for protein chemical synthesis. Angew. Chem. Int. Ed. 2015, 54, 2194-2198.
69. Bang, D.; Pentelute, B. L.; Kent, S. B., Kinetically controlled ligation for the convergent chemical synthesis of proteins. Angew. Chem. Int. Ed. 2006, 45, 3985-3988.
70. Dheur, J.; Ollivier, N.; Vallin, A.; Melnyk, O., Synthesis of peptide alkylthioesters using the intramolecular N, S-acyl shift properties of bis (2-sulfanylethyl) amido peptides. J. Org. Chem. 2011, 76, 3194-3202.
71. Zheng, J.-S.; Tang, S.; Qi, Y.-K.; Wang, Z.-P.; Liu, L., Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protoc. 2013, 8, 2483.
72. Pedersen, S. L.; Tofteng, A. P.; Malik, L.; Jensen, K. J., Microwave heating in solid-phase peptide synthesis. Chem. Soc. Rev. 2012, 41, 1826-1844.
73. Bacsa, B.; Horváti, K.; Bõsze, S.; Andreae, F.; Kappe, C. O., Solid-phase synthesis of difficult peptide sequences at elevated temperatures: a critical comparison of microwave and conventional heating technologies. J. Org. Chem. 2008, 73, 7532-7542.
74. 林芝蘭. 光解基團修飾之天門冬胺酸進行側鏈輔助天然化學連接法. 國立清華大學, 新竹市, 2017.
75. 王瑋皜. 以天然化學連接法合成不具有半胱胺酸單元之胜肽. 國立清華大學, 新竹市, 2015.
76. Bergman, P.; Termén, S.; Johansson, L.; Nyström, L.; Arenas, E.; Jonsson, A.-B.; Hökfelt, T.; Gudmundsson, G. H.; Agerberth, B., The antimicrobial peptide rCRAMP is present in the central nervous system of the rat. J. Neurochem. 2005, 93, 1132-1140.
77. Bizet, V.; Bolm, C., Sulfur Imidations by Light-Induced Ruthenium-Catalyzed Nitrene Transfer Reactions. Eur. J. Org. Chem. 2015, 2015, 2854-2860.
78. Szychowski, J.; Mahdavi, A.; Hodas, J. J.; Bagert, J. D.; Ngo, J. T.; Landgraf, P.; Dieterich, D. C.; Schuman, E. M.; Tirrell, D. A., Cleavable biotin probes for labeling of biomolecules via azide− alkyne cycloaddition. J. Am. Chem. Soc. 2010, 132, 18351-18360.
79. Taylor, C. M.; Weir, C. A., Synthesis of the Repeating Decapeptide Unit of Mefp1 in Orthogonally Protected Form. J. Org. Chem. 2000, 65, 1414-1421.
80. Zhang, Q.; Kulczynska, A.; Webb, D. J.; Megson, I. L.; Botting, N. P., A new class of NO-donor pro-drugs triggered by γ-glutamyl transpeptidase with potential for reno-selective vasodilatation. Chem. Commun. 2013, 49, 1389-1391.
81. Yokokawa, F.; Inaizumi, A.; Shioiri, T., Synthetic studies of the cyclic depsipeptides bearing the 3-amino-6-hydroxy-2-piperidone (Ahp) unit. Total synthesis of the proposed structure of micropeptin T-20. Tetrahedron 2005, 61, 1459-1480.