Polynucleotides labeled with dibenzorhodamine dyes
First Claim
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.
7 Assignments
0 Petitions
Accused Products
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.
-
Citations
34 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.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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.
- and/or
-
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. - View Dependent Claims (29)
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.
- fluorescence energy transfer occurs between the donor dye and acceptor dye;
-
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.- View Dependent Claims (31, 32, 33, 34)
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:
Specification