Cyanine dye compounds

• US 7,446,202 B2
• Filed: 12/06/2004
• Issued: 11/04/2008
• Est. Priority Date: 12/05/2003
• Status: Active Grant

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Huang, Shih-Jung, Yue, Stephen, Dallwig, Jason Alfred, Thomas, Gerald Alan, Hagen, David Carl
• Primary Examiner(s): SEAMAN, D MARGARET M
• Application Number:
  US11/005,860
• Publication Number:
  US 20050214810A1
• Time in Patent Office: 1,429 Days
• Field of Search: 546/153, 546/157, 546/159
• US Class Current: 546/159
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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

wherein W forms one or two fused 5- or 6-membered aromatic rings, a has a value of 0 or 1, n has a value of 0, or 1, X is O, S, or Se, and D is a pyridinium, or quinolinium moiety, provided that the compound has at least one negatively charged substituent.

Claims

1. A compound having the formula:

2. The compound according to claim 1, wherein the compound exhibits a fluorescence enhancement when non-covalently associated with a nucleic acid.

3. The compound according to claim 2, wherein the fluorescence enhancement is greater when the nucleic acid is DNA than when the nucleic acid is RNA.

4. The compound according to claim 1, wherein the negatively charged moiety 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.

5. The compound acccording to claim 1, wherein W comprises —

C or —

CR¹; and

wherein each R¹ is independently hydrogen, a reactive group, a carrier molecule, a solid support, carboxy, sulfo, phosphate, phosphonate, amino, hydroxy, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, trifluoromethyl, halogen, substituted alkyl, unsubstituted alkyl, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted dialkylamino, or unsubstituted dialkylamino.

6. The compound according to claim 1, wherein W is a substituted or unsubstituted phenyl ring.

7. The compound according to claim 1, wherein n is 0.

8. A compound according to claim 5, wherein D has the formula

9. The compound according to claim 8, wherein at least one of R³ and R⁴ is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkoxy, alkylthio, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.

10. The compound according to claim 8, wherein R³ is 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.

11. The compound according to claim 8, wherein R³ is 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.

12. The compound according to claim 8, having the formula

13. The compound according to claim 12, wherein R⁴ is hydrogen, alkyl, —

(CH₂)_a-aryl, or —

(CH₂)_a-heteroaryl, wherein a is an integer from 0 to about 6.

14. The compound according to claim 12, wherein R⁴ is an alkyl, phenyl, benzyl, alkylthio, indolyl, imidazolyl, or thiazolyl.

15. The compound according to claim 12, wherein at least one of R¹ and R² comprises a negatively charged substituent.

16. The compound according to claim 12, 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.

17. The compound according to claim 1, having the formula

18. The compound according to claim 12, 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.

19. The compound according to claim 12, 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.

20. The compound according to claim 12, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

21. The compound according to claim 12, 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.

22. The compound according to claim 12, 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.

23. The compound according to claim 12, 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.

24. The compound according to claim 12, 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.