Compounds and methods for fluorescent labeling

1. A compound having a formula selected from the group consisting of:

wherein

R 1 and R 1′ are each members independently selected from the group consisting of H, halogen, cyano, halo(C 1 –C 8 )alkyl, (C 1 –C 8 )alkyl, (C 1 –C 8 )alkylthio, (C 1 –C 8 )alkoxy, aryl and heteroaryl;

each R 5 and R 6 is independently selected from the group consisting of H, (C 1 –C 8 )alkyl, aryl, heteroaryl, aryl(C 1 –C 4 )alkyl and heteroaryl(C 1 –C 4 )alkyl;

wherein the alkyl portions of any of R 1 , R 1′ , R 5 and R 6 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′ , R 5 and R 6 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;

R A and R B are combined to form a substituted or unsubstituted fused ring system having from 1 to 4 five- or six-membered rings;

R x is selected from the group consisting of H and hydroxy protecting groups;

the subscript p is an integer of from 1 to 3;

W is a di-, tri- or tetravalent linker which is acyclic, cyclic, aromatic or a combination thereof, having from 4 to 50 atoms selected from the group consisting of C, N, O, P and S and exclusive of hydrogen atoms that fill available valences, and further having a nitrogen atom directly connected to the adjacent carbonyl group;

K is selected from the group consisting of H, OH, SH, NH, (C 1 –C 8 )alkyl, aryl, an amino protecting group and a hydroxy protecting group or is absent when W is O or S;

the subscript n is 0 or 1; and when n is 1, Y is a cleavable linking group and L is a solid support; and when n is 0, L is a phosphoramidite or reactive functional group; and tautomeric forms thereof.

2. A compound in accordance with claim 1 , wherein n is 1, and Y is selected from the group consisting of:

wherein the subscripts q and r are independently integers of from 1 to 15; and each R is independently (C 1 –C 8 )alkyl or (C 1 –C 8 )alkoxy.

3. A compound in accordance with claim 2 , wherein when R x is H said reagent has an emission wavelength of from 400 nm to about 850 nm.

4. A compound in accordance with claim 2 , wherein R A , R B and the ring to which each is attached forms a dye selected from the group consisting of a fluorescein, a benzocoumarin, a xanthene, a benzo[a]xanthene, a benzo[b]xanthene, a benzo[c]xanthene, a phenoxazine, a benzo[a]phenoxazine, a benzo[b]phenoxazine and a benzo[c]phenoxazine.

5. The compound in accordance with claim 1 , having a formula selected from the group consisting of:

wherein

is a member selected from the group consisting of ═N— and

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 2 –C 8 )alkenyl, (C 2 –C 8 )alkynyl, SO 3 H and CO 2 H, and optionally, any two adjacent X 1 through X 4 are combined to form an aromatic or heteroaromatic ring;

the moiety

is a member selected from the group consisting of:

optionally substituted with a member independently selected from the group consisting of halogen, cyano, halo(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 2 H and CO 2 H;

A 2 is O or NZ;

A 3 is hydroxyl, protected hydroxyl, amino, or protected amino;

Z is H or (C 1 –C 8 )alkyl, or is combined with either R 2 or R 4 and the atoms which attach them to form a 5-membered ring or a 6-membered ring, or is combined with both of R 2 and R 4 and the atoms which attach them to form two fused 6-membered rings; and

R 1 , R 2 , R 3 , R 4 and R 1′ are each independently selected from the group consisting of H, halogen, cyano, CF 3 , (C 1 –C 8 )alkyl, (C 1 –C 8 )alkylthio, (C 1 –C 8 )alkoxy, aryl and heteroaryl.

6. A compound in accordance with claim 1 , having the formula:

wherein

A 2 is O or N-Z in which Z is H or (C 1 –C 8 )alkyl;

R 1 , R 2 , R 3 , R 4 and R 1′ are each independently selected from the group consisting of H, halogen, cyano, CF 3 , (C 1 –C 8 )alkyl, (C 1 –C 8 )alkylthio, (C 1 –C 8 )alkoxy, aryl and heteroaryl;

each R 5 and R 6 is independently selected from the group consisting of H, (C 1 –C 8 )alkyl, aryl, heteroaryl, aryl(C 1 –C 4 )alkyl and heteroaryl(C 1 –C 4 )alkyl;

wherein the alkyl portions of any of 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 portions of any of 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;

optionally, R 2 taken together with R 3 form a fused aromatic ring that is optionally 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 and (C 1 –C 6 )alkoxy; and tautomeric forms thereof.

7. A compound in accordance with claim 6 , having the formula:

wherein R y is a protecting group.

8. A compound of claim 7 , having the formula:

9. The compound of claim 1 , having the formula:

wherein

R x and R y are H or independently selected protecting groups;

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 , (C 1 –C 8 )alkyl, (C 1 –C 8 )alkylthio, (C 1 –C 8 )alkoxy, aryl and heteroaryl;

each R 5 and R 6 is independently selected from the group consisting of H, (C 1 –C 8 )alkyl, aryl, heteroaryl, aryl(C 1 –C 4 )alkyl and heteroaryl(C 1 –C 4 )alkyl;

wherein the alkyl portions of any of R 1′ and R 1 through R 6 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 portions of any of R 1′ and R 1 through R 6 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; optionally, R 2 taken together with R 3 form a fused aromatic or heteroaromatic ring that is optionally 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 and (C 1 –C 6 )alkoxy;

the subscript p is an integer of from 1 to 3;

W is a di-, tri- or tetra-valent linker which is acyclic, cyclic, aromatic or a combination thereof, having from 4 to 50 atoms selected from the group consisting of C, N, O, P and S and exclusive of hydrogen atoms that fill available valences, and further having a nitrogen atom directly connected to the adjacent carbonyl group;

K is selected from the group consisting of H, OH, SH, NH, (C 1 –C 8 )alkyl, aryl, an amino protecting group and a hydroxy protecting group or is absent when W is O or S;

the subscript n is 0 or 1; and when n is 1, Y is a cleavable linking group and L is a solid support; and when n is 0, L is a phosphoramidite or reactive functional group;

wherein 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 2 –C 8 )alkenyl, (C 2 –C 8 )alkynyl, SO 3 H and CO 2 H, and optionally, any two adjacent X 1 through X 4 are combined to form an aromatic or heteroaromatic ring;

and tautomeric forms thereof.

10. A compound in accordance with claim 9 , wherein X 1 and X 4 are H, X 2 and X 3 are Cl; R 1 is Cl; R 5 and R 6 are H; and the subscript p is 2.

11. A compound in accordance with claim 9 , wherein R 2 is Cl; R 3 is H and R 4 is CH 3 .

12. A compound in accordance with claim 9 , having a formula selected from:

13. A compound in accordance with claim 9 , having a formula selected from: