Heteroarylcyanine dyes

• US 10,458,915 B2
• Filed: 05/20/2013
• Issued: 10/29/2019
• 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): Kosar, Andrew D
• Assistant Examiner(s): Kenyon, John S
• Application Number:
  US13/898,369
• Publication Number:
  US 20140005404A1
• Time in Patent Office: 2,353 Days
• Field of Search: 546256
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/08 more than three >CH- groups, e.g. polycarbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

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 or conjugate having the formula:

A-Q-B

wherein

Q has a formula which is a member selected from;

2. The compound or conjugate according to claim 1, wherein at least one of X³ and X^3′

is S.

3. The compound or conjugate according to claim 1, wherein at least one of X² and X^2′

is CH.

4. The compound or conjugate according to claim 1, wherein at least one of R⁶ and R^6′

is SO₃⁻

.

5. The compound or conjugate according to claim 1, wherein X², X^2′

, X³ and X^3′

are members independently selected from O, S, N, NH, CH, CH₂ and C(CH₃)₂.

6. The compound or conjugate according to claim 5, wherein at least one member selected from X², X^2′

, X³ and X^3′

is selected from NH, O and S.

7. The compound or conjugate according to claim 1, wherein not more than one member selected from X², X^2′

, X³ and X^3′

is O.

8. The compound or conjugate according to claim 7, wherein none of X², X^2′

, X³ and X^3′

is O.

9. The compound or conjugate according to claim 1, having a formula which is a member selected from:

10. The compound or conjugate according to claim 1, having a formula selected from:

11. The compound or conjugate according to claim 1, having a formula which is a member selected from:

12. The compound or conjugate according to claim 1, having the formula:

13. The compound or conjugate according to claim 1, having the formula:

14. The compound or conjugate according to claim 1, wherein R⁴, R⁵, R⁶, R⁷, R²² and R²³ are independently selected from:

15. The compound or conjugate according to claim 1, having a formula selected from:

16. The compound or conjugate 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, and an imidazole.

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

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

19. The compound or conjugate according to claim 1, having a formula which is a member selected from:

20. The compound or conjugate according to claim 19, having a formula selected from:

21. The compound or conjugate according to claim 1 having a formula selected from:

22. The compound or conjugate according to claim 1, wherein R²⁸ is substituted alkyl having from 1 to 20 carbon atoms.

23. The compound or conjugate according to claim 22, wherein said substituted alkyl is haloalkyl.

24. The compound or conjugate according to claim 23, wherein said haloalkyl is —

CF₃.

25. The compound or conjugate according to claim 1, wherein R^28′

is substituted alkyl having from 1 to 20 carbon atoms.

26. The compound or conjugate according to claim 25, wherein said substituted alkyl is haloalkyl.

27. The compound or conjugate according to claim 26, wherein said haloalkyl is —

CF₃.

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

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

(b) monitoring fluorescence of said reaction mixture.

29. The method according to claim 28 wherein, said enzyme is a DNA polymerase and said compound or conjugate comprises a nucleic acid moiety which is said substrate for said enzyme.

30. The method according to claim 28, wherein said enzyme reaction is template directed DNA synthesis.

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