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研究生: 黃于倫
Huang Yu-Lun
論文名稱: 開發新穎的生物正交反應並評量唾液酸蛋白在細胞表面的視覺化
Development and Biological Evaluation of New Bioorthogonal Reactions for Visualization of Sialoglycoproteins on the cell Surface
指導教授: 李文山
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 123
中文關鍵詞: 唾液酸生物正交反應
英文關鍵詞: sialic acid, bioorthogonal chemical reporter
論文種類: 學術論文
相關次數: 點閱:278下載:1
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  • 近年來逐漸發展出一種結合化學方法的生物標定成像工具,稱為Bioorthogonal chemical reporter,可以用於觀察一些生物分子在生物系統上的功能、代謝,例如蛋白質、核酸、醣類等。但這項工具應用於生理環境下存在著許多限制,由其對於標定醣類生物分子,至今只有疊氮化合物發展成很好的Bioorthogonal chemical reporter。
    於是我們利用click chemistry、Diels–Alder reaction and addition-elimination這三個反應設計為唾液酸生合成細胞標定的Bioorthogonal反應,於是分別設計合成帶有疊氮、1,2,4,5-tetrazine以及1,2 diketone的甘露醣胺衍生物為Bioorthogonal chemical reporter (化合物5、45和55),以及另一部份帶有螢光的反應化合物(16,39,48,52和62)。然而從這三個反應的合成過程中,成功突破了化合物35的反應性得到化合物45,也成功改善了化合物39的水溶性得到化合物48,但其中化合物48螢光基團對生物的緩衝液1X PBS溶解性不好而且對細胞染色並沒有專一性。不過最後成功合成出含有生物素的化合物52,可以穩定的存在於生物的環境中。接著和化合物45利用流式細胞儀去測試在MDA-MB-231乳癌細胞表面上的唾液酸標定量,但很可惜的並無成功的偵測到被標定的唾液酸,不過,藉由以上的經驗我們已經找到一個適合在生物環境中做Bioorthogonal reaction的方向,接下來只要在Bioorthogonal chemical reporter的設計上有所改良且能為唾液酸生合成之酵素所接受並催化,讓它能成功的表現在細胞表面,就可以順利表達出我們要標定的目標生物分子唾液酸。

    A bioorthogonal chemical reporter is a tool for visualizing and tagging biological molecules, observing their functional behaviour in living systems, however it is physiologically demanding and challenging for limited use in vivo. To our knowledge, few bioorthogonal chemical reporters (except azide) with an inert property toward cell’s metabolic enzymes have been reported so far.
    Here we describe the exploration and synthesis of azide-, 1,2,4,5-tetrazine-, and 1,2 diketone-containing N-alpha-acetylmannosamines (compounds 5, 45 and 55), which employ methods of click chemistry、Diels–Alder reaction and addition-elimination to facilitate their chemical ligations with fluorescent conjugate-partners (16, 39, 48, 52 and 62), and demonstrate their potential abilities of molecular imaging in vitro. Interestingly, the poor ligation with fluorescent conjugate-partners was initially found for 35; an improvement in this was successfully accomplished by the discovery of 45. On the other hand, an increase of solubility in aqueous buffer is solved by modifying fluorescent conjugate 39 into compound 48, which does display multiple/random recognitions toward bimolecular donors on the surface of cells. In order to provide an unique binding affinity toward 1,2,4,5-tetrazine-containing N-alpha-acetylmannosamines (45) rather than other bimolecular donors, we have identified and synthesized a biotin-norbornene conjugate 52 with suitable properties in cell culture. Building on these effective ligation and unique recognition studies in vitro (45 and 52), we want to investigate the ability of imaging sialic acid on cell surfaces by treating MDA-MB-231 cells with 1,2,4,5-tetrazine-containing N-alpha-acetylmannosamines (45), followed by sequential visualization (by reaction with 52) and analysis with flow cytometry. Unfortunately, this technique has not enabled visualization of sialic acid in living cells, not successful recognitions of 45 by these enzymes during the process of biosynthesis. However, the results demonstrated here should pave the way for the redesign of new chemical reporters in the near future.

    一.導論 1 1.1 唾液酸的結構及功能 1 1.2 唾液酸在細胞中的生合成及唾液酸轉移酶 3 1.3 Cell image的介紹 6 1.4 甚麼是生物正交反應 (Bioorthogonal reaction) 8 1.5 Bioorthogonal 反應的設計 9 1.6 Bioorthogonal 反應在生物系統上的應用 11 1.7 實驗設計 15 二、結果與討論 17 1. 以Click chemistry當作bioorthogonal reaction 17 1.1合成含疊氮基團甘露醣胺衍生物 17 1.2合成含三鍵螢光衍生物 19 1.3 Click chemistry (化合物5和化合物9)在體外的反應性 20 1.4合成水溶性的含三鍵螢光衍生物 23 1.5 Click chemistry (化合物5和化合物16)在體外的反應性 26 1.6結論Bioorthogonal reaction of click chemistry 27 2. 以Diels-alder反應當作bioorthogonal reaction 28 2.1合成含tetrazine基團的甘露醣胺衍生物 28 2.2合成含norbornene的螢光衍生物 32 2.3 Diels-alder (化合物35和化合物39)在體外的反應性 34 2.4 Diels-alder在體外的反應性 36 2.5合成具水溶性的norbornene螢光基團 39 2.6 Diels-alder (化合物45和化合物48)在體外的反應性 43 2.7合成具水溶性的norbornene 生物素 44 2.8 Diels-alder (化合物45和化合物52)在體外的反應性 45 2.9結論Bioorthogonal reaction of diels-alder reaction 46 3. 以1,2 Diamine-1,2 diketone反應當作bioorthogonal reaction 47 3.1合成含1,2 Diketone的甘露醣胺衍生物 47 3.2以Ester bond linkage合成含1,2 diamine基團的螢光衍生物 48 3.3以Amide bond linkage合成含1,2 diamine基團的螢光衍生物 50 3.4將1,2 Diamine和1,2 diketone (化合物67、55)在體外做反應測試 51 3.5以Ether bond linkage合成含1,2 diamine基團的螢光衍生物 52 3.6合成含1,2 diol基團的甘露醣胺衍生物 54 3.7結論Bioorthogonal reaction of 1,2 diamine-1,2 diketone reaction 54 4. 生物實驗 55 4.1化合物45在MDA-MB-231乳癌細胞的毒性測試 55 4.2化合物48在MDA-MB-231乳癌細胞的毒性測試 57 4.3化合物52在MDA-MB-231乳癌細胞的毒性測試 61 4.4 利用Diels-alder反應(化合物45和化合物52)偵測細胞株MDA-MB-231膜上的唾液酸 62 5. 結論 66 三、 實驗與光譜數據 67 1. 實驗儀器 67 2. 實驗藥品 68 3. 一般實驗方法 71 3.1 化合物5的合成31 71 3.2 化合物9的合成 72 3.3 化合物16的合成 74 3.4 化合物39的合成 76 3.5 化合物45的合成 78 3.6 化合物48的合成 81 3.7 化合物52的合成 83 3.8 化合物55的合成 84 3.9 化合物67的合成 85 3.10化合物78的合成 87 四、參考文獻 90 五. 光譜附錄 94

    1. Kiefel, M. J.; von Itzstein M., Recent Advances in the Synthesis of Sialic Acid Derivatives and Sialylmimetics as Biological Probes. Chem. Rev. 2002, 102, 471.
    2. Varki, A., Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 2007, 446, 1023.
    3. De Clercq, E., Antiviral agents active against influenza A viruses. Nat. Rev. Drug Discovery 2006, 5, 1015.
    4. Yarema1, K. J.; Goon, S.; Bertozzi, C. R., Metabolic selection of glycosylation defects in human cells. Nat. Biotechnol. 2001, 19, 533.
    5. Rao, F. V.; Rich, J. R.; Rakić, B.; Buddai, S.; Schwartz, M. F.; Johnson, K.; Bowe, C.; Wakarchuk, W. W.; DeFrees, S.; Withers, S. G.; Strynadka, N. C. J., Structural insight into mammalian sialyltransferases. Nat. Struct. Mol. Biol. 2009, 16, 1186.
    6. Hildebrandt, H.; Becker, C.; Gluer, S.; Rosner, H.; GerardySchahn, R.; Rahmann, H., Polysialic Acid on the Neural Cell Adhesion Molecule Correlates with Expression of Polysialyltransferases and Promotes Neuroblastoma Cell Growth. Cancer Res. 1998, 58, 779.
    7. Geßner, P.; Riedl, S.; Quentmaier, A.; Kemmer, W., Enhanced activity of CMP-NeuAc:Galβ1-4GlcNAc:α2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Lett. 1993, 75, 143.
    8. Burchell, J.; Poulsom, R.; Hanby, A.; Whitehouse, C.; Clausen, H.; Miles, D.; Taylor-Papadimitriou, J., An α2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas. Glycobiology 1999, 9, 1307.
    9. Dall’Olio, F.; Malagolini, N.; di Stefano, G.; Minni, F.; Marrano, D.; Serafini-Cessi, F., Increased CMP-NeuAc:Galβ1,4GlcNAc-R α2,6 sialyltransferase activity in human colorectal cancer tissues. Int. J. Cancer 1989, 44, 434.
    10. Dimitroff, C. J.; Pera, P.; Dall’Olio, F.; Matta, K. L.; Chandrasekaran, E. V.; Lau, J. T.; Bernacki, R. J., Metabolic Properties of Normal and Mutant Mannan-Binding Proteins in Mouse Plasma. Biochem. Biophys. Res. Commun. 1999, 256, 631.
    11. Picco, G.; Julien S.; Brockhausen, I.; Beatson, R.; Antonopoulos, A.; Haslam, S.; Mandel, U.; Dell, A.; Pinder, S.; Taylor-Papadimitriou, J.; Burchell, J.; Over-expression of ST3Gal-I promotes mammary tumorigenesis. Glycobiology 2010, 20, 1241.
    12. Chiang, C.-H.; Wang, C.-H.; Chang, H.-C.; More, S. V.; Li, W.-S.; Hung, W.-C., A novel sialyltransferase inhibitor AL10 suppresses invasion and metastasis of lung cancer cells by inhibiting integrin-mediated signaling. J. Cell. Physiol. 2010, 223, 492.
    13. Chen, J. Y.; Tang, Y. A.; Huang, S. M.; Juan, H. F.; Wu, L. W.; Sun, Y. C.; Wang, S. C.; Wu, K. W.; Balraj, G.; Chang, T. T.; Li, W. S.; Cheng, H. C.; Wang, Y. C., A Novel Sialyltransferase Inhibitor Suppresses FAK/Paxillin Signaling and Cancer Angiogenesis and Metastasis Pathways. Cancer Res. 2011, 71, 473.
    14. Laughlin, S. T.; Bertozzi, C. R., Imaging the glycome. PNAS 2008, 106, 12.
    15. Lippincott-Schwartz, J.; Patterson, G. H., Development and Use of Fluorescent Protein Markers in Living Cells. Science 2003, 300, 87.
    16. Hadjantonakis, A.-K.; Dickinson, M. E.; Fraser, S. E.; Papaioannou, V. E., Technicolour transgenics: imaging tools for functional genomics in the mouse. Nat. Rev. Genet. 2003, 4, 613.
    17. Prescher1, J. A.; Bertozzi, C. R., Chemistry in living systems. Nat. Chem. Biol. 2005, 1, 13.
    18. Ohtsubo, K.; Marth, J. D., Glycosylation in cellular mechanisms of health and disease. Cell 2006, 126, 855.
    19. Bishop, J. R.; Schuksz, M.; Esko, J. D., Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 2007, 446, 1030.
    20. Haltiwanger, R. S.; Lowe, J. B., Role of Glycosylation in Development. Annu. Rev. Biochem. 2004, 73, 491.
    21. Rosen, S. D., Ligands for L-selectin: Homing, inflammation, and beyond. Annu. Rev. Immunol. 2004, 22, 129.
    22. Fuster, M. M.; Esko, J. D., The sweet and sour of cancer: Glycans as novel therapeutic targets. Nat. Rev. Cancer 2005, 5, 526.
    23. Dennis, J. W.; Granovsky, M.; Warren, C. E., Glycoprotein glycosylation and cancer progression. Biochim. Biophys. Acta. 1999, 1473, 21.
    24. Jewetta, J. C.; Bertozzi, C. R., Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev 2010, 39, 1272.
    25. Laughlin, S. T.; Bertozzi, C. R., Imaging the glycome. PNAS 2009, 106, 12.
    26. 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.
    27. Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A., Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjugate Chem. 2008, 19, 2297.
    28. Pipkorn, R.; Waldeck, W.; Didinger, B.; Koch, M.; Mueller, G.; Wiessler M.; Braun, K., Inverse-electron-demand Diels-Alder reaction as a highly efficient chemoselective ligation procedure: Synthesis and function of a BioShuttle for temozolomide transport into prostate cancer cells . J. Pept. Sci. 2009, 15, 235.
    29. Jewett, J. C.; Bertozzi, C. R., Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 2010, 39, 1272.
    30. Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R., Copper-free click chemistry for dynamic in vivo imaging. PNAS 2007, 104, 16793.
    31. Laughlin, S. T.; Bertozzi, C. R., Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation. Nat. Protoc. 2007, 2, 2930.
    32. Langereis, S.; Kooistra, H.-A. T.; van Genderen, M. H. P.; Meijer, E. W., Probing the interaction of the biotin-avidin complex with the relaxivity of biotinylated Gd-DTPA. Org. Biomol. Chem. 2004, 2, 1271.
    33. More, S. V.; Sastry, M. N. V.; Yao, C.-F., Cerium (iv) ammonium nitrate (CAN) as a catalyst in tap water: A simple, proficient and green approach for the synthesis of quinoxalines. Green Chem. 2006, 8, 91.
    34. 謝世良。流式細胞技術Flow Cytometry第十七章。國立陽明大學免疫學研究中心。249-261。
    35. Krishnakumar B.; Velmurugan, R.; Jothivel, S.; Swaminathan, M., An efficient protocol for the green synthesis of quinoxaline and dipyridophenazine derivatives at room temperature using sulfated titania. Catal. Commun. 2010, 11, 997.

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