Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, 208 016, UP, India.
J Phys Chem A. 2011 Dec 1;115(47):13733-42. doi: 10.1021/jp206815t. Epub 2011 Nov 3.
Two green fluorescent protein (GFP) chromophore analogs (4Z)-4-(N,N-dimethylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DMPI) and (4Z)-4-(N,N-diphenylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DPMPI) were investigated using femtosecond fluorescence up-conversion spectroscopy and quantum chemical calculations with the results being substantiated by HPLC and NMR measurements. The femtosecond fluorescence transients are found to be biexponential in nature and the time constants exhibit a significant dependence on solvent viscosity and polarity. A multicoordinate relaxation mechanism is proposed for the excited state relaxation behavior of the model GFP analogs. The first time component (τ(1)) was assigned to the formation of twisted intramolecular charge transfer (TICT) state along the rotational coordinate of N-substituted amine group. Time resolved intensity normalized and area normalized emission spectra (TRES and TRANES) were constructed to authenticate the occurrence of TICT state in subpicosecond time scale. Another picosecond time component (τ(2)) was attributed to internal conversion via large amplitude motion along the exomethylenic double bond which has been enunciated by quantum chemical calculations. Quantum chemical calculation also forbids the involvement of hula-twist because of high activation barrier of twisting. HPLC profiles and proton-NMR measurements of the irradiated analogs confirm the presence of Z and E isomers, whose possibility of formation can be accomplished only by the rotation along the exomethylenic double bond. The present observations can be extended to p-HBDI in order to understand the role of protein scaffold in reducing the nonradiative pathways, leading to highly luminescent nature of GFP.
两种绿色荧光蛋白（GFP）发色团类似物（4Z）-4-（N，N-二甲基氨基苯亚甲基）-1-甲基-2-苯基-1,4-二氢-5H-咪唑啉-5-酮（DMPI）和（4Z）-4-（N，N-二苯基氨基苯亚甲基）-1-甲基-2-苯基-1,4-二氢-5H-咪唑啉-5-酮（DPMPI）使用飞秒荧光上转换光谱法和量子化学计算进行了研究，结果通过 HPLC 和 NMR 测量得到证实。飞秒荧光瞬变被发现具有双指数性质，时间常数表现出对溶剂粘度和极性的显著依赖性。提出了一个多坐标弛豫机制，用于模型 GFP 类似物的激发态弛豫行为。第一个时间常数（τ（1））被分配给沿着 N-取代胺基团的旋转坐标形成扭曲的分子内电荷转移（TICT）态。构建了时间分辨强度归一化和面积归一化发射光谱（TRES 和 TRANES），以在亚皮秒时间尺度上证实 TICT 态的发生。另一个皮秒时间常数（τ（2））归因于通过沿偕亚甲基双键的大振幅运动进行的内转换，这已通过量子化学计算阐明。量子化学计算还排除了呼啦圈扭曲的可能性，因为扭转的激活能垒很高。受激类似物的 HPLC 图谱和质子 NMR 测量证实了 Z 和 E 异构体的存在，只有通过沿偕亚甲基双键的旋转才能形成其异构体。目前的观察结果可以扩展到 p-HBDI，以了解蛋白质支架在减少非辐射途径中的作用，从而导致 GFP 具有高发光性质。
