MODIFIED CARBOCYANINE DYES AND THEIR CONJUGATES
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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.
128 Citations
17 Claims
- 1. A compound having the formula:
- 6. A compound having the formula:
-
9. A method of staining a biological sample, comprising:
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combining 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;
orR3 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;
R3 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;
ora member independently selected from R6 in combination with R7;
R7 in combination with R3; 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;
with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions. - View Dependent Claims (10, 11, 12, 13, 14)
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15. A method for forming a dye-conjugate of a protein, peptide or a nucleic acid polymer, said method comprising:
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combining a dye solution comprising a compound of 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, or a photoactivatable group;
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;
orR3 and R4 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;
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;
ora member independently selected from R6 in combination with R7;
R7 in combination with R3; 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;
with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed. - View Dependent Claims (16)
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17. A kit for fluorescent labeling of a biological sample, comprising:
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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;
orR3 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;
ora member independently selected from R6 in combination with R7;
R7 in combination with R3; and
R3 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.
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Specification