PHOTOSTABLE FLUORESCENT DYES FOR BIOCHEM/MICROSCOPY

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We propose to synthesize new reactive fluorescent stains based on an extraordinarily photostable ClaSS of naphthalimide dyes. These new stains will be applicable to many of the same uses as the currently available fluorescein, rhodamine, Cy3, coumarin and other labeling agents, and will have the additional advantage of combining large Stokes shift, high quantum yield, and suitably long lifetime to apply to fluorescence polarization immunoassays. In Phase I we will prepare several naphthalimide dyes, derivatize them with auxochromes to bathochromically shift their excitation and the emission wavelengths, and add a functional group which can react with bifunctional linkers to yield an array of reactive fluorescent probes. These probes will be tested for their ability to label amino groups in proteins such as actin and avidin. We will determine the effect of each modification of the dye on the photophysical constants (wavelength of excitation and emission, extinction coefficient, epsilon, and fluorescence quantum yield, (phiflu) and test the photostability of the dyes against commonly used fluorescent labels. We will also determine the degree of labeling of the proteins (# labels/protein molecule) and the phiflu of the label on the protein. The Phase I work will be designed to determine the feasibility of the approach to creating new photochemically stable labeling agents to replace existing dyes which have less desirable properties. PROPOSED COMMERCIAL APPLICATION: To the extent that our new fluorophores improve on existing fluorescent labeling agents, they will find a ready market. Because of their high photostability and high quantum yield, they will be particularly useful as fluorescent markers for light microscopy applications. Their large Stokes shifts will also make them suitable for fluorescence polarization immunoassays.