Phosphonate fluorescent dyes and conjugates

1. A fluorescent dye reagent having the formula:

wherein

Fl is a fluorescent dye component 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;

L f is a linking group having an attached member selected from the group consisting of a protected or unprotected functional group, a reactive group, a polyfunctional linking moiety, a phosphoramidite moiety and a solid support;

the subscript m is an integer of from 0 to 1;

P z is a zwitterionic phosphonate group having the formula (a) or a protected phosphonate group having formula (b) or (c):

wherein the wavy line indicates the direct attachment to a sp 2 carbon of said fluorescent dye component; L is a linking group; N a is an ammonium ion group; each of R A and R B is independently selected from the group consisting of H and a labile protecting group; R C is selected from the group consisting of H, (C 1 -C 8 )alkyl, aryl, aryl(C 1 -C 4 )alkyl, a labile protecting group or an alkylene linking group having a distal hydroxy or protected hydroxy group;

the subscript n is an integer of from 1 to 2;

and salts thereof.

2. A fluorescent dye reagent of claim 1 , 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, thiazole orange dyes and N-aryl-1,8-naphthalimide dyes.

3. A fluorescent dye reagent of claim 1 , wherein L is C 2 -C 8 alkylene.

4. A fluorescent dye reagent of claim 1 , wherein N a is an ammonium ion group having the formula —N + (R D ) 3 wherein each R D is independently selected from H, C 1 -C 8 alkyl, aryl and arylC 1 -C 4 alkyl.

5. A fluorescent dye reagent of claim 4 , wherein each R D is H or C 1 -C 8 alkyl.

6. A fluorescent dye reagent of claim 1 , wherein m is 0 and n is 1.

7. A fluorescent dye reagent of claim 1 , wherein m is 0 and n is 2.

8. A fluorescent dye reagent of claim 1 , wherein m is 1 and n is 1.

9. A fluorescent dye reagent of claim 2 , wherein said fluorescent dye component is a xanthene or benzoxanthene dye selected from the group consisting of fluorescein dyes, rhodamine dyes and rhodol dyes.

10. A fluorescent dye reagent of claim 9 , having formula I(a) or I(b):

wherein

each A is hydroxy, amino, protected hydroxy, or protected amino;

R 1′ , R 1″ , R 1 , R 2 , R 3 and R 4 are each independently selected from H, halogen, cyano, aryl, heteroaryl, CF 3 , (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 )alkoxy, L f and P z , or optionally two adjacent members of R 1′ , R 1″ , R 2 and R 3 are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic and with is optionally substituted with P z , 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, (C 1 -C 6 )alkoxy, L f and P z ;

X 1 , X 2 , X 3 and X 4 are each independently selected 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, L f and P z 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, L f and P z .

11. A fluorescent dye reagent of claim 1 , selected from compound:

12. A fluorescent dye reagent having the formula:

Fl-P 1

wherein

Fl is a fluorescent dye component 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; 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, aryl C 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.

13. A fluorescent dye reagent of claim 12 , wherein said fluorescent dye component is a xanthene or benzoxanthene dye selected from the group consisting of fluorescein dyes, rhodamine dyes and rhodol dyes.