Dark quenchers for donor-acceptor energy transfer

• US 9,018,369 B2
• Filed: 01/17/2014
• Issued: 04/28/2015
• Est. Priority Date: 05/09/2000
• Status: Expired due to Term

• Current Assignee: HSB Corporate Trust Company (UK) Limited
• Original Assignee: Biosearch Technologies Incorporated
• Inventors: Cook, Ronald M., Lyttle, Matt, Dick, Daren
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/158,521
• Publication Number:
  US 20140323703A1
• Time in Patent Office: 466 Days
• Field of Search: 536/26.6, 534/558
• US Class Current: 536/26.6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 245/08 with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
• CPC Class Codes: C07D 241/46 Phenazines
• CPC Class Codes: C07F 9/091 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 31/043 Amino-benzenes
• CPC Class Codes: C09B 56/00 Azo dyes containing other chromophoric systems
• CPC Class Codes: C09B 56/12 Anthraquinone-azo dyes from diazotised aminoanthracene C09B29/0022, azo dyes containing hydroxyl groups acylated with polyfunctional anthraquinone derivatives C09B43/26
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention provides a family of dark quenchers, termed Black Hole Quenchers (“BHQs”), that are efficient quenchers of excited state energy but which are themselves substantially non-fluorescent. Also provided are methods of using the BHQs, probes incorporating the BHQs and methods of using the probes.

Claims

1. A quencher of excited state energy having the structure:

2. The quencher of excited state energy according to claim 1, wherein

at least one of X and Y is —

NR′

R″

, wherein R′ and

R″

are members independently selected from unsubstituted alkyl and substituted alkyl,

with the proviso that at least one of R′ and

R″

is selected such that it bears a reactive functional group selected from carboxy, N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acyl imidazole, thioester, p-nitrophenyl ester, alkenyl ester, alkynyl ester, aromatic ester, —

NH₂, and phosphoramidite, or at least one of R′ and

R″

is covalently attached to a solid support or to a nucleic acid.

3. The quencher of excited state energy according to claim 2, wherein R′

is unsubstituted alkyl; and

R″

is substituted alkyl.

4. The quencher of excited state energy according to claim 3, wherein R″

is substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl.

5. The quencher of excited state energy according to claim 3, wherein R″

comprises a polyether.

6. The quencher of excited state energy according to claim 5, wherein said polyether is a member selected from poly(ethylene glycol), polypropylene glycol) and copolymers thereof.

7. The quencher of excited state energy according to claim 1, wherein said solid support is controlled pore glass (CPG).

8. A quencher of excited state energy having the structure:

9. The quencher of excited state energy according to claim 8, wherein

X is —

NR′

R″

, wherein, for X, R′ and

R″

are members independently selected from unsubstituted alkyl and substituted alkyl,

with the proviso that, for X, at least one of R′ and

R″

is selected such that it bears a reactive functional group selected from carboxy, N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acyl imidazole, thioester, p-nitrophenyl ester, alkenyl ester, alkynyl ester, aromatic ester, —

NH₂, and phosphoramidite, or at least one of R′ and

R″

is covalently attached to a solid support or to a nucleic acid.

10. The quencher of excited state energy according to claim 9, having the structure:

11. The quencher of excited state energy according to claim 9, wherein R′

is unsubstituted alkyl; and

R″

is substituted alkyl.

12. The quencher of excited state energy according to claim 11, wherein R″

is substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl.

13. The quencher of excited state energy according to claim 11, wherein R″

comprises a polyether.

14. The quencher of excited state energy according to claim 13, wherein said polyether is a member selected from poly(ethylene glycol), polypropylene glycol) and copolymers thereof.

15. The quencher of excited state energy according to claim 8, wherein said solid support is controlled pore glass (CPG).

16. A quencher of excited state energy having the structure:

17. The quencher of excited state energy according to claim 16, wherein R″

bears a reactive functional group selected from carboxy, N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acyl imidazole, thioester, p-nitrophenyl ester, alkyl ester, alkenyl ester, alkynyl ester, aromatic ester, hydroxy, —

NH₂, and phosphoramidite.

18. A quencher of excited state energy having the structure:

19. The quencher of excited state energy according to claim 18, having the structure:

20. The quencher of excited state energy according to claim 18, wherein, for X, R″

bears a reactive functional group selected from carboxy, N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acyl imidazole, thioester, p-nitrophenyl ester, alkyl ester, alkenyl ester, alkynyl ester, aromatic ester, hydroxy, —

NH₂, and phosphoramidite.

21. A quencher of excited state energy having the structure:

22. The quencher of excited state energy according to claim 21, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

23. The quencher of excited state energy according to claim 21, wherein R¹⁰ comprises a polyether.

24. The quencher of excited state energy according to claim 23, wherein said polyether is a member selected from poly(ethylene glycol), polypropylene glycol) and copolymers thereof.

25. A quencher of excited state energy having the structure:

26. The quencher of excited state energy according to claim 25, having the structure:

27. The quencher of excited state energy according to claim 25, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

28. The quencher of excited state energy according to claim 25, wherein R¹⁰ comprises a polyether.

29. The quencher of excited state energy according to claim 28, wherein said polyether is a member selected from poly(ethylene glycol), poly(propylene glycol) and copolymers thereof.

30. A quencher of excited state energy having the structure:

31. The quencher of excited state energy according to claim 30, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

32. A quencher of excited state energy having the structure:

33. The quencher of excited state energy according to claim 32, having the structure:

34. The quencher of excited state energy according to claim 32, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

35. A quencher of excited state energy having the structure:

36. The quencher of excited state energy according to claim 35, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

37. A quencher of excited state energy having the structure:

38. The quencher of excited state energy according to claim 37, having the structure:

39. The quencher of excited state energy according to claim 37, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.