Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis
Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis
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DOI：
10.1073/pnas.96.4.1193
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发表时间：
1999-02-16
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Furuta, T;Wang, SSH;Tsien, RY
如果光解可以由成对的IR光子而不是常规的单个UV光子提供动力，则在光散射介质中具有三维空间分辨率的生物活性信使的光化学释放（释放）将大大促进。光强度的二次依赖性将把光解限制在激光的焦点，并且较长的波长将受散射的影响小得多。然而，以前的笼中信使在IR区域的双光子激发的横截面非常小。我们现在表明，溴化的7-羟基香豆素-4-基甲基酯和氨基甲酸酯有效地释放羧酸盐和胺的光解，与单光子和双光子截面高达一个或两个数量级优于以前可用。这些优点在由N-（6-溴-7-羟基香豆素-4-基甲氧羰基）-L-谷氨酸（Bhc-glu）释放的谷氨酸兴奋的来自大鼠皮层和海马的脑切片中的神经元上得到证实。Bhc-glu的常规UV光解所需的强度小于先前最好的笼状谷氨酸盐之一γ-（α-羧基-2-硝基苄基）-L-谷氨酸盐（CNB-glu）所需强度的五分之一。光栅扫描飞秒红外脉冲的双光子光解给出了完整切片中神经元谷氨酸敏感性的第一个三维分辨图。Bhc-glu及其类似物不仅在细胞生物学和神经生物学中，而且在许多技术应用中，应该允许更有效和三维定位的解开和光切割。
Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one- and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-L-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, gamma-(alpha-carboxy-2-nitrobenzyl)-L-glutamate (CNB-glu). Two-photon photolysis with raster-scanned femtosecond IR pulses gives the first three-dimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.