Compounds and methods for fluorescent labeling

1. A method for preparing a fluorescent dye-labeled phosphoramidite reagent, said method comprising:

(a) contacting a fluorescent dye-fused lactone derivative with a linking group component to form an intermediate fluorescent dye-labeled linking group; and

(b) contacting said intermediate fluorescent dye-labeled linking group with a phosphoramidite moiety under conditions sufficient to covalently attach the phosphoramidite moiety to said fluorescent dye-labeled linking group and form said fluorescent dye-labeled phosphoramidite reagent.

2. A method in accordance with claim 1 , wherein said fluorescent dye-fused lactone derivative has a formula selected from the group consisting of:

wherein

A 1 represents O or N—Z in which Z is H or (C 1 –C 8 )alkyl, or is optionally combined with R 2 or R 4 to form a 5- or 6-membered ring or is combined with each of R 2 and R 4 to form two fused 6-membered rings; or is optionally combined with and adjacent X 0 to form a 5- or 6-membered ring or is combined with two adjacent X 0 to form two fused 6-membered rings;

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

R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from 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 X 0 , R 1′ and R 1 through R 9 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 X 0 , R 1′ and R 1 through R 9 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;

the subscript n is an integer of from 0 to 4:

each X 0 is a member 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 and CO 2 H;

R 0 is a member selected from the group consisting of halogen, cyano, CF 3 , (C 2 –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, SO 3 H and CO 2 H, and optionally, any two adjacent X 1 through X 5 are combined to form an aromatic or heteroaromatic ring.

3. A method in accordance with claim 1 , wherein fluorescent dye-fused lactone derivative has a formula selected from the group consisting of:

wherein

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

the subscript n is an integer of from 0 to 4;

each X 0 is a member 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 and CO 2 H;

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

R 5 , R 6 , R 7 and R 8 are each independently selected from H, (C 1 –C 8 )alkyl, aryl and aryl(C 1 –C 4 )alkyl; wherein the alkyl portions of any of X 0 , R 1′ and R 1 through R 8 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 X 0 , R 1′ and R 1 through R 8 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; and

optionally, any two adjacent substituents X 1 through X 4 can be taken together to form a fused 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 and (C 1 –C 6 )alkoxy.

4. A method in accordance with claim 1 , wherein said linking group component comprises two reactive functional groups selected from amino, hydroxy, hydrazino and thiol.

5. A method in accordance with claim 1 , wherein said linking group is linear or cyclic or a combination thereof.

6. A method in accordance with claim 1 , wherein said linking group comprises a (C 2 –C 20 )alkylene or (C 2 –C 20 )heteroalkylene group.

7. A method in accordance with claim 1 , wherein said linking group is cyclic and comprises a five-membered heterocycle.

8. A method in accordance with claim 7 , wherein said cyclic linking group is a prolinol linker.