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

Abstract

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.