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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王宗興 | zh_TW |
| dc.contributor.advisor | Tsung-Shing Andrew Wang | en |
| dc.contributor.author | 張曉瑜 | zh_TW |
| dc.contributor.author | Hsiao-Yu Chang | en |
| dc.date.accessioned | 2023-10-03T16:20:55Z | - |
| dc.date.available | 2023-11-10 | - |
| dc.date.copyright | 2023-10-03 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-08 | - |
| dc.identifier.citation | (1)Bird, R. E.; Lemmel, S. A.; Yu, X.; Zhou, Q. A. Bioorthogonal Chemistry and Its Applications. Bioconjugate Chemistry 2021, 32 (12), 2457–2479.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90496 | - |
| dc.description.abstract | 標靶型共價抑制劑(Targeted covalent inhibitors，TCIs)及蛋白降解靶向嵌合體(proteolysis targeting chimera，PROTAC)為近年來備受關注的癌症治療策略之一。TCI的優勢在於和標靶蛋白的不可逆共價抑制，可以增強藥效。但其中最大問題為脫靶效應(off-target effect)，一旦形成錯誤不可逆鍵結便會導致嚴重的細胞毒性。另一方面，PROTAC則可以加速標靶蛋白降解，可應用於降解難成藥靶的蛋白及具抗藥性的突變蛋白。然而，較大的PROTAC分子會降低細胞穿膜度，導致生物應用受限。
在本研究中，我們利用二種水相相容的接續反應生成TCI藥物及PROTAC。首先，藉由含拉電子基磷鹽(stable phosphorus ylides)和醛衍生物，可在生理條件下進行威悌反應(Wittig reaction)形成丙烯醯胺(acrylamide)，開啟共價抑制活性。若使用帶有疊氮基團的醛衍生物，則可更進一步和可點擊E3徵招分子進行SPAAC反應，組裝成PROTAC。利用布魯頓酪氨酸激酶(Bruton's tyrosine kinase, BTK) 抑制劑Ibrutinib、Spebrutinib的磷鹽做為測試模型，在試管中我們成功展示上述多樣性的組裝策略。同時在細胞實驗中，抑制劑磷鹽及醛衍生物可成功自組裝並共價標記上目標蛋白BTK。若一併加入可點擊E3徵招分子，可觀察到目標蛋白BTK的降解。利用此策略，多樣化的TCI及PROTAC分子可以在生理條件下簡易地組裝，並進一步進行生物活性測試。 | zh_TW |
| dc.description.abstract | Targeted covalent inhibitors (TCIs) and proteolysis targeting chimera (PROTACs) are currently attractive cancer therapeutic strategies. TCIs irreversibly bind to targeted proteins to enhance their bioactivity. However, nonspecific covalent bond formation might cause off-target cytotoxicity. On the other hand, PROTACs are able to accelerate targeted protein degradation. Moreover, PROTACs potentially target undruggable proteins and drug-resistant mutant proteins. Nevertheless, the larger PROTACs might have reduced cell permeability, hence limiting their biomedical applications.
In our work, we utilize two biocompatible reactions to assemble TCIs and PROTACs sequentially. First, under aqueous physiological conditions, stabilized phosphorus ylides and various functionalized aldehydes could undergo Wittig olefination to afford acrylamide inhibitors, which serve as the emerged electrophilic warheads for covalent inhibition. Furthermore, using azido-containing aldehydes, these acrylamide inhibitors could further ligate with clickable E3 ligase ligands through the strain-promoted alkyne-azide cycloaddition (SPAAC) to assemble PROTACs. Choosing Bruton's tyrosine kinase (BTK) inhibitors, Ibrutinib and Spebrutinib, as model compounds, we demonstrated our versatile assemble strategies of TCIs and PROTACs in vitro. In cellular conditions, the acrylamide-containing TCIs can be successfully assembled and in situ label BTK proteins. After ligating with clickable E3 ligase ligands, we can also observe degradation of BTK proteins. In short, through our versatile assemble strategy, various TCIs and PROTACs could be easily formed under mild conditions for subsequent bioactivity screening. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-10-03T16:20:55Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-10-03T16:20:55Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Acknowledgement ⅰ
Abstract (Chinese) ⅱ Abstract (English) ⅲ List of Figures ⅷ List of Schemes ⅺ Abbreviations ⅻ Chapter 1 Introduction 1 1-1 Aqueous Biocompatible Conjugation Reaction 1 1-1.1 Introduction of Aqueous Biocompatible Conjugation Reactions 1 1-1.2 The Applications of Biocompatible Conjugation Reactions 3 1-2 The Alkene Formation 4 1-2.1 The Wittig Type Reaction 4 1-2.2 The Optimization of Wittig Reaction under Aqueous Conditions 6 1-2.3 The Applications of Biocompatible Wittig Reaction 7 1-3 Targeted Covalent Inhibitors 9 1-3.1 Introduction of Targeted Covalent Inhibitors 9 1-3.2 α,β-unsaturated Carbonyl Warheads in Targeted Covalent Inhibitors 10 1-3.3 Introduction of Bruton’s Tyrosine Kinase 12 1-4 Proteolysis-Targeting Chimeras (PROTACs) 13 1-4.1 The Mechanism of Protein Degradation Through PROTACs 13 1-4.2 The Developments of PROTACs 14 Chapter 2 Results and Discussion 17 2-1 Molecular Design 17 2-2 Preparation of Phosphonium Drug Precursors 19 2-3 Screening the Carbonyl Substrates for Aqueous Wittig Reaction 20 2-4 Preparation of Azide-Containing Aldehydes 24 2-5 The Aqueous Wittig Reaction of Azide-Containing Aldehydes with Phosphonium Precursors 27 2-6 BTK Labeling Through Drug Precursors and Azide-Containing Aldehydes 31 2-7 Cytotoxicity of TCIs and TCI Derivatives 36 2-8 BTK Degradation Through Purified PROTACs and Two-Component PROTACs 40 2-9 Preparation of Glyoxamide-Containing Thalidomide 44 2-10 The Aqueous Wittig Reaction of AldeTH with Phosphonium Precursors 45 2-11 The Evaluation of BTK Degradation in Response to Ibr-OH-TH Treated in Ramos Cells 47 Chapter 3 Conclusions and Perspectives 49 Chapter 4 Material and Methods 50 4-1 General Methods 50 4-2 Synthesis and Characterization of Compounds 51 4-2.1 Synthesis of Drug Precursors – IbrPPh3 and SpePPh3 51 4-2.2 Synthesis of Azide-Containing Aldehydes 60 4-2.3 Synthesis of The TCI Derivative 67 4-2.4 Click Reaction of Ibr-9C-DBCOTH 68 4-2.5 Synthesis of Glyoxamide-Containing Thalidomide 69 4-2.6 Assembly of Ibr-OH-TH 75 4-3 In Vitro Experiments 76 4-3.1 The Wittig Product Formation Monitored by HPLC 76 4-3.2 General HPLC Conditions for Analytical Assays 77 4-4 Biological Experiments 77 4-4.1 Cell Culture 77 4-4.2 Treatment for BTK Labeling Experiments 78 4-4.3 Treatment for Cytotoxicity Experiments 79 4-4.4 Treatment for BTK Degradation Experiments 79 4-4.5 General Methods for Cell Lysis 80 4-4.6 General Methods for In-Gel Fluorescence 80 4-4.7 General Methods for AlamarBlue Cell Viability Assay 81 4-4.8 General Methods for Western Blotting 82 References 83 APPENDIX NMR Spectra and HPLC Characterization 90 | - |
| dc.language.iso | en | - |
| dc.subject | 威悌反應 | zh_TW |
| dc.subject | 水相相容反應 | zh_TW |
| dc.subject | 共價抑制劑 | zh_TW |
| dc.subject | 蛋白降解靶向嵌合體 | zh_TW |
| dc.subject | targeted covalent inhibitors | en |
| dc.subject | aqueous compatible reaction | en |
| dc.subject | Wittig reaction | en |
| dc.subject | PROTAC | en |
| dc.title | 利用水相威悌和點擊化學反應多樣化組裝共價抑制劑及蛋白降解靶向嵌合體 | zh_TW |
| dc.title | Versatile Assembly of Covalent Inhibitors and PROTACs via Aqueous Wittig and Click Reactions | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 謝俊結;朱忠瀚 | zh_TW |
| dc.contributor.oralexamcommittee | Jiun-Jie Shie;Chung-Han Chu | en |
| dc.subject.keyword | 威悌反應,水相相容反應,共價抑制劑,蛋白降解靶向嵌合體, | zh_TW |
| dc.subject.keyword | Wittig reaction,aqueous compatible reaction,targeted covalent inhibitors,PROTAC, | en |
| dc.relation.page | 128 | - |
| dc.identifier.doi | 10.6342/NTU202303020 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2023-08-09 | - |
| dc.contributor.author-college | 理學院 | - |
| dc.contributor.author-dept | 化學系 | - |
| dc.date.embargo-lift | 2028-08-10 | - |
| 顯示於系所單位： | 化學系 | |
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