葉酸結合蛋白在生物體內扮演著非常重要的角色，在醫學診斷與基礎生物學研究中，利用螢光探針來標記內源性葉酸結合蛋白是其中一種工具。為了得到優良且再現性佳的標記結果，對螢光標記探針穩定性與反應性的提升仍是非常具有挑戰的。我們成功設計出具有高穩定性與高選擇性的螢光標記探針用以標記葉酸結合蛋白，不僅能夠抵抗酯酶的水解，在DMSO中擁有良好的穩定性。除此之外，探針的設計使我們能夠簡易的更換螢光基團，並可於細胞裂解液與活體細胞中專一的標記和顯影葉酸結合蛋白。我們相信這個新穎的設計可以成為生物學研究、藥物發現和醫學診斷的重要工具之一。

In medical diagnosis and fundamental biological research, it is important to label endogenous folate binding proteins with fluorescent probes in living cells. To obtain optimal and reproducible labeling results for folate receptors, fine-tuning the stability and reactivity of reactive groups on the labeling probe is crucial and remains challenging. In this thesis, we have successfully designed a stable and selective folate binding protein fluorescent labeling probes which can resist catalytic hydrolysis by esterase and auto-hydrolysis in DMSO stock solution and aqueous buffer. Furthermore, our probe design can be customized with diverse fluorophores for the selective and traceless labeling and imaging of folate binding protein in cell lysates and living cells. We believed that our novel design can be a powerful tool in biological researches, drug discovery, and medical diagnosis for the study of folate binding proteins.

摘要 I
Abstract II
謝誌 III
第一章、緒論 1
1-1葉酸 (Folic Acid) 1
1-2葉酸結合蛋白 (Folate Binding Proteins, FBPs) 3
1-2-1 運輸型葉酸結合蛋白 3
1-2-2 酵素型葉酸結合蛋白 7
1-3 葉酸結合蛋白與癌症 8
第二章、文獻回顧 10
2-1 以西方墨點法偵測葉酸結合蛋白 10
2-2 螢光探針標記與偵測葉酸結合蛋白 12
2-2-1 TMP-Tag技術標記目標蛋白 12
2-2-2 非反應型螢光探針偵測葉酸受體 13
2-2-3 配體導向螢光探針標記葉酸受體 15
第三章、探針設計 17
3-1螢光探針之設計 17
3-2 氨甲蝶呤 (Methotrexate, MTX) 與葉酸結合蛋白 18
第四章、結果與討論 20
4-1 探針1-5 20
4-1-1 Cy5 染料的合成 20
4-1-2 MTX配體的合成 21
4-1-3 探針1-5的合成 22
4-2 探針1-5之反應性測試 28
4-3探針1-5之動力學測試 30
4-4探針1標記DHFR蛋白之質譜分析 32
4-5探針1之蛋白質標記選擇性測試 33
4-6探針1之穩定度測試 35
4-7探針1之細胞成像與細胞裂解液實驗 39
4-7-1 HeLa與MCF7細胞影像 39
4-7-2 MCF7與HeLa細胞裂解液實驗 42
第五章、實驗結論 44
第六章、實驗部分 45
6-1 實驗藥品與器材 45
6-2 有機合成及光譜資料 47
第七章、參考資料 94
附錄 100

1. Wigertz, K.; Svensson, U. K.; Jägerstad, M. Folate and folate-binding protein content in dairy products. J. Dairy Res. 1997, 64, 239-52.
2. Ebisch, I. M. W.; Thomas, C. M. G.; Peters, W. H. M.; Braat, D. D. M.; Steegers-Theunissen, R. P. M. The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility. Hum. Reprod. Update 2006, 13, 163-174.
3. Zheng, Y.; Cantley, L. C. Toward a better understanding of folate metabolism in health and disease. J. Exp. Med. 2019, 216, 253-266.
4. Sanderson, S. M.; Gao, X.; Dai, Z.; Locasale, J. W. Methionine metabolism in health and cancer: a nexus of diet and precision medicine. Nat. Rev. Cancer 2019, 19, 625-637.
5. Wan, Q.; Bennett, B. C.; Wilson, M. A.; Kovalevsky, A.; Langan, P.; Howell, E. E.; Dealwis, C. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. Proc. Natl. Acad. Sci. 2014, 111, 18225.
6. Lan, X.; Field, M. S.; Stover, P. J. Cell cycle regulation of folate-mediated one-carbon metabolism. Wiley Interdiscip. Rev. Syst. Biol. Med. 2018, 10, e1426.
7. Alam, C.; Hoque, M. T.; Finnell, R. H.; Goldman, I. D.; Bendayan, R. Regulation of reduced folate carrier (RFC) by vitamin D receptor at the blood-brain barrier. Mol. Pharm. 2017, 14 11, 3848-3858.
8. Zhao, R.; Najmi, M.; Aluri, S.; Spray, D. C.; Goldman, I. D. Concentrative transport of antifolates mediated by the proton-coupled folate transporter (SLC46A1); augmentation by a HEPES buffer. Mol. Pharmacol. 2018, 93, 208-215.
9. Desmoulin, S. K.; Hou, Z.; Gangjee, A.; Matherly, L. H. The human proton-coupled folate transporter: Biology and therapeutic applications to cancer. Cancer. Biol. Ther. 2012, 13, 1355-73.
10. Wilson, M. R.; Kugel, S.; Huang, J.; Wilson, L. J.; Wloszczynski, P. A.; Ye, J.; Matherly, L. H.; Hou, Z. Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6. Biochem. J. 2015, 469, 33-44.
11. Matherly, L. H.; Goldman, D. I. Membrane transport of folates. Vitam. Horm. 2003, 66, 403-56.
12. Matherly, L. H.; Hou, Z.; Deng, Y. Human reduced folate carrier: translation of basic biology to cancer etiology and therapy. Cancer Metastasis Rev. 2007, 26, 111-28.
13. Hou, Z.; Matherly, L. H. Biology of the major facilitative folate transporters SLC19A1 and SLC46A1. Curr. Top. Membr. 2014, 73, 175-204.
14. Ledermann, J. A.; Canevari, S.; Thigpen, T. Targeting the folate receptor: diagnostic and therapeutic approaches to personalize cancer treatments. Ann. Oncol. 2015, 26, 2034-2043.
15. Quici, S.; Casoni, A.; Foschi, F.; Armelao, L.; Bottaro, G.; Seraglia, R.; Bolzati, C.; Salvarese, N.; Carpanese, D.; Rosato, A. Folic acid-conjugated europium complexes as luminescent probes for selective targeting of cancer cells. J. Med. Chem. 2015, 58, 2003-2014.
16. Mironava, T.; Simon, M.; Rafailovich, M. H.; Rigas, B. Platinum folate nanoparticles toxicity: cancer vs. normal cells. Toxicol. In Vitro 2013, 27, 882-889.
17. Kelemen, L. E. The role of folate receptor alpha in cancer development, progression and treatment: cause, consequence or innocent bystander? Int. J. Cancer 2006, 119, 243-50.
18. O'Shannessy, D. J.; Somers, E. B.; Wang, L.-C.; Wang, H.; Hsu, R. Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers. J. Ovarian Res. 2015, 8, 29-29.
19. Cheung, A.; Bax, H. J.; Josephs, D. H.; Ilieva, K. M.; Pellizzari, G.; Opzoomer, J.; Bloomfield, J.; Fittall, M.; Grigoriadis, A.; Figini, M.; Canevari, S.; Spicer, J. F.; Tutt, A. N.; Karagiannis, S. N. Targeting folate receptor alpha for cancer treatment. Oncotarget 2016, 7, 52553-52574.
20. Braig, K.; Simon, M.; Furuya, F.; Hainfeld, J. F.; Horwich, A. L. A polypeptide bound by the chaperonin groEL is localized within a central cavity. Proc. Natl. Acad. Sci. 1993, 90, 3978.
21. Tam, P. E.; Schmidt, A. M.; Messner, R. P. Modified tissue pulverization technique and evaluation of dihydrofolate reductase amplification as a pan-tissue RT PCR control. PCR Methods Appl. 1993, 3, 71-2.
22. Campbell, I. G.; Jones, T. A.; Foulkes, W. D.; Trowsdale, J. Folate-binding protein is a marker for ovarian cancer. Clin. Cancer Res. 1991, 51, 5329-5338.
23. Toffoli, G.; Russo, A.; Gallo, A.; Cernigoi, C.; Miotti, S.; Sorio, R.; Boiocchi, M. Expression of folate binding protein as a prognostic factor for response to platinum-containing chemotherapy and survival in human ovarian cancer. Int. J. Cancer 1998, 79, 121-126.
24. Sun, L.; Wu, Q.; Peng, F.; Liu, L.; Gong, C. Strategies of polymeric nanoparticles for enhanced internalization in cancer therapy. Colloids Surf. B 2015, 135, 56-72.
25. Vlahov, I. R.; Leamon, C. P. Engineering folate-drug conjugates to target cancer: from chemistry to clinic. Bioconjugate Chem. 2012, 23, 1357-69.
26. Fernández, M.; Javaid, F.; Chudasama, V. Advances in targeting the folate receptor in the treatment/imaging of cancers. Chem. Sci. 2018, 9, 790-810.
27. Yu, Y.; Wang, J.; Kaul, S.; Wadhwa, R.; Miyako, E. Folic acid receptor-mediated targeting enhances the cytotoxicity, efficacy, and selectivity of withania somnifera feaf extract: in vitro and in vivo evidenceData_Sheet_1.docx. Front. Oncol. 2019, 9.
28. Jing, C.; Cornish, V. W. A fluorogenic TMP-tag for high signal-to-background intracellular live cell imaging. ACS Chem. Biol. 2013, 8, 1704-1712.
29. Gallagher, S. S.; Sable, J. E.; Sheetz, M. P.; Cornish, V. W. An in vivo covalent TMP-tag based on proximity-induced reactivity. ACS Chem. Biol. 2009, 4, 547-556.
30. Moon, W. K.; Lin, Y.; O'Loughlin, T.; Tang, Y.; Kim, D.-E.; Weissleder, R.; Tung, C.-H. Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate. Bioconjugate Chem. 2003, 14, 539-545.
31. Fujishima, S.-h.; Yasui, R.; Miki, T.; Ojida, A.; Hamachi, I. Ligand-directed acyl imidazole chemistry for labeling of membrane-bound proteins on live cells. J. Am. Chem. Soc. 2012, 134, 3961-3964.
32. Tsukiji, S.; Wang, H.; Miyagawa, M.; Tamura, T.; Takaoka, Y.; Hamachi, I. Quenched ligand-directed tosylate reagents for one-step construction of turn-on fluorescent biosensors. J. Am. Chem. Soc. 2009, 131, 9046-9054.
33. Takaoka, Y.; Nishikawa, Y.; Hashimoto, Y.; Sasaki, K.; Hamachi, I. Ligand-directed dibromophenyl benzoate chemistry for rapid and selective acylation of intracellular natural proteins. Chem. Sci. 2015, 6, 3217-3224.
34. Takaoka, Y.; Ojida, A.; Hamachi, I. Protein organic chemistry and applications for labeling and engineering in live-cell systems. Angew. Chem. Int. Ed. 2013, 52, 4088-4106.
35. Winocur, G.; Vardy, J.; Binns, M. A.; Kerr, L.; Tannock, I. The effects of the anti-cancer drugs, methotrexate and 5-fluorouracil, on cognitive function in mice. Pharmacol. Biochem. Behav. 2006, 85, 66-75.
36. Rajagopalan, P. T. R.; Zhang, Z.; McCourt, L.; Dwyer, M.; Benkovic, S. J.; Hammes, G. G. Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics. Proc. Natl. Acad. Sci. 2002, 99, 13481.
37. Alrobaian, M.; Al Azwari, S.; Belal, A.; Eldeab, H. An eco-friendly technique: solvent-free microwave synthesis and docking studies of some new pyridine nucleosides and their pharmacological significance. Molecules 2019, 24, 1969.
38. Blaxill, Z.; Holland-Crimmin, S.; Lifely, R. Stability through the ages: the GSK experience. J. Biomol. Screen 2009, 14, 547-56.
39. Lavis, L. D.; Chao, T.-Y.; Raines, R. T. Synthesis and utility of fluorogenic acetoxymethyl ethers. Chem. Sci. 2011, 2, 521-530.
40. Sonvico, F.; Dubernet, C.; Marsaud, V.; Appel, M.; Chacun, H.; Stella, B.; Renoir, M.; Colombo, P.; Couvreur, P. Establishment of an in vitro model expressing the folate receptor for the investigation of targeted delivery systems. J. Drug Deliv. Sci. Technol. 2005, 15, 407-410.