Dibenzorhodamine dyes

• US 6,221,606 B1
• Filed: 04/20/2000
• Issued: 04/24/2001
• Est. Priority Date: 11/25/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: The Perkin-Elmer Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Benson, Scott C., Menchen, Steven Michael
• Primary Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US09/556,040
• Time in Patent Office: 369 Days
• Field of Search: 435/6, 536/26.6, 549/382, 544/262, 544/333, 546/33
• US Class Current: 435/6.19
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 311/78 Ring systems having three or more relevant rings
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/02 derived from diarylmethanes
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Dibenzorhodamine compounds having the structure

are disclosed, including nitogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure

including nitrogen- and aryl-substituted forns thereof, and a second intermediate having the structure

including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alky, carboxylic acid, sulfonic acid, —CH₂OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Claims

1. An energy transfer dye comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

a linker for linking the donor dye and the acceptor dye, the linker serving to facilitate the efficient transfer of energy between the donor dye and the acceptor dye;

wherein at least one of the donor dye and acceptor dye is a dibenzorhodamine compound having the structure

including nitrogen- and aryl-substituted forms thereof.

2. The compound of claim 1 comprising a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members;

and/or

a second bridging group which when taken together with the C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring structure having from 4 to 7 members.

3. The compound of claim 2 wherein one or both of the first and second ring structures has five members.

4. The compound of claim 3 wherein the five membered ring structure includes one gem disubstituted carbon.

5. The compound of claim 4 wherein the gem substituents are lower alkyl (C₁-C₈).

6. The compound of claim 4 wherein the gem substituents are methyl.

7. The compound of claim 4 wherein the five membered ring is not aromatic.

8. The compound of claim 4 wherein the five membered ring is substituted with a linking group or a water-solubilizing group.

9. The compound of claim 2 wherein the first and second ring structures are the same.

10. The compound of claim 2 wherein the first and second ring structures are different.

11. The compound of claim 1 comprising a C-7 substituent selected from the group consisting of acetylene, lower alkyl (C₁-C₈), lower alkene (C₂-C₈), cyano, phenyl, heterocyclic aromatic, electron-rich heterocycle, and substituted forms thereof.

12. The compound of claim 11 wherein the C-7 substituent is hydrogen.

13. The compound of claim 11 wherein the C-7 substituent is phenyl having the structure:

including aryl-substituted forms thereof.

14. The compound of claim 13 wherein substituents at any one of positions C-14 to C-18 are selected from the group consisting of chlorine, fluorine, lower alkyl (C₁-C₈), carboxyl, sulfonate, —

CH₂OH, alkoxy, phenoxy, linking group, water solubilizing group, and substituted forms thereof.

15. The compound of claim 13 wherein a substituent at position C-18 is selected from the group consisting of carboxyl and sulfonate.

16. The compound of claim 13 wherein a substituent at position C-18 is carboxyl.

17. The compound of claim 13 wherein a substituent at positions C-14 and C-17 is chlorine.

18. The compound of claim 13 wherein a substituent at positions C-14 to C-17 is chlorine.

19. The compound of claim 13 wherein a substituent at positions C-14 to C-17 is fluorine.

20. The compound of claim 13 wherein a substituent at one of positions C-15 and C-16 is linking group and the other is hydrogen, a substituent at positions C-14 and C-17 is chlorine, and a substituent at position C-18 is carboxyl.

21. The compound of claim 1 comprising one or more nitrogen substituents selected from the group consisting of lower alkyl (C₁-C₈), lower alkene (C₂-C₈), lower alkyne (C₂-C₈), phenyl, benzyl, aryl, heterocycle, polycyclic aromatic, water-solubilizing group, and iring group, including substituted forms thereof.

22. The compound of claim 21 wherein one or more substituents is alkylsulfonate.

23. The compound of claim 21 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, activated ester, pentafluorophenyl ester, maleimide, haloacetyl, iodoacetamide, and N-hydroxysuccinimidyl ester.

24. The compound of claim 21 wherein the water-solubilizing group is carboxylate, sulfonate, sulfate, phosphonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

25. The compound of claim 1 comprising a third bridging group which when taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a third ring structure having from 5 to 7 members;

and/or

a fourth bridging group which when taken together with the C-2-bonded nitrogen and the C-2 and C-3 carbons forms a fourth ring structure having from 5 to 7 members.

26. The compound of claim 25 wherein one or both of the third and fourth ring structures has six members.

27. The compound of claim 26 wherein the six membered ring structure includes one gem disubstituted carbon.

28. The compound of claim 27 wherein the gem substituents are lower alkyl (C₁-C₈).

29. The compound of claim 27 wherein the gem substituents are methyl.

30. The compound of claim 26 wherein the six membered ring is not aromatic.

31. The compound of claim 25 wherein one or both of the third and fourth ring structures has five members.

32. The compound of claim 31 wherein the five membered ring structure includes one gem disubstituted carbon.

33. The compound of claim 32 wherein the gem substituents are lower alkyl (C₁-C₈).

34. The compound of claim 32 wherein the gem substituents are methyl.

35. The compound of claim 1 comprising one or more substituents at one or more of positions C-1, C-3 to C-6, C-8 to C-11, and C-13, selected from the group consisting of fluorine, chlorine, lower alkyl (C₁-C₈), lower alkene (C₂-C₈), lower alkyne (C₂-C₈), sulfate, sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, phenyl, aryl, polycyclic aromatic, heterocycle, water-solubilizing group, and linking group, including substituted forms thereof.

36. The compound of claim 35 wherein the one or more substituents is alkylsulfonate.

37. The compound of claim 1 wherein the substituents at positions C-3 to C-6 and C-8 to C-11 are hydrogen.

38. The compound of claim 37 wherein the C-12-bonded-nitrogen and the C-12 and C-13 carbons form a five membered ring and the C-2-bonded-nitrogen and the C-1 and C-2 carbons form a five membered ring.

39. The compound of claim 1 wherein the acceptor dye is a dibenzorhodamine and the donor dye is a cyanine, a phthalocyanine, a squaraine, a bodipy, a fluorescein, or a dibenzorhodamine dye.

40. The compound of claim 1 wherein the acceptor dye is a dibenzorhodamine and the donor dye is linked to the dibenzorhodamine and to a polynucleotide.

41. The compound of claim 40 wherein the donor dye is linked to the 5′

-terminus of the polynucleotide.

42. The compound of claim 40 wherein the donor dye is linked to the 3′

-terminus of the polynucleotide.

43. The compound of claim 40 wherein the donor dye is linked to a nucleobase of the polynucleotide, wherein if the nucleobase is a purine, the linker is attached at the 8-position, if the nucleobase is a 7-deazapurine, the linker is attached at the 7-position or 8-position, and if the nucleobase is a pyrimidine, the linker is attached at the 5-position.

44. The compound of claim 1 wherein the linker has the structure

wherein

Z₁ is selected from the group consisting of —

NH, sulfur and oxygen;

R₂₁ is a lower alkyl attached to the donor dye;

R₂₂ is a substituent selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond and a fused ring structure which is attached to the carbonyl carbon; and

R₂₈ includes a functional group which attaches the linker to the acceptor dye.

45. The compound of claim 44 wherein the linker has the structure

wherein

Z₂ is selected from the group consisting of —

NH, sulfur and oxygen; and

R₂₉ is a lower alkyl.

46. The compound of claim 44 wherein R₂₂ is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

47. The compound of claim 1 wherein the linker has the structure

48. The compound of claim 1 wherein the linker has the structure

49. The compound of claim 1 wherein the linker has the structure

50. The compound of claim 49 wherein the DONOR is a fluorescein and the ACCEPTOR is a dibenzorhodamine.

51. The compound of claim 1 wherein the linker is attached at one of positions C-15 or C-16 on the C-7 phenyl substituent of the dibenzorhodamine dye.

52. The compound of claim 1 wherein the linker is attached at one of positions C-2-bonded nitrogen or C-12-bonded nitrogen of the dibenzorhodamine dye.