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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 方俊民(Jim-Min Fang) | |
| dc.contributor.author | Pin-Hsuan Chiu | en |
| dc.contributor.author | 邱品瑄 | zh_TW |
| dc.date.accessioned | 2022-11-25T05:36:50Z | - |
| dc.date.available | 2026-09-29 | |
| dc.date.copyright | 2021-11-06 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-29 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82145 | - |
| dc.description.abstract | 甲型流感病毒經常引起季節性的呼吸道疾病，甚至導致嚴重的全球性大流行。由於核蛋白（NP）在流感病毒生命週期中扮演重要的角色，又因其基因序列不易變異的特點，使核蛋白成為抗流感藥物的目標蛋白之一。流感病毒核蛋白通常以三聚體（trimer）的形態存在，核蛋白單體（monomer）構成三聚體時，其尾端套環（tail-loop，胺基酸402到428）將插入至另一個核蛋白單體的主體結構域（body domain）之尾端套環結合區（tail loop binding pocket）中。已知E339...R416鹽橋的相互作用對於核蛋白三聚體的形成至關重要。先前已發現化合物A對流感病毒具有良好的抑制活性，其通過破壞核蛋白單體間E339...R416鹽橋作用力，進而抑制流感病毒的複製。 基於先導化合物A的結構，我們的團隊設計出一個同時具有疊氮芳香基（aryl azide）和疊氮烷基（alkyl azide)的雙功能化合物JMF4496，致力於進行結構與活性關係（structure−activity relationship，SAR）的研究。該雙功能化合物對流感病毒（H1N1）也具有良好的抑制活性（EC50 = 9.06 μM）。 疊氮基團可以透過UV光照射而產生高反應性的氮烯活性中間體，並與鄰近的胺基酸殘基生成共價鍵結。鑑於此雙功能光親和探針之疊氮芳香基在304奈米處表現出最大吸收，其波長長於疊氮烷基的吸收波峰（259奈米）。此雙功能化合物上的疊氮芳香基被選擇性照光活化，使其嵌入核蛋白的活性位點附近之殘基；而另一端的疊氮烷基則與帶有末端炔烴的生物素標籤分子進行銅（I）催化炔−疊氮化物環加成（CuAAC）反應，並在胰蛋白酶（trypsin）的消化反應（digestion）後，進行胜肽鏈富集、純化與隨後的質譜分析。 透過交叉比對奈流液相層析串聯質譜（nano LC-MS/MS）在 Mascot 分析中的結果，我們識別出五個候選的目標胜肽片段。分子嵌合（molecular docking）實驗表明光親和探針JMF4496可與目標片段之一的401ASSGQISIQPTFSVQR416結合，干擾尾端套環插入另一個核蛋白單體的主體結構域，從而破壞核蛋白的三聚化。本研究結合化學合成、光化學、生物化學和質譜方法來驗證尾端套環上的目標胜肽以及R416胺基酸殘基在核蛋白三聚體中的作用。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T05:36:50Z (GMT). No. of bitstreams: 1 U0001-2809202123595400.pdf: 6305343 bytes, checksum: 1f0fbb55e2cd28ada1c9e0bd538ff16f (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 摘要 I Abstract III Table of Contents V Index of Figures IX Index of Schemes XIII Index of Tables XIV Abbreviations XV Chapter 1. Introduction 1 1.1 Introduction of influenza A virus 1 1.1.1 Epidemiology and pathophysiology 1 1.1.2 Structure of influenza A virion 2 1.1.3 Life cycle of the influenza A virus 3 1.2 Antiviral drug target 6 1.2.1 Influenza drugs 6 1.2.2 Nucleoprotein as a druggable target 9 1.3 Identification of drug targets 14 1.3.1 Activity-based protein profiling (ABPP) 14 1.3.2 Photoaffinity labeling (PAL) 15 1.3.2.1 General design of photoaffinity probes 15 1.3.2.2 General experimental workflow of photoaffinity labeling 22 1.3.3 Tandem photoaffinity labeling–bioorthogonal conjugation 25 1.3.4 Protein identification by mass spectrometry 26 1.3.4.1 Peptide mass fingerprinting (PMF) 26 1.3.4.2 Identification by tandem mass spectrometry (MS/MS) 27 1.4 Design of this research 28 1.4.1 Research motivation 28 1.4.2 Inspiration of the design of photoaffinity probe 30 Chapter 2. Results and Discussion 34 2.1 Synthesis of photoaffinity probe 34 2.2 Properties of photoaffinity probe 37 2.2.1. Inhibitory activity of photoaffinity probe JMF4496 37 2.2.2 Ultraviolet–visible spectral analysis 38 2.3 Cell lysate photoaffinity labeling experiment 40 2.4 Selective bifunctional experiment of JMF4496 44 2.4.1 Determine the photochemical efficiency of photoaffinity probe 44 2.4.2 Selective activation of aryl azide on JMF4496 49 2.5 H1N1 viral NP photoaffinity labeling experiment 52 2.5.1 PAL experiments of influenza NP 52 2.5.2 Improved PAL experiments of influenza NP 54 2.6 Prediction of modifications on the labeled peptides in Mascot searching 56 2.7 Result of nano LC-MS/MS analysis with Mascot searching 59 2.8 Examination of the peptide candidates 66 2.8.1 Molecular docking 67 2.8.1.1 Glide module 67 2.8.1.2 UCSF Chimera and AutoDock Vina 71 2.8.2 Basic local alignment search tool (BLAST) 79 Chapter 3. Conclusion 82 Chapter 4. Experimental Section 84 4.1 General part 84 4.2 Instrumentation 84 4.3 Synthetic procedure and characterization of compounds 85 4.4 Procedure of bioassay 98 4.4.1 Materials and methods 98 4.4.2 Determination of EC50 of NP inhibitors 98 4.5 General procedure for photoaffinity labeling and CuAAC reaction 99 4.5.1 Example 1 for PAL and CuAAC 100 4.5.2 Example 2 for PAL and CuAAC 100 4.5.3 Example 3 for PAL and CuAAC 101 4.6 Affinity purification 101 4.7 SDS-PAGE 102 4.8 Silver staining 103 4.9 Western blot analysis 104 4.10 In-gel trypsin digestion 105 4.11 In-solution trypsin digestion 107 4.12 Zip Tip C18 protocol 107 4.13 Nano LC-MS/MS analysis 108 4.14 Mascot protocol 109 4.15 Molecular docking study 110 4.16 Complex energy minimization and binding free energy analysis 111 4.17 AutoDock Vina in UCSF Chimera 111 Chapter 5. References 114 Appendix 123 | |
| dc.language.iso | en | |
| dc.subject | 點擊化學 | zh_TW |
| dc.subject | 流行性感冒 | zh_TW |
| dc.subject | 病毒 | zh_TW |
| dc.subject | 核蛋白 | zh_TW |
| dc.subject | 鹽橋作用力 | zh_TW |
| dc.subject | 光親和探針 | zh_TW |
| dc.subject | 雙功能 | zh_TW |
| dc.subject | 疊氮苯 | zh_TW |
| dc.subject | 選擇性活化 | zh_TW |
| dc.subject | photoaffinity probe | en |
| dc.subject | bifunctional | en |
| dc.subject | influenza | en |
| dc.subject | virus | en |
| dc.subject | nucleoprotein | en |
| dc.subject | salt bridge interaction | en |
| dc.subject | click chemistry | en |
| dc.subject | selective activation | en |
| dc.subject | aryl azide | en |
| dc.title | 設計標記流感之核蛋白三聚體的雙功能二疊氮光親和探針 | zh_TW |
| dc.title | Design of Bifunctional Diazido Photoaffinity Probe for Labeling Influenza Nucleoprotein Trimer | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林榮信(Hsin-Tsai Liu),徐丞志(Chih-Yang Tseng),鄭婷仁 | |
| dc.subject.keyword | 流行性感冒,病毒,核蛋白,鹽橋作用力,光親和探針,雙功能,疊氮苯,選擇性活化,點擊化學, | zh_TW |
| dc.subject.keyword | influenza,virus,nucleoprotein,salt bridge interaction,photoaffinity probe,bifunctional,aryl azide,selective activation,click chemistry, | en |
| dc.relation.page | 139 | |
| dc.identifier.doi | 10.6342/NTU202103442 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-10-01 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-09-29 | - |
| 顯示於系所單位： | 化學系 | |
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