Fluorescent dye composition and target labeled therewith

• US 7,553,959 B2
• Filed: 01/23/2004
• Issued: 06/30/2009
• Est. Priority Date: 03/12/2002
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Rabbani, Elazar
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/764,417
• Publication Number:
  US 20040192893A1
• Time in Patent Office: 1,985 Days
• Field of Search: 536/23.1, 536/26.6, 546/1, 546/10, 546/26, 435/6
• US Class Current: 536/26.6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: C09B 47/00 Porphines; Azaporphines non-dyeing compounds C07D487/22
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.

Claims

1. A dye composition of the formula

R—

Fluorescent Dye

wherein R comprises a reactive group covalently linked to said Fluorescent Dye wherein said Fluorescent Dye comprises a combination of two or more members selected from;

a) unsaturated aliphatic groups comprising an alkene or alkyne;

b) unsaturated heterocyclic groups;

c) aromatic groups;

and wherein R provides a conjugated system or an electron delocalized system with said Fluorescent Dye and said composition optionally comprises a charged or polar R′

group and wherein said composition further comprises a reactive group R_x attached to either said fluorescent dye, said R group or said polar R′

group, wherein said reactive group R_x comprises sulfhydryl, hydroxyl, amine, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonylhalide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde.

2. The dye composition of claim 1, wherein said aromatic groups comprise a phenyl group, an aryl group or an aromatic heterocyclic group.

3. The dye composition of claim 2, wherein said unsaturated aliphatic groups or aromatic groups are substituted.

4. The dye composition of claim 3, wherein said substituted unsaturated aliphatic groups or substituted aromatic groups comprise alkyl groups, aryl groups, alkoxy groups, phenoxy groups, amines, amino groups, amido groups, carboxy groups, nitrates, nitrites, sulfonates, sulfhydryl groups or phosphates.

5. The dye composition of claim 3, wherein said substituted aromatic groups comprise a fused ring structure.

6. The dye composition of claim 5, wherein said fused ring structure is a naphthalene, anthracene or a phenanthrene.

7. The dye composition of claim 1, wherein said combination comprises two members of the same group or of different groups.

8. The dye composition of claim 7, wherein said different groups comprise

an unsaturated aliphatic group (a) and an unsaturated heterocyclic group (b);

an unsaturated aliphatic group (a) and an aromatic group (c);

or

an unsaturated heterocyclic group (b) and an aromatic group (c).

9. The dye composition of claim 1, wherein said fluorescent dye comprises an anthracene, a xanthene, a cyanine, a porphyrin, a coumarin or a composite dye.

10. The dye composition of claim 1, wherein said charged or polar R′

group increases aqueous solubility of said composition.

11. The dye composition of claim 1, further comprising a linker arm attaching said reactive group R_x to said fluorescent dye, said R group or said R′

group.

12. A dye composition of the formula

R—

Fluorescent Dye

wherein R comprises a reactive group covalently linked to said Fluorescent Dye wherein said Fluorescent Dye comprises a combination of two or more members selected from;

a) unsaturated aliphatic groups;

b) unsaturated heterocyclic groups;

c) aromatic groups;

and wherein R provides a conjugated system or an electron delocalized system with said Fluorescent Dye and wherein said reactive group R comprises an alkene group, an alkyne group, a halogenated compound or a metallo-organic compound, said R group having the ability to form a carbon-carbon linkage with a target.

13. A labeled target having the structure

[R]—

Dye target

wherein said Dye is a fluorescent dye, wherein R comprises a reactive group covalently linked to said Dye, and wherein R comprises two or more members selected from;

a) unsaturated aliphatic groups;

b) unsaturated heterocyclic groups;

c) aromatic groups;

wherein R provides a conjugated system or an electron delocalized system with said Dye, and wherein said target is covalently attached to said Dye or said R.

14. The labeled target of claim 13, wherein said unsaturated aliphatic groups comprise an alkene or an alkyne.

15. The labeled target of claim 13, wherein said aromatic groups comprise a phenyl group, an aryl group or an aromatic heterocyclic group.

16. The labeled target of claims 14 or 15, wherein said unsaturated aliphatic groups or aromatic groups are substituted.

17. The labeled target of claim 13, wherein said substituted unsaturated aliphatic groups or substituted aromatic groups comprise alkyl groups, aryl groups, alkoxy groups, phenoxy groups, amines, amino groups, amido groups, carboxy groups, nitrates, nitrites, sulfonates, sulfhydryl groups or phosphates.

18. The labeled target of claim 16, wherein said substituted aromatic groups comprise a fused ring structure.

19. The labeled target of claim 18, wherein said fused ring structure is a naphthalene, anthracene or a phenanthrene.

20. The labeled target of claim 13, wherein said combination comprises two members of the same group or of different groups.

21. The labeled target of claim 20, wherein said different groups comprise

an unsaturated aliphatic group (a) and an unsaturated heterocyclic group (b);

an unsaturated aliphatic group (a) and an aromatic group (c);

or

an unsaturated heterocyclic group (b) and an aromatic group (c).

22. The labeled target of claim 13, wherein said fluorescent dye comprises an anthracene, a xanthene, a cyanine, a porphyrin, a coumarin or a composite dye.

23. The labeled target of claim 13, further comprising a charged or polar R′

group.

24. The labeled target of claim 13, wherein said charged or polar R′

group increases aqueous solubility of said composition.

25. The labeled target of claim 23, further comprising a linker arm attaching said target to said fluorescent dye, said R group or said R′

group.

26. The labeled target of claim 13, wherein said target comprises a protein, a peptide, a nucleic acid, a nucleotide or a nucleotide analog, a receptor, a natural or synthetic drug, a synthetic oligomer, a synthetic polymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, an antigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- or polysaccharide.

27. The labeled target of claim 26, wherein said ligand comprises biotin, iminobiotin, digoxygenin or fluorescein, and the dye comprises a fluorescent dye.