Fluorescent compounds

Abstract

The present invention relates to fluorescent dyes in general. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis, and/or treatment of disease conditions.

What is claimed is: 
     
         1 - 65 . (canceled) 
     
     
         66 . A compound of Formula III: 
       
         
           
           
               
               
           
         
         wherein 
         Z is —H, alkyl, —CF 3 , —CN or a radical of the formula: 
       
       
         
           
           
               
               
           
         
         X 1  is ═O, ═NH 2   +  or ═NR 6 R 7   + ; 
         X 2  is —OH, NH 2 , or —NR 8 R 9 ; 
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are each independently (R) p -(L) q -; 
         each R of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  is independently i) a reactive group capable of forming a covalent bond upon reacting with a reaction substrate; ii) a water soluble polymer group; iii) an alkyl group, a trifluoroalkyl group, a halogen group, a sulfonate group or a sulfonamido group; or iv) —H; 
         each L of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  is independently a linking moiety formed of one or more chemical bonds and containing about 1-100 atoms; 
         each p of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  is independently an integer ranging from 1 to 20; 
         each q of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  is independently an integer ranging from 0 to 20; 
         a, b, c, d and e are independently 0, 1, 2, 3 or 4; 
         at least one R of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  is a reactive moiety; and 
         at least one R of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  is a water soluble polymer group. 
       
     
     
         67 . The compound of  claim 66  wherein at least one of R 6 , R 7 , R 8  or R 9  is combined with a neighboring R 1  or R 2  and any intervening atoms in a ring to which the neighboring R 1  or R 2  is attached, to form a 5- or 6-membered ring which is unsubstituted or substituted by one or more (R) p -(L) q -. 
     
     
         68 . (canceled) 
     
     
         69 . (canceled) 
     
     
         70 . The compound of  claim 66 , wherein X 1  is ═NH 2   +  or ═O. 
     
     
         71 . (canceled) 
     
     
         72 . The compound of  claim 66 , wherein X 2  is —OH or —NH 2 . 
     
     
         73 . (canceled) 
     
     
         74 . (canceled) 
     
     
         75 . The compound of  claim 66 , wherein a maximal fluorescence emission wavelength of the compound ranges from about 470 to 800 nm. 
     
     
         76 . (canceled) 
     
     
         77 . A kit comprising:
 i) the compound of  claim 66 ; ii) a buffer; iii) materials or devices for purifying conjugation products; and iv) instructions instructing use of the compound.   
     
     
         78 . A biomolecule comprising the compound of  claim 66 , wherein the at least one reactive moiety of the compound has undergone a reaction which attaches the compound to the biomolecule. 
     
     
         79 . The biomolecule of  claim 78  wherein the biomolecule comprises a polynucleotide or a polypeptide. 
     
     
         80 . (canceled) 
     
     
         81 . (canceled) 
     
     
         82 . The biomolecule of  claim 79 , wherein the polypeptide is a whole immunoglobulin or a Fab fragment. 
     
     
         83 . (canceled) 
     
     
         84 . An immunoglobin comprising the compound of  claim 66 , wherein the at least one reactive moiety of the compound has undergone a reaction which attaches the compound to the immunoglobin, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell. 
     
     
         85 . (canceled) 
     
     
         86 . A method of preparing a labeled biomolecule comprising reacting the compound of  claim 66  and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule. 
     
     
         87 . The method of  claim 86 , wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof. 
     
     
         88 . (canceled) 
     
     
         89 . A method for labeling a cell within a population of cells whereby the cell is differentially labeled relative to neighboring cells within the population, the method comprising contacting the cell with the biomolecule of  claim 78 , wherein the biomolecule comprises a targeting moiety that binds to a binding partner that is indicative of the cell, and thereby differentially labeling the cell relative to neighboring cells within the population. 
     
     
         90 . The method of  claim 89 , further comprising the step of imaging the cell, the imaging step comprising:
 i) directing exciting wavelength to the cell; and   ii) detecting emitted fluorescence from the cell.   
     
     
         91 . (canceled) 
     
     
         92 . (canceled) 
     
     
         93 . An immunoglobulin labeled with a fluorescent compound comprising a polyalkylene oxide and a fluorophore that has an absorption maximal wavelength equal to or greater than 685 nm, wherein the fluorescent compound is the compound of  claim 66 . 
     
     
         94 . (canceled) 
     
     
         95 . The immunoglobulin of  claim 93 , wherein the immunoglobulin retains binding specificity to a target upon conjugation to the fluorescent compound. 
     
     
         96 . The immunoglobulin of  claim 95 , wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell. 
     
     
         97 . (canceled) 
     
     
         98 . (canceled) 
     
     
         99 . A polypeptide labeled with a fluorescent compound, the polypeptide exhibiting a serum half-life no shorter than that of a corresponding polypeptide that lacks the fluorescent compound, wherein the fluorescent compound is the compound of  claim 66 . 
     
     
         100 . A method of labeling a polypeptide comprising: forming a complex that comprises the polypeptide and a binding agent, wherein the binding agent comprises the compound of  claim 66 , wherein the at least one reactive moiety of the compound has undergone a reaction which attaches the compound to the binding agent. 
     
     
         101 .- 104 . (canceled) 
     
     
         105 . The method of  claim 100 , wherein the complex yields a signal to noise ratio greater than about 100, wherein the signal to noise ratio is calculated by the formula: (fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent). 
     
     
         106 .- 113 . (canceled)