Intermediates and the synthesis of modified carbocyanine dyes and their conjugates

1. A compound having the formula:

and its salts, wherein

R 3 is -L-R x ; or -L-S C ;

R 4 is -L-R x ; or -L-S C , 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 -L-R x ; or -L-S C ;

L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O, and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds;

R x is a reactive group that is 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;

S c is a conjugated substance that is an antibody, an amino acid, an avidin or streptavidin, a biotin, a blood component protein, a blood vessel proliferation inhibition factor, a dextran, an enzyme, an enzyme inhibitor, a hormone, an lgG-binding protein, an on-complexing moiety, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phospholipid-binding protein, a small-molecule drug, a structural protein, or a tyramide;

Y represents the atoms necessary to form an aromatic ring having 6 atoms in the ring, which atoms are selected from —CH, —C, and —CR 1 , and each R 1 is independently -L-R x , -L-S c , 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, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

R 2 is -L-R x , -L-S c , 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; and

α is 0 or 1.

2. The compound according to claim 1 , wherein the atoms of Y are —CH or —CR 1 and R 1 is C 1 -C 6 alkyl optionally substituted by sulfo.

3. The compound according to claim 1 , wherein R 1 is sulfo or sulfoalkyl.

4. The compound according to claim 1 , wherein

R 2 is alkyl or sulfoalkyl;

R 3 is carboxyalkyl; and

R 4 is alkyl.

5. A compound having the formula:

and its salts, wherein

R 3 is -L-R x ;

R 4 is a C 1 -C 22 alkyl; or

R 3 and R 4 taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by -L-R x ;

L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O, and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds;

R x is a reactive group that is an amine or a carboxylic acid;

Y represents the atoms necessary to form an aromatic ring having 6 atoms in the ring, which atoms are selected from —CH, —C, and —CR 1 , and each R 1 is independently 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, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

R 2 is an alkyl or sulfoalkyl; and

α is 0 or 1.

6. A compound having the formula:

and its salts, wherein

R 3 is -L-R x ; or -L-S c ;

R 4 is a C 1 -C 22 alkyl, 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 -L-R x or -L-S c ; and

L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O, and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds;

R x is a reactive group that is 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;

S c is a conjugated substance that is an antibody, an amino acid, an avidin or streptavidin, a biotin, a blood component protein, a blood vessel proliferation inhibition factor, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, an ion-complexing moiety, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a non -biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phospholipid-binding protein, a small-molecule drug, a structural protein, or a tyramide;

R 6 through R 9 are independently H, -L-R x , -L-S c , 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, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy; and

R 2 is a C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted by sulfo.

7. The compound according to claim 6 , having the structure:

and its salts, wherein

R 2 is an alkyl or sulfoalkyl;

R 7 is an alkyl or sulfo;

R 4 is a C 1 -C 22 alkyl; and

R 3 is -L-R x ;

wherein L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O, and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds; and

R x is a reactive group that is an amine or a carboxylic acid.