Design and synthesis of an enzyme-cleavable sensor molecule for phosphodiesterase activity based on fluorescence resonance energy transfer

Ratiometric measurement is a technique that can provide precise data and even quantitative detection. To carry out ratiometric measurements, it is necessary that the sensor molecule exhibits a large shift in its emission or excitation spectrum after reaction with the target molecule. Fluorescence resonance energy transfer (FRET) is one mechanism used to obtain a large spectral shift. In this study, our aim was to develop a ratiometric fluorescent sensor molecule for phosphodiesterase activity based on FRET. We designed and synthesized CPF4 with a coumarin donor, a fluorescein acceptor, and two phenyl linkers having the phosphodiester moiety interposed between them. In the emission spectrum of CPF4 in aqueous buffer excited at 370 nm, the emission of the coumarin donor was strongly quenched and the emission of the fluorescein acceptor was observed. This emission spectrum demonstrates that energy transfer from the coumarin donor to the fluorescein acceptor proceeds efficiently. Addition of a phosphodiesterase to an aqueous solution of CPF4 resulted in an increase in the donor fluorescence and a decrease in the acceptor fluorescence. CPF4 exhibited a large shift in its emission spectrum after the hydrolysis of the phosphodiester group by the enzyme. This large shift of the emission spectrum indicates that ratiometric measurements can be made by using CPF4. The method described in this paper for designing enzyme-cleavable sensor molecules based on FRET should be readily applicable to other hydrolytic enzymes.