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首页> 外文期刊>Chemistry: A European journal >A Novel Design Method of Ratiometric Fluorescent Probes Based on Fluorescence Resonance Energy Transfer Switching by spectral Overlap Integral
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A Novel Design Method of Ratiometric Fluorescent Probes Based on Fluorescence Resonance Energy Transfer Switching by spectral Overlap Integral

机译：基于光谱重叠积分的荧光共振能量转移切换的比例式荧光探针设计新方法

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摘要

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and it suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescence probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other appropriate enzyme-cleavable groups into the fluoresce in acceptor.
机译：比率测量，即同时记录两个波长的荧光强度并计算它们的比率，比单个波长的测量具有更高的精确度，并且适用于细胞成像研究。在这里，我们描述了一种新型的设计探针的方法，该探针基于荧光共振能量转移（FRET）的转换来按比例测量水解酶活性。该方法使用具有3'-O，6'-O-保护的荧光素受体的荧光探针，该受体通过连接体部分与香豆素供体连接。由于在香豆素发射和荧光素吸收之间没有光谱重叠积分，所以荧光素部分不能接受供体部分的激发能并且可以观察到供体荧光。在通过水解酶切割保护基之后，荧光素部分在香豆素发射区显示出强吸收，然后观察到由于FRET引起的受体荧光。基于此机制，我们已经开发了新型的用于蛋白质酪氨酸磷酸酶（PTP）活性的比例荧光探针（1-3）。与PTP反应后，它们的发射波长发生很大变化。通过使用香豆素-环己烷-荧光素FRET盒部分，可以克服FRET探针通常发生的荧光猝灭问题，其中两种染料的紧密接触受到阻碍。在研究了它们的化学和动力学性质之后，我们得出结论，带有刚性环己烷接头的化合物1和2实际上可用于比例测量PTP活性。本文所述的设计概念，通过光谱重叠积分和防止两种染料紧密接触的刚性连接，使用FRET转换，也应适用于受体中发荧光的其他合适的酶可裂解基团。

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