Compounds and methods for fluorescent labeling
Dye reagents useful in labeling biological materials are provided along with methods for their use.
1. A method for preparing a fluorescent dye-labeled conjugate, said method comprising:
(a) contacting a fused lactone derivative of a benzo[c]xanthene 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 ligand under conditions sufficient to covalently attach the ligand to said fluorescent dye-labeled linking group and form said fluorescent dye-labeled conjugate.
2. A method in accordance with claim 1 , wherein said linking group component comprises two reactive functional groups selected from amino, hydroxy, hydrazino and thiol.
3. A method in accordance with claim 1 , wherein said linking group is linear or cyclic or a combination thereof.
4. 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.
5. A method in accordance with claim 1 , wherein said linking group is cyclic and comprises a five-membered heterocycle.
6. A method in accordance with claim 5 , wherein said cyclic linking group is a prolinol linker.
7. A method in accordance with claim 1 , wherein said ligand is selected from the group consisting of a phosphoramidite moiety, a solid support and a biological agent wherein the biological agent is selected from the group consisting of a nucleic acid, a peptide, a protein, an aminocarbohydrate, an oligosaccharide, a glycosylated protein and analogs thereof.
8. A method in accordance with claim 7 , wherein said biological agent is an oligonucleotide having one or more modified bases.
9. A method in accordance with claim 1 , wherein said fused-lactone derivative of a benzo[c]xanthene has the formula:
wherein
A 1 is O or N-Z;
Z is H or (C 1 -C 8 )alkyl, or is optionally combined with an adjacent X 0 to form a 5- or 6- membered ring or is combined with two adjacent X 0 groups to form two fused 6- membered rings;
R 1 , R 1 ′, R 2 ,and R 3 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;
R 5 , R 6 , R 7 and R 8 are each 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 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 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. the subscript n is an integer of from 0 to 3; each X 0 is a member independently selected from the group consisting of 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 1 -C 8 )alkynyl, aryl, heteroaryl, SO3H and CO2H; 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, SO3H and CO2H, and optionally, any two adjacent X 1 through X 5 are combined to form an aromatic or heteroaromatic ring.
10. A method in accordance with claim 9 , wherein said fused-lactone derivative of a benzo[c]xanthene 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 3;
each X 0 is a member independently selected from the group consisting of 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, SO3H and CO2H;
R 1 ′, R 1 , R 2 and R 3 are each independently selected from the group consisting of 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 the group consisting of H, C 1 -C 8 )alkyl, aryl and aryl(C 1 -C 4 )alkyl; wherein the alkyl portions of any of 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 1to 6 carbon atoms; and the aryl portions of any of R 5 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 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.
11. A method in accordance with claim 10 , wherein said fluorescent dye-fused lactone derivative has formula IVa;
A is hydroxy or a protected hydroxy;
R 5 through R 8 are each H; and
each R 1 , R 2 , R 3 and R 1 ′ are independently selected from the group consisting of H, halogen, cyano and CF 3 .
12. A conjugate 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, 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 , 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 (C1- C6)alkoxy;
R A and R B and the ring to which each is attached forms a benzo[c]xanthene dye;
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; and
L is a ligand is selected from the group consisting of a solid support and a biological agent wherein the biological agent is selected from the group consisting of a nucleic acid, a peptide, a protein, an aminocarbohydrate, an oligosaccharide, a glycosylated protein and analogs thereof.
13. A conjugate in accordance with claim 12 , having the formula:
wherein
A 1 is O or N-Z;
Z is H or (C 1 ,-C 8 )alkyl, or is optionally combined with an adjacent X 0 to form a 5- or 6- membered ring or is combined with two adjacent X 0 groups to form two fused 6- membered rings;
R 1 , R 1 ′, R 2 and R 3 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;
R 5 , R 6 , R 7 and R 8 are each 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 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 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;
the subscript n is an integer of from 0 to 3;
each X 0 is a member independently selected from the group consisting of 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 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:
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.
14. A conjugate in accordance with claim 13 , having the 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 3;
each X 0 is a member independently selected from the group consisting of 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 , and R 2 , R 3 are each independently selected from the group consisting of 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 the group consisting of H, (C 1 -C 8 )alkyl, aryl and aryl(C 1 -C 4 )alkyl; wherein the alkyl portions of any of 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 R 5 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 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
L is a ligand is selected from the group consisting of a solid support and a biological agent wherein the biological agent is selected from the group consisting of a nucleic acid, a peptide, a protein, an aminocarbohydrate, an oligosaccharide, a glycosylated protein and analogs thereof.
15. A conjugate in accordance with claim 14 , having the formula:
16. A conjugate in accordance with claim 15 , wherein R 1 is Cl; and R 5 and R 6 are H.
17. A conjugate having a formula selected from:
wherein
R 1 and R 1 ′ are each members 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 ,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 and the ring to which each is attached forms a benzo[c]xanthene dye;
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;
the subscript n′ is 0 or 1;
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 exculsive 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 a lone pair of electrons, H, OH, SH, NH, (C 1 -C 8 ) alkyl, aryl, an amino protecting group and a hydroxy protecting group;
Y is a cleavable linker; and
L is a ligand is selected from the group consisting of a phosphoramidite moiety, a solid support and a biological agent wherein the biological agent is selected from the group consisting of a nucleic acid, a peptide, a protein, an aminocarbohydrate, an oligosaccharide, a glycosylated protein and analogs thereof.
18. A conjugate in accordance with claim 17 , 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.
19. A conjugate in accordance with claim 18 , having the formula:
wherein
A 1 is O or N-Z;
Z is H or (C 1 -C 8 )alkyl, or is optionally combined with an adjacent X 0 to form a 5- or 6-membered ring or is combined with two adjacent X 0 groups to form two fused 6-membered rings;
R 1 , R 1 ′, R 2 and R 3 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;
R 5 ,R 6 ,R 7 and R 8 are each independently selected from the group consisting of H, (C1- 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 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 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;
the subscript n is an integer of from 0 to 3;
each X 0 is a member independently selected from the group consisting of 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 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:
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.
20. A conjugate in accordance with claim 19 , having the 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 3;
each X 0 is a member independently selected from the group consisting of 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 and R 3 are each independently selected from the group consisting of 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 the group consisting of H, (C 1 -C 8 )alkyl, aryl and aryl(C 1 -C 4 )alkyl; wherein the alkyl portions of any of 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 R 5 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 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
L is a ligand is selected from the group consisting of a phosphoramidite moiety, a solid support and a biological agent wherein the biological agent is selected from the group consisting of a nucleic acid, a peptide, a protein, an aminocarbohydrate, an oligosaccharide, a glycosylated protein and analogs thereof.
21. A conjugate in accordance with claim 20 having the formula:
22. A conjugate in accordance with claim 21 , wherein R 1 is Cl; and R 5 and R 6 are-H.