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研究生:莊惠鈞
研究生(外文):Hui-Chun Chuang
論文名稱:以增強電子施受體強度使類綠螢光蛋白發光團螢光紅移之研究
論文名稱(外文):Fluorescence Red-Shifting of GFP-like Chromophores by Enhancing Electronic Donor-Acceptor Strength
指導教授:楊吉水
指導教授(外文):Jye-Shane Yang
口試委員:鄭原忠孫世勝
口試委員(外文):Yuan-Chung ChengShih-Sheng Sun
口試日期:2016-07-20
學位類別:碩士
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:2016
畢業學年度:104
語文別:中文
論文頁數:141
中文關鍵詞:類綠色螢光蛋白發光團螢光紅移電子施受體
外文關鍵詞:GFP-like chromophoresfluorescence red-shiftingelectronic donor-acceptor
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本實驗室過去已發表一系列間位胺基取代類綠螢光蛋白發光團能有效地提高螢光量子產率(m-DMABDI,Φf = 0.46 in n-Hex),且具有在質子性溶劑中螢光被淬滅的特性,具有潛力應用在即時細胞顯影,這一類的發光團也被成功的應用至人類乳腺上皮細胞(MCF-10A)做細胞顯影,在細胞中呈現綠色螢光。
現階段紅色螢光的類綠螢光蛋白仍相當少見,然而紅色螢光應用在細胞顯影上更具有優勢,本論文的研究動機即為將m-DMABDI做修飾使其放光更加紅移,在結構設計上保留了間位胺基取代效應來提高螢光量子產率以及在非質子、質子性環境中具有螢光量子產率的高度對比差異,期望能得到放光紅移卻且具有高亮度的發光團以利應用於即時細胞顯影。本論文所採用的三個策略分別為:(1)利用互變異構物之放光,在鄰位接上羥基(o-OH),希望藉由ESIPT(excited-state intramolecularproton transfer)所致的互變異構物使放光紅移;(2)為增強施予體與授體的強度,在鄰位和對位上引入甲氧基(o-OMe及p-OMe);(3)則是結合延長共軛及加強施予體與授體的強度的方式,以dicyanomethylene此拉電子基團來增強授體的強度且延長共軛(m-DMABDI-2CN)。目前的研究結果顯示出(1) o-OH的放光並非ESIPT造成的互變異構物放光,且其氫鍵會引致螢光焠熄。(2) o-OMe由於具有較強的charge transfer能力,可有效達到紅移的效果,比起m-DMABDI大約紅移50-60 nm。(3)由於p-OMe的基態結構具有明顯meta-amino扭轉的現象,使得meta-amino effect效用降低,以致放光波長紅移效應不佳且螢光量子產率相對較低。

Meta-Amino substituted green fluorescence protein chromophores (GFPc) such as m-DMABDI display strong fluorescence in non-polar aprotic solvent (Φf = 0.46 in n-Hex)as a result of “meta-amino substituent effect” but undergo fluorescence quehching in protic solvents due to solvent–solute H-bond interactions. This type of chromophore has been applied on bioimaging, which showed green fluorescence in human mammary epithelial cells.However, fluorescent dyes with green emission maybe interfered by autofluorescence of biological sample. In order to avoid this probleme, developing chromophores of red-shifted emission is necessary.
In this thesis, we have three strategies to red-shift the emission of m-DMABDI :(1) introducingo-hydroxyl group into m-DMABDI, to induce tautomer emission throughexcited-state intramolecularproton transfer(ESIPT)(o-OH); (2) introducing strong electron-donating(methoxy)groupinto the donor of m-DMABDIto enhance the push-pull strength of chromophore (o-OMe and p-OMe); (3) introducingelectron-accepting(dicyanomethylene)groupinto the acceptor moiety of m-DMABDIto not only enhance the donor-acceptorstrength but also to extend the conjugation length (m-DMABDI-2CN).
Our results show that the emission maximum of o-OMe is red-shifted as compared with that of m-DMABDIby 50-60 nm.

摘要 i
Abstract iv
目錄 v
圖目錄 viii
表目錄 x
附圖目錄 xi
第一章引言 1
1-1 電磁輻射簡介 1
1-2 光物理行為 2
1-2.1 分子吸收 3
1-2.2 螢光量子產率 3
1-2.3 生命期 4
1-2.4 溶劑化顯色效應(Solvatochromism) 4
1-3 有機分子之光化學行為 5
1-3.1 二苯乙烯與光異構化 6
1-4 綠色螢光蛋白(Green fluorescent protein, GFP) 7
1-5 綠色螢光蛋白發光團衍生物(類GFPc) 9
1-5.1 鎖式結構 10
1-5.2 環境效應 11
1-5.3 電子效應 12
1-6 胺基取代之類綠色螢光蛋白發光團 13
1-7 類綠螢光蛋白發光團衍生物之細胞顯影應用 15
1-8 使類GFPc放光紅移之策略 17
1-8.1 利用互變異構物之放光(tautomer emission) 17
1-8.2 延長共軛 19
1-8.3 增加施予體與授體的強度 21
1-9 研究動機 22
第二章結果與討論 25
2-1 目標化合物之合成與結構 25
2-1.1 o-OH、o-OMe、p-OMe和m-DMABDI-2CN之合成 25
2-1.2 目標化合物之結構 32
2-2 o-OH之光物理性質探討 36
2-3 OMe系列之目標化合物光物理性質 41
2-3.1 吸收光譜 41
2-3.2 螢光光譜 44
2-3.3 螢光量子產率與生命期 46
2-3.4 聚集引致發光測試 50
第三章結論 52
第四章實驗儀器與方法 53
4-1 實驗藥品 53
4-2 實驗儀器與方法 55
4-2.1 核磁共振光譜儀(Nuclear Magnetic Resonance,400MHz) 55
4-2.2 紫外光/可見光吸收光譜儀 (Ultraviolet/Visible Spectrophotometer) 55
4-2.3 螢光光譜儀 (Fluorescence Spectrometer) 55
4-2.4 螢光量子產率測量步驟 55
4-2.5 聚集引致發光測量步驟 56
4-2.6 高效能液相層析儀(High-Performance Liduid Chromotography) 57
4-2.7 紅外線吸收光譜儀(FT-Infrared Spectrometer) 57
4-2.8 高解析度質譜儀(High resolution Mass) 57
4-2.9 X-ray單晶繞射體(Single-crystal X-ray Spectrometer) 57
4-2.10 溶點測定儀 58
4-2.11 溶劑純化系統 58
4-3 化合物之合成步驟 58
第五章參考資料 70


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