環境敏感性螢光分子被廣泛應用於偵測蛋白質微環境、細胞染色以及生物活體體內蛋白質標記。此類型螢光分子具有較大的斯托克斯位移，將有利於放光光譜之偵測而不受吸收波長干擾。在本研究中，利用1,4-甲苯磺醯三唑具有拉電子基團甲苯磺醯基因容易經由戴氏重排與重氮亞氨基官能基產生平衡反應式，再經由加熱或一價銅催化催化反應脫除一分子氮氣，生成高活性親電性中間體，乙烯亞胺。最後透過迪爾斯-阿爾德反應生成具環境敏感性之螢光分子，二氫喹啉-4-亞胺 (DQI)。
針對 DQI 具有特殊螢光性質，本研究對其深入探討此分子之光物理特性，藉由理論計算確立其放光機制，並將其結果，利用推、拉電子基團效應調控最高占據分子軌域和最低未占分子軌域之間的能階差，達到調整此分子光色之目的。DQI分子於二氯甲烷中量子產率為62%，但進入質子溶劑甲醇後下降至6%，依此推測此分子具有環境敏感之特性。依據此特性，設計感興趣蛋白質之開關，並利用碳酸酐酶與其高效率受質磺胺為模型，合成螢光分子開關磺胺-二氫喹啉亞胺 (SA-DQI)。DQI分子藉由磺胺基之引導，由親水性環境帶入疏水性環境，使得螢光量子產率提升15倍。接著將其轉移至細胞系統。利用轉染技術，將293 T細胞細胞膜表面表達出碳酸酐酶，並在未清洗背景的條件下，觀察碳酸酐酶在293 T細胞上確切位置。最後合成水向分子，磺胺-三甘醇-二氫喹啉亞胺 (SA-TEG-DQI)，將此分子應用於觀測斑馬魚發展耳石過程中，其體內碳酸酐酶之蛋白質生成位置之追蹤，期望在斑馬魚發育學上做出貢獻。

Environmental sensitive fluorophores are widely used to detect protein microenvironment, cell staining, and protein labeling in living organisms. This type of fluorescent molecule has a large Stokes shift, which will facilitate the detection of the emission spectrum without interference from the absorption wavelength. In this research, we used 1,4-tosyltriazole under heating or Cu (I) catalyst to generate high electrophilic center, keteneimine, induced denitrogenative annulation for the synthesis of dihydroquinoline-4-imine (DQI).
After DFT simulation, we can modify electron donating or withdrawing group on benzene ring to modulate HOMO and LUMO’s energy gap to change the emission wavelength. DQI in different polarity solvent system showed dramatic change, which shifts of the fluorescence emission from 573 nm in methanol to 478 nm in n-hexane, as well as diminishes quantum yield from 62% in dichloromethane to 6% in methanol. It matches environmental sensitive properties. In order to apply DQI fluorophore for biological system, SA-DQI was synthesized successfully and used to detect human carbonic anhydrase II (hCAII) with a fifteen-fold enhancement of fluorescence intensity. We also utilized SA-DQI to stain transfected HEK 293T cell. It shows fluorescence signal on cell membrane. Finally, more water soluble aryl-suldonamide conjugated DQI (SA-TEG-DQI) molecule was successfully track CA in vivo in zebrafish.

論文審定書…………………………………………………………………………… i
致謝……………………………………………………………………………………ii
中英文關鍵字………………………………………………………………………...iv
中文摘要………………………………………………………………………………v
英文摘要……………………………………………………………………………...vi
圖目錄…………………………………………………………………………………x
光譜目錄…………………………………………………………………………….xiii
表目錄………………………………………………………………………………..xx
流程目錄…………………………………………………………………………….xxi
縮寫表………………………………………………………………………………xxii
第 一 章 緒論………………………………………………………………………..1
1.1有機螢光小分子及其應用…………………………………………………..1
1.1.1異硫氰酸螢光素 (fluorescein isothiocyante, FITC) ………………...4
1.1.2羅丹明 (rhodamine) ………………………………………………….7
1.1.3 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY……………...12
1.1.4 花青 (Cyanines, Cy) ……………………………………………….15
1.2 環境敏感之有機螢光小分子介紹及其應用……………………………...22
1.2.1溶劑化變色染料 -分子內電荷轉移………………………………..23
1.2.2溶劑化變色染料 -激發態分子內具有質子轉移…………………..28
1.2.3螢光染料 - 構象變化………………………………………………31
1.2.4螢光染料 - 分子之異構化…………………………………………34
1.2.5螢光染料 -分子聚集型態…………………………………………..34
1.2.5.1螢光染料 -分子聚集型態 - 聚集導致淬息………………..34
1.2.5.2螢光染料 -分子聚集型態 - 聚集誘導放光………………..37
第 二 章 研究動機及目的…………………………………………………………40
2.1 實驗室過去研究發展介紹………………………………………………...40
2.2 研究目標…………………………………………………………………...48
第 三 章 結果與討論………………………………………………………………49
3.1 以甲苯磺醯基三唑為起始物於加熱 (無金屬) 條件下合成二氫喹啉-4-亞
胺…………………………………………………………………………..49
3.2 以一價銅金屬催化1,4-甲苯磺醯基三唑合成螢光分子 DQI…………...57
3.3 DQI螢光分子光物理性質之探討…………………………………………61
3.4 DQI螢光分子應用於低背景生物螢光影像………………………………73
3.4.1 碳酸酐酶之簡介及其與磺胺之專一性結合………………………73
3.4.2 SA-DQI之合成與其應用於蛋白質螢光標記……………………..74
3.4.3 SA-DQI應用於HEK 293-T細胞…………………………………..77
3.4.4 SA-DQI 應用於追蹤斑馬魚體內CAII……………………………81
第 四 章 結論………………………………………………………………………89
第 五 章 參考文獻…………………………………………………………………91
第 六 章 實驗步驟與光譜數據……………………………………………………98
6.1 儀器設備與藥品材料……………………………………………………...98
6.2 合成步驟與數據…………………………………………………………...99
6.2.1 Indole synthesis via Rh(II) catalyst…..…………..…………………...99
6.2.2 Thermally induced annulation to synthesize DQI analogues………..111
6.2.3 Synthesis of SA-DQI & SA-TEG-DQI……………………………...119
6.3 Absorption spectra, emission spectra and the determination of fluorescence Quantum yield determination of DQI moleclules….…………………….133
6.4 Transfection of 293T cells………………………………………….……148
6.5 Fluorescence imaging of transfected 293T cells..………….…………....149
6.6 CAII In vivo imaging on zebrafish……….......………………………….149
第 七 章 光譜目錄………………………………………………………………..151
第 八 章 發表文獻 (以第一作者身分)………………………………………….299

(1) Onken, M.; Eichelberg, M.; Riesmeier, J.; Jensch, P. Digital Imaging and Communications in Medicine
(2) Xu, C. -N.; Watanabe, T.; Akiyama, M.; Zheng, X. -G. Appl. Phys. Lett. 1999, 74, 2414.
(3) Schuster, G. B. Acc. Chem. Res. 1979, 12, 366.
(4) Davidson, M. W. Laboratory Medicine 2009, 40, 694.
(5) Pakhomov, A. A.; Martynov, V. I. Chem. Biol. 2008, 15, 755.
(6) Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509.
(7) Dean, K. M.; Palmer, A. E. Nat. Chem. Biol. 2014, 10, 512.
(8) Kubitscheck, U. Fluorescence Microscopy : From Principles to Biological Applications
(9) Valeur B.; Berberan-Santos, M. N. J. Chem. Educ. 2011, 88, 731.
(10) www.hhmi.org/news/new-fluorescent-dyes-could-advance-biological- imaging.
(11) Herschel, J. F. W. Philos. Trans. Roy. Soc. London 1845, 135, 147.
(12) Stokes, G. G. Phil. Trans. R. Soc. Lond. 1852, 142, 463.
(13) Holme, I. Color. Technol. 2006, 122, 235.
(14) Glushko, V. N.; Blokhina, L. I.; Sadovskaya, N. Y. Russ. J. Gen. Chem. 2015, 85, 2458.
(15) Cooksey, C. J. Biotech. Histochem. 2016, 91, 71.
(16) Coons, A. H.; Creech, H. J.; Jones, R. N.; Berliner, E. J. Immunol. 1942, 45, 159.
(17) Coons, A. H.; Kaplan, M. H. J. Exp. Med. 1950, 91, 1.
(18) Samuel, D.; Patt, R. J.; Abuknesha R. A. J. Immunol. Methods 1988, 107, 217.
(19) Rotman, B.; Papermaster, B. W. Biochemistry 1966, 55, 134.
(20) https://en.wikipedia.org/wiki/Fluorochromasia
(21) Wang, H. -Y.; Hua, X. -W.; Jia, H. -R.; Li, C.; Lin, F.; Chen, Z.; Wu, F. -G. ACS Biomater. Sci. Eng. 2016, 2, 987.
(22) Kamena, F.; Tamborrini M.; Liu X.; Kwon, Y. -U.; Thompson, F.; Pluschke, G.; Seeberger, P. H. Nat. Chem. Biol. 2008, 4, 238.
(23) Veeranarayanan, S.; Poulose A. C.; Mohamed S.; Aravind, A.; Nagaoka, Y.; Yoshida, Y.; Maekawa, T.; Kumar, D. S. J. Fluoresc. 2012, 22, 537.
(24) Cho, H. K.; Lone, S.; Kim, D. D.; Choi, J. H.; Choi, S. W.; Cho, J. H.; Kim, J. H.; Cheong, I. W. Polymer 2009, 50, 2357.
(25) Drexhage, K. H. J. Res. Natl. Bur. Stand. 1976, 80A, 421.
(26) Crosby, G. A.; Demas, J. N. J. Phys. Chem. 1971, 75, 991.
(27) Fonseca, T.; Relo’gio, P.; Martinho, J. M. G.; Farinha, J. P. S. Langmuir 2007, 23, 5727.
(28) Nicolas, J.; San Miguel, V.; Mantovani, G.; Haddleton, D. M. Chem. Commun. 2006, 4697.
(29) Prazeres, T. J. V.; Santos, A. M.; Martinho, J. M. G. Langmuir 2004, 20, 6834.
(30) Bossi, M.; Belov, V.; Polyakova, S. Hell, S. W. Angew. Chem. Int. Ed. 2006, 45, 7462.
(31) Shiraishi, Y.; Miyamoto, R.; Zhang, X.; Hirai, T. Org. Lett. 2007, 9, 3921.
(32) Schlick, T. L.; Ding, Z. B.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 3718–3723.
(33) Moon, H.; Park, J.; Tae, J. Chem. Rev. 2016, 16, 124.
(34) Goncalves, M. S. Chem. Rev. 2009, 109, 190.
(35) Titus, J. A.; Haugland, R.; Sharrow, S. O.; Segal, D. M. J. Immunol. Methods 1982, 50, 193.
(36) Woiwode, U.; Sievers-Engler, A.; Lammerhofer, M. J. Pharmaceut. Biomed. 2016, 121, 307.
(37) Wang, Y.; Hao, D.; Stein, W. D.; Yang, L. Biochim. Biophys. Acta 2006, 1758, 1671.
(38) Meng, Q.; Yu, M.; Zhang, H.; Ren, J.; Huang, D. Dyes Pigm. 2007, 73, 254.
(39) Popp, M. W.; Antos, J. M.; Grotenbreg, G. M.; Spooner, E.; Ploegh, H. L. Nat. Chem. Biol. 2007, 3, 707.
(40) Ko, S. -K.; Yang,Y. -K.; Tae, J.; Shin, I. J. Am. Chem. Soc. 2006, 128, 14150.
(41) Yang, Y. -K.; Ko, S. -K.; Shin, I.; Tae, J. Nat. Protoc. 2007, 2, 1740.
(42) Yang, Y. -K.; Ko, S. -K.; Shin, I.; Tae, J. Org. Biomol. Chem. 2009, 7, 4590.
(43) Moon, K. -S.; Yang, Y. -K.; Ji, S.; Tae, J. Tetrahedron Lett. 2010, 51, 3290.
(44) Yang, Y. -K.; Lee, S.; Tae, J. Org. Lett. 2009, 11, 5610.
(45) Treibs, A.; Kreuzer, F. -H. Liebigs Ann. Chem. 1968, 718, 203.
(46) Haugland, R. P. Handbook of Fluorescent Probes and Research Products; Molecular Probes: Eugene, OR, 2002.
(47) Haugland, R. P.; Kang, H. C. U.S. Patent 4,774,339, 1988.
(48) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891.
(49) Yang, J.; Seckute, J.; Cole, C. M.; Devaraj, N. K. Angew. Chem. Int. Ed. 2012, 124, 7594.
(50) Maier, G. Angew. Chem. 1967, 79, 446.
(51) Carlson, J. C. T.; Meimetis, L. G.; Hilderbrand, S. A.; Weissleder, R. Angew. Chem. Int. Ed. 2013, 52, 6917.
(52) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.; Mazitschek, R.; Weissleder, R. Angew. Chem. Int. Ed. 2010, 49, 2869.
(53) Almeida, P. Quimica 1999, 73, 9.
(54) Williams, D. J. Angew. Chem., Int. Ed. 1984, 23, 690.
(55) Tung, C.-H. Biopolymers 2004, 76, 391.
(56) Patonay, G.; Salon, J.; Sowell, J.; Strekowski, L. Molecules 2004, 9, 40.
(57) Mitra, R. D.; Shendure, J.; Olejnik, J.; Krzymanska-Olejnik, E.; Church, G. M. Anal. Biochem. 2003, 320, 55.
(58) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123.
(59) Tonge, R.; Shaw, J.; Middleton, B.; Rowlinson, R.; Rayner, S.; Young, J.; Pognan, F.; Hawkins, E.; Currie, I.; Davison, M. Proteomics 2001, 1, 337.
(60) Narayanan, N.; Patonay, G. J. Org. Chem. 1995, 60, 2391.
(61) Flanagan, J. H.; Khan, S. H.; Menchen, S.; Soper, S. A.; Hammer, R. P. Bioconjugate Chem. 1997, 8, 751.
(62) Strekowski, L.; Lipowska, M.; Patonay, G. J. Org. Chem. 1992, 57, 4578.
(63) Strekowski, L.; Mason, J. C.; Lee, H.; Say, M.; Patonay, G. J. Heterocyclic Chem. 2004, 41, 227.
(64) Vilsmeier, A.; Haack, A. Ber. 1927, 60, 119.
(65) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J. -H.; Yoon, J. J. Am. Chem. Soc. 2014, 136, 5351.
(66) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Chem. Soc. Rev. 2014, 43, 16.
(67) Grate, J. W.; Mo, K. -F.; Shin, Y.; Vasdekis, A.; Warner, M. G.; Kelly, R. T.; Orr, G.; Hu, D.; Dehoff, K. J.; Brockman, F. J.; Wilkins, M. J. Bioconjugate Chem. 2015, 26, 593.
(68) Grimm, J. B.; English, B. P.; Choi, H.; Muthusamy, A. K.; Mehl, B. P.; Dong, P.; Brown, T. A.; Lippincott-Schwartz, J.; Liu, Z.; Lionnet, T.; Lavis, L. D. Nat. Methods 2016, 13, 985.
(69) Furse, K. E.; Corcelli, S. A. J. Am. Chem. Soc. 2008, 130, 13103.
(70) Kulakova, A. N.; Hobbs, D.; Smithen, M.; Pavlov, E.; Gilbert, J. A.; Quinn, J. P.; McGrath, J. W. Environ. Sci. Technol. 2011, 45, 7799.
(71) Grimm, J. B.; Muthusamy, A. K.; Liang, Y.; Brown, T. A.; Lemon, W. C.; Patel, R.; Lu, R.; Macklin, J. J.; Keller, P. J.; Ji, N.; Lavis, L. D. Nat. Methods 2017, 14, 987.
(72) Shweta, H.; Singh, M. K.; Yadav, K.; Verma, S. D.; Pal, N.; Sen, S. J. Phys. Chem. B 2017, 121, 10735.
(73) Klymchenko, A. S. Acc. Chem. Res. 2017, 50, 366.
(74) Loving, G. S.; Sainlos, M.; Imperiali, B. Trends Biotechnol. 2010, 28, 73.
(75) Kucherak, O. A.; Didier, P.; Mely, Y.; Klymchenko, A. A. J. Phys. Chem. Lett. 2010, 1, 616.
(76) Zhuang, Y. D.; Chiang, P. Y.; Wang, C. W.; Tan, K. T. Angew. Chem. Int. Ed. 2013, 52, 8124.
(77) Klymchenko, A. S.; Mely, Y. Prog. Mol. Biol. Transl. Sci. 2013, 113, 35.
(78) Shynkar, V. V.; Klymchenko, A. S.; Kunzelmann, C.; Duportail, G.; Muller, C. D.; Demchenko, A. P.; Freyssinet, J. M.; Mely, Y. J. Am. Chem. Soc. 2007, 129, 2187.
(79) Long, L.; Wu, Y.; Wang, L.; Gong, A.; Hu, F.; Zhang, C. Chem. Commun. 2015, 51, 10435.
(80) Lukinavicius, G.; Reymond, L.; Umezawa, K.; Sallin, O.; D’Este, E.; Gottfert, F.; Ta, H.; Hell, S. W.; Urano, Y.; Johnsson, K. J. Am. Chem. Soc. 2016, 138, 9365.
(81) Karpenko, I. A.; Collot, M.; Richert, L.; Valencia, C.; Villa, P.; Mély, Y.; Hibert, M.; Bonnet, D.; Klymchenko, A. S. J. Am. Chem. Soc. 2015, 137, 405.
(82) Kwok, R. T.; Leung, C. W.; Lam, J. W.; Tang, B. Z. Chem. Soc. Rev. 2015, 44, 4228.
(83) Yuan, Y.; Xu, S.; Cheng, X.; Cai, X.; Liu, B. Angew. Chem. Int. Ed. 2016, 55, 6457.
(84) Hermes, M. E.; Marsh, F. D. J. Am. Chem. Soc. 1967, 89, 4760.
(85) Huisgen, R. Angew. Chem. Int. Ed. 1963, 89, 565
(86) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew Chem Int Ed. 2002, 41, 2596.
(87) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
(88) Cho, S. H.; Yoo, E. J.; Bae, I.; Chang, S. J. Am. Chem. Soc. 2005, 127, 16046.
(89) Raushel, J. Fokin, V. V. Org. Lett. 2010, 12, 4952.
(90) Miura, T.; Biyajima, T.; Fujii, T.; Murakami, M. J. Am. Chem.Soc. 2012, 134, 194.
(91) Rajagopal, B.; Chen, Y. –Y.; Chen, C. –C.; Liu, X. –Y.; Wang, H. –R.; Lin, P. –C. J. Org. Chem. 2014, 79, 1254.
(92) Rajagopal, B.; Chou, C. –H.; Chung, C. –C.; Lin, P. –C. Org. Lett. 2014, 16, 3752.
(93) Alford, J. S.; Davies, H. M. L. Org. Lett. 2012, 14, 6020.
(94) Alford, J. S.; Davies, H. M. L. J. Am. Chem. Soc. 2014, 136, 10266.
(95) Chauhan, D. P.; Varma, S. J.; Vijeta, A.; Banerjee, P.; Talukdar, P. Chem. Commun. 2014, 50, 323.
(96) Chou, C. –H.; Chen, Y. –Y.; Rajagopal, B.; Tu, H. –C.; Chen, K. –L.; Wang, S. –F.; Liang, C. –F.; Tyan, Y. –C.; Lin, P. –C. Chem. Asian J. 2016, 11, 757.
(97) Chou, C. –H.; Rajagopal, B.; Liang, C. –F.; Chen, K. –L.; Jin, D.-Y.; Chen, H. –Y.; Tu, H. –C.; Shen, Y. –Y.; Lin, P. –C. Chem. Eur. J. 2018, 24, 1112.
(98) Adamo, C.; Barone, V. J. Chem. Phys. 1999, 110, 6158.
(99) Yanai, T., Tew, D.; Handy, N. Chem. Phys. Lett. 2004, 393, 51.
(100) Zhao, Y.; Truhlar, D. G. J. Chem. Phys. 2006, 126, 194101.
(101) Chai, J .D.; Head-Gordon, M. Phys. Chem. Chem. Phys. 2008, 10, 6615.
(102) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. J. Chem. Phys. 2010, 132, 154104.
(103) Marenich, A. V.; Cramer, C. J.; Truhlar, D .G. J. Phys. Chem. B 2009, 113, 6378.
(104) Frisch, M.J. et al. Gaussian 09, revision D.01; Gaussian, Inc.: Wallingford, CT, 2009.
(105) Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Dev. Dyn. 1995, 203, 253.
(106) Matsumoto1, H.; Fujiwara1, S.; Miyagi, H.; Nakamura, N.; Shiga1, Y.; Ohta1, T.; Tsuzuki, M. Mar. Biotechnol. 2017, 19, 430.
(107) Gilmour, K. M.; Thomas, K.; Esbaugh, A. J.; Perry, S. F. J. Exp. Biol. 2009, 212, 3837.
(108) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664.
(109) Lin, T. –Y,; Liao, B. –K.; Horng, J. –L.; Yan, J. –J.; Hsiao, C. –D.; Hwang, P. –P. Am J. Physiol Cell Physiol 2008, 294, 1250.
(110) Jones II, G.; Jackson, W. R., Choi, C. -Y., Bergmark, W. R. J. Phys. Chem. 1985, 89, 294.
(111) Handbook of organic solvent properties, Ian M. Smallwood; Halsted Press; John Wdey & Sons Inc. 1996.