Novel Fluorescent Dyes, Labeled Conjugates and Analytical Methods

Abstract

There are described novel fluorescent dyes, conjugates which include a radical of a dye and a biological or a synthetic moiety and diagnostic and in vivo assays utilizing such conjugates and other products including the dyes and conjugates.

What is claimed is: 
     
         1 . A dye compound represented by the formula 
       
         
           
           
               
               
           
         
         wherein 
         D is selected from —C—R 5 , —C-(A)q-R 6 , E + (G − ), and 
       
       
         
           
           
               
               
           
         
         X is selected from oxygen, sulfur, selenium, N—B, and —C(═O)O—; 
         B is selected from hydrogen, alkyl having from 1 to 10 carbon atoms, aryl, heteroaryl, acyl, aroyl having from 1 to 10 carbon atoms and heteroaroyl having from 1 to 10 carbon atoms); 
         Z is carbon or nitrogen; 
         Z a  is selected from carbon, nitrogen and 
         —C-(A)q-R 6  ; 
         Z b  is selected from carbon, N-alkyl ad N-aryl; 
         A is a divalent linking group having alternating single and multiple bonds; 
         E +  is oxygen or N + —R 7 , where R 7  is hydrogen, cyano, alkyl, linear or branched, preferably having from 1 to 10 carbon atoms, arylalkyl, preferably having from 1 to 10 carbon atoms or heteroarylalkyl, preferably having from 1 to 10 carbon atoms; 
         (G − ) is a counterion; 
         Y is a proton transfer group; 
         Ar is an aromatic or a heteroaromatic moiety; 
         PG is a protective group; 
         R 1 , R 2  and R 3  each independently is selected from hydrogen, an electron withdrawing group and an electron releasing group; or 
         R 1  and R 2  taken together with the carbon atoms to which they are attached represent the carbon atoms necessary to form a substituted or unsubstituted unsaturated carbocycic or heteroaromatic ring; 
         R 4  is selected from hydrogen, an electron withdrawing group and an electron releasing group; 
         R 5  is selected from hydrogen and a 5 or 6 member aromatic or heteroaromatic; 
         or 
         R 2  and R 5  taken together with the carbon atoms to which they are attached represent the carbon atoms necessary to form an unsaturated carbocyclic or heteroaromatic ring; 
         R 6  is 
       
       
         
           
           
               
               
           
         
         Where R 11  is H, alkyl, aryl, heteroaryl, H 2 W or R 13 R 14 ; 
         R 12  is hydrogen, alkyl, aryl or heteroaryl; 
         R 13  is hydrogen; 
         R 11  and R 12  taken together with the nitrogen and oxygen atoms to which they are attached represent the carbon atoms necessary to form a substituted or unsubstituted, saturated or unsaturated ring as shown in the examples below. 
       
       
         
           
           
               
               
           
         
         R 14  is alkyl, aryl or heteroaryl; 
         W is an anion; 
         n is an integer of from 0 to 4; and 
         q is 0 or 1 
         provided that at least Z a  or D is —C-(A)q-R 6 . 
       
     
     
         2 . A dye compound as defined in  claim 1  which is represented by the formula 
       
         
           
           
               
               
           
         
         wherein 
         R 15  is selected from hydrogen, an electron withdrawing group and an electron releasing group; 
         R 16  is selected from hydrogen, an electron withdrawing group and an electron releasing group; 
         Z b  is —C-(A) q —R 6 ; 
         m is 0 or 1; 
         is an integer of from 0 to 4; and 
         E, G, X, Y, Z, R 4 , R 6 , q and n are as defined in  claim 1 . 
       
     
     
         3 . A dye compound as defined in  claim 1  which is represented by the formula 
       
         
           
           
               
               
           
         
         where X and y are as defined in  claim 1   
       
     
     
         4 . A fluorescent conjugate comprising a radical of a dye molecule of  claim 1  and a radical of a biological or a synthetic organic or inorganic moiety 
     
     
         5 . A fluorescent conjugate as defined in  claim 4  wherein said radical of a dye molecule is a radical of a dye molecule of  claim 2 . 
     
     
         6 . A fluorescent conjugate as defined in  claim 4  wherein said radical of a dye molecule is a radical of a dye molecule of  claim 3 . 
     
     
         7 . A fluorescent conjugate as defined in  claim 4  wherein said radical of a dye molecule is attached to said radical of a biological or a synthetic organic or inorganic moiety through a non-conjugated divalent achromophoric linking group. 
     
     
         8 . A fluorescent conjugate as defined in  claim 7  wherein said radical of a dye molecule is attached to a radical of an amino acid or a polymer of amino acids. 
     
     
         9 . A fluorescent conjugate as defined in  claim 7  wherein said radical of a dye molecule is attached to a biological moiety selected from a nucleic acid base, a nucleoside, a nucleotide and a nucleic acid polymer. 
     
     
         10 . A fluorescent conjugate as defined in  claim 7  wherein said biological moiety comprises a nucleic acid polymer having fewer than 50 nucleotides. 
     
     
         11 . A fluorescent conjugate as defined in  claim 7  wherein said radical of a dye molecule is attached to a carbohydrate or lipid moiety. 
     
     
         12 . A fluorescent conjugate as defined in  claim 7  wherein said radical of a dye molecule is attached to a radical of a drug or a toxin. 
     
     
         13 . A diagnostic assay method for determining a component in a biological fluid comprising contacting a biological fluid with a fluorescent conjugate as defined in  claim 7  to produce a detectable change which is a function of a component in said biological fluid; and detecting said change.