Methods of labeling polynucleotide with dibenzorhodamine dyes

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, —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 - 105 . (canceled) 
     
     
         106 . A method of identifying polynucleotide classes comprising the steps of:
 providing multiple classes of polynucleotides distributed among locations in an array;   wherein a first class of polynucleotide comprises a dibenzorhodamine dye having a structure of the formula:   
       
         
           
           
               
               
           
         
         including nitrogen- and aryl-substituted forms thereof; 
         and further wherein other classes of polynucleotides are labeled with dyes configured to be spectrally resolvable from the dibenzorhodamine dye of the first class of polynucleotide and from each other; 
         illuminating the array with an illumination beam configured to cause the dyes to fluoresce; and 
         identifying the classes of the polynucleotides in the array by the fluorescence spectrum of the dyes. 
       
     
     
         107 . The method of  claim 106  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. 
 
     
     
         108 . The method of  claim 107  wherein one or both of the first and second ring structures has five members. 
     
     
         109 . The method of  claim 108  wherein the five membered ring structure includes one gem disubstituted carbon. 
     
     
         110 . The method of  claim 109  wherein the gem substituents are lower alkyl. 
     
     
         111 . The method of  claim 110  wherein the gem substituents are methyl. 
     
     
         112 . The method of  claim 108  wherein the five membered ring is not aromatic. 
     
     
         113 . The method of  claim 107  wherein the first and second ring structures are the same. 
     
     
         114 . The method of  claim 107  wherein the first and second ring structures are different. 
     
     
         115 . The method of  claim 106  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. 
     
     
         116 . The method of  claim 115  wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof. 
     
     
         117 . The method of  claim 115  wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group or sulfonate. 
     
     
         118 . The method of  claim 115  wherein the linking group is isothiocyanate, sulfonyl chloride, 4, 6, dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl or iodoacetamide. 
     
     
         119 . The method of  claim 115  wherein the linking group is carboxylate. 
     
     
         120 . The method of  claim 115  wherein the nitrogen substituents are water-solubilizing group. 
     
     
         121 . The method of  claim 115  wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer. 
     
     
         122 . The method of  claim 106  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. 
     
     
         123 . The method of  claim 122  wherein the linkage is attached to the linking group substituent of the dibenzorhodamine dye. 
     
     
         124 . The method of  claim 122  wherein the linkage is an acetylenic amido or alkenic amido linkage. 
     
     
         125 . The method of  claim 124  wherein the linkage has a structure of one of the following formulae: 
       
         
           
           
               
               
           
         
         wherein NUC is a nucleobase of the polynucleotide; D is the dibenzorhodamine dye; R 3  is H or C 1 -C 8  alkyl; and 
         X is a moiety having a structure of one of the following formulae: 
       
       
         
           
           
               
               
           
         
         wherein R 1  is H or C 1 -C 8  alkyl, and n is an integer of 1 to 5. 
       
     
     
         126 . The method of  claim 106  wherein the dibenzorhodamine dye has a structure of the following formula: 
       
         
           
           
               
               
           
         
         including aryl-substituted forms thereof; 
         wherein R 1  and R 2  are independently selected from lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof; and 
         the dibenzorhodamine dye is covalently attached to the polynucleotide by a linkage connected to a linking group substituent of R 1  or R 2 .