Heteroarylcyanine dyes with sulfonic acid substituents

1. A fluorescent cyanine compound, or a covalent conjugate thereof, having a formula selected from:

wherein

R 1 , R 2 and R 3 are independently CH 2 R 7 ;

wherein

R 4 , R 5 , R 6 , R 6′ , and R 7 are members independently selected from H, R 28 , R 28 Z 2 , SO 3 H, CO 2 H, R 28 SO 3 H, C(O)NHR 28 SO 3 H, CONHR 28 C(O)Z 2 ,

wherein

Z 2 is selected from OH, O—, a reactive functional group, and a linker covalently bound to a carrier molecule;

R g1 and R g2 are members independently selected from H, R 38 , R 38 Z 3 , SO 3 H, CO 2 H, R 38 SO 3 H, CONHR 38 SO 3 H, CONHR 38 C(O)Z 2 ,

wherein

 R 28 and R 38 are independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;

 Z 3 is selected from OH, O—, a reactive functional group, and a linker covalently bound to a carrier molecule;

wherein

R 1′ , R 2′ and R 3′ are independently CH 2 R 7′ ;

wherein

R 4′ , R 5′ , and R 7′ are members independently selected from H, R 28′ , R 28′ Z 2′ , SO 3 H, CO 2 H, R 28′ SO 3 H, CONHR 28′ SO 3 H, CONHR 28′ C(O)Z 2′ , and

wherein

Z 2′ is selected from OH, O—, a reactive functional group, and a linker covalently bound to a carrier molecule;

R g1′ and R g2′ are members independently selected from H, R 38′ , R 38′ Z 3′ , SO 3 H, CO 2 H, R 38′ SO 3 H, CONHR 38′ SO 3 H, CONHR 38′ C(O)Z 3′ ,

wherein

 R 28′ and R 38′ are independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; and

 Z 3′ is selected from OH, O—, a reactive functional group, and a linker covalently bound to a carrier molecule,

at least one of Z 2 , Z 2′ , Z 3 , Z 3′ , R 1 , R 1′ , R 2 , R 2′ , R 3 , R 3′ , R 4 , R 4′ , R 5 , R 5′ , R 6 and R 6′ is a reactive functional group, or is a linker covalently bound to a carrier molecule; and

said compound comprises at least three SO 3 H moieties.

2. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 4 and R 5 are each H.

3. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 6 is a member selected from SO 3 H and CO 2 H.

4. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 3 is a member selected from R 28 SO 3 H and R 28 CO 2 H.

5. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 6 is H.

6. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 4 and R 5 are independently selected from H and C(O)NHR 28 SO 3 H.

7. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 1 and R 2 are independently selected from CH 3 and R 28 SO 3 H.

8. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 5′ is H.

9. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 4′ is:

wherein

R g1′ and R g2′ are C(O)NHR 38′ SO 3 H.

10. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 1′ and R 2′ are independently selected from CH 3 and R 28′ SO 3 H.

11. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 3′ is R 28′ SO 3 H.

12. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R 5′ is H.

13. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R 3′ is R 28′ SO 3 H.

14. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R 6 is:

wherein

R g1 and R g2 are C(O)NHR 38 SO 3 H.

15. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R 1 and R 2 are independently selected from CH 3 and R 28 SO 3 H.

16. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R 4′ is:

wherein

R g1′ and R g2′ are C(O)NHR 38′ SO 3 H.

17. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 3′ is R 28′ SO 3 H.

18. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R 1′ and R 2′ are independently selected from CH 3 and R 28′ SO 3 H.

19. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 , wherein said reactive functional group is a member selected from an amine, a hydroxy, an aldehyde, a ketone, a hydroxylamine, a sulfhydryl, an isocyanate, an isothiocyanate, a haloacetamide, a hydrazine, an ester, an anhydride, an acyl azide, an acyl halide, a sulfonyl halide, a maleimide, a carbodiimide, a phosphoramidite, fluorinated azide and an imidazole.

20. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 , wherein said reactive functional group is a member selected from:

wherein

each r is independently selected from the integers from 1 to 10;

G is a halogen; and

R 30 and R 31 are members independently selected from H and halogen and at least one of R 30 and R 31 is halogen.

21. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 , wherein said linker includes a linkage fragment of a formula which is a member selected from:

—(CH 2 ) t S(CH 2 ) z —, —(CH 2 ) x SC(O)NR(CH 2 ) z —, —(CH 2 ) x SC(O)O(CH 2 ) z —, —(CH 2 ) x NR(CH 2 ) z —, —(CH 2 ) x NRC(O)(CH 2 ) z —, —(CH 2 ) x NRC(O)O(CH 2 ) z —, —(CH 2 ) x O(CH 2 ) z —, —(CH 2 ) x C(O)(CH 2 ) z S—, —S-maleimide-N—, —RNC(O)NR—, —RNS(O)NR—, —S(O) 2 NR—, —C(O)NH—, —C(O)O—, and —NH—, wherein

R is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

and

t, x and z are independently selected from the integers from 0 to 10.

22. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 , wherein said carrier molecule comprises the structure:

wherein

u is selected from the integers from 1 to 8;

J is a member selected from H, OH, and OMe; and

Y is a nucleobase.

23. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 22 , wherein the linker binding the fluorescent compound to said carrier molecule has the formula:

wherein L D is a member selected from:

24. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 , which is a trimethine cyanine compound having an emission maximum of at least about 635 nm, said compound having the formula:

25. A method of monitoring an enzyme reaction, said method comprising:

(a) forming a reaction mixture by contacting said enzyme with said fluorescent cyanine compound, or a covalent conjugate thereof, according to claim 1 wherein said dye is a substrate for said enzyme under conditions sufficient for said enzyme and said dye to react; and

(b) monitoring fluorescence of said reaction mixture.

26. The method according to claim 25 wherein, said enzyme is a DNA polymerase and said dye comprises a nucleic acid moiety which is said substrate for said enzyme.

27. The method according to claim 26 , wherein said enzyme reaction is template directed DNA synthesis.

28. The method according to claim 26 , wherein said reaction is a component of a single molecule DNA sequencing analysis.

29. A fluorescent cyanine compound, or a covalent conjugate thereof, having a formula selected from:

wherein

R 1 , R 2 and R 3 are independently CH 2 R 7 ;

wherein

R 4 , R 5 , R 6 , R 6′ , and R 7 are members independently selected from H, R 28 , R 28 Z 2 , SO 3 H, CO 2 H, R 28 SO 3 H, C(O)NHR 28 SO 3 H, CONHR 28 C(O)Z 2 ,

wherein

Z 2 is selected from OH, O—, COOH, an activated carboxyl moiety, and a linker covalently bound to a carrier molecule;

R g1 and R g2 are members independently selected from H, R 38 , R 38 Z 3 , SO 3 H, CO 2 H, R 38 SO 3 H, CONHR 38 SO 3 H, CONHR 38 C(O)Z 2 ,

wherein

 R 28 and R 38 are independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;

 Z 3 is selected from OH, O—, COOH, an activated carboxyl moiety, and a linker covalently bound to a carrier molecule;

in which wherein

R 1′ , R 2′ and R 3′ are independently CH 2 R 7′ ;

wherein

R 4′ , R 5′ , and R 7′ are members independently selected from H, R 28′ , R 28′ Z 2′ , SO 3 H, CO 2 H, R 28′ SO 3 H, CONHR 28′ SO 3 H, CONHR 28′ C(O)Z 2′ , and

wherein

Z 2′ is selected from OH, O—, COOH, an activated carboxyl moiety, and a linker covalently bound to a carrier molecule;

R g1′ and R g2′ are members independently selected from H, R 38′ , R 38′ Z 3′ , SO 3 H, CO 2 H, R 38′ SO 3 H, CONHR 38′ SO 3 H, CONHR 38′ C(O)Z 3′ ,

wherein

 R 28′ and R 38′ are independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; and

 Z 3′ is selected from OH, O—, COOH, an activated carboxyl moiety, a reactive functional group, and a linker covalently bound to a carrier molecule,

at least one of Z 2 , Z 2′ , Z 3 , Z 3′ , R 1 , R 1′ , R 2 , R 2′ , R 3 , R 3′ , R 4 , R 4′ , R 5 , R 5′ , R 6 and R 6′ is a linker covalently bound to a carrier molecule; and

said compound comprises at least three SO 3 H moieties.