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論文中文名稱:可偵測對羥基苯甲酸酶之新型隱藏式螢光探的針合成 [以論文名稱查詢館藏系統]
論文英文名稱:Synthesis of a New para-Hydroxybenzoate Hydroxylase Targeted Latent Fluorophore [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:98
出版年度:99
中文姓名:羅祥銘
英文姓名:Hsiang-Ming Lo
研究生學號:97688020
學位類別:碩士
語文別:英文
口試日期:2010-07-13
論文頁數:78
指導教授中文名:黃聲東
指導教授英文名:Sheng-Tung Huang
口試委員中文名:郭憲壽;汪昆立
口試委員英文名:Hsien-Shou Kuo;KUN-LI WANG
中文關鍵詞:隱藏式螢光探針、對羥基苯甲酸酶、雙酵素系統
英文關鍵詞:Latent fluorophore, para-hydroxybenzoate hydroxylase, bienzyme system
論文中文摘要:對羥基苯甲酸酶(PHBH)是一種黃素酵素,此酵素可以藉由NADPH與氧氣催化對羥基苯甲酸進行氧化反應。本研究主題是合成一新型的隱藏式螢光探針,可用於即時偵測對羥基苯甲酸酶(PHBH)的酵素活性。螢光受質羥基苯甲酸(HBLF)是利用對羥基苯甲酸與螢光分子經化學合成行成共價接合,降低其螢光分子之螢光強度形成隱藏性螢光探針HBLF。新型隱藏式螢光探針HBLF可以被對羥基苯甲酸酶催化,對羥基苯甲酸酶會在對羥基苯甲酸的間位進行氧化反應,利用間位上氧基電子的轉移進行 quinon-methide-type rearrangement reaction 釋放其螢光分子。本研究主要是研究可行的合成途徑合成HBLF。本合成經由8步驟完成,總產率 1.77 %,其中HBLF結構中的羥基苯甲酸酯是由丹尼謝夫斯基雙烯(Danishefsky diene)與丙炔醇衍生物進行Diels-Alder反應完成,然後進行SN2反應接上一已知螢光分子,最後在經由水解酯類得到最終產物。本論文也討論失敗的合成途徑,在完成HBLF的實驗總共設計了三條途徑。最後本研究也測試新HBLF與對羥基苯甲酸酶和NADPH+反應後螢光釋放強度,也證實HBLF可以運用於即時偵測對羥基苯甲酸酶。
論文英文摘要:Pare-hydroxybenzoate hydroxylase (PHBH) is an extensively studied enzyme as a model for single-component flavoprotein monooxygenase. PHBH in the presence NADPH and O2 catalyst the hydroxylation of para-hydroxybenzoic acid. We designed and synthesised a novel latent fluorophore, HBLF, for p-hydroxybenzoate hydroxylase. We slected 2-benzothiazoyl cyanocoumarin as a flourophore to be masked. The fluorigenic chemical transformation of HBLF triggered by PHBH in the presence of NADPH and O2 is through a cascade reaction, hydroxylation and quinon-methide-type rearrangement reaction. In this thesis, we successfully prepared HBLF in 8 steps with 1.77 % yield. Para-hydroxybenzoate moiety in the HBLF is obtained from condense Danishefsky diene with the acetylene derivate through Diels-Alder reaction. Eventually, benzoic ester was converted into benzoic acid with trifluoroacetic acid. We also discussed the futile approaches toward the synthesis of HBLF. Finally, we also characterized the fluorescence responce of HBLF with PHBH in the presence of NADPH. PHBH with the presence of NADPH under aerobic condition catalyzes the release of fluorogenic cyanocoumarin.
論文目次:Content



摘要 i

Abstract ii

誌謝 iii

Content iv

Scheme vi

Figure vii

Appendix viii

Chapter 1 INTRODUCTION 1

1.1 p-Hydroxybenzoate hydroxylase 1

1.2 PHBH applications 2

1.2.1 PHBH application based on electrochemical
 method 2

1.2.2 A method used PHBH with HBLF to detect
 para-hydroxybenzoic acid 4

1.3 A useful fluorimetric indicator 5

1.3.1 Latent Fluorophores 7

1.3.2 Fluorescent probe based on quinone-methide
 -type reaction 8

1.4 Goal and approaches 9

Chapter 2 EXPERIMENTAL 11

2.1 Measurement 11

2.2 Materials 12

2.3 Synthetic Procedure 13

2.3.1 Synthesis of compound, 15 13

2.3.1.1 Ethyl 4-(tosyloxy)but-2-ynoate, 14 13

2.3.1.2 Ethyl 4-(tosyloxy)but-2-ynoate, 15 14

2.3.2 Synthesis of compound, 10 15

2.3.2.1 3-[(tert-butyldimethylsilyl)oxy]-1-propyne, 7 16

2.3.2.2 Ethyl 4-(tert-butyldimethylsilyloxy)-2-butynoate, 6 16

2.3.2.3 4-(tert-Butyldimethylsilyloxy)but-2-ynal, 6a 17

2.3.2.4 Trans-1-Methoxy-3-trimethylsilyloxy-1,3-butadiene
 (Danishefsky’s diene), 19 17

2.3.2.5 Ethyl 2-((tert-butyldimethylsilyloxy)methyl)
 -4-hydroxybenzoate, 10 18

2.3.2.6 2-((tert-butyldimethylsilyloxy)methyl)
 -4 hydroxybenzaldehyde, 10a 18

2.3.3 Synthesis of compound, 3 19

2.3.3.1 Ethyl 4-hydroxy-2-(hydroxymethyl)benzoate, 17 20

2.3.3.2 Ethyl 2-(chloromethyl)-4-hydroxybenzoate, 18 20

2.3.3.3 Ethyl 4-acetoxy-2-(chloromethyl)benzoate, 9 21

2.3.3.4 Fluorogenic substrate of 4-hydroxybenzoic easter,3 21

2.3.4 Synthesis of compound, HBLF (1) 22

2.3.4.1 Fluorogenic substrate of hydroxybenzoic easter, 1a 23

2.3.4.2 Fluorogenic substrate of hydroxybenzoic acid, HBLF (1) 23

2.4 Chemicals 24

2.5 Photophysical of compound 1a with or without 
 PHBH 24

2.6 Photophysical of HBLF with or without PHBH 24

Chapter 3 RESULT AND DISCUSSION 25

3.1 Synthesis strategy of HBLF 25

3.1.1 Retrosynthestic analysis of 1a 26

3.1.2 Synthesis of 15 27

3.2 Synthesis strategy 2 28

3.2.1 Retrosynthestic analysis of 3 28

3.2.2 Synthesis of 10 and 10a 29

3.2.3 H-NMR analysis of 10 30

3.2.4 The regioselectivity in Diels-Alder reaction 31

3.2.5 2D-NMR analysis of 10 32

3.2.6 Synthesis of 17 and 18 36

3.2.7 Synthesis of 9 and 3 37

3.3 Base hydrolysis of 3 38

3.4 Absorption and emission of coumarin 20a 40

3.5 Other method for deprotect group 41

3.6 Acid hydrolysis of 3 41

3.7 Fluorescence response of HBLF 43

Chapter 4 CONCLUSION 47

Reference 48

Appendix 1.1 1H NMR of compound 10. 52

Appendix 1.2. 1H NMR of compound 10a 53

Appendix 1.3. 1H NMR of compound 17 54

Appendix 1.4. 1H NMR of compound 15 55

Appendix 1.5. 1H NMR of compound 17a 56

Appendix 1.6. 1H NMR of compound 20a 57

Appendix 1.7. 1H NMR of compound 18 58

Appendix 1.8. 1H NMR of compound 9 59

Appendix 1.9. 1H NMR of compound 3 60

Appendix 1.10. 1H NMR of compound 1a 61

Appendix 1.11. 1H NMR of compound HBLF 62

Appendix 1.14. 1H NMR of compound HBLF 63

Appendix 2.1. 13C NMR of compound 17 64

Appendix 2.2. 13C NMR of compound 18 65

Appendix 2.3. 13C NMR of compound 9 66

Appendix 2.4. 13C NMR of compound 3 67

Appendix 2.5. 13C NMR of compound 1a 68

Appendix 2.6. 13C NMR of compound 10 69

Appendix 2.7. 13C NMR of compound 20a 70

Appendix 3.1. Mass spectrometry of compound 10 71

Appendix 3.2. Mass spectrometry of compound 17 72

Appendix 3.3. Mass spectrometry of compound 18 73

Appendix 3.4. Mass spectrometry of compound 9 74

Appendix 3.5. Mass spectrometry of compound 3 75

Appendix 3.6. Mass spectrometry of compound 1a 76

Appendix 3.7. Mass spectrometry of compound HBLF (1) 77

Appendix 3.8. Mass spectrometry of compound 1c 78
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論文全文使用權限:同意授權於2011-08-24起公開