Fluorescent dyes are widely used as tracers for localization of biological structures by fluorescence microscopy, for quantification of analytes by fluorescence immunoassay, for flow cytometric analysis of cells, for measurement of physiological state of cells and other applications. Their primary advantages over other types of absorption dyes include the visibility of emission at a wavelength distinct from the excitation, the orders of magnitude greater detectability of fluorescence emission over light absorption, the generally low level of fluorescence background in most biological samples and the measurable intrinsic spectral properties of fluorescence polarization, life-time and excited state energy transfer.
For many applications that utilize fluorescent dyes as tracers, it is necessary to chemically react the dye with a biologically active component, such as a cell, tissue, protein, antibody, enzyme, or a biomolecule such as a drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecules, to make a fluorescent composite or to react the dye with natural or synthetic macromolecules or polymers. With these synthetic probes, the biomolecule frequently confers a specificity for a biochemical interaction that is under investigation and the fluorescent dye provides the method for detection and/or quantification of the interaction. Chemically reactive synthetic fluorescent dyes have long been recognized as essential for tracking these interactions. It is often desirable to employ a fluorescent dye which is significantly different from the background fluorescence, such as a protein which has green fluorescence to avoid interference. It is also frequently desirable to employ more than one fluorescent conjugate simultaneously and to quantify the conjugates independently, requiring selective detection of each fluorescent probe. It is also desirable that the dye does not have negative charges or positive charges so that it does not impede entry into cells for detecting the interactions inside the cell. The dyes in common use are limited to a relatively small number of aromatic structures. It is an object of this invention to provide fluorescent tracers which can be used to the green fluorescein background or in conjunction with fluorescein and other commonly used fluorescent probes. It is further an object of this invention to provide dyes with the chemical reactivity necessary for conjugation to the functional groups commonly found in biomolecules, drugs, and natural and synthetic macromolecules or polymers. It is further an object of this invention to provide dyes whose fluorescence has low sensitivity to solution pH, whose composition has high solubility in aqueous medium, and whose structure has no negative or positive charges to allow approach to the cell surface or access to the cell entry.
Virtually all fluorescence microscopes are equipped with excitation sources and filters optimized to excite and detect fluorescein emission. Fluorescein has broad emission in the visible portion of the spectrum beginning at approximately 480 nm, peaking at about 514 nm and decreasing to 10% of the peak intensity at approximately 580 nm (FIGS. 3 and 4). However, the commonly used green fluorescent protein in some cases causes interference as the background noise. There is a need for suitable fluorophores which can shift away from the green emission wavelength region to avoid the interference with fluorescein and with green fluorescent protein. And there is also a recognized need for suitable fluorophores for applications in multi-color microscopy, flow cytometry, immnoassays, and DNA sequencing. Since fluorescein has essentially no fluorescence below 490 nm, there is a clear opportunity to detect suitable fluorophores that have strong emission below this wavelength (FIG. 1). The desirable dyes would have the following properties:                1. A high fluorescence quantum yield with a reasonably narrow emission band at wavelengths sufficiently shorter than that of fluorescein so the longest wavelength components of the dye emission have minimal spectral overlap with the fluorescein emission band.        2. A high absorptivity as measured by extinction coefficient. Preferred are dyes that can be excited with the most intense emission lines of the common excitation sources such as the 365 nm line or longer. Excitation shorter than 365 nm is less desirable since it can result in cell injury or death in applications where fluorescence measurements are performed on living cells. Furthermore, auto-fluorescence of proteins, nucleic acids and other biomolecules present in cells is also increased with shorter wavelength excitation. Use of wavelengths longer than 350 nm also permits use of less expensive glass optics instead of quartz optics.        3. High solubility of the dye and its reactive derivatives in aqueous solution to enhance the utility of the dye for modification of cells, biopolymers, macromolecules and other biomolecules to be used in an aqueous environment.        4. High stability of the dye to excitation light, enhancing the utility of the dye for quantitative measurements and permitting extended illumination time and higher lamp intensities without significant photobleaching.        5. For quantitative measurements, low sensitivity of the emission intensity to properties of the solution is necessary so that the measured signal is proportional only to the absolute quantity of dye present and is independent of environmental effects such as pH, viscosity and polarity.        6. Suitability of the dye for preparation of reactive derivatives of several different types which exhibit reactivity toward a variety of chemically reactive sites.        7. Intrinsically low biological activity or toxicity of the dye.        8. The non-ionic structure of the dye, which avoids undesired electrostatic interactions with other components of the system.        
Pyrene derivatives such as pyrene-1-butyrate is quite lipophilic and insoluble in the aqueous solution. The property of low aqueous solubility hinders the fluorescent labeling of proteins, polysaccharides and other biomolecules, thereby making it practically unusable in consequent biological application. The Cascade Blue (Haugland et al. U.S. Pat. No. 5,132,432), a commercially available sulfonated pyrene derivative, has three negative charges whose anionic character compromises cell membrane-permeability which can only be used extracellularly. A need clearly exists for new fluorescent dyes that have good aqueous solubility and in particular a non-ionic structure that can be used for both intracellular and extracellular detection. The high solubility of the pyrenyloxy sulfonamide dyes, that are the subject of this invention, results in fluorescent derivatives that are highly water soluble. This uncharged nature of the molecule and high aqueous solubility of the dyes enhance their usefulness as fluorescent tracers for hydrophilic environments and increase their suitability for use in applications requiring not only extracellular detection, but also intracellular detection. This solubility property also facilitates the coupling, in aqueous solution, of the fluorescent dye with a protein, drug, or other component of interest.
In conclusion, the dyes which are the subject of this invention exhibit all of the desirable properties described above, namely:                1. Large Stokes' shifts (over 40 nm).        2. Little spectral overlap with fluorescein.        3. High extinction coefficient.        4. Excellent photostability.        5. High water solubility.        6. Low sensitivity to pH.        7. Reactivity with many of the functional groups found in biomolecules.        8. Compatibility with common excitation sources.        9. Uncharged structure useful for extracelluar, and more importantly, intracellular detection.        