間位胺基取代綠色螢光蛋白發光團於溶液態中螢光特性與機制之研究

Abstract

順反異構化經常是造成綠色螢光蛋白發光團及衍生物之低螢光量子產率的原因之一，如p-HBDI及p-ABDI於非質子性溶劑中皆表現出低螢光量子產率 (Фf < 10-3於正己烷)。相反地，本實驗室之間位胺基之綠螢光蛋白發光團 m-DMABDI在非質子性溶劑中具有高螢光量子產率，此效應稱為”間位胺基取代效應”。然而，隨著溶劑極性的增加，m-DMABDI之放光有顯著的紅位移卻同時急遽下降之螢光量子產率 (Фf =0.46至0.05，正己烷至乙腈)，而此現象並不利於細胞顯影之應用。先前的研究中我們觀察到m-DMABDI在高極性之非質子性溶劑中具有非正常之螢光生命期對溫度之關係，暗示”扭轉致分子內電荷轉移”的非輻射螢光淬滅路徑有可能參與其激發態過程，然而Фf +2 ФZE於高極性溶劑中仍接近1，意味著所有去活化路徑最後是透過螢光放光與異構化來回到基態。由於先前研究中之m-B-DMABDI發光團亦具有類似的反常螢光生命期對溫度之關係，目前已排除是胺基部分C-N單鍵扭轉的可能性。 為了更了解m-DMABDI在激發態之螢光機制與特性，我們設計出限制C-C單鍵扭轉之m-DMABDI-B。研究結果顯示出m-DMABDI-B具有與m-DMABDI迥異之螢光性質，且在高極性非質子性溶劑中具有高螢光量子產率 (Фf = 0.29)，暗示著”扭轉致分子內電荷轉移”在此系統中被抑制的結果。根據Фf +2 ФZE ~ 1，我們進一步推測m-DMABDI於高極性溶劑中之激發態為”扭轉致分子內電荷轉移”與” 順反異構化”相互耦合的過程，導致其低螢光量子產率的結果。另外，我們根據理論計算的結果設計出m-4ABDI，使得其螢光放光達到較m-DMABDI紅移達16 nm而不減低螢光量子產率，並期待未來能設計出新一代有利於生物細胞顯影之綠螢光發光團衍生物。

The Z-E photoisomerization (C=C bond torsion) is generally responsible for the low fluorescence quantum yield (Фf < 10-3 in hexane) of green fluorescence protein chromophore and its analoges (GFPc) , such as the wild-type GFPc p-HBDI and p-ABDI. We previously reported that meta-amino substituted GFPc (m-DMABDI) display high Фf, as a result of “meta-amino substituent effect”. However, m-DMABDI exhibits red-shifted emission but low Фf in polar solvent (e.g., 0.46 to 0.05 from Hex to MeCN) , which limits their application as dyes for bioimaging. We previously observed that m-DMABDI exhibits unusual temperature-dependence of fluorescence lifetime in polar solvents, which implies that twisted intramolecular charge transfer (TICT) is taking place. In addition, the possibility of C-N bond torsion to account for the TICT state was excluded, because m-B-DMABDI also exhibits abnormal temperature effects on the fluorescence lifetime. In this work, in order to understand the intrinsic excited state behavior of m-DMABDI, we designed m-4ABDI and m-DMABDI-B, in which the amino-inversion and C-C bond rotation is restricted, respectively. Our results show that m-DMABDI-B possesses an opposite solvent effect on fluorescence quantum yield. On the basis of Фf +2 ФZE ~ 1, we conclude that “TICT couples with Z/E isomerization” in polar solvents. Also, based on the theoretical calculation, m-4ABDI was designed and found to exhibit a red shift of 16 nm in the fluorescence maximum relative to m-DMABDI, which might be useful for the applications.of bioimaging.