Modified carbocyanine dyes and their conjugates

A compound having the formula:

• and its salts, wherein
• R3 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;
• R4 is a C1-C22 alkyl or C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, or C2-C12 dialkylamino, or C3-C18 trialkylammonium;
• or
• R3 and R4 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;
• R2 is a C1-C22 alkyl or C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, or C3-C18 trialkylammonium;
• R6 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R7 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R8 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R9 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R16 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R17 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R18 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 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
• R6 in combination with R7;
• R7 in combination with R8;
• R8 in combination with R9;
• R16 in combination with R17;
• and
• R17 in combination with R18;
• together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR1 wherein R1 is amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 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;
• Ru is C1-C22 alkyl or C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, or C3-C18 trialkylammonium;
• R13 is a C1-C22 alkyl and C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, and C3-C18 trialkylammonium;
• R14 is a C1-C22 alkyl and C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, and C3-C18 trialkylammonium,
• or R13 and R14 taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted.

A kit for fluorescent labeling of a biological sample, comprising:

• a dye solution comprising a compound having the formula:
• and its salts, wherein
• R3 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;
• R4 is a C1-C22 alkyl or C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, or C2-C12 dialkylamino, or C3-C18 trialkylammonium;
• or
• R3 and R4 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;
• R2 is a C1-C22 alkyl or C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, or C3-C18 trialkylammonium;
• R6 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R7 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R8 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 dialkylamino, each alkyl portion of which is optionally further substituted by substituents selected from the group consisting of carboxy, sulfo, amino, or hydroxyl;
• R9 is H, amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 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
• R6 in combination with R7;
• R7 in combination with R8;
• and
• R8 in combination with R9;
• together with the atoms to which they are joined, form an aromatic ring comprising —CH or —CR1 wherein R1 is amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C2-C12 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, —CR1′, and —N(R12)β′, where β′ is 0 or 1, but no more than one of said atoms in W is —N(R12)β′, and each R1′ is amino, sulfo, trifluoromethyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, or C2-C12 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;
• R12 is C1-C22 alkyl or C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, or C3-C18 trialkylammonium;
• R13 is a C1-C22 alkyl and C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, and C3-C18 trialkylammonium;
• R14 is a C1-C22 alkyl and C7-C22 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, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, and C3-C18 trialkylammonium,
• or R13 and R14 taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted;
• and
• a buffer.