Topochemistry and Photomechanical Effects in Crystals of Green Fluorescent Protein-like Chromophores: Effects of Hydrogen Bonding and Crystal Packing

To obtain insight into the effects of the environment on the photophysics and photochemistry of the green fluorescence protein (GFP), eight crystal structures of six synthetic aryl-substituted analogues (2-fluoro, 2-methyl, 3-hydroxy, 3-methoxy, 2,4-dimethyl and 2,5-dimethyl) of the GFP chromophore (4-hydroxy-benzylidenedimethylimidazolinone) were determined and correlated with their two-dimensional steady-state and time-resolved solid-state excitation−emission spectra. The stacking between the molecules greatly affected the emission energy and the lifetime of the emission of the chromophore, implying that ππ interactions could be critical for the photophysics of GFP. The reaction pathways were dependent on the excitation energy, resulting either in [2 + 2] photodimerization at the bridging double bond (UV excitation) or flipping of the imidazolone ring (visible excitation). The meta-hydroxy chromophore (3-HOBDI) was the only GFP−chromophore analogue that was obtained as more than one stable polymorph in the pure state thus far. Due to the asymmetric substitution with hydrogen bond donors and acceptors, 3-HOBDI is tetramorphic, the forms showing distinctly different structure and behavior: (1) while one of the polymorphs (3-HOBDI-A), having multilayer structure with alternating stereochemistry of linear hydrogen-bonded motifs, undergoes photodimerization under UV light, (2) another (3-HOBDI-C), which has dimeric head-to-tail structure, shows Z-to-E isomerization via τ-one-bond flip of the imidazolone ring by excitation in the visible region. X-ray diffraction analysis of a partially reacted single crystal of 3-HOBDI-C provided the first direct evidence of τ-one-bond flip occurring in a GFP-like compound. Moreover, the cooperative action of the photodimerization of 3-HOBDI-A appears as a photomechanical effect of unprecedented magnitude for a single crystalline specimen, where photoexcited single crystals bend to more than 90° without breaking.

为深入探究环境对绿色荧光蛋白（green fluorescence protein, GFP）的光物理与光化学过程的影响，本研究测定了6种合成芳基取代GFP生色团（4-羟基亚苄基二甲基咪唑啉酮）的8种晶体结构，所涉衍生物分别为2-氟、2-甲基、3-羟基、3-甲氧基、2,4-二甲基及2,5-二甲基取代物，并将其与二维稳态及时间分辨固态激发发射光谱进行关联分析。分子间的堆叠作用对生色团的发射能量与发射寿命具有显著影响，这表明π-π相互作用可能对GFP的光物理过程至关重要。反应路径取决于激发能量：在紫外激发下会于桥连双键处发生[2+2]光环二聚化，而在可见激发下则会引发咪唑啉酮环的翻转。截至目前，间位羟基生色团（meta-hydroxy chromophore, 3-HOBDI）是唯一一种可在纯态下获得多种稳定多晶型的GFP生色类似物。由于同时带有氢键供体与受体的不对称取代，3-HOBDI呈现四晶型特征，不同晶型展现出截然不同的结构与行为：(1) 其中一种晶型（3-HOBDI-A）具有多层结构，其线性氢键基序呈现交替立体化学排布，在紫外光照下会发生光二聚化反应；(2) 另一种晶型（3-HOBDI-C）则为二聚体头尾相接结构，在可见区域激发下可通过咪唑啉酮环的τ单键翻转实现Z到E异构化。对部分反应的3-HOBDI-C单晶进行X射线衍射分析，首次直接证实了类似GFP的化合物中存在τ单键翻转过程。此外，3-HOBDI-A的光二聚化协同作用展现出单晶样品前所未有的光机械效应：受光激发的单晶可弯折至90°以上且不会发生断裂。