Methods of labelling polynucleotides with dibenzorhodamine dyes
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, —CH 2 OH, 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.
1 . 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.
6 . The compound of claim 5 wherein the gem substituents are methyl.
7 . The compound of claim 3 wherein the five membered ring is not aromatic.
8 . The compound of claim 3 wherein the five membered ring is substituted with linking group or 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, lower alkene, cyano, phenyl, heterocyclic aromatic, electron-rich heterocycle, and substituted forms thereof.
12 . The compound of claim 11 wherein the C-7 substituent is phenyl having the structure
including aryl-substituted forms thereof.
13 . The compound of claim 12 wherein substituents at any one of positions C-14 to C-18 are selected from the group consisting of chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH 2 OH, alkoxy, phenoxy, linking group, water solubilizing group, and substituted forms thereof.
14 . The compound of claim 11 wherein a substituent at position C-18 is selected from the group consisting of carboxylic acid and sulfonate.
15 . The compound of claim 13 wherein a substituent at position C-18 is carboxylic acid.
16 . The compound of claim 12 wherein a substituent at positions C-14 and C-17 is chlorine.
17 . The compound of claim 12 wherein a substituent at positions C-14 to C-17 is chlorine.
18 . The compound of claim 12 wherein a substituent at positions C-14 to C-17 is fluorine.
19 . The compound of claim 12 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 carboxy.
20 . The compound of claim 1 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.
21 - 106 . (canceled)