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COMMUNICATIONS 3438 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000 1433-7851/00/3919-3438 $ 17.50+.50/0 Angew. Chem. Int. Ed. 2000, 39, No. 19 Design and Synthesis of Intramolecular Resonance-Energy Transfer Probes for Use in Ratiometric Measurements in Aqueous Solution Yasutomo Kawanishi, Kazuya Kikuchi, Hideo Takakusa, Shin Mizukami, Yasuteru Urano, Tsunehiko Higuchi, and Tetsuo Nagano* In recent years, many fluorescent probes have been developed to study biological phenomena in living cells. By using fluorescent probes, it is possible to follow a phenom- enon in real time with high sensitivity. However, the fluorescence intensity of the probes is influenced by many factors, such as changes of environment around the probe (pH, polarity, temperature, and so forth), changes in the probe concentration, and changes in the excitation intensity. To reduce the influence of such factors, ratiometric measure- ments are utilized, [1] namely, observation of changes in the ratio of the fluorescence intensities at two wavelengths. This technique allows more precise measurement and, with some probes, quantitative detection is possible. Many probes have been developed for ratiometric measurements, [1] such as the calcium indicator Fura-2 [2] and the pH indicator SNARF-1. [3] To carry out ratiometric measurements, the probe must exhibit a large shift in its emission or excitation spectrum after it reacts (or binds) with the target molecule. Resonance- energy transfer (RET) is one mechanism used to obtain a large shift in the spectral peak. RET is a radiationless transmission of an energy quantum between two fluorophores in close proximity. [4] If the bond between two linked fluorophores, which have an overlap in their spectra, is cleaved, we observe the emission of the acceptor, due to RET, before the cleavage and the emission of the donor after. This results in a large shift in the emission peak, which can be used for ratiometric measurements. Little work has been done on chemically synthesized intramolecular RET probes except for the probe for detecting b-lactamase activity [5] reported by Tsien and co-workers. This is because, even if two fluorophores are located in close proximity, self-quenching occurs due to the close contact of the two hydrophobic fluorophores in an aqueous solution. [6] If we could observe the emission of the acceptor in chemically synthesized RET probes, we would be able to design probes for ratiometric measurements of many enzymes, by binding two fluorophores at the ends of a peptide substrate (or another cleavable linker), which could be cleaved selectively by the enzyme. Herein, we present a method to observe the emission of the acceptor caused by intramolecular RET, applicable for ratiometric measurements in aqueous solution. This method is based upon the idea that self-quenching between the two fluorophores can be blocked and the emission of the acceptor can be observed by restricting the flexibility of the linker between the two fluorophores, so that they can not readily come into close contact. To investigate the relation between the flexibility of the linker and the emission spectrum, we designed and synthe- sized coumarin phosphodiester-linked fluoresceins (CPFs). CPFs have two fluorophores, coumarin as a donor and fluorescein as an acceptor. These two fluorophores are known to have a large overlap in their spectra and exhibit efficient emission in an aqueous solution. The phosphodiester moiety was introduced to enhance the aqueous solubility of the probe and was placed as a linker between the two fluorophores. Ethylene was chosen as a linker extension with high flexibility and cyclohexane as one with low flexibility. Three molecules with different linkers were synthesized; CPF1 with two P OH O O O O O Cl HO O HO O COOH P O OH O O H N N H O O N H H N O HO O COOH N H O O O O Cl HO H N O O O HO O COOH N H HO O O O Cl HO H N OH O P OH O O HO O COOH O O O Cl HO O CPF2 CPF3 control CPF1 ethylene linkers, CPF2 with two cyclohexane linkers, and CPF3 with one ethylene and one cyclohexane linker. We used a mixture of the amide form of coumarin and fluorescein as the control. CPFs were synthesized as shown in Scheme 1. The emission spectra of CPFs in an aqueous buffer excited at 370 nm (the excitation wavelength, l ex ,of the coumarin moiety) are shown in Figure 1 a. Efficient RET was observed with all CPFs, as more than 90% of the emission of the coumarin donor (l em 450 nm) was quenched. A strong emission of the fluorescein acceptor (l em 515 nm) was observed only in CPF2. The fluorescein acceptor of CPF2 emitted 54 % of the photons that excited the donor. CPF1 and CPF3 had only weak emission and showed self-quenching. [*] Prof. T. Nagano, Y. Kawanishi, Dr. K. Kikuchi, H. Takakusa, S. Mizukami, Dr. Y. Urano, Dr. T. Higuchi Graduate School of Pharmaceutical Sciences The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) Fax: ( 81) 3-5841-4855 E-mail: [email protected]

Design and Synthesis of Intramolecular Resonance-Energy Transfer Probes for Use in Ratiometric Measurements in Aqueous Solution

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