Phosphonylated fluorescent dyes and conjugates

Abstract

Reagents are provided for the introduction of phosphonate groups into fluorescent dyes. Methods are also provided for preparing dye conjugates.

1 .- 20 . (canceled) 
     
     
         21 . A fluorescent dye reagent having the formula:
   Fl—P 1      
       wherein
 Fl is a fluorescent dye component; and 
 P 1  is functionalized phosphonate group having the formula: 
 
       
         
           
           
               
               
           
         
         wherein the wavy line indicates the direct attachment to a sp 2  carbon of said fluorescent dye component; 
         L 1  is a linking group; 
         R is a member selected from the group consisting of H, C 1 -C 8  alkyl, aryl, arylC 1 -C 4  alkyl, -L a -N a , and -L a -NR A R B ;
 wherein L a  is an alkylene linking group, N a  is an ammonium ion group, and each of R A  and R B  is independently selected from the group consisting of H and a labile protecting group; 
 
         and 
         P a  is a functional group component selected from the group consisting of a phosphoramidite moiety, a mono-, di- or tri-functional linking group having at least one terminal functional group or protected functional group, a solid support and a reactive group; 
         and salts thereof. 
       
     
     
         22 . A fluorescent dye reagent of  claim 21 , wherein said fluorescent dye component is selected from the group consisting of coumarins, benzocoumarins, xanthenes, benzo[a]xanthenes, benzo[b]xanthenes, benzo[c]xanthenes, cyanines, acridines, dipyrrometheneboron difluorides, phenoxazines, benzo[a]phenoxazines, benzo[b]phenoxazines and benzo[c]phenoxazines. 
     
     
         23 . A fluorescent dye reagent of  claim 21 , wherein said fluorescent dye component is selected from the group consisting of coumarin dyes, resorufin dyes, dipyrrometheneboron difluoride dyes, ruthenium bipyridyl dyes, energy transfer dyes, polymethine dyes, thiazole orange dyes and N-aryl-1,8-naphthalimide dyes. 
     
     
         24 . A fluorescent dye reagent of  claim 21 , wherein said fluorescent dye component is a xanthene or benzoxanthene dye selected from the group consisting of fluorescein dyes, rhodamine dyes and rhodol dyes. 
     
     
         25 .- 33 . (canceled) 
     
     
         34 . An oligonucleotide probe having an attached fluorescent dye reagent of  claim 21 , wherein attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on pa. 
     
     
         35 . An oligonucleotide probe having an attached quencher and an attached fluorescent dye reagent of  claim 21 , wherein attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on pa. 
     
     
         36 . An oligonucleotide probe having an attached quencher, minor groove binding agent, and an attached fluorescent dye reagent of  claim 21 , wherein attachment of the fluorescent dye reagent to the oligonucleotide is through a functional group present on P a . 
     
     
         37 .- 42 . (canceled) 
     
     
         43 . A fluorescent dye reagent of  claim 24 , wherein said fluorescent dye component has formula I(a): 
       
         
           
           
               
               
           
         
       
       wherein
 A 1  and A 2  are each independently OR′ or N(Z) 2 , wherein at each occurrence Z is independently hydrogen, (C 1 -C 8 )alkyl, aryl-(C 1 -C 8 )alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of each Z group are optionally substituted with halogen and (C 1 -C 4 )alkyl; or optionally the Z groups of A 1  at each occurrence, independently are combined with R 2  or R 4  to form a fused 5- to 7-membered ring, and optionally the Z groups of A 2  at each occurrence, independently are combined with R 1  or R 1″  to form a fused 5- to 7-membered ring, and each of the resultant fused 5- to 7-membered rings is optionally substituted with halogen and (C 1 -C 4 )alkyl; and R′ is selected from H, (C 1 -C 8 )alkyl, aryl, aryl(C 1 -C 4 )alkyl and a protecting group; 
 R 1′ , R 1″ , R 1 , R 2 , R 3  and R 4  are each independently selected from H, halogen, cyano, sulfo, aryl, heteroaryl, CF 3 , (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylthio and (C 1 -C 8 )alkoxy, wherein the alkyl portions of any of R 1′ , R 1″  and R 1  through R 4  are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms and the aryl or heteroaryl portions of any of R 1′ , R 1″  and R 1  through R 4  are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C 1 -C 6 )alkylamino, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkylthio and (C 1 -C 6 )alkoxy; 
 X 1 , X 2 , X 3  and X 4  are each independently selected from the group consisting of H, halogen, cyano, CF 3 , (C 1 -C 8 )alkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, (C 1 -C 8 )alkenyl, (C 1 -C 8 )alkynyl, SO 3 H, PO 3 H, CO 2 H, and P 1  and optionally, any two adjacent X 1  through X 4  are combined to form an aromatic or heteroaromatic ring that is optionally further substituted with from one to four substituents selected from halogen cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C 1 -C 6 )alkylamino, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkylthio, (C 1 -C 6 )alkoxy. 
 
     
     
         44 . A fluorescent dye reagent of  claim 43 , wherein A 1  and A 2  are each OR′. 
     
     
         45 . A fluorescent dye reagent of  claim 43 , wherein A 1  is OR′ and A 2  is N(Z) 2 . 
     
     
         46 . A fluorescent dye reagent of  claim 43 , wherein A 1  and A 2  are each N(Z) 2 . 
     
     
         47 . A fluorescent dye reagent of  claim 44 , wherein each R′ is selected from the group consisting of H and C 1 -C 8  alkyl. 
     
     
         48 . A fluorescent dye reagent of  claim 21 , wherein L 1  is an alkylene linking group. 
     
     
         49 . A fluorescent dye reagent of  claim 21 , wherein P a  is a reactive electrophilic group. 
     
     
         50 . A fluorescent dye reagent of  claim 49 , wherein said reactive electrophilic group is an activated ester group. 
     
     
         51 . A fluorescent dye reagent of  claim 50 , wherein said activated ester group is selected from the group consisting of pentafluorophenyl esters and N-hydroxysuccinimide esters.