Polynucleotides labeled with dibenzorhodamine dyes

• US 6,326,153 B2
• Filed: 02/14/2001
• Issued: 12/04/2001
• Est. Priority Date: 11/25/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Lam, Joe Y. L., Benson, Scott C., Menchen, Steven Michael
• Primary Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US09/784,943
• Publication Number:
  US 20010011139A1
• Time in Patent Office: 293 Days
• Field of Search: 435/6, 536/26.6, 544/262, 544/333, 546/33, 549/382
• 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 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 labeled polynucleotide comprising a polynucleotide covalently attached to a dibenzorhodamine dye of the structure:

including nitrogen- and aryl-substituted forms thereof;

wherein the 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 7-deazapurine nucleobase, the 5-position of a pyrimidine nucleobase, the 5′

terminus, the 3′

terminus, and the phosphodiester backbone.

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

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

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

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

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

8. The labeled polynucleotide of claim 4 wherein the five membered ring is substituted with a linkage.

9. The labeled polynucleotide of claim 4 wherein the five membered ring is substituted with a water-solubilizing group.

10. The labeled polynucleotide of claim 1 wherein the linkage is attached at one position selected from the C-2-bonded nitrogen and the C-12-bonded nitrogen of the dibenzorhodamine dye.

11. The labeled polynucleotide of claim 1 wherein the linkage is an aminohexyl linkage attached to the polynucleotide at the 5′

terminus.

12. The labeled polynucleotide of claim 1 comprising one or more nitrogen substituents selected from (C₁-C₈) alkyl, (C₂-C₈) alkene, (C₂-C₈) alkyne, phenyl, benzyl, aryl, heterocycle, polycyclic aromatic, water-solubilizing group, and a linkage, including substituted forms thereof.

13. The labeled polynucleotide of claim 12 wherein one or more nitrogen substituents is alkylsulfonate.

14. The labeled polynucleotide of claim 12 wherein the water-solubilizing group is carboxylate, sulfonate, sulfate, phosphonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

15. The labeled polynucleotide 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.

16. The labeled polynucleotide of claim 15 wherein one or both of the third and fourth ring structures has six members.

17. The labeled polynucleotide of claim 16 wherein the six membered ring structure includes one gem disubstituted carbon.

18. The labeled polynucleotide of claim 17 wherein the gem substituents are (C₁-C₈) alkyl.

19. The labeled polynucleotide of claim 17 wherein the gem substituents are methyl.

20. The labeled polynucleotide of claim 16 wherein the six membered ring is not aromatic.

21. The labeled polynucleotide of claim 15 wherein one or both of the third and fourth ring structures has five members.

22. The labeled polynucleotide of claim 21 wherein the five membered ring structure includes one gem disubstituted carbon.

23. The labeled polynucleotide of claim 22 wherein the gem substituents are (C₁-C₈) alkyl.

24. The labeled polynucleotide of claim 22 wherein the gem substituents are methyl.

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

26. The labeled polynucleotide of claim 25 wherein the one or more aryl substituents is alkylsulfonate.

27. A labeled polynucleotide comprising a polynucleotide covalently attached to a dibenzorhodamine dye of the structure:

including aryl-substituted forms thereof;

wherein R₁ and R₂ are nitrogen substituents taken separately from (C₁-C₈) alkyl, (C₂-C₈) alkene, (C₂-C₈) alkyne, phenyl, benzyl, aryl, heterocycle, polycyclic aromatic, water-solubilizing group, and a linkage, including substituted forms thereof;

wherein the linkage is attached to the polynucleotide at a position selected from the 8-position of a purine nucleobase, the 7- or 8-position of a 7-deazapurine nucleobase, the 5-position of a pyrimidine nucleobase, the 5′

terminus, the 3′

terminus, and the phosphodiester backbone.

28. A labeled polynucleotide comprising a polynucleotide covalently attached to multiple dyes wherein the dyes comprise a donor dye and an acceptor dye;

fluorescence energy transfer occurs between the donor dye and acceptor dye;

the donor dye is attached to the polynucleotide by a first linkage and the acceptor dye is attached to the polynucleotide by a second linkage; and

at least one of the donor dye and acceptor dye is a dibenzorhodamine dye of the structure;

including nitrogen- and aryl-substituted forms thereof.

29. The labeled polynucleotide of claim 28 wherein the first linkage and the second linkage taken independently are attached to a nucleobase of the polynucleotide at positions selected from the 8-position of a purine nucleobase, the 7- or 8-position of a 7-deazapurine nucleobase, and the 5-position of a pyrimidine nucleobase;

and the first or second linkage is selected from the structures;

wherein NUC is the nucleobase;

D is a dye;

R₃ is H and (C₁-C₈) alkyl, and X is selected from the structures;

 

wherein R₁ is H or (C₁-C₈) alkyl, and n ranges from 1 to 5.

30. A labeled polynucleotide comprising a polynucleotide covalently attached by a linkage to 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 dye of the structure;

 

including nitrogen- and aryl-substituted forms thereof.

31. The labeled polynucleotide of claim 30 wherein the linker has the structure:

wherein

Z₁ is selected from the group consisting of —

NH, sulfur and oxygen;

R₂, is a (C₁-C₈) alkyl attached to the donor dye;

R₂₂ is a substituent selected from 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.

32. The labeled polynucleotide of claim 31 wherein R₂₂ is a five or six membered ring selected from cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

33. The labeled polynucleotide of claim 31 wherein the linker has the structure:

wherein

Z₂ is selected from the group consisting of —

NH, sulfur and oxygen; and

R₂₉ is a (C₁-C₈) alkyl.

34. The labeled polynucleotide of claim 31 wherein the linker has the structure: