HETEROARYLCYANINE DYES

• US 20140080127A1
• Filed: 11/20/2013
• Published: 03/20/2014
• 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
• Granted Patent: US 9,315,864 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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

The present invention provides heteroaryl functionalized cyanine dyes including a reactive functional moiety, or which are conjugated to a carrier molecule.

Claims

1. A fluorescent compound having the formula:

A-Q—

B

wherein

Q has a formula which is a member selected from;

2. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R⁴ and R⁵ are each H.

3. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R⁶ is a member selected from SO₃H and CO₂H.

4. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R³ is a member selected from R²⁸SO₃H and R²⁸CO₂H.

5. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R⁶ is H.

6. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R⁴ and R⁵ are independently selected from H and C(O)NHR²⁸SO₃H.

7. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R¹ and R² are independently selected from CH₃ and R²⁸SO₃H.

8. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R^5′

is H.

9. The compound according to claim 1, wherein B is a moiety according to Formula X¹ and R^4′

is;

10. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R^1′

and R^2′

are independently selected from CH₃ and R^28′

SO₃H.

11. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R^3′

is R^28′

SO₃H.

12. The compound according to claim 1, wherein B is a moiety according to Formula XII;

and R^5′

is H.

13. The compound according to claim 1, wherein B is a moiety according to Formula XII;

and R^3′

is R^28′

SO₃H.

14. The compound according to claim 1, wherein B is a moiety according to Formula XII;

and R⁶ is;

15. The compound according to claim 1, wherein B is a moiety according to Formula XII;

and R¹ and R² are independently selected from CH₃ and R²⁸SO₃H.

16. The compound according to claim 1, wherein B is a moiety according to Formula XII;

and R^4′

is;

17. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R^3′

is R^28′

SO₃H.

18. The compound according to claim 1, wherein B is a moiety according to Formula XI;

and R^1′

and R^2′

are independently selected from CH₃ and R^28′

SO₃H.

19. The compound 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 compound according to claim 1, wherein said reactive functional group is a member selected from:

21. The compound according to claim 1, wherein said linkage fragment has 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)_oT-PEG-, —

(CH₂)_xC(O)(CH₂)_zS—

, —

S-maleimide-N—

, —

RNC(O)NR—

, —

RNS(O)NR—

, —

S(O)₂NR—

wherein

T is a member selected from S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH, and O;

o is an integer from 1 to 50; and

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

22. The compound according to claim 1, wherein said carrier molecule comprises the structure:

23. The compound according to claim 1, wherein the linkage fragment binding the fluorescent compound to said carrier molecule has the formula:

24. A compound 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 a fluorescent dye 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 26 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 any of claim 26, wherein said reaction is a component of a single molecule DNA sequencing analysis.