蛋白質的化學修飾為化學生物學相關研究的重要工具。為了避免蛋白質活性的降低，蛋白質位向專一的修飾在近幾年中以被廣泛的探討。本論文主要是專注於蛋白質位向專一的修飾，並研究蛋白質透過氟標記以及DNA標記固化在固相載體之活性。
氟化學為近年快速發展的領域，其特性為氟分子之間有很強的作用力，此特殊的作用力可用來抑制蛋白質固化時所造成的非專一性吸附，因此於本篇論文中，我們發展了兩種位向專一修飾氟標記於蛋白質的策略；其中，目標蛋白－綠色螢光蛋白、麥芽糖結合蛋白以及麩胺基硫轉移酶利用 intein 蛋白質表現系統表達，之後透過自然化學鍵結反應，將含有磺基丙胺酸的氟探針修飾在蛋白質的 C 端。另一方面，anti-蓖麻毒素抗體則是透過硼酸可與二元醇反應形成硼酯的特性，將含有硼酸的氟探針修飾在抗體的醣體上。修飾氟探針的蛋白質，可透過簡單的混合，固化在固相載體上。氟-氟非共價鍵作用力相當穩定，除了可以承受不斷的清洗之外，亦可有效地抑制蛋白質的非專一性吸附。
由於 DNA 生物檢測技術具有快速且低成本的優勢，近年來有越來越多的科學家投入其研究當中。於本論文中，透過 DNA 鹼基對之間的作用力，固化蛋白質於磁性奈米粒子，並探討固化前後蛋白質的活性差異。目標酵素－磷酸葡萄糖胺胸苷轉移酶及半乳醣激酶可透過 2-氰基苯並噻唑與半胱胺酸進行縮和反應，專一地於蛋白質的 C 端建構 DNA 分子，除此之外，修飾 DNA之酵素可透過 DNA 互補對之間的作用力，固化在磁性奈米粒子；當高於解構溫度時，固化之酵素會從磁性奈米粒子中釋放出來。其中，修飾 DNA 之磷酸葡萄糖胸苷轉移酶的活性最佳，較直接固化以及透過 DNA 固化在磁性奈米粒子的活性好；另外，無論是以直接固化或是透過 DNA 固化的方式，半乳糖激酶的活性都相似。透過加熱從奈米粒子釋放出具有 DNA 之酵素，可利用磁性奈米粒子再回收利用。

Chemical modification of protein is an important tool for studying protein structure and function. To avoid the loss of protein activity, site-specific protein modification has been extensively studied in last decades. The studies of this thesis focused on the developments of site-specific protein modification and immobilization of modified protein on solid support by fluorous- or DNA-tagged protein.
The unique affinity interaction between fluorous molecules has been applied in many fields. We took advantage of the resistance of non-specific interaction by fluorous surface on protein microarray fabrication. Two strategies for site-specific modification of proteins with a fluorous tag were developed in this thesis. First, the target protein, enhance green fluorescent protein (eGFP), maltose binding protein (MBP), and glutathione transferase (GST), were expressed by intein expression system and their C-terminus were conjugated with cysteic acid contained fluorous tag by native chemical ligation (NCL). Second, the anti-RAC antibody was labeled with boronic acid contained fluorous tag through boronic acid-diol interaction. The fluorinated protein were site-specifically immobilized on fluorous solid support by simply mixing the fluorinated protein and solid support. The non-covalent fluorous-fluorous interaction were stable enough to withstand continuous washing and presented excellent performance to suppress the non-specfic adsorption.
DNA biosensor technologies are currently under intense investigation owing to their great promise for rapid and low-cost detection of specific DNA sequence. In this thesis, the specific interaction between DNA base pairs was applied on the protein immobilization on the magnetic nanoparticles to investigation of the activity difference between free and immobilized enzymes. The target enzyme, RmlA and GalK, were site specifically modified with DNA at their C-terminus using 2-cyanobenzothiazole (CBT)-cysteine (Cys) condensation reaction to give Enzyme-DNA. Then, these enzymes were assembled on DNA@MNPs through the sequence-specific hybridization properties of DNA. The captured enzymes were released from DNA@MNPs when the incubation temperature was higher than Tm of dsDNA. The results showed that the activity of RmlA-DNA is higher than those of RmlA-DNA-DNA@MNP and directly immobilized RmlA@MNP. However, the activity of GalK is identical as those of GalK-DNA-DNA@MNP and directly immobilized GalK@MNP. The enzyme-DNA was easily recovered by incubation with DNA@MNP and can be re-used after released from MNP by heating.

1. Prescher, J. A.; Bertozzi, C. R. Chemistry in living systems. Nat. Chem. Biol. 2005, 1, 13-21.
2. Shu-Min Zhou, L. C., Shu-Juan Guo, Heng Zhu, Sheng-Ce Tao unctional protein microarray: an ideal platform for investigating protein binding property. Front. Biol. 2012, 7, 336-349.
3. Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Global Analysis of Protein Activities Using Proteome Chips. Science 2001, 293, 2101-2105.
4. Chen, C.-S.; Korobkova, E.; Chen, H.; Zhu, J.; Jian, X.; Tao, S.-C.; He, C.; Zhu, H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat. Methods 2008, 5, 69-74.
5. Hall, D. A.; Zhu, H.; Zhu, X.; Royce, T.; Gerstein, M.; Snyder, M. Regulation of Gene Expression by a Metabolic Enzyme. Science 2004, 306, 482-484.
6. Hu, S.; Xie, Z.; Onishi, A.; Yu, X.; Jiang, L.; Lin, J.; Rho, H.-s.; Woodard, C.; Wang, H.; Jeong, J.-S.; Long, S.; He, X.; Wade, H.; Blackshaw, S.; Qian, J.; Zhu, H. Profiling the Human Protein-DNA nteractome Reveals ERK2 as a Transcriptional Repressor of Interferon Signaling. Cell 2009, 139, 610-622.
7. Moravcevic, K.; Mendrola, J. M.; Schmitz, K. R.; Wang, Y.-H.; Slochower, D.; Janmey, P. A.; Lemmon, M. A. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids. Cell 2010, 143, 966-977.
8. Huang, J.; Zhu, H.; Haggarty, S. J.; Spring, D. R.; Hwang, H.; Jin, F.; Snyder, M.; Schreiber, S. L. Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16594-16599.
9. 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.
10. Chun, E.; Thompson, Aaron A.; Liu, W.; Roth, Christopher B.; Griffith, Mark T.; Katritch, V.; Kunken, J.; Xu, F.; Cherezov, V.; Hanson, Michael A.; Stevens, Raymond C. Fusion Partner Toolchest for the Stabilization and Crystallization of G Protein-Coupled Receptors. Structure 2012, 20, 967-976.
11. Tamura, T.; Tsukiji, S.; Hamachi, I. Native FKBP12 Engineering by Ligand-Directed Tosyl Chemistry: Labeling Properties and Application to Photo-Cross-Linking of Protein Complexes in Vitro and in Living Cells. J. Am. Chem. Soc. 2012, 134, 2216-2226.
12. Carlsson, J.; Drevin, H.; Axén, R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 1978, 173, 723-737.
13. Patterson, D. P.; Schwarz, B.; Waters, R. S.; Gedeon, T.; Douglas, T. Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle. ACS Chem. Biol. 2014, 9, 359-365.
14. Cuatrecasas, P. Protein Purification by Affinity Chromatography: DERIVATIZATIONS OF AGAROSE AND POLYACRYLAMIDE BEADS. J. Biol. Chem. 1970, 245, 3059-3065.
15. Lin, P.-C.; Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Wang, K.-Y.; Chen, Y.-J.; Lin, C.-C. Ethylene Glycol-Protected Magnetic Nanoparticles for a Multiplexed Immunoassay in Human Plasma. Small 2006, 2, 485-489.
16. Yu, C.-C.; Lin, P.-C.; Lin, C.-C. Site-specific immobilization of CMP-sialic acid synthetase on magnetic nanoparticles and its use in the synthesis of CMP-sialic acid. Chem. Commun. 2008, 1308-1310.
17. Hahn, M. E.; Pellois, J.-P.; Vila-Perelló, M.; Muir, T. W. Tunable Photoactivation of a Post-translationally Modified Signaling Protein and its Unmodified Counterpart in Live Cells. ChemBioChem 2007, 8, 2100-2105.
18. Oya, T.; Hattori, N.; Mizuno, Y.; Miyata, S.; Maeda, S.; Osawa, T.; Uchida, K. Methylglyoxal Modification of Protein: Chemical and Immunochemical Characterization of Methylglyoxal-Arginine Adducts. J. Biol. Chem. 1999, 274, 18492-18502.
19. Chen, G.; Heim, A.; Riether, D.; Yee, D.; Milgrom, Y.; Gawinowicz, M. A.; Sames, D. Reactivity of Functional Groups on the Protein Surface:  Development of Epoxide Probes for Protein Labeling. J. Am. Chem. Soc. 2003, 125, 8130-8133.
20. Foettinger, A.; Melmer, M.; Leitner, A.; Lindner, W. Reaction of the Indole Group with Malondialdehyde:  Application for the Derivatization of Tryptophan Residues in Peptides. Bioconjugate Chem. 2007, 18, 1678-1683.
21. Gilles, M. A.; Hudson, A. Q.; Borders Jr, C. L. Stability of water-soluble carbodiimides in aqueous solution. Anal. Biochem. 1990, 184, 244-248.
22. Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126, 15942-15943.
23. 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. Bioconjugate Chem. 2008, 19, 786-791.
24. Bernardes, G. J. L.; Grayson, E. J.; Thompson, S.; Chalker, J. M.; Errey, J. C.; El Oualid, F.; Claridge, T. D. W.; Davis, B. G. From Disulfide- to Thioether-Linked Glycoproteins. Angew. Chem., Int. Ed. 2008, 120, 2276-2279.
25. Tilley, S. D.; Joshi, N. S.; Francis, M. B.; Begley, T. P., Proteins: Chemistry and Chemical Reactivity. In Wiley Encyclopedia of Chemical Biology, John Wiley & Sons, Inc.: 2007.
26. Lim, R. K. V.; Lin, Q. Bioorthogonal chemistry: recent progress and future directions. Chem. Commun. 2010, 46, 1589-1600.
27. Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Engineering Chemical Reactivity on Cell Surfaces Through Oligosaccharide Biosynthesis. Science 1997, 276, 1125-1128.
28. Gaertner, H. F.; Rose, K.; Cotton, R.; Timms, D.; Camble, R.; Offord, R. E. Construction of protein analogs by site-specific condensation of unprotected fragments. Bioconjugate Chem. 1992, 3, 262-268.
29. Saxon, E.; Bertozzi, C. R. Cell Surface Engineering by a Modified Staudinger Reaction. Science 2000, 287, 2007-2010.
30. Kolb, H. C.; Sharpless, K. B. The growing impact of click chemistry on drug discovery. Drug Discov. Today 2003, 8, 1128-1137.
31. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126, 15046-15047.
32. Blackman, M. L.; Royzen, M.; Fox, J. M. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity. J. Am. Chem. Soc. 2008, 130, 13518-13519.
33. Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D. J.; Blackman, M. L.; Fox, J. M.; Chin, J. W. Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions. J. Am. Chem. Soc. 2012, 134, 10317-10320.
34. Ning, X.; Temming, R. P.; Dommerholt, J.; Guo, J.; Ania, D. B.; Debets, M. F.; Wolfert, M. A.; Boons, G.-J.; van Delft, F. L. Protein Modification by Strain-Promoted Alkyne–Nitrone Cycloaddition. Angew. Chem., Int. Ed. 2010, 49, 3065-3068.
35. Lin, Y. A.; Boutureira, O.; Lercher, L.; Bhushan, B.; Paton, R. S.; Davis, B. G. Rapid Cross-Metathesis for Reversible Protein Modifications via Chemical Access to Se-Allyl-selenocysteine in Proteins. J. Am. Chem. Soc. 2013, 135, 12156-12159.
36. Ren, H.; Xiao, F.; Zhan, K.; Kim, Y.-P.; Xie, H.; Xia, Z.; Rao, J. A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins. Angew. Chem., Int. Ed. 2009, 48, 9658-9662.
37. Springsteen, G.; Wang, B. A detailed examination of boronic acid– diol complexation. Tetrahedron 2002, 58, 5291-5300.
38. Lang, K.; Chin, J. W. Bioorthogonal Reactions for Labeling Proteins. ACS Chem. Biol. 2014, 9, 16-20.
39. 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, 906-18.
40. Lippincott-Schwartz, J.; Patterson, G. H. Development and use of fluorescent protein markers in living cells. Science 2003, 300, 87-91.
41. Xu, M. Q.; Evans, T. C., Jr. Intein-mediated ligation and cyclization of expressed proteins. Methods 2001, 24, 257-77.
42. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science 1994, 266, 776-9.
43. Dawson, P. E.; Kent, S. B. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 2000, 69, 923-60.
44. 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, 6705-10.
45. Muir, T. W. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 2003, 72, 249-89.
46. 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, 4642-6.
47. 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, 4116-25.
48. 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, 7383-7.
49. 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, 9248-52.
50. Wan, Q.; Chen, J.; Yuan, Y.; Danishefsky, S. J. Oxo-ester mediated native chemical ligation: concept and applications. J Am Chem Soc 2008, 130, 15814-6.
51. 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, 15026-33.
52. Brik, A.; Yang, Y. Y.; Ficht, S.; Wong, C. H. Sugar-assisted glycopeptide ligation. J Am Chem Soc 2006, 128, 5626-7.
53. 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, 687-90.
54. 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, 8521-4.
55. Haase, C.; Rohde, H.; Seitz, O. Native chemical ligation at valine. Angew. Chem., Int. Ed. 2008, 47, 6807-10.
56. Crich, D.; Banerjee, A. Native chemical ligation at phenylalanine. J Am Chem Soc 2007, 129, 10064-5.
57. 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, 4916-8.
58. Dawson, P.; Muir, T.; Clark-Lewis, I.; Kent, S. Synthesis of proteins by native chemical ligation. Science 1994, 266, 776-779.
59. 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.
60. Muralidharan, V.; Muir, T. W. Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat. Methods 2006, 3, 429-38.
61. 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, 3923-6.
62. 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, 590-6.
63. 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, 8768-9.
64. 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, 14730-1.
65. 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, 1726-9.
66. Muir, T. W. Semisynthesis of Proteins by Expressed Protein Ligation. Annu. Rev. Biochem. 2003, 72, 249-289.
67. Feng, J.-J.; Zhao, G.; Xu, J.-J.; Chen, H.-Y. Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan. Anal. Biochem. 2005, 342, 280-286.
68. Wang, Y.; Caruso, F. Mesoporous Silica Spheres as Supports for Enzyme Immobilization and Encapsulation. Chem. Mater. 2005, 17, 953-961.
69. Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. Noncovalent Sidewall Functionalization of Single-Walled Carbon Nanotubes for Protein Immobilization. J. Am. Chem. Soc. 2001, 123, 3838-3839.
70. Gahoi, N.; Ray, S.; Srivastava, S. Array-based proteomic approaches to study signal transduction pathways: Prospects, merits and challenges. Proteomics 2015, 15, 218-231.
71. Zhang, J.; Zhang, F.; Yang, H.; Huang, X.; Liu, H.; Zhang, J.; Guo, S. Graphene Oxide as a Matrix for Enzyme Immobilization. Langmuir 2010, 26, 6083-6085.
72. Deng, Y.; Deng, C.; Qi, D.; Liu, C.; Liu, J.; Zhang, X.; Zhao, D. Synthesis of Core/Shell Colloidal Magnetic Zeolite Microspheres for the Immobilization of Trypsin. Adv. Mater. 2009, 21, 1377-1382.
73. Alivisatos, A. P. Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science 1996, 271, 933-937.
74. Yu, C.-C.; Kuo, Y.-Y.; Liang, C.-F.; Chien, W.-T.; Wu, H.-T.; Chang, T.-C.; Jan, F.-D.; Lin, C.-C. Site-Specific Immobilization of Enzymes on Magnetic Nanoparticles and Their Use in Organic Synthesis. Bioconjugate Chem. 2012, 23, 714-724.
75. Heyes, C. D.; Groll, J.; Moller, M.; Nienhaus, G. U. Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces. Molecular BioSystems 2007, 3, 419-430.
76. Jiang, S.; Cao, Z. Ultralow-Fouling, Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications. Adv. Mater. 2010, 22, 920-932.
77. Jonkheijm, P.; Weinrich, D.; Schröder, H.; Niemeyer, C. M.; Waldmann, H. Chemical Strategies for Generating Protein Biochips. Angew. Chem., Int. Ed. 2008, 47, 9618-9647.
78. Handbook of Fluorous Chemistry. Wiley-VCH Verlag GmbH & Co. KGaA: 2004.
79. Horvath, I. T.; Rabai, J. Facile catalyst separation without water: fluorous biphase hydroformylation of olefins. Science 1994, 266, 72-5.
80. 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.
81. 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, 119, 7023-7026.
82. 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.
83. 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.
84. 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.
85. Carrel, F. R.; Geyer, K.; Codée, J. D. C.; Seeberger, P. H. Oligosaccharide Synthesis in Microreactors. Org. Lett. 2007, 9, 2285-2288.
86. Miura, T.; Goto, K.; Waragai, H.; Matsumoto, H.; Hirose, Y.; Ohmae, M.; Ishida, H.-k.; Satoh, A.; Inazu, T. Rapid Oligosaccharide Synthesis Using a Fluorous Protective Group. J. Org. Chem. 2004, 69, 5348-5353.
87. Liu, B.; Zhang, F.; Zhang, Y.; Liu, G. A new approach for the synthesis of O-glycopeptides through a combination of solid-phase glycosylation and fluorous tagging chemistry (SHGPFT). Org. Biomol. Chem. 2014, 12, 1892-1896.
88. 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.
89. Gladysz, J. A.; Curran, D. P. Fluorous chemistry: from biphasic catalysis to a parallel chemical universe and beyond. Tetrahedron 2002, 58, 3823-3825.
90. Curran, D. P.; Luo, Z. 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.
91. Edwards, H. D.; Nagappayya, S. K.; Pohl, N. L. B. Probing the limitations of the fluorous content for tag-mediated microarray formation. Chem. Commun. 2012, 48, 510-512.
92. 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.
93. Yu, M. S.; Curran, D. P.; Nagashima, T. Increasing Fluorous Partition Coefficients by Solvent Tuning. Org. Lett. 2005, 7, 3677-3680.
94. Chu, Q.; Yu, M. S.; Curran, D. P. New fluorous/organic biphasic systems achieved by solvent tuning. Tetrahedron 2007, 63, 9890-9895.
95. Curran, D. P. Fluorous chemistry in Pittsburgh: 1996–2008. J. Fluorine Chem. 2008, 129, 898-902.
96. Ko, K.-S.; Jaipuri, F. A.; Pohl, N. L. Fluorous-Based Carbohydrate Microarrays. J. Am. Chem. Soc. 2005, 127, 13162-13163.
97. Jaipuri, F. A.; Collet, B. Y. M.; Pohl, N. L. Synthesis and quantitative evaluation of glycero-D-manno-heptose binding to concanavalin A by fluorous-tag assistance. Angew. Chem., Int. Ed. 2008, 47, 1707-1710.
98. Vegas, A. J.; Bradner, J. E.; Tang, W.; McPherson, O. M.; Greenberg, E. F.; Koehler, A. N.; Schreiber, S. L. Fluorous-based small-molecule microarrays for the discovery of histone deacetylase inhibitors. Angew. Chem., Int. Ed. 2007, 46, 7960-7964.
99. Nicholson, R. L.; Ladlow, M. L.; Spring, D. R. Fluorous tagged small molecule microarrays. Chem. Commun. 2007, 3906-3908.
100.Hong, M.; Zhou, X.; Lu, Z.; Zhu, J. Nanoparticle-Based, Fluorous-Tag-Driven DNA Detection. Angew. Chem., Int. Ed. 2009, 121, 9667-9670.
101.Collet, B. Y. M.; Nagashima, T.; Yu, M. S.; Pohl, N. L. B. Fluorous-based peptide microarrays for protease screening. J. Fluorine Chem. 2009, 130, 1042-1048.
102.Rapp, H. M.; Bacher, S.; Ahrens, A.; Rapp, W.; Kammerer, B.; Nienhaus, G. U.; Bannwarth, W. Attachment of Proteins to Surfaces by Fluorous–Fluorous Interactions Restoring Their Structure and Activity. ChemPlusChem 2012, 77, 1066-1070.
103.Dioumaev, V. K.; Bullock, R. M. A recyclable catalyst that precipitates at the end of the reaction. Nature 2000, 424, 530-532.
104.Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry. Nat Biotech 2005, 23, 463-468.
105.Zhao, M.; Deng, C. Designed synthesis of fluorous-functionalized magnetic mesoporous microspheres for specific enrichment of phosphopeptides with fluorous derivatization. Proteomics 2016, 16, 1051-1058.
106.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.
107.Deng, K.; George, K. W.; Reindl, W.; Keasling, J. D.; Adams, P. D.; Lee, T. S.; Singh, A. K.; Northen, T. R. Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme. Rapid Commun. Mass Sp. 2012, 26, 611-615.
108.Dietrich, C.; Schmitt, L.; Tampé, R. Molecular organization of histidine-tagged biomolecules at self-assembled lipid interfaces using a novel class of chelator lipids. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9014-9018.
109.Lebeau, L.; Lach, F.; Vénien-Bryan, C.; Renault, A.; Dietrich, J.; Jahn, T.; Palmgren, M. G.; Kühlbrandt, W.; Mioskowski, C. Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer1. J. Mol. Biol. 2001, 308, 639-647.
110. Hussein, W. M.; Ross, B. P.; Landsberg, M. J.; Lévy, D.; Hankamer, B.; McGeary, R. P. Synthesis of Nickel-Chelating Fluorinated Lipids for Protein Monolayer Crystallizations. J. Org. Chem. 2009, 74, 1473-1479.
111. Malik, L.; Nygaard, J.; Hoiberg-Nielsen, R.; Arleth, L.; Hoeg-Jensen, T.; Jensen, K. J. Perfluoroalkyl Chains Direct Novel Self-Assembly of Insulin. Langmuir 2012, 28, 593-603.
112. Durairaj, C.; Kadam, R. S.; Chandler, J. W.; Hutcherson, S. L.; Kompella, U. B. Nanosized Dendritic Polyguanidilyated Translocators for Enhanced Solubility, Permeability, and Delivery of Gatifloxacin. Invest. Ophth. Vis. Sci. 2010, 51, 5804-5816.
113. Hsiao, H.-Y.; Chen, M.-L.; Wu, H.-T.; Huang, L.-D.; Chien, W.-T.; Yu, C.-C.; Jan, F.-D.; Sahabuddin, S.; Chang, T.-C.; Lin, C.-C. Fabrication of carbohydrate microarrays through boronate formation. Chem. Commun. 2011, 47, 1187-1189.
114. Chen, M.-L.; Adak, A. K.; Yeh, N.-C.; Yang, W.-B.; Chuang, Y.-J.; Wong, C.-H.; Hwang, K.-C.; Hwu, J.-R. R.; Hsieh, S.-L.; Lin, C.-C. Fabrication of an Oriented Fc-Fused Lectin Microarray through Boronate Formation. Angew. Chem., Int. Ed. 2008, 47, 8627-8630.
115. Lin, P.-C.; Chen, S.-H.; Wang, K.-Y.; Chen, M.-L.; Adak, A. K.; Hwu, J.-R. R.; Chen, Y.-J.; Lin, C.-C. Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application. Analytical Chemistry 2009, 81, 8774-8782.
116. Jin, M.-J.; Lee, D.-H. A Practical Heterogeneous Catalyst for the Suzuki, Sonogashira, and Stille Coupling Reactions of Unreactive Aryl Chlorides. Angew. Chem., Int. Ed. 2010, 49, 1119-1122.
117. Wang, K.-Y.; Chuang, S.-A.; Lin, P.-C.; Huang, L.-S.; Chen, S.-H.; Ouarda, S.; Pan, W.-H.; Lee, P.-Y.; Lin, C.-C.; Chen, Y.-J. Multiplexed Immunoassay: Quantitation and Profiling of Serum Biomarkers Using Magnetic Nanoprobes and MALDI-TOF MS. Analytical Chemistry 2008, 80, 6159-6167.
118. 王筱嬋 2-氰基苯並噻唑縮合反應於位向選擇性蛋白質固化之應 用. 民國 101 年, 國立清華大學化學研究所 碩士論文.
119. Maliakel, B. P.; Schmid, W. Chemo-enzymatic synthesis of natural products: synthesis of sphydrofuran. Tetrahedron Lett. 1992, 33, 3297-3300.
120.Monsan, P.; Paul, F. Enzymatic synthesis of oligosaccharides. FEMS Microbiol. Rev. 1995, 16, 187-192.
121.Holzberger, B.; Marx, A. Enzymatic synthesis of perfluoroalkylated DNA. Bioorg. Med. Chem. 2009, 17, 3653-3658.
122.Huang, C.-Y.; Thayer, D. A.; Chang, A. Y.; Best, M. D.; Hoffmann, J.; Head, S.; Wong, C.-H. Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15-20.
123.Jones, M. N. Physico-chemical Basis of Specific Interactions Involving CarbohydratesCarbohydrate-mediated liposomal targeting and drug delivery. Adv. Drug Deliver Rev 1994, 13, 215-249.
124.Yu, H.; Chen, X. One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates. Org. Biomol. Chem. 2016, 14, 2809-2818.
125.侯凱齡 利用氟標記輔助化學及酵素合成寡醣. 民國102年, 國立 清華大學化學研究所 碩士論文.
126.李泗芃 探討噬熱菌 Meiothermus taiwanensis ATCC BAA-400 之半乳糖激酶酵素動力學及應用於合成Pk抗原類似物. 民國101 年, 國立清華大學化學研究所 碩士論文.
127.郭力禎 酵素唾液酸化 Globo- 系列醣體. 民國104年, 國立清華 大學化學研究所 碩士論文.
128.蕭偉鎮 結合化學與酵素方法合成多醣體. 民國 103年, 國立清 華大學化學研究所 博士論文.
129.簡薇庭 階段型合成醣核苷與其應用於合成聚 N-乙醯乳醣胺. 民 國 101 年, 國立清華大學化學研究所 博士論文.
130.Bülter, T.; Elling, L. Enzymatic synthesis of UDP-galactose on a gram scale. J. Mol. Catal. B: Enzym. 2000, 8, 281-284.
131.Liu, Z.; Zhang, J.; Chen, X.; Wang, P. G. Combined Biosynthetic Pathway For De Novo Production of UDP-Galactose: Catalysis with Multiple Enzymes Immobilized on Agarose Beads. ChemBioChem 2002, 3, 348-355.
132.Lee, J.-H.; Chung, S.-W.; Lee, H.-J.; Jang, K.-S.; Lee, S.-G.; Kim, B.-G. Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose. Bioproc. Biosyst. Eng. 2009, 33, 71-78.
133.Wong, C. H.; Haynie, S. L.; Whitesides, G. M. Enzyme-catalyzed synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose. J. Org. Chem. 1982, 47, 5416-5418.
134.Zervosen, A.; Elling, L. A Novel Three-Enzyme Reaction Cycle for the Synthesis of N-Acetyllactosamine with in Situ Regeneration of Uridine 5‘-Diphosphate Glucose and Uridine 5‘-Diphosphate Galactose. J. Am. Chem. Soc. 1996, 118, 1836-1840.
135.Koizumi, S.; Endo, T.; Tabata, K.; Ozaki, A. Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria. Nat. Biotechnol. 1998, 16, 847-850.
136.Chen, X.; Zhang, J.; Kowal, P.; Liu, Z.; Andreana, P. R.; Lu, Y.; Wang, P. G. Transferring a Biosynthetic Cycle into a Productive Escherichia coli Strain:  Large-Scale Synthesis of Galactosides. J. Am. Chem. Soc. 2001, 123, 8866-8867.
137.Liu, J.; Zou, Y.; Guan, W.; Zhai, Y.; Xue, M.; Jin, L.; Zhao, X.; Dong, J.; Wang, W.; Shen, J.; Wang, P. G.; Chen, M. Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP). Bioorg. Med. Chem. Lett. 2013, 23, 3764-3768.
138.Timmons, S. C.; Mosher, R. H.; Knowles, S. A.; Jakeman, D. L. Exploiting Nucleotidylyltransferases To Prepare Sugar Nucleotides. Org. Lett. 2007, 9, 857-860.
139.Guo, Y.; Fang, J.; Li, T.; Li, X.; Ma, C.; Wang, X.; Wang, P. G.; Li, L. Comparing substrate specificity of two UDP-sugar pyrophosphorylases and efficient one-pot enzymatic synthesis of UDP-GlcA and UDP-GalA. Carbohydr. Res. 2015, 411, 1-5.
140.Litterer, L. A.; Schnurr, J. A.; Plaisance, K. L.; Storey, K. K.; Gronwald, J. W.; Somers, D. A. Characterization and expression of Arabidopsis UDP-sugar pyrophosphorylase. Plant Physiol. Bioch. 2006, 44, 171-180.
141.Bulyk, M. L. DNA microarray technologies for measuring protein– DNA interactions. Current Opin. Biotech. 2006, 17, 422-430.
142.Stoltenburg, R.; Reinemann, C.; Strehlitz, B. SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 2007, 24, 381-403.
143.Xia, F.; Zuo, X.; Yang, R.; Xiao, Y.; Kang, D.; Vallée-Bélisle, A.; Gong, X.; Yuen, J. D.; Hsu, B. B. Y.; Heeger, A. J.; Plaxco, K. W. Colorimetric detection of DNA, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10837-10841.
144.Mahmuh, J.; Rownd, R.; Schildkbatjt, C. L., Denaturation and Renaturation of Deoxyribonucleic Acid. In Progress in Nucleic Acid Research and Molecular Biology, Davidson, J. N.; Waldo, E. C., Eds. Academic Press: 1963; Vol. Volume 1, pp 231-300.
145.Owczarzy, R.; Moreira, B. G.; You, Y.; Behlke, M. A.; Walder, J. A. Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations. Biochem. 2008, 47, 5336-5353.
146.In Situ Hybridization. https://lifescience.roche.com/shop/en/tw/products/in-situ-hybridizatio n.
147.Levine, L.; Gordon, J. A.; Jencks, W. P. The Relationship of Structure to the Effectiveness of Denaturing Agents for Deoxyribonucleic Acid. Biochem. 1963, 2, 168-175.
148.Wetmur, J. G.; Davidson, N. Kinetics of renaturation of DNA. J. Mol. Biol. 1968, 31, 349-370.
149.Wetmur, J. G. Acceleration of DNA renaturation rates. Biopolymers 1975, 14, 2517-2524.
150.Herne, T. M.; Tarlov, M. J. Characterization of DNA Probes Immobilized on Gold Surfaces. J. Am. Chem. Soc. 1997, 119, 8916-8920.
151.Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Maximizing DNA Loading on a Range of Gold Nanoparticle Sizes. Analytical Chemistry 2006, 78, 8313-8318.
152.Meredith, G. D.; Wu, H. Y.; Allbritton, N. L. Targeted Protein Functionalization Using His-Tags. Bioconjugate Chem. 2004, 15, 969-982.
153.Niemeyer, C. M.; Sano, T.; Smith, C. L.; Cantor, C. R. Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA—streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucleic Acids Res. 1994, 22, 5530-5539.
154.Lovrinovic, M.; Seidel, R.; Wacker, R.; Schroeder, H.; Seitz, O.; Engelhard, M.; Goody, R. S.; Niemeyer, C. M. Synthesis of protein-nucleic acid conjugates by expressed protein ligation. Chem. Commun. 2003, 822-823.
155.Takeda, S.; Tsukiji, S.; Nagamune, T. Site-specific conjugation of oligonucleotides to the C-terminus of recombinant protein by expressed protein ligation. Bioorg. Med. Chem. Lett. 2004, 14, 2407-2410.
156.Burbulis, I.; Yamaguchi, K.; Gordon, A.; Carlson, R.; Brent, R. Using protein-DNA chimeras to detect and count small numbers of molecules. Nat. Methods 2005, 2, 31-37.
157.Lovrinovic, M.; Niemeyer, C. M. Microtiter Plate-Based Screening for the Optimization of DNA–Protein Conjugate Synthesis by Means of Expressed Protein Ligation. ChemBioChem 2007, 8, 61-67.
158.Takeda, S.; Tsukiji, S.; Ueda, H.; Nagamune, T. Covalent split protein fragment-DNA hybrids generated through N-terminus-specific modification of proteins by oligonucleotides. Org. Biomol. Chem. 2008, 6, 2187-2194.
159.Duckworth, B. P.; Chen, Y.; Wollack, J. W.; Sham, Y.; Mueller, J. D.; Taton, T. A.; Distefano, M. D. A Universal Method for the Preparation of Covalent Protein–DNA Conjugates for Use in Creating Protein Nanostructures. Angew. Chem., Int. Ed. 2007, 46, 8819-8822.
160.Jongsma, M. A.; Litjens, R. H. G. M. Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address. Proteomics 2006, 6, 2650-2655.
161.Niemeyer, C. M. Semisynthetic DNA–Protein Conjugates for Biosensing and Nanofabrication. Angew. Chem., Int. Ed. 2010, 49, 1200-1216.
162.Chu, B. C. F.; Wahl, G. M.; Orgel, L. E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 1983, 11, 6513-6529.
163.Qin, P. Z.; Pyle, A. M. Site-Specific Labeling of RNA with Fluorophores and Other Structural Probes. Methods 1999, 18, 60-70.
164.Wang, T.-P.; Chiou, Y.-J.; Chen, Y.; Wang, E.-C.; Hwang, L.-C.; Chen, B.-H.; Chen, Y.-H.; Ko, C.-H. Versatile Phosphoramidation Reactions for Nucleic Acid Conjugations with Peptides, Proteins, Chromophores, and Biotin Derivatives. Bioconjugate Chem. 2010, 21, 1642-1655.
165.Wang, T.-P.; Ko, N. C.; Su, Y.-C.; Wang, E.-C.; Severance, S.; Hwang, C.-C.; Shih, Y. T.; Wu, M. H.; Chen, Y.-H. Advanced Aqueous-Phase Phosphoramidation Reactions for Effectively Synthesizing Peptide– Oligonucleotide Conjugates Trafficked into a Human Cell Line. Bioconjugate Chem. 2012, 23, 2417-2433.
166.Kotera, M.; Dheu, M.-L.; Milet, A.; Lhomme, J.; Laayoun, A. Pyrenyldiazomethane, a versatile reagent for nucleotide phosphate alkylation. Bioorg. Med. Chem. Lett. 2005, 15, 705-708.
167.Gampe, C. M.; Hollis-Symynkywicz, M.; Zécri, F. Covalent Chemical 5'-Functionalization of RNA with Diazo Reagents. Angew. Chem., Int. Ed. 2016, ASAP.
168.Wang, H.-C.; Yu, C.-C.; Liang, C.-F.; Huang, L.-D.; Hwu, J.-R.; Lin, C.-C. Site-Selective Protein Immobilization through 2-Cyanobenzothiazole-Cysteine Condensation. ChemBioChem 2014, 15, 829-835.
169.Urdea, M. S.; Warner, B. D.; Running, J. A.; Stempien, M.; Clyne, J.; Horn, T. A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 1988, 16, 4937-4956.
170.Michael Adler, R. W., Essy Booltink, Bernhard Manz & Christof M Niemeyer Detection of femtogram amounts of biogenic amines using self-assembled DNA-protein nanostructures. Nat. Methods 2005, 2, 147-149.
171.Niemeyer, C. M.; Adler, M.; Pignataro, B.; Lenhert, S.; Gao, S.; Lifeng, C.; Fuchs, H.; Dietmar, B. Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR. Nucleic Acids Res. 1999, 27, 4553-4561.
172.Liang, G.; Ren, H.; Rao, J. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2010, 2, 54-60.
173.Bruce, I. J.; Sen, T. Surface Modification of Magnetic Nanoparticles with Alkoxysilanes and Their Application in Magnetic Bioseparations. Langmuir 2005, 21, 7029-7035.
174.Tsukiji, S.; Miyagawa, M.; Takaoka, Y.; Tamura, T.; Hamachi, I. Ligand-directed tosyl chemistry for protein labeling in vivo. Nat Chem Biol 2009, 5, 341-343.
175.Goodman, R. P.; Erben, C. M.; Malo, J.; Ho, W. M.; McKee, M. L.; Kapanidis, A. N.; Turberfield, A. J. A Facile Method for Reversibly Linking a Recombinant Protein to DNA. ChemBioChem 2009, 10, 1551-1557.
176.Rudiuk, S.; Venancio-Marques, A.; Baigl, D. Enhancement and Modulation of Enzymatic Activity through Higher-Order Structural Changes of Giant DNA–Protein Multibranch Conjugates. Angew. Chem. Int. Ed. 2012, 51, 12694-12698.
177.Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. What Controls the Melting Properties of DNA-Linked Gold Nanoparticle Assemblies? J. Am. Chem. Soc. 2003, 125, 1643-1654.
178.Howarth, N. M.; Lindsell, W. E.; Murray, E.; Preston, P. N. Lipophilic peptide nucleic acids containing a 1,3-diyne function: synthesis, characterization and production of derived polydiacetylene liposomes. Tetrahedron 2005, 61, 8875-8887.
179.Moreau, J.; Marchand-Brynaert, J. Modular Synthesis of Bifunctional Linkers for Materials Science. Eur. J. Org. Chem. 2011, 2011, 1641-1644.