氟化學為近年快速發展的領域，其特性為氟化合物與氟化合物之間有很強的作用力。這個特性可以用來抑制蛋白質的非專一性吸附；因此本論文結合 intein 蛋白質表達系統、Native Chemical Ligation（NCL）反應機制以及氟化學，將蛋白質專一性的固化在奈米粒子和晶片上。目標蛋白－麥芽糖結合蛋白和綠色螢光蛋白以 intein 蛋白質表達系統表現，之後透過 NCL 的方式，將含有乙二醇的氟化物與蛋白質的C-端鍵結。C-端修飾氟標記的蛋白質，可透過簡單混合，固化在固體載體上。C-端修飾氟標記的蛋白質可以專一性的固化在氟標記磁性奈米粒子和氟晶片上，未修飾氟的蛋白質則不能固化在氟晶片和氟標記磁性奈米粒子，證實氟－氟之間的特殊作用力可有效降低非專一性吸附的發生。

1. Wittke, S.; Dunnwald, M.; Albertsen, M.; Johnsson, N., Recognition of a subset of signal sequences by Ssh1p, a Sec61p-related protein in the membrane of endoplasmic reticulum of yeast Saccharomyces cerevisiae. Mol. Biol. Cell 2002, 13, 2223-2232.
2. Kingsmore, S. F., Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat. Rev. Drug Discov. 2006, 5, 310-320.
3. Zheng, G.; Patolsky, F.; Cui, Y.; Wang, W. U.; Lieber, C. M., Multiplexed electrical detection of cancer markers with nanowire sensor arrays. Nat. Biotechnol. 2005, 23, 1294-1301.
4. Drager, G.; Kiss, C.; Kunz, U.; Kirschning, A., Enzyme-purification and catalytic transformations in a microstructured PASSflow reactor using a new tyrosine-based Ni-NTA linker system attached to a polyvinylpyrrolidinone-based matrix. Org. Biomol. Chem. 2007, 5, 3657-3664.
5. Dyal, A.; Loos, K.; Noto, M.; Chang, S. W.; Spagnoli, C.; Shafi, K. V.; Ulman, A.; Cowman, M.; Gross, R. A., Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles. J. Am. Chem. Soc. 2003, 125, 1684-1685.
6. Rossi, L. M.; Quach, A. D.; Rosenzweig, Z., Glucose oxidase-magnetite nanoparticle bioconjugate for glucose sensing. Anal. Bioanal. Chem. 2004, 380, 606-613.
7. Li, Y.; Xu, X.; Deng, C.; Yang, P.; Zhang, X., Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis. J. Proteome Res. 2007, 6, 3849-3855.
8. Kim, Y.; Ho, S. O.; Gassman, N. R.; Korlann, Y.; Landorf, E. V.; Collart, F. R.; Weiss, S., Efficient site-specific labeling of proteins via cysteines. Bioconjug. Chem. 2008, 19, 786-791.
9. Bernardes, G. J.; Grayson, E. J.; Thompson, S.; Chalker, J. M.; Errey, J. C.; El Oualid, F.; Claridge, T. D.; Davis, B. G., From disulfide- to thioether-linked glycoproteins. Angew. Chem. Int. Ed. 2008, 47, 2244-2247.
10. Antos, J. M.; Francis, M. B., Transition metal catalyzed methods for site-selective protein modification. Curr. Opin. Chem. Biol. 2006, 10, 253-262.
11. Lim, R. K.; Lin, Q., Bioorthogonal chemistry: recent progress and future directions. Chem. Commun. 2010, 46, 1589-1600.
12. Prescher, J. A.; Bertozzi, C. R., Chemistry in living systems. Nat. Chem. Biol. 2005, 1, 13-21.
13. Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R., Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997, 276, 1125-1128.
14. Sadamoto, R.; Niikura, K.; Ueda, T.; Monde, K.; Fukuhara, N.; Nishimura, S., Control of bacteria adhesion by cell-wall engineering. J. Am. Chem. Soc. 2004, 126, 3755-3761.
15. Gaertner, H. F.; Rose, K.; Cotton, R.; Timms, D.; Camble, R.; Offord, R. E., Construction of protein analogues by site-specific condensation of unprotected fragments. Bioconjug. Chem. 1992, 3, 262-268.
16. Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y., Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 2002, 3, 906-918.
17. Lippincott-Schwartz, J.; Patterson, G. H., Development and use of fluorescent protein markers in living cells. Science 2003, 300, 87-91.
18. Xu, M. Q.; Evans, T. C., Jr., Intein-mediated ligation and cyclization of expressed proteins. Methods 2001, 24, 257-277.
19. Foley, T. L.; Burkart, M. D., Site-specific protein modification: advances and applications. Curr. Opin. Chem. Biol. 2007, 11, 12-19.
20. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B., Synthesis of proteins by native chemical ligation. Science 1994, 266, 776-779.
21. Dawson, P. E.; Kent, S. B., Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 2000, 69, 923-960.
22. Muir, T. W.; Sondhi, D.; Cole, P. A., Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 6705-6710.
23. Muir, T. W., Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 2003, 72, 249-289.
24. Offer, J.; Boddy, C. N.; Dawson, P. E., Extending synthetic access to proteins with a removable acyl transfer auxiliary. J. Am. Chem. Soc. 2002, 124, 4642-4646.
25. Wu, B.; Chen, J.; Warren, J. D.; Chen, G.; Hua, Z.; Danishefsky, S. J., Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol. Angew. Chem. Int. Ed. 2006, 45, 4116-4125.
26. Chen, G.; Wan, Q.; Tan, Z.; Kan, C.; Hua, Z.; Ranganathan, K.; Danishefsky, S. J., Development of efficient methods for accomplishing cysteine-free peptide and glycopeptide coupling. Angew. Chem. Int. Ed. 2007, 46, 7383-7387.
27. 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.
28. Wan, Q.; Chen, J.; Yuan, Y.; Danishefsky, S. J., Oxo-ester mediated native chemical ligation: concept and applications. J. Am. Chem. Soc. 2008, 130, 15814-15816.
29. Brik, A.; Ficht, S.; Yang, Y. Y.; Bennett, C. S.; 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.
30. Brik, A.; Yang, Y. Y.; Ficht, S.; Wong, C. H., Sugar-assisted glycopeptide ligation. J. Am. Chem. Soc. 2006, 128, 5626-5627.
31. Pentelute, B. L.; Kent, S. B., Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation. Org. Lett. 2007, 9, 687-690.
32. 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.
33. Haase, C.; Rohde, H.; Seitz, O., Native chemical ligation at valine. Angew. Chem. Int. Ed. 2008, 47, 6807-6810.
34. Crich, D.; Banerjee, A., Native chemical ligation at phenylalanine. J. Am. Chem. Soc. 2007, 129, 10064-10065.
35. Machova, Z.; von Eggelkraut-Gottanka, R.; Wehofsky, N.; Bordusa, F.; Beck-Sickinger, A. G., Expressed enzymatic ligation for the semisynthesis of chemically modified proteins. Angew. Chem. Int. Ed. 2003, 42, 4916-4918.
36. Noren, C. J.; Wang, J.; Perler, F. B., Dissecting the Chemistry of Protein Splicing and Its Applications. Angew. Chem. Int. Ed. 2000, 39, 450-466.
37. Muralidharan, V.; Muir, T. W., Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat. Methods 2006, 3, 429-438.
38. Evans, T. C., Jr.; Benner, J.; Xu, M. Q., The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum. J. Biol. Chem. 1999, 274, 3923-3926.
39. Reulen, S. W.; Brusselaars, W. W.; Langereis, S.; Mulder, W. J.; Breurken, M.; Merkx, M., Protein-liposome conjugates using cysteine-lipids and native chemical ligation. Bioconjug. Chem. 2007, 18, 590-596.
40. Lesaicherre, M. L.; Lue, R. Y.; Chen, G. Y.; Zhu, Q.; Yao, S. Q., Intein-mediated biotinylation of proteins and its application in a protein microarray. J. Am. Chem. Soc. 2002, 124, 8768-8769.
41. Camarero, J. A.; Kwon, Y.; Coleman, M. A., Chemoselective attachment of biologically active proteins to surfaces by expressed protein ligation and its application for "protein chip" fabrication. J. Am. Chem. Soc. 2004, 126, 14730-14731.
42. Kwon, Y.; Coleman, M. A.; Camarero, J. A., Selective immobilization of proteins onto solid supports through split-intein-mediated protein trans-splicing. Angew. Chem. Int. Ed. 2006, 45, 1726-1729.
43. Brennan, J. L.; Hatzakis, N. S.; Tshikhudo, T. R.; Dirvianskyte, N.; Razumas, V.; Patkar, S.; Vind, J.; Svendsen, A.; Nolte, R. J.; Rowan, A. E.; Brust, M., Bionanoconjugation via click chemistry: The creation of functional hybrids of lipases and gold nanoparticles. Bioconjug. Chem. 2006, 17, 1373-1375.
44. You, C. C.; Agasti, S. S.; De, M.; Knapp, M. J.; Rotello, V. M., Modulation of the catalytic behavior of alpha-chymotrypsin at monolayer-protected nanoparticle surfaces. J. Am. Chem. Soc. 2006, 128, 14612-14618.
45. Mirzabekov, T.; Kontos, H.; Farzan, M.; Marasco, W.; Sodroski, J., Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5. Nat. Biotechnol. 2000, 18, 649-654.
46. Willner, I.; Katz, E., Magnetic control of electrocatalytic and bioelectrocatalytic processes. Angew. Chem. Int. Ed. 2003, 42, 4576-4588.
47. Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J., Entrapping enzyme in a functionalized nanoporous support. J. Am. Chem. Soc. 2002, 124, 11242-11243.
48. Kim, J.; Lee, J.; Na, H. B.; Kim, B. C.; Youn, J. K.; Kwak, J. H.; Moon, K.; Lee, E.; Park, J.; Dohnalkova, A.; Park, H. G.; Gu, M. B.; Chang, H. N.; Grate, J. W.; Hyeon, T., A magnetically separable, highly stable enzyme system based on nanocomposites of enzymes and magnetic nanoparticles shipped in hierarchically ordered, mesocellular, mesoporous silica. Small 2005, 1, 1203-1207.
49. Lee, J.; Lee, D.; Oh, E.; Kim, J.; Kim, Y. P.; Jin, S.; Kim, H. S.; Hwang, Y.; Kwak, J. H.; Park, J. G.; Shin, C. H.; Hyeon, T., Preparation of a magnetically switchable bio-electrocatalytic system employing cross-linked enzyme aggregates in magnetic mesocellular carbon foam. Angew. Chem. Int. Ed. 2005, 44, 7427-7432.
50. Zhang, J.; Zhang, F.; Yang, H.; Huang, X.; Liu, H.; Guo, S., Graphene Oxide as a Matrix for Enzyme Immobilization. Langmuir 2010, 26, 6083-6085.
51. Alivisatos, A. P., Semiconductor clusters, nanocrystals, and quantum dots. Science 1996, 271, 933-937.
52. Gladysz, J. A., Curran, D. P., Horvath, I. T., Eds., Handbook of fluorous chemistry. Wiley-VCH: Weinheim 2004.
53. Horvath, I. T.; Rabai, J., Facile Catalyst Separation Without Water: Fluorous Biphase Hydroformylation of Olefins. Science 1994, 266, 72-75.
54. Curran, D. P.; Hadida, S.; He, M., Thermal allylations of aldehydes with a fluorous allylstannane. Separation of organic and fluorous products by solid phase extraction with fluorous reverse phase silica gel. J. Org. Chem. 1997, 62, 6714-6715.
55. Luo, Z.; Williams, J.; Read, R. W.; Curran, D. P., Fluorous Boc ((F)Boc) carbamates: new amine protecting groups for use in fluorous synthesis. J. Org. Chem. 2001, 66, 4261-4266.
56. Zhang, W.; Lu, Y.; Chen, C. H.; Curran, D. P.; Geib, S., Fluorous Synthesis of Hydantoin-, Piperazinedione-, and Benzodiazepinedione-Fused Tricyclic and Tetracyclic Ring Systems. European J. Org. Chem. 2006, 2055-2059.
57. Dakas, P. Y.; Barluenga, S.; Totzke, F.; Zirrgiebel, U.; Winssinger, N., Modular synthesis of radicicol A and related resorcylic acid lactones, potent kinase inhibitors. Angew. Chem. Int. Ed. 2007, 46, 6899-6902.
58. Luo, Z.; Zhang, Q.; Oderaotoshi, Y.; Curran, D. P., Fluorous mixture synthesis: a fluorous-tagging strategy for the synthesis and separation of mixtures of organic compounds. Science 2001, 291, 1766-1769.
59. Zhang, Q.; Rivkin, A.; Curran, D. P., Quasiracemic synthesis: concepts and implementation with a fluorous tagging strategy to make both enantiomers of pyridovericin and mappicine. J. Am. Chem. Soc. 2002, 124, 5774-5781.
60. Curran, D. P.; Zhang, Q.; Richard, C.; Lu, H.; Gudipati, V.; Wilcox, C. S., Total synthesis of a 28-member stereoisomer library of murisolins. J. Am. Chem. Soc. 2006, 128, 9561-9573.
61. Curran, D. P.; Moura-Letts, G.; Pohlman, M., Solution-phase mixture synthesis with fluorous tagging en route: total synthesis of an eight-member stereoisomer library of passifloricins. Angew. Chem. Int. Ed. 2006, 45, 2423-2426.
62. Montanari, V.; Kumar, K., Just add water: A new fluorous capping reagent for facile purification of peptides synthesized on the solid phase. J. Am. Chem. Soc. 2004, 126, 9528-9529.
63. Miura, T.; Goto, K.; Waragai, H.; Matsumoto, H.; Hirose, Y.; Ohmae, M.; Ishida, H.; Satoh, A.; Inazu, T., Rapid oligosaccharide synthesis using a fluorous protective group. J. Org. Chem. 2004, 69, 5348-5353.
64. Carrel, F. R.; Geyer, K.; Codee, J. D.; Seeberger, P. H., Oligosaccharide synthesis in microreactors. Org. Lett. 2007, 9, 2285-2288.
65. Mizuno, M.; Goto, K.; Miura, T., Fluorous glycopeptide synthesis without protection of sugar hydroxy groups. Chem. Lett. 2005, 34, 426-427.
66. Pearson, W. H.; Berry, D. A.; Stoy, P.; Jung, K. Y.; Sercel, A. D., Fluorous affinity purification of oligonucleotides. J. Org. Chem. 2005, 70, 7114-7122.
67. Gladysz, J. A.; Curran, D. P., Introduction - Fluorous chemistry: from biphasic catalysis to a parallel chemical universe and beyond. Tetrahedron 2002, 58, 3823-3825.
68. Jaipuri, F. A.; Pohl, N. L., Toward solution-phase automated iterative synthesis: fluorous-tag assisted solution-phase synthesis of linear and branched mannose oligomers. Org. Biomol. Chem. 2008, 6, 2686-2691.
69. Curran, D. P.; Luo, Z. Y., Fluorous synthesis with fewer fluorines (light fluorous synthesis): separation of tagged from untagged products by solid-phase extraction with fluorous reverse-phase silica gel. J. Am. Chem. Soc. 1999, 121, 9069-9072.
70. Matsugi, M.; Curran, D. P., Reverse fluorous solid-phase extraction: a new technique for rapid separation of fluorous compounds. Org. Lett. 2004, 6, 2717-2720.
71. Yu, M. S.; Curran, D. P.; Nagashima, T., Increasing fluorous partition coefficients by solvent tuning. Org. Lett. 2005, 7, 3677-3680.
72. Chu, Q.; Yu, M. S.; Curran, D. P., New fluorous/organic biphasic systems achieved by solvent tuning. Tetrahedron 2007, 63, 9890-9895.
73. Curran, D. P., Fluorous Chemistry in Pittsburgh: 1996-2008. J. Fluor. Chem. 2008, 129, 898-902.
74. Zhang, F.; Zhang, W.; Zhang, Y.; Curran, D. P.; Liu, G., Synthesis and applications of a light-fluorous glycosyl donor. J. Org. Chem. 2009, 74, 2594-2597.
75. Ko, K. S.; Jaipuri, F. A.; Pohl, N. L., Fluorous-based carbohydrate microarrays. J. Am. Chem. Soc. 2005, 127, 13162-13163.
76. Chen, G. S.; Pohl, N. L., Synthesis of fluorous tags for incorporation of reducing sugars into a quantitative microarray platform. Org. Lett. 2008, 10, 785-788.
77. Nicholson, R. L.; Ladlow, M. L.; Spring, D. R., Fluorous tagged small molecule microarrays. Chem. Commun. 2007, 3906-3908.
78. Min Hong, X. Z., Zhiqiang Lu, and Jin Zhu, Nanoparticle-Based, Fluorous-Tag-Driven DNA Detection. Angew. Chem. Int. Ed. 2009, 48, 1-5.
79. Beatrice Y.M. Collet , T. N., Marvin S. Yu , Nicola L.B. Pohl, Fluorous-based peptide microarrays for protease screening. J. Fluor. Chem. 2009, 130, 1042-1048.
80. Northen, T. R.; Lee, J. C.; Hoang, L.; Raymond, J.; Hwang, D. R.; Yannone, S. M.; Wong, C. H.; Siuzdak, G., A nanostructure-initiator mass spectrometry-based enzyme activity assay. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3678-3683.
81. Maltese, M., Reductive demercuration in deprotection of trityl thioethers, trityl amines, and trityl ethers. J. Org. Chem. 2001, 66, 7615-7625.
82. Girish, A.; Sun, H.; Yeo, D. S.; Chen, G. Y.; Chua, T. K.; Yao, S. Q., Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing. Bioorg. Med. Chem. Lett. 2005, 15, 2447-2451.
83. 郭昱瑩, α-2,3-唾液酸轉移酶之 C-端修飾與固化. 國立清華大學 碩士班論文 2008.
84. Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680-685.
85. Schagger, H.; von Jagow, G., Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368-379.
86. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
87. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C., Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76-85.
88. Hungerhoff, B.; Sonnenschein, H.; Theil, F., Combining lipase-catalyzed enantiomer-selective acylation with fluorous phase labeling: a new method for the resolution of racemic alcohols. J. Org. Chem. 2002, 67, 1781-1785.
89. Martin Braun, X. C., Otto Vostrowsky, Andreas Hirsch, Emil Endreß,; Thomas M. Bayerl, O. B., and Günter Gauglitz, Synthesis of a Biotinated Lipofullerene as a New Type of Transmembrane Anchor. Eur. J. Org. Chem. 2000, 1173-1181.
90. Mohamed A. Ghanem , J.-M. C., Aleksandra Pinczewska , Jeremy D. Kilburn and Philip N. Bartlett Covalent modification of glassy carbon surface with organic redox probes through diamine linkers using electrochemical and solid-phase synthesis methodologies. J. Mater. Chem. 2008, 18, 4917-4927.
91. Seibel, J.; Hellmuth, H.; Hofer, B.; Kicinska, A. M.; Schmalbruch, B., Identification of new acceptor specificities of glycosyltransferase R with the aid of substrate microarrays. ChemBioChem 2006, 7, 310-320.