具有功能性分子修飾的抗體經常作為研究試劑，應用於生物檢測或醫療研究上，因此發展出一套便利的抗體修飾方法十分重要。在抗體修飾過程中，為了維持抗體本身的活性，需盡量避免將功能分子修飾於抗體的抗原結合區域。
為了達到這個目的，在本研究中，利用抗體Fc區域上的醣體作為目標，開發出含疊氮基之硼酸導向親和性探針，對抗體進行位置專一性的標記。由於親和性探針標記的效率並非100%，因此我們利用生物素作為純化用的標籤，透過表面具有卵白素的磁珠純化，移除未修飾的抗體。之後藉由紫外光照射，使光解基團發生斷裂反應並將抗體由磁珠表面釋放至溶液中，以得到純化的疊氮基修飾抗體。
藉由上述方法，本論文成功對四種不同的抗體 (h-Ab、anti-HER2-Ab、anti-ConA-Ab及anti-CETP-Ab) 進行修飾，並在修飾完成後，以ELISA binding affinity assay檢測修飾前後抗體對於抗原及Fcγ接收蛋白的辨認能力，結果顯示此修飾方法並不會影響抗體活性。此外，藉由修飾於抗體表面的疊氮基團，我們能透過CuAAC或SPAAC click反應將抗體與多種不同基團結合。
首先，我們將修飾後的抗體分別與生物素和螢光分子Cy3進行結合，並透過西方墨點法及膠體電泳分析，證明此種標記方法的可行性；此外，我們也將其與螢光分子四苯乙烯 (TPE) 結合，藉由螢光訊號的消長證實修飾位置位於目標醣體的附近；最後，我們將修飾的anti-HER2-Ab與藥物doxorubicin 結合作為抗體藥物，應用於癌細胞抑制實驗中，並藉由MTT assay分析MCF-7細胞的活性，結果顯示此抗體藥物可抑制MCF-7細胞生長。
綜合以上，本論文開發出簡單且容易進行的抗體標記方法，並成功應用於各種抗體後修飾。此方法不須藉由複雜的基因工程技術，即可製備不同的修飾抗體。

Modified antibodies have emerged as an efficient tool for biological research. Therefore, it is important to develop a straightforward method for the antibody modification. To maintain the bioactivity of the modified antibody, it demands to site-specifically install a tag located away from antigen binding site of antibody.
To achieve site-specific modification of an antibody, we developed a boronic acid (BA)-directed labeling strategy. By interacting with glycan of the Fc moiety of an antibody, BA-ligand probe attracts activated thioester and follows by nucleophilic acyl substitution to achieve covalently conjugation. However, the labeling efficiency is not always quantitative. Thus, a photo-cleavable linker and a biotin purification tag were incorporated in the designed labeling probe. The labeled antibody can be captured by avidin coated magnetic beads. After irradiation of bead complexes by UV light, the labeled antibody was released from the beads. By this strategy, high purity site-specific azide-labeled antibody was obtained.
Four different antibodies (h-Ab, anti-HER2-Ab, anti-ConA-Ab and anti-CETP-Ab) were successfully modified by BA-directed labeling strategy. The binding affinities of the labeled antibodies with corresponding antigens or Fcγ receptor are the same as those intact antibodies, indicating that the developed labeling method does not affect the original activities of antibodies.
The installed azide groups on the modified antibodies were conjugated with several different alkynated groups by Cu(Ⅰ)-catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. Antibodies conjugated with biotin and Cy3 were analyzed by SDS-PAGE and western blotting to conform the azide tag on antibodies. Moreover, the antibody conjugated with TPE fluorophore showed the fluorescence change, indicatng that the labeling site is close to the glycan on Fc moiety. Azide-labeled anti-HER2-Ab was further conjugated with doxorubicin and the resulting complex was used to treat with HER2-expressed MCF-7 cancer cell line. The MTT assay shows the cytotoxicity of anti-HER2-Ab-Dox conjugate.
　　Overall, the newly developed BA-directed labeling method is simple for modification of intact antibodies. Most importantly, it does not require genetic engineering to install a biorthogonal group on the antibody. Thus, the method is suitable for labeling any intact antibody containing glycan on its Fc domain.

目錄
摘要 I
Abstract III
縮寫對照表 (abbreviation) V
圖目錄 XI
流程圖目錄 XIII
表目錄 XIV
壹. 緒論 1
1.1 抗體 1
1.1.1 抗體的結構 2
1.1.2 哺乳類動物抗體的種型 5
1.1.3 抗體的功能 7
1.1.4 抗體的修飾 8
1.2 化學探針 19
1.2.1 配位基 (ligand) 20
1.2.2 連接劑 (linker) 21
1.2.3 標籤 (tag) 22
1.2.4 反應基團 26
1.3 硼酸 29
1.3.1 硼酯 (boronate) 30
1.3.2 硼酸的應用 32
1.4 光解基團 (photocleavable group) 37
1.5 研究動機 39
貳. 硼酸連接DMAP探針的合成與應用 40
2.1 硼酸連接DMAP探針的合成與標記 41
2.1.1 硼酸連結DMAP探針之合成 44
2.1.2 醯基予體 (acyl donor) 47
2.1.3 標記結果與討論 49
2.1.4 可光解acyl donor的設計與合成 57
2.1.5 光解acyl donor標記結果與討論 63
2.1.6 標記效率與純化 68
2.2 應用 73
2.2.1 不同抗體之修飾前後之binding affinity測試 73
2.2.2 生物素與螢光分子 77
2.2.3 四苯乙烯螢光分子 78
2.2.4 抗體藥物 82
參. 總結 87
肆. 實驗部分 89
4.1 General procedures 89
4.2 Synthetic procedures and characterizations 91
4.3 Experiments of antibody modification 121
伍. 參考文獻 130
附錄

1. Jain, N., Smith, S.W., Ghone, S. Current ADC Linker Chemistry. Pharm. Res. 2015, 32, 3526-3540.
2. Zhang, Z.; Guan, Y.; Li, M.; Zhao, A.; Ren, J.; Qu, X. Highly stable and reusable imprinted artificial antibody used for in situ detection and disinfection of pathogens. Chem. Sci. 2015, 6, 2822-2826.
3. Maverakis, E.; Kim K.; Shimoda, M.; Gershwin, M. E.; Patel, F.; Wilken, R.; Raychaudhuri, S.; Ruhaak, L. R.; Lebrilla, C. B. Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: a critical review. J. Autoimmun. 2015, 57, 1-13.
4. Harpaz Y, Chothia C. Many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains. J. Mol. Biol. 1994, 238, 528-539.
5. Borghesi, L.; Milcarek, C. From B cell to plasma cell: regulation of V(D)J recombination and antibody secretion. Immunol. Res. 2006, 36, 27-32.
6. Litman, G.W.; Rast, J. P.; Shamblott, M. J.; Haire, R. N.; Hulst, M.; Roess, W.; Litman, R. T.; Hinds-Frey, K. R.; Zilch, A.; Amemiya, C. T. Phylogenetic diversification of immunoglobulin genes and the antibody repertoire. Mol. Biol. Evol. 1993, 10, 60-72.
7. Arora, S.; Saxena, V.; Ayyar, B. V. Affinity chromatography: A versatile technique for antibody purification. Methods 2017, 116, 84-94.
8. Janeway, C. A. J.; Travers, P.; Walport, M.; Shlomchik, M. J. Immunobiology: The Immune System in Health and Disease. 5th edition. (Garland Science, New York. 2001).
9. Woof, J. M.; Burton, D. R. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat. Rev. Immunol. 2004, 4, 89-99.
10. Nemazee, D. Receptor editing in lymphocyte development and central tolerance. Nat. Rev. Immunol. 2006, 6, 728-740.
11. Lin, C.-W.; Tsai, M.-H.; Li, S.-T.; Tsai, T.-I.; Chu, K.-C.; Liu, Y.-C.; Lai, M.-Y.; Wu, C.-Y.; Tseng, Y.-C.; Shivatare, S. S.; Wang, C.-H.; Chao, P.; Wang, S.-Y.; Shih, H.-W.; Zeng, Y.-F.; You, T.-H.; Liao, J.-Y.; Tu, Y.-C.; Lin, Y.-S.; Chuang H.-Y.; Chen, C.-L.; Tsai, C.-S.; Huang, C.-C.; Lin, N.-H.; Ma, C.; Wu, C.-Y.; Wong, C.-H. A common glycan structure on immunoglobulin G for enhancement of effector functions. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 10611-10616.
12. MBL Life Science. The role of antibodies. http://ruo.mbl.co.jp/bio/g/support/method/antibody-role.html. (accessed 05/23/2018).
13. Lu, L. L.; Suscovich, T. J.; Fortune, S. M.; Alter, G. Beyond binding: antibody effector functions in infectious diseases. Nat. Rev. Immunol. 2018, 18, 46-61.
14. Underdown, B.; Schiff, J. Immunoglobulin A: strategic defense initiative at the mucosal surface. Annu. Rev. Immunol. 1986, 4, 389-417.
15. Pier, G. B.; Lyczak, J. B.; Wetzler, L. M. Immunology, Infection, and Immunity; ASM Press : Washington, DC, 2004.
16. Geisberger, R.; Lamers, M.; Achatz, G. The riddle of the dual expression of IgM and IgD. J. Immunol. 2006, 118, 429-437.
17. Chen, K.; Xu, W.; Wilson, M.; He, B.; Miller, N. W.; Bengtén, E.; Edholm, E. S.; Santini, P. A.; Rath, P.; Chiu, A.; Cattalini, M.; Litzman, J. B.; Bussel, J.; Huang, B.; Meini, A.; Riesbeck, K.; Cunningham-Rundles, C.; Plebani, A.; Cerutti, A. Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils. Nat. Immunol. 2009, 10, 889-898.
18. Noris, M.; Remuzzi, G. Overview of Complement Activation and Regulation. Semin. Nephrol. 2013, 33, 479-492.
19. Hermanson, G.T. Bioconjugate Techniques; Academic Press: New York, NY, USA, 2008.
20. Mueller, B. M.; Wrasidlo, W. A.; Reisfeld, R. A. Determination of the number of e-amino groups available for conjugation of effector molecules to monoclonal antibodies. Hybridoma 1988, 7, 453-456.
21. Widdison, W. C.; Wilhelm, S. D.; Cavanagh, E. E.; Whiteman, K. R.; Leece, B. A.; Kovtun, Y.; Goldmacher, V. S.; Xie, H.; Steeves, R. M.; Lutz, R. J.; Zhao, R.; Wang, L.; Blättler, W. A.; Chari, R. V. J. Semisynthetic maytansine analogues for the targeted treatment of cancer. J. Med. Chem. 2006, 49, 4392-4408.
22. Wakankar, A. A.; Feeney, M. B.; Rivera, J.; Chen, Y.; Kim, M.; Sharma, V. K.; Wang, Y. J. Physicochemical stability of the antibody-drug conjugate Trastuzumab-DM1 : Changes due to modification and conjugation processes. Bioconjugate Chem. 2010, 21, 1588-1595.
23. Wang, L.; Amphlett, G.; Blattler, W. A.; Lambert, J. M.; Zhang, W. Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci. 2005, 14, 2436-2446. Protein Sci. 2005, 14, 2436-2446.
24. Adamczyk, M.; Gebler, J.; Shreder, K.; Wu, J. Region-selective labeling of antibodies as determined by electrospray ionization-mass spectrometry (ESI-MS). Bioconjugate Chem. 2000, 11, 557-563.
25. Peters, C.; Brown, S. Antibody-drug conjugates as novel anti-cancer chemotherapeutics. Biosci. Rep. 2015, 35, e00225.
26. Liu, H.; Chumsae, C.; Gaza-Bulseco, G.; Hurkmans, K.; Radziejewski, C. H. Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC-MS analysis. Anal. Chem. 2010, 82, 5219-5226.
27. Sun, M. M.; Beam, K. S.; Cerveny, C. G.; Hamblett, K. J.; Blackmore, R. S.; Torgov, M. Y.; Handley, F. G.; Ihle, N. C.; Senter, P. D.; Alley, S. C. Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjugate Chem. 2005, 16, 1282-1290.
28. Krall, N.; Cruz, F. P.; Boutureira, O.; Bernardes, G. J. L. Site-selective protein-modification chemistry for basic biology and drug development. Nature Chemistry 2016, 8, 103-113. Nat. Chem. 2016, 8, 103-113.
29. Badescu, G.; Bryant, P.; Bird, M.; Henseleit, K.; Swierkosz, J.; Parekh, V.; Tommasi, R.; Pawlisz, E.; Jurlewicz, K.; Farys, M.; Camper, N.; Sheng, X. B.; Fisher, M.; Grygorash, R.; Kyle, A.; Abhilash, A.; Frigerio, M.; Edwards, J.; Godwin, A. Bridging disulfides for stable and defined antibody drug conjugates. Bioconjugate Chem. 2014, 25, 1124-1136.
30. Nunes, J. P.; Morais, M.; Vassileva, V.; Robinson, E.; Rajkumar, V. S.; Smith, M. E.; Pedley, R. B.; Caddick, S.; Baker, J. R.; Chudasama, V. Functional native disulfide bridging enables delivery of a potent, stable and targeted antibody-drug conjugate (ADC). Chem. Commun. 2015, 51, 10624-10627.
31. Josten, A.; Haalck, L.; Spener, F.; Meusel, M. Use of microbial transglutaminase for the enzymatic biotinylation of antibodies. J. Immunol. Methods 2000, 240, 47-54. J. Immunol. Methods 2000, 240, 47-54.
32. Jeger, S.; Zimmermann, K.; Blanc, A.; Gruenberg, J.; Honer, M.; Hunziker, P.; Struthers, H.; Schibli, R. Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew. Chem. Int. Ed. 2010, 49, 9995-9997.
33. Junutula, J. R.; Raab, H.; Clark, S.; Bhakta, S.; Leipold, D. D.; Weir, S.; Chen, Y.; Simpson, M.; Tsai, S. P.; Dennis, M. S.; Lu, Y.; Meng, Y. G.; Ng, C.; Yang, J.; Lee, C. C.; Duenas, E.; Gorrell, J.; Katta, V.; Kim, A.; McDorman, K.; Flagella, K.; Venook, R.; Ross, S.; Spencer, S. D.; Wong, W. L.; Lowman, H. B.; Vandlen, R.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Mallet, W. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 2008, 26, 925-932.
34. Caron, P. C.; Laird, W.; Co, M. S.; Avdalovic, N. M.; Queen, C.; Scheinberg, D. A. Engineered humanized dimeric forms of IgG are more effective antibodies. J. Exp. Med. 1992, 176, 1191-1195.
35. Shopes, B. A genetically engineered human IgG with limited flexibility fully initiates cytolysis via complement. Mol. Immunol. 1993, 30, 603-609.
36. Adumeau, P.; Davydova, M.; Zeglis, B. M. Thiol-Reactive Bifunctional Chelators for the Creation of Site-Selectively Modified Radioimmunoconjugates with Improved Stability. Bioconjugate Chem. 2018, 29, 1364-1372.
37. Shen, B. Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K.L.; Tien, J.; Yu, S. F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 2012, 30, 184-189.
38. Xiao, H.; Chatterjee, A.; Choi, S.-H.; Bajjuri, K. M.; Sinha, S. C.; Schultz, P. G. Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew. Chem. Int. Ed. 2013, 52, 14080-14083.
39. Axup, J.; Bajjuri, K.; Ritland, M.; Hutchins, B.; Kim, C.; Kazane, S.; Halder, R.; Forsyth, J.; Santidrian, A.; Stafin, K.; Lu, Y.; Tran, H.; Seller, A. J.; Biroc, S. L.; Szydlik, A.; Pinkstaff, J. K.; Tian, F.; Sinha, S. C.; Felding-Habermann, B.; Smider, V. V.; Schultz, P. G. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 16101-16106.
40. Jackson, D.; Atkinson, J.; Guevara, C. I.; Zhang, C.; Kery, V.; Moon, S.-J. J.; Virata, C.; Yang, P.; Lowe, C.; Pinkstaff, J.; Cho, H.; Knudsen, N.; Manibusan, A.; Tian, F.; Sun, Y.; Lu, Y.; Sellers, A.; Jia, X.-C.; Joseph, I., Anand, B.; Morrison, K.; Pereira, D. S.; Stover, D. In vitro and in vivo evaluation of cysteine and site-specific conjugated herceptin antibody-drug conjugates. PloS One 2014, 9, e83865.
41. Park, Y.-J.; Liang, J.-F.; Song, H.; Li, Y. T.; Naik, S.; Yang, V. C. ATTEMPTS: A heparin/protamine-based triggered release system for the delivery of enzyme drugs without associated side-effects. Adv. Drug Deliv. Rev. 2003, 55, 251-265.
42. Lai, C.-H. , Lim, S. C.; Wu, L.-C.; Wang, C.-F.; Tsai, W.-S.; Wu, H.-C.; Chang, Y.-C. Site-specific antibody modification and immobilization on a microfluidic chip to promote the capture of circulating tumor cells and microemboli. Chem. Commun. 2017, 53, 4152-4155.
43. Zuberbühler, K.; Casi, G.; Bernardes, G. J.; Neri, D. Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format. Chem. Commun. 2012, 48, 7100-7102.
44. Dennler, P.; Fischer, E.; Schibli, R. Antibody Conjugates: From Heterogeneous Populations to Defined Reagents. Antibodies 2015, 4, 197-224.
45. Solomon, B.; Koppel, R.; Schwartz, F.; Fleminger, G. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes. J. Chromatogr. A 1990, 510, 321-329.
46. Li, X.; Fang, T.; Boons, G. J. Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew. Chem. Int. Ed. 2014, 53, 7179-7182.
47. Boeggeman, E.; Ramakrishnan, B.; Kilgore, C.; Khidekel, N.; Hsieh-Wilson, L. C.; Simpson, J. T.; Qasba, P. K. Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method. Bioconjugate Chem. 2007, 18, 806-814.
48. van Geel, R.; Wijdeven, M. A.; Heesbeen, R.; Verkade, J. M.; Wasiel, A. A.; van Berkel, S. S.; van Delft, F. L. Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates. Bioconjugate Chem. 2015, 26, 2233-2242.
49. Zhu, Z.; Ramakrishnan, B.; Li, J.; Wang, Y.; Feng, Y.; Prabakaran, P.; Colantonio, S.; Dyba, M. A.; Qasba, P. K.; Dimitrov, D. S. Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. mAbs 2014, 6, 1190-1200.
50. Boeggeman, E.; Ramakrishnan, B.; Pasek, M.; Manzoni, M.; Puri, A.; Loomis, K. H.; Waybright, T. J.; Qasba, P. K. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: Application for cell surface antigen detection. Bioconjug. Chem. 2009, 20, 1228-1236. Bioconjugate Chem. 2009, 20, 1228-1236.
51. Ohata, J.; Ball, Z. T. A Hexa-rhodium Metallopeptide Catalyst for Site-Specific Functionalization of Natural Antibodies. J. Am. Chem. Soc. 2017, 139, 12617-12622.
52. Rosen, C. B.; Kodal, A. L. B.; Nielsen, J. S.; Schaffert, D. H.; Scavenius, C.; Okholm, A. H.; Voigt, N. V.; Enghild, J. J.; Kjems, J.; Tørring, T.; Gothelf, K. V. Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins. Nat. Chem. 2014, 6, 804-809.
53. Lodish, H.; Berk, A.; Zipursky, S. L.; Matsudaira, P.; Baltimore, D.; Darnell, J. Molecular Cell Biology, 4th ed. (W. H. Freeman, New York, 2000).
54. Lee, F. S.; Shapiro, R.; Vallee, B. L. Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor. Biochem. 1989, 28, 225-230.
55. 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.
56. Lis, H.; Sharon, N., Lectins:Carbohydrate-Specific Proteins That Mediate Cellular Recognition. Chem. Rev. 1998, 98, 637-674.
57. Chang, T. C.; Lai, C. H.; Chien, C. W.; Liang, C. F.; Adak, A. K.; Chuang, Y. J.; Chen, Y. J.; Lin, C. C., Synthesis and Evaluation of a Photoactive Probe with a Multivalent Carbohydrate for Capturing Carbohydrate-Lectin Interactions. Bioconjugate Chem. 2013, 24, 1895-1906.
58. 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.
59. Hang, H. C.; Yu, C.; Kato, D. L.; Bertozzi, C. R. A Metabolic Labeling Approach towards Proteomic Analysis of Mucin-type O-linked Glycosylation. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14846-14851.
60. Lang, K.; Chin, J. W. Bioorthogonal Reactions for Labeling Proteins. ACS Chem. Biol. 2014, 9, 16-20.
61. Ganesan, R.; Kratz, K.; Lendlein, A., Multicomponent protein patterning of material surfaces. J. Mater. Chem. 2010, 20, 7322-7331.
62. Miksa, B. Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy. Med. Chem. 2016, 6, 611-639.
63. Hirsch, J. D.; Eslamizar, L.; Filanoski, B. J.; Malekzadeh, N.; Haugland, R. P.; Beechem, J. M.; Haugland, R. P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. 2002, 308, 343-357.
64. Magalhães, M. L. B.; Czekster, C. M.; Guan, R.; Malashkevich, V. N.; Almo, S. C.; Levy, M. Evolved streptavidin mutants reveal key role of loop residue in high‐affinity binding. Protein Sci. 2011, 20, 1145-1154.
65. Hamachi, I.; Nagase, T.; Shinkai, S. A General Semisynthetic Method for Fluorescent Saccharide-Biosensors Based on a Lectin. J. Am. Chem. Soc. 2000, 122, 12065-12066.
66. Takaoka, Y.; Tsutsumi, H.; Kasagi, N.; Nakata, E.; Hamachi, I. One-Pot and Sequential Organic Chemistry on an Enzyme Surface to Tether a Fluorescent Probe at the Proximity of the Active Site with Restoring Enzyme Activity. J. Am. Chem. Soc. 2006, 128, 3273-3280.
67. Hughes, C. C.; Yang, Y.-L.; Liu, W.-T.; Dorrestein, P. C.; Clair, J. J. L.; Fenical, W. Marinopyrrole A Target Elucidation by Acyl Dye Transfer. J. Am. Chem. Soc. 2009, 131, 12094-12096.
68. 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.
69. Fujishima, S.; 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.
70. Matsuo, K.; Nishikawa, Y.; Masuda, M.; Hamachi, I. Live‐Cell Protein Sulfonylation Based on Proximity‐driven N‐Sulfonyl Pyridone Chemistry. Angew. Chem. Int. Ed. 2018, 57, 659-662.
71. Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. Target-Specific Chemical Acylation of Lectins by Ligand-Tethered DMAP Catalysts. J. Am. Chem. Soc. 2008, 130, 245-251.
72. Tamura, T.; Song, Z.; Amaike, K.; Lee, S.; Yin, S.; Kiyonaka, S.; Hamachi, I. Affinity-Guided Oxime Chemistry for Selective Protein Acylation in Live Tissue Systems. J. Am. Chem. Soc. 2017, 139, 14181-14191.
73. Kunishima, M.; Nakanishi, S.; Nishida, J.; Tanaka, H.; Morisaki, D.; Hioki, K.; Nomoto, H. Convenient modular method for affinity labeling (MoAL method) based on a catalytic amidation. Chem. Commun. 2009, 0, 5597-5599.
74. Chen, Z.; Popp, B. V.; Bovet, C. L.; Ball, Z. T. Site-Specific Protein Modification with a Dirhodium Metallopeptide Catalyst. ACS Chem. Biol. 2011, 6, 920-925.
75. Cambre, J. N.; Sumerlin, B. S. Biomedical applications of boronic acid polymers. Polymer 2011, 52, 4631-4643.
76. Whyte, G. F.; Vilar, R.; Woscholski, R. Molecular recognition with boronic acids-applications in chemical biology. J. Chem. Biol. 2013, 6, 161-174.
77. Pappin, B.; Kiefel, M. J.; Houston, T. A. Boron-Carbohydrate Interactions. IntechOpen, 2012.
78. Lorand, J. P.; Edwards, J. O., Polyol Complexes and Structure of the Benzeneboronate Ion. J. Org. Chem. 1959, 24, 769-774.
79. Springsteen, G.; Wang, B., A detailed examination of boronic acid-diol complexation. Tetrahedron 2002, 58, 5291-5300.
80. Lü, C.; Li, H.; Wang, H.; Liu, Z. Probing the Interactions between Boronic Acids and cis-Diol-Containing Biomolecules by Affinity Capillary Electrophoresis. Anal. Chem. 2013, 85, 2361-2369.
81. 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.
82. 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.
83. Adak, A. K.; Li, B.-Y.; Huang, L.-D.; Lin, T.-W.; Chang, T.-C.; Hwang, K. C.; Lin, C.-C. Fabrication of Antibody Microarrays by Light-Induced Covalent and Oriented Immobilization. ACS Appl. Mater. Interfaces 2014, 6, 10452-10460.
84. 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. Anal. Chem. 2009, 81, 8774-8782.
85. Djanashvili, K.; Frullano, L.; Peters, J. A. Molecular Recognition of Sialic Acid End Groups by Phenylboronates. Chem. Eur. J. 2005, 11, 4010-4018.
86. Liu, H.; Li Y.; Sun, K.; Fan, J.; Zhang, P.; Meng, J.; Wang, S., Jiang, L. Dual-Responsive Surfaces Modified with Phenylboronic Acid-Containing Polymer Brush To Reversibly Capture and Release Cancer Cells. J. Am. Chem. Soc. 2013, 135, 7603-7609.
87. Meng, H.; Zheng, J.; Wen, X.; Cai, Z.; Zhang, J.; Chen, T. pH- and Sugar-Induced Shape Memory Hydrogel Based on Reversible Phenylboronic Acid-Diol Ester Bonds. Macromol. Rapid Commun. 2015, 36, 533-537.
88. Leriche, G.; Chisholm, L.; Wagner, A. Cleavable linkers in chemical biology. Bioorg. Med. Chem. 2012, 20, 571-582.
89. Holmes, C. P. Model Studies for New o-Nitrobenzyl Photolabile Linkers:  Substituent Effects on the Rates of Photochemical Cleavage. J. Org. Chem. 1997, 62, 2370-2380.
90. Shopes, B. A genetically engineered human IgG with limited flexibility fully initiates cytolysis via complement. Mol. Immunol. 1993, 30, 603-609.
91. Choe, W.; Durgannavar, T. A.; Chung, S. J. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials 2016, 9, 994.
92. Lemmers, R. F. H.; Vilaj, M.; Urda, D.; Agakov, F.; Šimurina, M.; Klaric, L.; Rudan, I.; Campbell, H.; Hayward, C.; Wilson, J. F.; Lieverse, A. G.; Gornik, O.; Sijbrands, E. J.G.; Lauc, G.; Hoek, M. IgG glycan patterns are associated with type 2 diabetes in independent European populations. Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 2240-2249.
93. 蔡佳靜, 合成配位基導向探針並運用於探討蛋白質-蛋白質間作用力. 國立清華大學化學研究所 碩士論文, 民國103年.
94. Song, Z.; Takaoka, Y.; Kioi, Y.; Komatsu, K.; Tamura, T.; Miki, T.; Hamachi, I. Extended Affinity-guided DMAP Chemistry with a Finely Tuned Acyl Donor for Intracellular FKBP12 Labeling. Chem. Lett. 2015, 44, 333-335.
95. Verma, K. D.; Massing, J. O.; Kamper, S. G.; Carney, C. E.; MacRenaris, K. W.; Basilion, J. P.; Meade, T. J. Synthesis and evaluation of MR probes for targeted-reporter imaging. Chem. Sci. 2017, 8, 5764-5768.
96. Szychowski, J.; Mahdavi, A.; Hodas, J. J. L.; Bagert, J. D.; Ngo, J. T.; Landgraf, P.; Dieterich, D. C.; Schuman, E. M.; Tirrell, D. A. Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition. J. Am. Chem. Soc. 2010, 132, 18351-18360.
97. Chang, T.-C.; Adak, A. K.; Lin, T.-W.; Li, P.-J.; Chen, Y.-J.; Lai, C.-H.; Liang, C.-F.; Chen, Y.-J.; Lin, C.-C. A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein. Bioorg. Med. Chem. 2016, 24, 1216–1224.
98. 林亭維, 開發Triple-click模組化之化學探針合成平台與其於蛋白質標記與蛋白質修飾的應用. 國立清華大學化學研究所 博士論文, 民國106年.
99. Liaw, Y.-W.; Lin, C.-Y.; Lai, Y.-S.; Yang, T.-C.; Wang, C.-J.; Whang-Peng, J.; Liu, L. F.; Lin, C.-P.; Nieh, S.; Lu, S.-C.; Hwang, J.-L. A Vaccine Targeted at CETP Alleviates High Fat and High Cholesterol Diet-Induced Atherosclerosis and Non-Alcoholic Steatohepatitis in Rabbit. PLoS One 2014, 9, e111529.
100. Maruani, A.; Smith, M. E. B.; Miranda, E.; Chester, K. A.; Chudasama, V.; Caddick, S. A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat. Commun. 2015, 6, 6645.
101. Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718-11940.
102. Thorn, C, F.; Oshiro, C.; Marsh, S.; Hernandez-Boussard, T.; McLeod, H.; Klein, T. E.; Altman, R. B. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet. Genomics 2011, 21, 440-446.
103. Li, X.; Fang, T.; Boons, G.-J. Preparation of Well‐Defined Antibody-Drug Conjugates through Glycan Remodeling and Strain‐Promoted Azide-Alkyne Cycloadditions. Angew. Chem. Int. Ed. 2014, 53, 7179-7182.
104. Lee, M. H.; Kim, E.-J.; Lee, H.; Park, S. Y.; Hong, K. S.; Kim, J. S.; Sessler, J. L. Acid-triggered release of doxorubicin from a hydrazone-linked Gd3+-texaphyrin conjugate. Chem. Commun. 2016, 52, 10551-10554.
105. Subik, K.; Lee, J.-F.; Baxter, L.; Strzepek, T.; Costello, D.; Crowley, P.; Xing, L.; Hung, M.-C.; Bonfiglio, T.; Hicks, D. G.; Tang, P. The Expression Patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by Immunohistochemical Analysis in Breast Cancer Cell Lines. Breast Cancer (Auckl) 2010, 4, 35-41.
106. Wang, H.; Koshi, Y.; Minato, D.; Nonaka, H.; Kiyonaka, S.; Mori, Y.; Tsukiji, S.; Hamachi, I. Chemical Cell-Surface Receptor Engineering Using Affinity-Guided, Multivalent Organocatalysts. J. Am. Chem. Soc. 2011, 133, 12220-12228.
107. Oves-Costales, D.; Kadi, N.; Fogg, M. J.; Song, L.; Wilson, K. S.; Challis, G. L. Enzymatic Logic of Anthrax Stealth Siderophore Biosynthesis: AsbA Catalyzes ATP-Dependent Condensation of Citric Acid and Spermidine. J. Am. Chem. Soc. 2007, 129, 8416-8417.
108. Fixon-Owooa, S.; Levasseura, F.; Williamsb, K.; Sabadob, T. N.; Lowec, M.; Klosec, M.; Mercierc, A. J.; Fieldsd, P.; Atkinsona, J. Preparation and biological assessment of hydroxycinnamic acid amides of polyamines. Phytochem. 2003, 63, 315-334.
109. Delaney, E. J.; Wood, L. E.; Klotz, I. M. Poly(ethylenimines) with Alternative (Alkylamino)pyridines as Nucleophilic Catalysts. J. Am. Chem. Soc. 1982, 104, 799-807.
110. DeForest, C. A.; Anseth, K. S. Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions. Nat. Chem. 2011, 3, 925-931.
111. Becht, J. M.; Wagner, A.; Mioskowski, C. Facile introduction of SH group on aromatic substrates via electrophilic substitution reactions. J. Org. Chem. 2003, 68, 5758-5761.
112. Isaad, A. L. C.; Barbetti, F.; Rovero, P.; D’Ursi, A. M.; Chelli, M.; Chorev, M.; Papini, A. M. Nα-Fmoc-Protected ω-Azido- and ω-Alkynyl-L-amino Acids as Building Blocks for the Synthesis of “Clickable” Peptides. Eur. J. Org. Chem. 2008, 31, 5308-5314.
113. Decuypere, E.; Specklin, S.; Gabillet, S.; Audisio, D.; Liu, H.; Plougastel, L.; Kolodych, S.; Taran, F. Copper(I)-Catalyzed Cycloaddition of 4-Bromosydnones and Alkynes for the Regioselective Synthesis of 1,4,5-Trisubstituted Pyrazoles. Org. Lett. 2015, 17, 362-365.
114. Shukla, N. M.; Malladi, S. S.; Mutz, C. A.; Balakrishna, R.; David, S. A. Structure-Activity Relationships in Human Toll-Like Receptor 7-Active Imidazoquinoline Analogues. J. Med. Chem. 2010, 53, 4450-4465.