Phosphonylated fluorescent dyes and conjugates

• US 20060199955A1
• Filed: 02/21/2006
• Published: 09/07/2006
• Est. Priority Date: 08/13/2004
• Status: Active Grant

• Current Assignee: Elitechgroup MDX LLC
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Lukhtanov, Eugeny, Vorobiev, Alexei
• Granted Patent: US 7,767,834 B2
• Time in Patent Office: Days
• US Class Current: 544/75
• CPC Class Codes: C07F 9/1411 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/5728 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/5765 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/58 Pyridine rings
• CPC Class Codes: C07F 9/6533 Six-membered rings
• CPC Class Codes: C07F 9/65335 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6541 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6547 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/65583 each of the hetero rings containing nitrogen as ring hetero atom
• CPC Class Codes: C07F 9/65586 at least one of the hetero rings does not contain nitrogen as ring hetero atom
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• CPC Class Codes: C07F 9/65842 Cyclic amide derivatives of acids of phosphorus, in which one nitrogen atom belongs to the ring
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution

Abstract

Reagents are provided for the introduction of phosphonate groups into fluorescent dyes. Methods are also provided for preparing dye conjugates.

Claims

1. A fluorescent dye reagent having the formula:

2. A fluorescent dye reagent of claim 1, wherein said fluorescent dye component is selected from the group consisting of coumarins, benzocoumarins, xanthenes, benzo[a]xanthenes, benzo[b]xanthenes, benzo[c]xanthenes, cyanines, acridines, dipyrrometheneboron difluorides, phenoxazines, benzo[a]phenoxazines, benzo[b]phenoxazines and benzo[c]phenoxazines.

3. A fluorescent dye reagent of claim 1, wherein said fluorescent dye component is selected from the group consisting of coumarin dyes, resorufin dyes, dipyrrometheneboron difluoride dyes, ruthenium bipyridyl dyes, energy transfer dyes, thiazole orange dyes, polymethines and N-aryl-1,8-naphthalimide dyes.

4. A fluorescent dye reagent of claim 1, wherein L is C₂-C₈ alkylene.

5. A fluorescent dye reagent of claim 1, wherein N^a is an ammonium ion group having the formula —

N⁺(R^D)₃ wherein each R^D is independently selected from H, (C₁-C₈)alkyl, aryl and aryl(C₁-C₄)alkyl.

6. A fluorescent dye reagent of claim 5, wherein each R^D is H or 9C₁-C₈)alkyl.

7. A fluorescent dye reagent of claim 1, wherein m is 0 and n is 1.

8. A fluorescent dye reagent of claim 1, wherein m is 0 and n is 2.

9. A fluorescent dye reagent of claim 1, wherein m is 1 and n is 1.

10. A fluorescent dye reagent of claim 2, wherein said fluorescent dye component is a xanthene or benzoxanthene dye selected from the group consisting of fluorescein dyes, rhodamine dyes and rhodol dyes.

11. A fluorescent dye reagent of claim 10, having formula I:

12. A fluorescent dye reagent of claim 11, having formula I(a) or I(b):

13. A fluorescent dye reagent of claim 11, having formula Ic:

14. A fluorescent dye reagent of claim 13, selected from the group consisting of

15. A fluorescent dye reagent of claim 1, having formula:

16. A fluorescent dye reagent of claim 1, having formula:

17. A fluorescent dye reagent of claim 1, having the formula:

18. A fluorescent dye reagent of claim 17, wherein Q is a conjugated linking system selected from the group consisting of:

19. A fluorescent dye reagent of claim 1, having the formula:

20. A fluorescent dye reagent of claim 1, selected from compounds 8, 9, 10, 11, 17, 18, 19, 25, 26, 27, 37, 38, 39, 64, 65 and 66.

31. An oligonucleotide probe having an attached fluorescent dye reagent of claim 1, wherein m is 1 and attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on L^f.

32. An oligonucleotide probe having an attached quencher and an attached fluorescent dye reagent of claim 1, wherein m is 1 and attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on L^f.

33. An oligonucleotide probe having an attached quencher, minor groove binding agent, and an attached fluorescent dye reagent of claim 1, wherein m is 1 and attachment of the dye reagent to the oligonucleotide is through a functional group present on L^f.

21. A fluorescent dye reagent having the formula:

Fl-P¹

wherein

Fl is a fluorescent dye component; and

P¹ is functionalized phosphonate group having the formula;

wherein the wavy line indicates the direct attachment to a sp² carbon of said fluorescent dye component;

L¹ is a linking group;

R is a member selected from the group consisting of H, C₁-C₈ alkyl, aryl, arylC₁-C₄ alkyl, -L^a-N^a, and -L^a-NR^AR^B;

wherein L^a is an alkylene linking group, N^a is an ammonium ion group, and each of R^A and R^B is independently selected from the group consisting of H and a labile protecting group; and

P^a is a functional group component selected from the group consisting of a phosphoramidite moiety, a mono-, di- or tri-functional linking group having at least one terminal functional group or protected functional group, a solid support and a reactive group;

and salts thereof.

22. A fluorescent dye reagent of claim 21, wherein said fluorescent dye component is selected from the group consisting of coumarins, benzocoumarins, xanthenes, benzo[a]xanthenes, benzo[b]xanthenes, benzo[c]xanthenes, cyanines, acridines, dipyrrometheneboron difluorides, phenoxazines, benzo[a]phenoxazines, benzo[b]phenoxazines and benzo[c]phenoxazines.

23. A fluorescent dye reagent of claim 21, wherein said fluorescent dye component is selected from the group consisting of coumarin dyes, resorufin dyes, dipyrrometheneboron difluoride dyes, ruthenium bipyridyl dyes, energy transfer dyes, polymethine dyes, thiazole orange dyes and N-aryl-1,8-naphthalimide dyes.

24. A fluorescent dye reagent of claim 21, wherein said fluorescent dye component is a xanthene or benzoxanthene dye selected from the group consisting of fluorescein dyes, rhodamine dyes and rhodol dyes.

25. A fluorescent dye reagent of claim 24, selected from the group consisting of compound 44, 45, 46, 51, 52, 53, 56, 57, 58 and 59.

34. An oligonucleotide probe having an attached fluorescent dye reagent of claim 21, wherein attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on P^a.

35. An oligonucleotide probe having an attached quencher and an attached fluorescent dye reagent of claim 21, wherein attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on P^a.

36. An oligonucleotide probe having an attached quencher, minor groove binding agent, and an attached fluorescent dye reagent of claim 21, wherein attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on P^a.

26. A method of preparing a phosphonate-fluorescent dye derivative, said method comprising contacting a halo-fluorescent dye substrate having at least one halogen atom attached to an aromatic ring carbon atom, with a phosphite reagent under conditions sufficient to remove said halogen atom and covalently attach a phosphonate group to said aromatic ring carbon atom to form said phosphonate-fluorescent dye derivative.

27. A method in accordance with claim 26, wherein said halo-fluorescent dye substrate is selected from the group consisting of a halo coumarins, a halo benzocoumarin, a halo xanthenes, a halo benzo[a]xanthene, a halo benzo[b]xanthene, a halo benzo[c]xanthene, a halo cyanine, a halo acridine, a halo dipyrrometheneboron difluoride, a halo phenoxazine, a halo polymethine, a halo benzo[a]phenoxazine, a halo benzo[b]phenoxazine and a halo benzo[c]phenoxazines.

28. A method in accordance with claim 26, wherein said phosphite reagent is a member selected from the group consisting of diphenyl phosphite, diethyl phosphite and bis(4-(2,2,2-trifluoroacetamido)butyl)phosphite.

29. A method in accordance with claim 26, wherein said phosphite reagent is bis(4-(2,2,2-trifluoroacetamido)butyl)phosphite or compound 41.

30. A phosphonate fluorescent dye derivative prepared according to the method of claim 29.

37. A compound having the formula:

38. A compound of claim 37, wherein R is tertiary butyl group.

39. A compound of claim 37, wherein R is -L-NR^AR^B, and X is —

NR^AR^B.

40. A compound of claim 39, wherein each R^A is H and each R^B is a protecting group selected from the group consisting of —

C(O)CF₃, —

C(O)OtBu, FMOC, phthalimide, monomethoxytrityl and —

C(O)O-benzyl.

41. A compound of claim 37, wherein X is O-dimethoxytrityl.

42. A compound of claim 37, selected from the group consisting of