A Novel Design Method of Ratiometric Fluorescent Probes Based on Fluorescence Resonance Energy Transfer Switching by Spectral Overlap Integral

Abstract 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 is 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 fluorescent 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 ratiometric 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 hydrolytic enzymes by introducing other appropriate enzyme‐cleavable groups into the fluorescein acceptor.