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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 方俊民(Jim-Min Fang) | |
dc.contributor.author | Hsin-Chuan Hsu | en |
dc.contributor.author | 徐欣傳 | zh_TW |
dc.date.accessioned | 2021-06-16T09:20:59Z | - |
dc.date.available | 2022-07-07 | |
dc.date.copyright | 2017-07-07 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-06-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59342 | - |
dc.description.abstract | 細菌轉醣酶的位置在細胞膜外,是合成細胞壁不可或缺的酵素,而且人體裡沒有轉醣酶的存在,所以針對其設計抑制劑是發展抗生素中極有潛力的一種方法。雖然目前已發現一些轉醣酶抑制劑,但它們還未被研發成適合人所使用的抗生素;另外目前轉醣酶的抑制活性測試速度仍不足以用來開發更多有潛力的抗生素,因此發展出更快速且有效率的的高通量篩選方式是非常急迫的。
我們選擇4′-(4-N,N-二甲胺基苯基)-2,2′:6′,2″-三聯吡啶和鋅離子錯合形成受體9來作為追蹤轉醣酶反應的感測器。為了測試受體9在高通量篩選的表現,我們合成了GlcNAc–GPP (23) 來代替較難合成的lipid II,另外用可以購買得到的FPP來作為C55-PP的替代,來檢視受體9與它們的錯合結果。 實驗結果顯示在HEPES (pH 7.4) 緩衝溶液下,受體9與FPP錯合會在波長650奈米附近產生螢光,相對地, GlcNAc–GPP (23)與受體9結合並不會有螢光的變化。接著我們在轉醣酶活性測試中加入受體9,實驗分為反應組與對照組,NBD-lipid II首先進行轉醣反應後加入受體9來觀測螢光變化,結果顯示NBD-lipid II與受體9的錯合造成NBD意外的螢光淬熄與受體9的放光,因此不易觀察C55-PP–受體9的螢光。儘管如此,藉由受體9造成對照組NBD的螢光淬熄,轉醣酶反應仍然可以由觀察波長550奈米處的變化來追蹤反應。 我們成功發展出使用受體9來偵測轉醣酶反應的系統,並且在未來有機會應用在開發轉醣酶抑制劑的高通量篩選上。 | zh_TW |
dc.description.abstract | Transglycosylase (TGase) is an attractive target for development of new antibiotics due to its location outside the membrane of bacteria and essential role in peptidoglycan synthesis. Some TGase inhibitors have been identified, but none of them have been developed into antibiotics for human. The existing TGase assays are not fast enough to develop new antibiotics. Therefore a continuous, quantitative, and high-throughput assay for TGase will facilitate the discovery of new and effective inhibitors.
We chose 4′-(4-N,N-dimethylaminophenyl)-2,2′:6′,2″-terpyridine as the ligand (L) of Zn(II) ion to form a complex ZnCl2L (9) as the TGase probe, which has been reported as a fluorescent sensor of inorganic pyrophosphate. To establish a model system, we first synthesized GlcNAc–GPP (23) instead of lipid II, and the commercially available farnesyl pyrophosphate (FPP) was used as the truncated analogue of undecaprenyl pyrophosphate (C55-PP) to compare the binding behavior with sensor 9. The experiment showed that FPP was selectively bound with 9 to emit fluorescence at 650 nm. In contrast, GlcNAc–GPP 23–9 did not emit fluorescence. Next, the sensor 9 was applied in TGase assay which was in a more complex solution system. NBD–lipid II was utilized as the substrate for the TGase assay, and sensor 9 was added to monitor the change of fluorescence between the reacted group and unreacted group. The result showed an unexpected fluorescence quenching effect of NBD-lipid II at 550 nm, but enhancement at a longer wavelength of 650 nm that was presumably caused by complexation of NBD-lipid II with 9. Although the result was opposite to our prediction, the TGase catalyzed reaction could still be monitored by fluorescence at 550 nm. In summary, we successfully developed a TGase assay in which sensor 9 was utilized to track the enzymatic reaction. This system could be operated as a high-throughput assay for screening TGase inhibitors in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:20:59Z (GMT). No. of bitstreams: 1 ntu-106-R03223215-1.pdf: 6026561 bytes, checksum: 79b8b3e813a568702440e43639a8bb22 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | Table of Contents
Abstract in Chinese………………………………………………………………………….………I Abstract in English……………………………………………………………………………..…III Table of Contents………………………………………………………………...…………………V Index of Figures…………………………………………………………………………….……VIII Index of Schemes…………………………………………………………………………………XII Index of Tables……………………………………………………………………………..……XIII Index of Equations………………………………………………………………………………XIII Appendix…………………………………………………………………………………………XIV Abbreviations………………………………………………………………………….…………XVI Chapter 1: Introduction 1.1 Molecular Recognition……………………………………………………………..…..1 1.1.1 Introduction of chemosensors…………………………………………..……1 1.1.2 Optical signals of sensors...…………………………………………….……..2 1.1.3 General mechanism of fluorescent sensors…………………………….……2 1.1.3.1 Photoinduced electron transfer (PET) ……………………………2 1.1.3.2 Photoinduced charge transfer (PCT) ……………………………..3 1.1.3.3 Förster resonance electron transfer (FRET) ……………………..4 1.2 Detection of Organic and Inorganic Pyrophosphates...………………………………5 1.3 Bacterial Cell Wall……………………………………………………………...………9 1.4 Peptidoglycan glycosyltransferases, PGTs………………………………………...…11 1.4.1 Crystal structure of peptidoglycan glycosyltransferases…………….……11 1.4.2 Mechanism of transglycosylation…………………………………………...12 1.5 Current Methods for Detection of Transglycosylase Reaction………………..……15 1.5.1 Surface plasma resonance…………………………………………...………15 1.5.2 Fluorescence anisotropy…………………………………………………..…16 1.5.3 High-performance liquid chromatography…………………………..…….16 1.5.4 Förster resonance energy transfer (FRET) ………………………………..18 1.6 Goals and Design of Sensors……………………………………………………….….19 Chapter 2: Results and Discussion 2.1. Synthesis of Sensors…………………………………………………………………...21 2.1.1 Synthesis of 4′-(4-N,N′-dimethylaminophenyl)-2,2′:6′,2″-terpyridine zinc complex 9…………………………………………………………………….21 2.1.2 Synthesis of bilaterally methylated terpyridine zinc complex 13………...21 2.1.3 Synthesis of unilaterally methylated terpyridine zinc complex 16…….....23 2.2. Synthesis of Substrates……………………………………………………………......24 2.2.1 Synthesis of pyrophosphate diester GlcNAc–GPP (23)……………..........25 2.2.2 Synthesis of GPP (24)…………………………………………………....…..26 2.3. Titration of Terpyridine–Zn2+ Sensor 9 with Substrates……………………….…..29 2.3.1 Titration with inorganic pyrophosphate…………………………….......…29 2.3.2 Titration with farnesyl pyrophosphate……………………………………..31 2.3.3 Titration with GlcNAc–GPP (23)…………………………………………...38 2.4. Detection in TGase Assay…………………………………………………………….40 2.5. Synthesis of coumarin-lipid II…………………………………………………..……46 2.6. Titration with Coumarin-Park Nucleotide (30)……………………………………..47 Chapter 3: Conclusion………………………………………………………………………...…..51 Chapter 4: Experimental Section 4.1 General………………………………………………………………………………....52 4.2 Material……………………………………………………………...…………………53 4.3 UV-Vis Titraion Studies………………………………………………………….……53 4.4 Fluorescent Titration Studies………………………………………………………....53 4.5 Job Plot…………………………………………………………………………………53 4.6 Evaluation of TGase Catalyzed Reaction………………………………………….…54 4.7 Synthetic Procedures and Characterization of Compounds…………………..…....54 Chapter 5: References………………………………………………………………………...…...64 Appendix: 1H, 13C and 31P NMR spectra…………………………………………………………68 | |
dc.language.iso | en | |
dc.title | 合成三聯吡啶衍生物作為篩選轉醣酶抑制劑之感測器 | zh_TW |
dc.title | Synthesis of Terpyridine Derivatives as Sensors for Screening of Transglycosylase Inhibitors | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭婷仁(Ting-Jen Cheng),楊吉水(Jye-Shane Yang),王宗興(Tsung-Shing Andrew Wang),孫世勝(Shih-Sheng Sun) | |
dc.subject.keyword | 轉醣?,抑制劑,感測器, | zh_TW |
dc.subject.keyword | Transglycosylase,Inhibitors,Sensors, | en |
dc.relation.page | 83 | |
dc.identifier.doi | 10.6342/NTU201701211 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-06-30 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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