Cyanine dye compounds
First Claim
Patent Images
1. A compound having the formula:
- wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.
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Abstract
Cyanine dye compounds having a negatively charged substituent that are nucleic acid stains, particularly for fluorescent staining of DNA, including compounds having the formula
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Citations
59 Claims
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1. A compound having the formula:
-
wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
wherein R3 is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl; - unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;
R4 is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;
unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;
R5 is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;
unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;
R6 is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;
unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;
ora member selected from R5 in combination with R6 R4 in combination with R5;
R4 in combination with R3;
R4 in combination with R6;
R3 in combination with R6 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered aryl or a substituted 5-, 6- or 7-membered aryl.
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10. The compound according to claim 9, wherein at least one of R3 and R4is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkoxy, alkylthio, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.
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11. The compound according to claim 9, wherein R3is alkyl, substituted alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, alkylthio, substituted alkylthio, indolyl, imidazolyl, or thiazolyl;
- and,
R4 is alkyl, substituted alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, alkylthio, substituted alkylthio, indolyl, imidazolyl, or thiazolyl.
- and,
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12. The compound according to claim 9, wherein R3is hydrogen, alkyl, substituted alkyl, —
- (CH2)a-aryl or —
(CH2)a-heteroaryl; and
,R4 is hydrogen, alkyl, substituted alkyl, —
(CH2)a-aryl or —
(CH2)a-heteroaryl;
wherein a is an integer from 0 to about 6.
- (CH2)a-aryl or —
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13. The compound according to claim 1, having the formula
wherein each R1a is independently hydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, fused benzene, substituted fused benzene, trifluomethyl, halogen, reactive group, solid support or carrier molecule; -
each R1b is independently hydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, fused benzene, substituted fused benzene, trifluomethyl, halogen, reactive group, solid support or carrier molecule;
t is an integer from 1 to 4;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
n=0 or 1;
R3 is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;
unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule; and
R4 is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;
unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule.
-
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14. The compound according to claim 13, wherein R4 is hydrogen, alkyl, —
- (CH2)a-aryl, or —
(CH2)a-heteroaryl, wherein a is an integer from 0 to about 6.
- (CH2)a-aryl, or —
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15. The compound according to claim 13, wherein R4 is an alkyl, phenyl, benzyl, alkylthio, indolyl, imidazolyl, or thiazolyl.
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16. The compound according to claim 13, wherein at least one of R1 and R2 comprises a negatively charged substituent.
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17. The compound according to claim 13, wherein at least one R1 and R2 is sulfo, carboxy, phosphate, phosphonate, an alkyl group substituted by sulfo, an alkyl group substituted by a carboxy, an alkyl group substituted by phosphate, or an alkyl group substituted by phosphonate.
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18. The compound according to claim 1, having the formula
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19. The compound according to claim 1, wherein the reactive group, solid support and carrier molecule comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;
- and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.
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20. The compound according to claim 1, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group.
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21. The compound according to claim 1, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.
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22. The compound according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.
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23. The compound according to claim 1, wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.
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24. The compound according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.
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25. The compound according to claim 1, wherein the solid support is sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.
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26. A composition of matter comprising a nucleic acid reporter molecule and a sample, wherein the nucleic acid reporter molecule has the formula:
-
wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH. - View Dependent Claims (27, 28, 33, 34)
-
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29. A complex comprising a nucleic acid reporter molecule non-covalently associated with a nucleic acid molecule, wherein the nucleic acid molecule has the formula:
-
wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH. - View Dependent Claims (30, 31, 32)
-
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35. A method for detecting the presence or absence of nucleic acid in a sample, wherein the method comprises:
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a. combining a nucleic acid reporter molecule with the sample to prepare a labeling mixture, wherein the nucleic acid reporter molecule is according to the formula;
wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH;
b. incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule to associate with nucleic acid in the sample to form an incubated mixture;
c. illuminating the incubated sample with an appropriate wavelength to form an illuminated mixture; and
,d. observing the illuminated mixture whereby the presence or absence of the nucleic acid in a sample is detected. - View Dependent Claims (36, 37, 38, 39, 40)
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41. A method for detecting DNA in the presence of RNA, wherein the method comprises the steps:
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a. combining a nucleic acid reporter molecule with a sample to prepare a labeling mixture, wherein the nucleic acid reporter molecule has a DNA/RNA ratio of fluorescence enhancement greater than about one wherein the nucleic acid reporter molecule is according to the formula wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH;
b. incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule to associate with DNA in the sample to form an incubated mixture;
c. illuminating the incubated mixture with an appropriate wavelength to form an illuminated mixture; and
,d. observing the illuminated mixture whereby the DNA is detected in the presence of RNA. - View Dependent Claims (42, 43, 44, 45)
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46. A kit for detecting nucleic acid in a sample, wherein the kit comprises a nucleic acid reporter molecule according to the formula:
wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH. - View Dependent Claims (47, 49, 51, 52, 53, 54, 56, 57, 58, 59)
- 48. The kit according to claim 48, further comprising instructions for quantitating DNA from about 1 ng to about 2000 ng.
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55. A staining solution comprising a nucleic acid reporter molecule and a detergent, wherein the nucleic acid reporter molecule has the formula:
wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;
R2 is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;
α
is 0 or 1;
n is 0 or 1;
X is O, S, or Se;
D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;
with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.
Specification