Modified carbocyanine dyes and their conjugates

Abstract

Chemically reactive carbocyanine dyes incorporating an indolium ring moiety that is substituted at the 3-position by a reactive group or by a conjugated substance, and their uses, are described. Conjugation through this position results in spectral properties that are uniformly superior to those of conjugates of spectrally similar dyes wherein attachment is at a different position. The invention includes derivative compounds having one or more benzo nitrogens.

What is claimed is: 
     
         1 . A compound having the formula: 
       
         
           
           
               
               
           
         
       
       and its salts, wherein
 R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
 R 4  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; or 
 R 3  and R 4  taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by a group comprising an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
 R 2  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 6  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 7  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 8  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 9  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; or
 a member independently selected from 
 R 6  in combination with R 7 ; 
 R 7  in combination with R 8 ; and 
 R 8  in combination with R 9 ; 
 together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR 1  wherein R 1  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxyl; 
 
 n is 0, 1, 2 or 3; 
 W represents the atoms necessary to form one to two fused aromatic rings having 5 or 6 atoms in each ring, wherein said W atoms are selected from the group consisting of —CH, —C, —CR 1′ , and —N(R 12 ) β ′, where βp′ is 0 or 1, but no more than one of said atoms in W is —N(R 12 ) β ′, and each R 1′  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxy;
 δ is 0 or 1, and δ+β′=1; 
 
 R 12  is C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 13  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium; 
 R 14  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium, 
 or R 13  and R 14  taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted. 
 
     
     
         2 . The compound according to  claim 1 , wherein R 3  comprises a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide. 
     
     
         3 . The compound according to  claim 1 , wherein R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group. 
     
     
         4 . The compound according to  claim 1 , wherein R 3  comprises an antibody or fragment thereof, a blood component protein, a blood vessel proliferation inhibition factor, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a peptide toxin, a phospholipid-binding protein, or a structural protein. 
     
     
         5 . The compound according to  claim 1 , wherein the atoms of W are —CH, —C, —CR 1 , and —N(R 12 ) β ′, where β′ is 1, but no more than one of said atoms in W is —N(R 12 ) β ′, and each R 1′  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12 dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxy; 
     
     
         6 . A compound having the formula: 
       
         
           
           
               
               
           
         
         and its salts, wherein 
         R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
         R 4  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; or 
         R 3  and R 4  taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by a group comprising an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
         R 2  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
         R 6  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
         R 7  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
         R 8  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
         R 9  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
         R 16  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
         R 17  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
         R 18  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; or
 a member independently selected from 
 R 6  in combination with R 7 ; 
 R 7  in combination with R 8 ; 
 R 8  in combination with R 9 ; 
 R 16  in combination with R 17 ; and 
 R 17  in combination with R 18 ; 
 together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR 1  wherein R 1  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxyl; 
 
         n is 0, 1, 2 or 3; 
         R 12  is C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
         R 13  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium; 
         R 14  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium, 
         or R 13  and R 14  taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted. 
       
     
     
         7 . The compound according to  claim 6 , wherein R 3  comprises a carboxylic acid, a succinimidyl ester of a carboxylic acid, hydrazide, a maleimide, an antibody or fragment thereof, a fluorescent protein, a lectin, a nucleotide, an oligonucleotide, a peptide, protein, a small-molecule drug, or a tyramide. 
     
     
         8 . The compound according to  claim 6 , wherein
 R 4 , R 13  and R 14  are each methyl;   R 6 , R 8 , R 9 , R 16 , R 17  and R 18  are each H;   R 7  is sulfo;   R 2  and R 12  are independently methyl or sulfopropyl; and   n is 2.   
     
     
         9 . A method of staining a biological sample, comprising:
 combining a dye solution comprising a compound having the formula:   
       
         
           
           
               
               
           
         
       
       and its salts, wherein
 R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
 R 4  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; or 
 R 3  and R 4  taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by a group comprising an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
 R 2  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 6  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 7  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 8  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 9  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; or
 a member independently selected from 
 R 6  in combination with R 7 ; 
 R 7  in combination with R 8 ; and 
 R 8  in combination with R 9 ; 
 together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR 1  wherein R 1  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxyl; 
 
 n is 0, 1, 2 or 3; 
 W represents the atoms necessary to form one to two fused aromatic rings having 5 or 6 atoms in each ring, wherein said W atoms are selected from the group consisting of —CH, —C, —CR 1′ , and —N(R 12 ) β ′, where β′ is 0 or 1, but no more than one of said atoms in W is —N(R 12 ) β ′, and each R 1′  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxy;
 δ is 0 or 1, and δ+β′=1; 
 
 R 12  is C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 13  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium; 
 R 14  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium,
 or R 13  and R 14  taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted; 
 
 with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions. 
 
     
     
         10 . The method according to  claim 9 , wherein R 3  comprises a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide. 
     
     
         11 . The method according to  claim 9 , wherein R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group. 
     
     
         12 . The method according to  claim 9 , wherein R 3  comprises an antibody or fragment thereof, a blood component protein, a blood vessel proliferation inhibition factor, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a peptide toxin, a phospholipid-binding protein, or a structural protein. 
     
     
         13 . The method according to  claim 9 , wherein the sample comprises cells, proteins, or nucleic acid polymers. 
     
     
         14 . The method according to  claim 9 , wherein the sample is contain in or on a solid or semi-solid matrix this a membranem an elctrophoretic gel, silicon chip, a glass slide, a microwell plate or a microfluidic chip. 
     
     
         15 . A method for forming a dye-conjugate of a protein, peptide or a nucleic acid polymer, said method comprising:
 combining a dye solution comprising a compound of the formula   
       
         
           
           
               
               
           
         
         and its salts, wherein
 R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group; 
 R 4  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; or 
 R 3  and R 4  taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group; 
 R 2  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 6  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 7  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 8  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 9  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; or
 a member independently selected from 
 R 6  in combination with R 7 ; 
 R 7  in combination with R 8 ; and 
 R 8  in combination with R 9 ; 
 together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR 1  wherein R 1  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxyl; 
 
 n is 0, 1, 2 or 3; 
 W represents the atoms necessary to form one to two fused aromatic rings having 5 or 6 atoms in each ring, wherein said W atoms are selected from the group consisting of —CH, —C, —CR 1′ , and —N(R 12 ) β ′, where β′ is 0 or 1, but no more than one of said atoms in W is —N(R 12 ) β ′, and each R 1′  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxy;
 δ is 0 or 1, and δ+β′=1; 
 
 R 12  is C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 13  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium; 
 
         R 14  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium,
 or R 13  and R 14  taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted; 
 
         with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed. 
       
     
     
         16 . The method according to  claim 15 , wherein R 3  comprises a carboxylic acid, an amine, an activated ester of a carboxylic acid, a halotriazine, a hydrazine, a maleimide, a reactive platinum complex. 
     
     
         17 . A kit for fluorescent labeling of a biological sample, comprising:
 a dye solution comprising a compound having the formula:   
       
         
           
           
               
               
           
         
         and its salts, wherein
 R 3  comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
 R 4  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; or 
 R 3  and R 4  taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by a group comprising an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, a photoactivatable group, a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide; 
 R 2  is a C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 6  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 7  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 8  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; 
 R 9  is H, amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl; or
 a member independently selected from 
 R 6  in combination with R 7 ; 
 R 7  in combination with R 8 ; and 
 R 8  in combination with R 9 ; 
 together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR 1  wherein R 1  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, and C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxyl; 
 
 n is 0, 1, 2 or 3; 
 W represents the atoms necessary to form one to two fused aromatic rings having 5 or 6 atoms in each ring, wherein said W atoms are selected from the group consisting of —CH, —C, —CR 1 , and —N(R 12 ) β ′, where β′ is 0 or 1, but no more than one of said atoms in W is —N(R 12 ) β ′, and each R 1′  is amino, sulfo, trifluoromethyl, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, or C 2 -C 12  dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxy;
 δ is 0 or 1, and δ+β′=1; 
 
 R 12  is C 1 -C 22  alkyl or C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, or C 3 -C 18  trialkylammonium; 
 R 13  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium; 
 R 14  is a C 1 -C 22  alkyl and C 7 -C 22  arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from the group consisting of N, O and S, and each alkyl portion of which is independently and optionally substituted one or more times by substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, and C 3 -C 18  trialkylammonium, 
 or R 13  and R 14  taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted; and 
 a buffer.