Methods of labelling polynucleotides with dibenzorhodamine dyes

• US 7,833,756 B2
• Filed: 07/08/2008
• Issued: 11/16/2010
• Est. Priority Date: 11/25/1997
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Benson, Scott C., Menchen, Steven Michael
• Primary Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US12/169,624
• Publication Number:
  US 20090068751A1
• Time in Patent Office: 861 Days
• Field of Search: None
• US Class Current: 435/91.1
• 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 nitrogen- 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 forms 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 alkyl, 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. A method of identifying polynucleotide classes comprising the steps of:

providing multiple classes of polynucleotides distributed among locations in a spatially addressable hybridization array;

wherein a first class of polynucleotide comprises a dibenzorhodamine dye having a structure of the formula;

2. The method of claim 1 wherein the dibenzorhodamine dye comprises 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 method of claim 2 wherein one or both of the first and second ring structures has five members.

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

5. The method of claim 4 wherein the gem substituents are lower alkyl.

6. The method of claim 5 wherein the gem substituents are methyl.

7. The method of claim 3 wherein the five membered ring is not aromatic.

8. The method of claim 2 wherein the first and second ring structures are the same.

9. The method of claim 2 wherein the first and second ring structures are different.

10. The method of claim 1 wherein the dibenzorhodamine dye comprising one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof.

11. The method of claim 10 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.

12. The method of claim 10 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.

13. The method of claim 10 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.

14. The method of claim 10 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl, or iodoacetamide.

15. The method of claim 10 wherein the linking group is carboxylate.

16. The method of claim 10 wherein the nitrogen substituents are water-solubilizing group.

17. The method of claim 10 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

18. The method of claim 1 wherein the dibenzorhodamine dye and the polynucleotide are connected by a linkage at a position on the polynucleotide selected from the 8-position of a purine nucleobase, the 7- or 8-position of a deazapurine nucleobase, the 5-position of a pyrimidine nucleus, the 5′

terminus, the 3′

terminus, and the phosphodiester backbone.

19. The method of claim 18 wherein the linkage is attached to the linking group substituent of the dibenzorhodamine dye.

20. The method of claim 18 wherein the linkage is an acetylenic amido or alkenic amido linkage.

21. The method of claim 20 wherein the linkage has a structure of one of the following formulae:

22. The method of claim 1 wherein the dibenzorhodamine dye has a structure of the following formula:

23. A method of identifying polynucleotide classes comprising the steps of:

providing multiple classes of polynucleotides distributed among locations in a spatially addressable hybridization array;

wherein a first class of polynucleotide comprises an energy transfer dye comprising;

a donor dye configured to absorb light at a first wavelength and emit excitation energy in response;

an acceptor dye configured to absorb the excitation energy emitted by the donor dye and fluoresce 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 dye having a structure of the formula;

24. The method of claim 23 wherein the first wavelength is greater than about 600 nm.

25. The method of claim 23 wherein the dibenzorhodamine dye comprises 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.

26. The method of claim 25 wherein one or both of the first and second ring structures has five members.

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

28. The method of claim 27 wherein the gem substituents are lower alkyl.

29. The method of claim 28 wherein the gem substituents are methyl.

30. The method of claim 26 wherein the five membered ring is not aromatic.

31. The method of claim 25 wherein the first and second ring structures are the same.

32. The method of claim 25 wherein the first and second ring structures are different.

33. The method of claim 23 wherein the dibenzorhodamine dye comprises one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, and linking group, including substituted forms thereof.

34. The method of claim 33 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.

35. The method of claim 33 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.

36. The method of claim 35 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl or iodoacetamide.

37. The method of claim 35 wherein the linking group is carboxylate.

38. The method of claim 33 wherein one nitrogen substituent is water-solubilizing group.

39. The method of claim 33 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

40. The method of claim 33 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.

41. The method of claim 23 wherein the linker has the structure

42. The method of claim 41 wherein R₂₂ is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, triazine, pyrazine, oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

43. The method of claim 23 wherein the linker has the structure

44. The method of claim 23 wherein the linker has the structure

45. The method of claim 23 wherein the energy transfer dye and the polynucleotide are connected by a linkage at a position on the polynucleotide selected from the 8-position of a purine nucleobase, the 7- or 8-position of a deazapurine nucleobase, the 5-position of a pyrimidine nucleus, the 5′

terminus, the 3′

terminus, and the phosphodiester backbone.

46. The method of claim 45 wherein the linkage is attached to a linking group substituent of the dibenzorhodamine dye.

47. The method of claim 45 wherein the linkage is an acetylenic amido or alkenic amido linkage.

48. The method of claim 47 wherein the linkage has a structure of one of the following formulae:

49. The method of claim 23 wherein the dibenzorhodamine dye has a structure of the following formula:

50. The method of claim 23 wherein the donor or the acceptor dye is a dibenzorhodamine dye and the donor or acceptor dye is a fluorescein dye.

51. A method of identifying polynucleotide classes comprising the steps of:

providing multiple classes of polynucleotides distributed among locations in a spatially addressable hybridization array;

wherein a first class of polynucleotide comprises a dibenzorhodamine dye having a structure of the formula;

52. The method of claim 51 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

53. The method of claim 51 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl or iodoacetamide.

54. The method of claim 51 wherein the linking group is carboxylate.

55. The method of claim 51 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.