Phosphonate fluorescent dyes and conjugates

1. A fluorescent dye reagent having the formula:

wherein

Fl is a fluorescent dye component;

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, (C 1 -C 8 )alkyl, 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, polymethines and N-aryl-1,8-naphthalimide dyes.

3. A fluorescent dye reagent of claim 1 , having formula I:

wherein,

the symbol , represents a single or double bond;

the Ar ring, represented by the symbol

 is selected from the group consisting of:

A 1 in Ar 1 represents O, N—Z, or N + (Z) 2 , and A 1 in Ar 2 represents 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 the Z group are optionally substituted with halogen, (C 1 -C 4 )alkyl, aryl, L f or P z ; or optionally the Z group, at each occurrence, independently is combined with R 2 or R 4 to form a fused 5- to 7-membered ring, and the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, (C 1 -C 4 )alkyl, L f or P z ; R, is selected from H, (C 1 -C 8 )alkyl, aryl, aryl(C 1 -C 4 )alkyl, protecting group and L f ;

A 2 represents OR or N(Z) 2 , wherein each 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 the Z group are optionally substituted with halogen, (C 1 -C 4 )alkyl, aryl, L f or P z ; or optionally the Z group, at each occurrence, independently is combined with R 1 or R 1″ to form a fused 5- to 7-membered ring wherein the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, C 1 -C 4 alkyl, aryl, L f or P z ; and the substituent R is selected from H, (C 1 -C 8 )alkyl, aryl, aryl(C 1 -C 4 )alkyl, a protecting group and L f ;

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

R 0 is halogen, cyano, CF 3 , (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, substituted or unsubstituted heteroaryl or aryl having the formula:

wherein X 1 , X 2 , X 3 , X 4 and X 5 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 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, aryl, heteroaryl, SO 3 H, PO 3 H 2 , CO 2 H, L f and P z and optionally, any two adjacent substituents of X 1 to X 5 are combined to form an aromatic or heteroaromatic ring; wherein the aryl or heteroaryl portions of R 0 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 ;

R 0′ in formula I is absent or is (C 1 -C 6 )alkoxy, hydroxy, (C 1 -C 6 )alkylamino, or, di(C 1 -C 6 )alkylamino, or optionally, the R 0 and R 0′ groups are combined to form a 5- to 6-membered heterocyclic ring;

and wherein in formula I, there will be from 0 to 1 L f groups and from 1 to 4 P z groups, preferably 1 to 2 P z groups.

4. A fluorescent dye reagent of claim 3 , having formula I(a) or I(b):

wherein

each A 1 and A 2 are each independently OR or N(Z) 2 , wherein R and Z are as set forth in claim 3 ;

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, (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, sulfa, 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 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, 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 .

5. A fluorescent dye reagent of claim 3 , having formula Ic:

wherein the group Z′ or Z″, at each occurrence, is independently hydrogen, (C 1 -C 8 )alkyl, aryl-C 1 -C 8 alkyl or aryl, wherein the aliphatic or aryl portions of the Z′ or Z″ groups are optionally substituted with halogen, (C 1 -C 4 )alkyl, L f or P z ; optionally the Z′ group, at each occurrence is independently combined with R 2 or R 4 to form a fused 5- or 6-membered ring, and optionally, the Z″ group, at each occurrence is independently combined with R 1 or R 1″ to form a fused 5- or 6-membered ring; wherein if present, said fused 5- or 6-membered ring is optionally fuse to an aryl ring and is substituted with halogen, C 1 -C 4 alkyl, L f or P z ; and the R 1′ , R 1″ , R 1 , R 2 , R 3 , R 4 and R 0 groups in formula Ic are as described in claim 3 .

6. A fluorescent dye reagent of claim 1 , having formula:

wherein

A 1 represents O, N—Z or N + (Z) 2 in which Z is independently H, (C 1 -C 8 )alkyl, C 8 )-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C 1 -C 4 )alkyl, aryl, L f or P z ; wherein the Z group, at each occurrence, is optionally combined with R 2 or R 4 to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused to an aryl ring and is optionally substituted with halogen, (C 1 -C 4 )alkyl, aryl, L f or P z ;

A′ represents OR or N(Z) 2 in which Z is independently H, (C 1 -C 8 )alkyl, aryl-(C 1 -C 8 )-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C 1 -C 4 )alkyl, aryl L f or P z ; wherein the Z group, at each occurrence, is optionally combined with R 1 or R 1′ to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused to an aryl ring and is optionally substituted with halogen, (C 1 -C 4 )alkyl, aryl, L f or P z ; and R is selected from the group consisting of H, a protecting group, (C 1 -C 8 )alkyl, aryl, aryl(C 1 -C 4 )alkyl and L f , or is optionally combined with R 1 or R 1″ to form a fused 5- to 7-membered ring;

R 1′ , R 1″ , R 1 , R 2 , R 3 and R 4 are each independently selected from H, halogen, cyano, CF 3 , sulfo, (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 )alkoxy, aryl, heteroaryl, L f and 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 .

7. A fluorescent the reagent of claim 1 , having formula:

wherein

R 1 , R 1′ , R 1″ , R 2 and R 3 are each independently selected from the group consisting of H, halogen, cyano, sulfo, CF 3 , (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 )alkoxy, aryl, heteroaryl, L f and P z ;

wherein the alkyl portions of any of R 1′ , R 1″ and R 1 through R 3 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 ; and

A 1 represents OR, N(Z) 2 , in which 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 the Z group are optionally substituted with halogen, (C 1 -C 4 )alkyl, L f or P z ; or optionally the Z group at teach occurrence is independently combined with R 1 or R 1″ to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally substituted with halogen, (C 1 -C 4 )alkyl, L f or P z , and is optionally fused with an aryl or heteroaryl ring wherein the aryl or heteroaryl ring is optionally substituted with halogen, (C 1 -C 4 )alkyl, L f or P z ; and the R substituent in A 1 is hydrogen, (C 1 -C 8 )alkyl, aryl, aryl(C 1 -C 4 )alkyl, a protecting group or L f .

8. A fluorescent dye reagent of claim 1 , having the formula:

wherein

Q 1 and Q 2 are independently selected from O, S, N and CR a R b or —C(═CH 2 )— wherein R a and R b are independently selected from the group consisting of H, methyl, ethyl and L f ;

the rings, A x and A y , each independently represents a condensed substituted or unsubstituted aryl or heteroaryl rings wherein said rings are optionally substituted with halogen, CN, CF, (C 1 -C 8 )alkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, (C 1 -C 8 )alkylamino, di(C 1 -C 8 )alkylamino, an aryl or heteroaryl that is optionally substituted with L f or P z ;

Q represents a conjugated linking system;

R G and R G′ are independently selected from the group consisting of H, C 1 -C 8 alkyl, heteroalkyl, alkylene sulfonic acid, alkylene phosphoric acid, alkylene phosphonic acid, alkylene aryl, substituted alkylene aryl, alkylene carboxylic acid, L f and P z , wherein said aryl group in R G or R G′ is optionally substituted with from about one to two P z groups and from about 0 to about 2 L f groups.

9. A fluorescent dye reagent of claim 1 , having the formula:

wherein X 1 , X 2 , X 3 and X 4 are each independently selected from the group consisting of H, halogen, cyano, CF 1 , (C 1 -C 8 )alkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, (C 2 -C 8 )alkenyl, (C 2 -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 a non-aromatic, aromatic or heteroaromatic five or six-membered 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 ; and wherein there is present from 0 to 1 L f groups and from 1 to 2 P z groups.

10. 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 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, (C 1 -C 8 )alkyl, 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.

11. A fluorescent dye reagent of claim 10 , 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, polymethines and N-aryl-1,8-naphthalimide dyes.

12. A conjugated substance comprising one or more phosphonylated fluorescent dyes coupled through a linking group to a conjugated substance or biological agent, wherein the phosphonylated fluorescent dyes have structures selected from the group of consisting of:

wherein

Fl is a fluorescent dye component;

L f is a linking group connecting said conjugated substance and said phosphonylated fluorescent dye;

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, (C 1 -C 8 )alkyl, 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 0 to 4; or

Fl-(P 1 ) n ,

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, (C 1 -C 8 )alkyl, and a labile protecting group; and

P a is a conjugated substance;

and salts thereof.