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研究生:戴志軒
研究生(外文):Chih-Hsuan Tai
論文名稱:探索發報基團在化學探針新應用之研究
論文名稱(外文):Manipulations of the Reporter Group for Versatile Applications of Chemical Probes
指導教授:羅禮強
指導教授(外文):Lee-Chiang Lo
口試委員:陳昭岑蔡蘊明林敬哲林照雄洪上程
口試委員(外文):Chao-Tsen ChenYeun-Min TsaiJing-Jer LinChao-Hsiung LinShang-Cheng Hung
口試日期:2015-03-25
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:169
中文關鍵詞:活性探針硫酸酯水解酵素螢光探針共軛焦顯微鏡氟[碳]相催化劑微波反應綠色化學絲胺酸水解型酵素同位素編碼親和標籤
外文關鍵詞:activity-based probessulfatasefluorogenic probesconfocal fluorescence microscopyfluorous catalystmicrowave reactiongreen chemistryserine hydrolaseICAT
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活性探針獨特的活性標示能力已被廣泛地應用於蛋白質體學之研究,運用其在生物系統中專一性地反應特定目標蛋白分子的活性,以研究生物系統在特定生理狀態下的酵素活性調控。而本論文的工作致力於化學探針發報端的開發,增加其研究的應用,共進行了三種不同的開發方向,第一個是設計、合成與應用硫酸酯水解酵素活性螢光探針,第二為發展與應用氟[碳]相催化劑,第三為發展具有同位素編碼親和標籤之絲胺酸水解型酵素活性探針。在第一個部分,我們設計一組螢光探針,具備辨識與標記硫酸酯水解酵素的能力,並於標記後開啟螢光訊號,配合共軛焦顯微鏡,可應用於細胞中硫酸酯水解酵素之活性顯影。第二部份的研究為合成與應用氟[碳]相有機金屬與有機催化劑,利用氟[碳]相化合物具備容易純化的特性,發展氟[碳]相催化劑,搭配微波反應進行催化,並且有效率地純化產物與回收催化劑,達到綠色化學的訴求。最後一部分的研究為針對絲胺酸水解型酵素發展具備同位素標籤之活性探針,結合質譜儀系統,增進在複雜系統中酵素活性的定量研究。

The activity-based probes have been widely used in proteomic researches due to its ability to reflect specific enzyme’s activity under certain physiological conditions. We have put a lot of effort into reporter group manipulation for versatile application of chemical probes. We reported three kinds of developments in this dissertation. First, design, synthesis and application of turn-on fluorescent probes for imaging steroid sulfatase activities in cells. Secondly, development and application of fluorous catalysts. Thirdly, development of isotope-coded ABPs for quantitative profiling of serine hydrolase. In the first part, we have designed two turn-on fluorescent probes targeting steroid sulfatase which are designed to generate immobilized fluorescent signal upon hydrolysis. Confocal fluorescence imaging experiments were carried out and compared the results to commercially available imaging reagent. The second part includes development of organo- and organometallic fluorous catalysts. Due to the characters of easy separation, fluorous chemistry is widely utilized in organic synthesis. We have developed new fluorous-taged catalysts and carried catalytic reactions under microwave irradiation. These catalysts did offer better efficiency in purification and matched the statements of green chemistry. The last part is the development of isotope-coded ABPs which applied for quantitative profiling of SHs. We envisioned this design would benefit from MS analysis platform that ensure the in-depth study of complex proteomes.

Contents
誌謝 i
中文摘要 ii
Abstract iii
Contents iv
List of Abbreviations xii
Chapter 1 Introduction 1
1.1 Introduction 1
1.2 General tools for proteomic study 1
1.3 General design of activity-based probes 4
1.4 Applications of ABPs 9
1.5 Research aim 11
1.6 References 12
Chapter 2 Synthesis and evaluation of turn-on fluorescent probes for imaging steroid sulfatase activities in cells† 15
2.1 Introduction 15
2.1.1 General introduction of sulfatases 15
2.1.2 Structural biology of sulfatases 16
2.1.3 Catalytic mechanisms of sulfatases 18
2.1.4 Steroid sulfatase and breast cancer 20
2.1.5 Activity-based probes for sulfatases 22
2.1.6 Probe development 26
2.2 Results and discussions 30
2.2.1 Synthesis of probe 1a and 1b 30
2.2.2 Synthesis of compound 11 33
2.2.3 STS expression and fractionation 34
2.2.4 Probe stability test 34
2.2.5 Evaluation of time-dependence catalytic reaction of probes 35
2.2.6 Evaluating the fluorogenic properties of probes 38
2.2.7 Fluorogenic properties of compound 6 and 11 characterization 39
2.2.8 Evaluation of the self-immobilizing fluorogenic imaging 40
2.3 Conclusion 43
2.4 Future directions 44
2.5 Experiments and reagents 46
2.5.1 Instruments and reagents 46
2.5.2 Synthetic procedures and compound characterizations 46
2.6 References 56
Chapter 3 Design and synthesis of fluorous catalysts 61
3.1 Introduction 61
3.1.1 General introduction of fluorous chemistry 61
3.1.3 Properties of fluorous compounds 62
3.1.4 Application of fluorous chemistry 67
3.1.5 Development of organometallic and organocatalyst catalysts 71
3.2 Results and discussions 82
3.2.1 Part I synthesis and evaluation of fluoroorganometallic Catalyst† 82
3.2.2 Part II synthesis and evaluation of fluoroorganocatalyst 85
3.3 Conclusion 91
3.4 Future directions 92
3.4.1 Fluorous palladacycle 92
3.4.2 Organocatalyst 92
3.5 Experiments 93
3.5.1 Synthetic procedures and compound characterizations 93
3.6 References 101
Chapter 4 Development of isotope-coded activity-based probes for quantitative profiling of serine hydrolase 105
4.1 Introduction 105
4.1.1 General introduction of serine hydrolase 105
4.1.2 Activity-based probes for serine hydrolases 107
4.1.3 Fluorophosphonate group 109
4.1.4 Benzyl fluorophosphonate probe 110
4.2 Probe development 111
4.3 Results and discussion 114
4.3.1 Synthetic route analysis of probe 4H-1 and 4D-1 114
4.3.2 Synthesis of probe 4H-1 and 4D-1 115
4.3.3 Synthesis of probe 8H-1 and 8D-1 121
4.3.4 Examination of probes labeling performance 124
4.4 Conclusion 129
4.5 Future direction 130
4.6 Experiments and reagents 131
4.6.1 Instruments and reagents 131
4.6.2 Synthetic procedures and compound characterization 131
4.7 Reference 134
Chapter 5 Conclusion 137
Appendix 140


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