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| 研究生： |
林冠宇 Lin, Kuan-Yu. |
|---|---|
| 論文名稱： |
離去基與立體效應在蛋白質標記探針的相互作用 The Interplay of Leaving Group and Steric Effect in Protein Labeling Probes |
| 指導教授： |
陳貴通
Tan, Kui-Thong |
| 口試委員: |
林俊成
Lin, Chun-Cheng 王宗興 Wang, Tsung-Shing |
| 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： | 2019 |
| 畢業學年度： | 107 |
| 語文別： | 中文 |
| 論文頁數： | 247 |
| 中文關鍵詞： | 蛋白質標記探針 、配體導向螢光探針 、人類碳酸酐酶 、細胞成像 、癌細胞標記 、離去基 |
| 外文關鍵詞： | Protein Labeling Probes, ligand-directed fluorescent probe, human carbonic anhydrase, hCA, cell imaging, cancer cell labeling, leaving Group |
| 相關次數： | 點閱：3 下載：0 |
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利用螢光探針在複雜的生物環境中保持優良選擇性和有效的標記內源性蛋白質到現在仍是具有挑戰性的。理論來說，擁有高度反應速率常數的生物相容性親電子基團對於確實的標記蛋白質來說是非常必要的。但是過高的反應性往往會表現出較低的穩定度和顯著的非特異性反應。在本實驗中，我們藉由在配體引導探針上引入新型的二氟苯基新戊酸酯作為反應親電子端來解決這些問題。探針在儲備溶液和緩衝溶液中是非常穩定的，並且因為二氟苯酚基團適當的pka和新戊酸酯基團的大空間阻位使探針對於酯酶催化水解有強大的抵抗能力。用不同的螢光團和蛋白質配體對探針進行修飾來有效標記細胞裂解物、血清和動物活體細胞中的不同靶蛋白，在含有高達7000倍非靶蛋白的環境下仍然可以達到6奈克的偵測極限。通過這種設計，基礎及缺氧環境下所誘導產生的碳酸酐酶同功酶可以使用活體細胞成像和凝膠螢光分析來揭示其特性及相對水平。我們相信這種二氟苯基新戊酸酯可以成為藥物發現、醫學診斷和基礎生物研究的重要工具。
The selective and efficient labeling of native proteins with small molecules in complex biological environments remains challenging. Ideally, biocompatible electrophiles with large reaction rate constants are highly desirable for efficient protein labeling. However, too high of reactivity always result in low stability and significant nonspecific reactions. In this paper, we overcome this problem by introducing difluorophenyl pivalate as a novel reactive electrophile on ligand-guided protein labeling probes. The probes are stable in stock solutions, aqueous buffer and are resistant to catalytic hydrolysis by esterase owing to the moderate pKa of difluorophenol and the large steric hindrance by the pivalate group. The probes can be modified with diverse fluorophores and protein ligands to label selectively and efficiently different target proteins in cell lysates, blood serum and living mammalian cells, achieving detection limits as low as 6 ng protein in the presence of up to 7000-fold non-target proteins. With this design, the identity and relative levels of basal and hypoxia-induced carbonic anhydrase isozymes can be revealed by live cell imaging and in-gel fluorescence analysis. We believe that this difluorophenyl pivalate approach can become an important tool in drug-discovery, medical diagnosis and basic biology research.
[1] C. D. Williams, B. Oxon, H. Lond., Bull. W.H.O. 2003, 81, 912.
[2] Lalonde, R.; Dumont, M.; Staufenbiel, M.; Sturchler-Pierrat, C.; Strazielle, C., Brain Res. 2002, 956, 36-44.
[3] Ghosh, R.; Gilda, J. E.; Gomes, A. V., Expert Rev. Proteomics. 2014, 11, 549–560.
[4] Esteban, J. I.; Shih, J. W.; Tai, C. C.; Bodner, A. J.; Kay, J. W.; Alter, H. J.; Lancet. 1985, 2, 1083-1086.
[5] Porcario, C.; Hall, S. M.; Martucci, F.; Corona, C.; Iulini, B.; Perazzini, A. Z.; Acutis, P.; Hamir, A. N.; Loiacono, C. M.; Greenlee, J. J.; Richt, J. A.; Caramelli, M.; Casalone, C.; BMC. Res. Notes. 2011, 4, 376.
[6] Thâmara, Aline. Bertoni.; Maysa, Cláudia. Zolin. Perenha-Viana.; Eliana, Valéria. Patussi.; Rosilene, Fressatti. Cardoso.; Terezinha, Inez. Estivalet. Svidzinski., Clin. Vaccine Immunol. 2012, 19, 1887-1888.
[7] Alwine, J. C.; Kemp, D. J.; Stark, G. R., Proc. Natl. Acad. Sci. U. S. A. 1977, 74, 5350-5354.
[8] Liu, H. W.; Chen, L.; Xu, C.; Li, Z.; Zhang, H.; Zhang, X. B.; Tan, W., Chem. Soc. Rev. 2018, 47, 7140-7180.
[9] Lozano-Torres, B.; Galiana, I.; Rovira, M.; Garrido, E.; Chaib, S.; Bernardos, A.; Muñoz-Espín, D.; Serrano, M.; Martínez-Máñez, R.; Sancenón, F., J. Am. Chem. Soc. 2017, 139, 8808-8811.
[10] Xu, S.; Liu, H. W.; Hu, X. X.; Huan, S. Y.; Zhang, J.; Liu, Y. C.; Yuan, L.; Qu, F. L.; Zhang, X. B.; Tan, W., Anal. Chem. 2017, 89, 7641-7648.
[11] Zhan, C.; Cheng, J.; Li, B.; Huang, S.; Zeng, F.; Wu, S., Anal. Chem. 2018, 90, 8807-8815.
[12] Bretscher, M. S., Proc. Natl. Acad. Sci. U. S. A. 1983, 80, 454–458.
[13] De, Las. Rivas. J.; Fontanillo, C., PLoS Comput. Biol. 2010, 6, e1000807.
[14] Tsien, R.Y., Annu. Rev. Biochem. 1998, 67, 509-544.
[15] Shimomura, O., Biol. Bull. 1995, 189, 1-5.
[16] Prasher, D. C.; Eckenrode, V. K.; Ward, W. W.; Prendergast, F. G.; Cormier, M. J., Gene 1992, 111, 229-233.
[17] Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C., Science 1994, 263, 802-805.
[18] Yang, F.; Moss, L. G.; Phillips. G. N. Jr., Nat. Biotechnol. 1996, 14, 1246-1251.
[19] Hochuli, E.; Döbeli, H.; Schacher, A., J. Chromatogr. 1987, 411, 177-184.
[20] Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K., Nat. Biotechnol. 2003, 21, 86-89.
[21] Miller, L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W., Nat. Methods 2005, 2, 255-257.
[22] Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V., ACS. Chem. Biol. 2008, 3, 373-382.
[23] Hong, Y.-R.; Lam, C. H.; Tan, K.-T., Bioconjugate Chem. 2017, 28, 2895-2902.
[24] Gong, Y.; Pan, L. Tetrahedron Lett. 2015, 56, 2123-2132.
[25] Tamura, T.; Hamachi, I., J. Am. Chem. Soc. 2019, 141, 2782−2799.
[26] Takaoka, Y.; Ojida, A.; Hamachi, I., Angew. Chem. Int. Ed. 2013, 15, 4088-4106.
[27] Liu, Y.; Patricelli, M. P.; Cravatt, B. F., Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 14694-14699.
[28] Tsukiji, S.; Miyagawa, M.; Takaoka, Y.; Tamura, T.; Hamachi, I., Nat. Chem. Biol. 2009, 5, 341-343.
[29] Miki, T.; Fujishima, S. H.; Komatsu, K.; Kuwata, K.; Kiyonaka, S.; Hamachi, I., Chem. Biol. 2014, 21, 1013-1022.
[30] Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I., Nat. Commun. 2018, 9, 1870.
[31] Takaoka, Y.; Nishikawa, Y.; Hashimoto, Y.; Sasaki, K.; Hamachi, I., Chem. Sci. 2015, 6, 3217-3224.
[32] Blaxill, Z.; Holland-Crimmin, S.; Lifely, R., J. Biomol. Screen. 2009, 14, 547-56.
[33] Di Fiore, A.; Maresca, A.; Supuran, C. T.; De Simone, G., Chem. Commun. 2012, 48, 8838-8840.
[34] Fisher, Z.; Boone C. D.; Biswas, S. M.; Venkatakrishnan, B.; Aggarwal, M.; Tu, C.; Agbandje-McKenna, M.; Silverman, D.; McKenna, R., Protein Eng. Des. Sel. 2012, 25, 347-355.
[35] Han, J.; Tao, F. M., J. Phys. Chem. A. 2006, 110, 257-263.
[36] Tan, X.; Song, Z., RSC. Advances 2014, 4, 3263-3271.
[37] Zini, R.; d'Athis, P.; Hoareau, A.; Tillement, J. P., Eur. J. Clin. Pharmacol. 1976, 10, 139-145.
[38] Blaxill, Z.; Holland-Crimmin, S.; Lifely, R., J. Biomol. Screen. 2009, 14, 547-56.
[39] Luke, D. Lavis.; Tzu-Yuan, Chao.; Ronald, T. Raines., Chem. Sci. 2011, 2, 521-530.
[40] Wilchek, M.; Bayer, E. A., Methods Enzymol. 1990, 184, 5-13.
[41] Chiche, J.; Ilc, K.; Laferrière, J.; Trottier, E.; Dayan, F.; Mazure, N. M.; Brahimi-Horn, M. C.; Pouysségur, J., Cancer research 2009, 69, 358-368.
[42] Watson, P. H.; Chia, S. K.; Wykoff, C. C.; Han, C.; Leek, R. D.; Sly, W. S.; Gatter, K. C.; Ratcliffe, P.; Harris, A. L. Br. J., Cancer 2003, 88, 1065-1070.
[43] Chen, Z.; Ai, L.; Mboge, M. Y.; Tu, C.; McKenna, R.; Brown, K. D.; Heldermon, C. D.; Frost, S. C., PLoS One. 2018, 13, e0199476.
[44] Yuan, Y.; Hilliard, G.; Ferguson, T.; Millhorn, D. E., J. Biol. Chem. 2003, 278, 15911-15916.
[45] Wykoff, C. C.; Beasley, N. J. P.; Watson, P. H.; Turner, K. J.; Pastorek, J.; Sibtain, A.; Wilson, G. D.; Turley, H.; Talks, K. L.; Maxwell, P. H.; Pugh, C. W.; Ratcliffe, P. J.; Harris, A. L., Cancer research 2000, 60, 7075-7083.
[46] Ilie, M.; Hofman, V.; Zangari, J.; Chiche, J.; Mouroux, J.; Mazure, N. M.; Pouysségur, J.; Brest, P.; Hofman, P., Lung Cancer 2013, 82, 16-23.
[47] Waterman, E. A.; Cross, N. A.; Lippitt, J. M.; Cross, S. S.; Rehman, I.; Holen, I.; Hamdy, F. C.; Eaton, C. L., Int. J. Cancer 2007, 121, 1958-1966.
[48] Qattan, A. T.; Mulvey, C.; Crawford, M.; Natale, D. A.; Godovac-Zimmermann, J., J. Proteome Res. 2010, 9, 495-508.