Heteroarylcyanine dyes with sulfonic acid substituents

• US 9,315,864 B2
• Filed: 11/20/2013
• Issued: 04/19/2016
• Est. Priority Date: 05/18/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Yue, Stephen, Shen, Gene, Sun, Wei-Chuan
• Primary Examiner(s): Andres, Janet L
• Assistant Examiner(s): Michelson, Ben S
• Application Number:
  US14/085,616
• Publication Number:
  US 20140080127A1
• Time in Patent Office: 881 Days
• Field of Search: 546/276.7, 546/277.4, 546/277.7, 546/256, 546/80, 435/6.11, 548/430
• US Class Current: 1/1
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Heteroaryl cyanine dyes bearing sulfonic acid substituents are of use in various assays, including single molecule nucleic acid sequencing. Exemplary heteroaryl cyanines dyes include or more reactive functional group, which is of use to covalently conjugate the cyanine dye to a carrier molecule. An exemplary carrier molecule is an analyte molecule or other molecule of interest, for example, a nucleotide oligophosphate.

Claims

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

2. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R⁴ and R⁵ are each H.

3. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R⁶ is a member selected from SO₃H and CO₂H.

4. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R³ is a member selected from R²⁸SO₃H and R²⁸CO₂H.

5. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R⁶ is H.

6. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R⁴ and R⁵ are independently selected from H and C(O)NHR²⁸SO₃H.

7. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XX, wherein R¹ and R² are independently selected from CH₃ and R²⁸SO₃H.

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;

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 CH₃ and R^28′

SO₃H.

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

is R^28′

SO₃H.

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′

SO₃H.

14. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R⁶ is:

15. The fluorescent cyanine compound, or covalent conjugate thereof, according to claim 1 according to Formula XXI, wherein R¹ and R² are independently selected from CH₃ and R²⁸SO₃H.

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

is;

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

is R^28′

SO₃H.

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 CH₃ and R^28′

SO₃H.

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:

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:

—

(CH₂)_tS(CH₂)_z—

, —

(CH₂)_xSC(O)NR(CH₂)_z—

, —

(CH₂)_xSC(O)O(CH₂)_z—

, —

(CH₂)_xNR(CH₂)_z—

, —

(CH₂)_xNRC(O)(CH₂)_z—

, —

(CH₂)_xNRC(O)O(CH₂)_z—

, —

(CH₂)_xO(CH₂)_z—

, —

(CH₂)_xC(O)(CH₂)_zS—

, —

S-maleimide-N—

, —

RNC(O)NR—

, —

RNS(O)NR—

, —

S(O)₂NR—

, —

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:

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:

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: