IP Library › Granted Patent US 9,018,369

Granted Patent B2

US 9,018,369 · App. 14/158,521 · Granted Apr 28, 2015

Dark quenchers for donor-acceptor energy transfer

Inventors: Ronald M. Cook (Novato, CA); Matt Lyttle (San Rafael, CA); Daren Dick (San Rafael, CA)

C07C245/08 C07B2200/11 C07D241/46 C07F9/091 C07F9/2408 C07H21/00 C09B31/043 C09B56/00 C12Q1/6818 C40B40/00 G01N33/542 G01N33/582

View Patent ↗

Loading inventors, assignments & file history…

Monitor This Case

Get email alerts when status or documents change.

Order Certified Copies

Most orders are placed with the USPTO same day — all within 24 business hours.

Order via The Patent Place →

Pre-filled with this patent's details

Quick Facts

Patent No.: US 9,018,369
App. No.: 14/158,521
Granted: Apr 28, 2015
Kind: B2

External Links

USPTO Patent Center↗ Full Patent PDF↗

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 (188)

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

wherein

X and Y are members independently selected from reactive functional groups;

wherein at least one of X and Y is a substituted alkyl amine comprising 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, —NH 2 , and phosphoramidite; or

at least one of X and Y is a substituted alkyl amine comprising a covalent bond to a solid support or to a nucleic acid;

m is a number selected from 0 to 4, inclusive, such that when m is greater than 1, the X groups are the same or different;

v is a number from 1 to 10 and when v is greater than 1, the R 2 groups are the same or different; and

R 1 , R 2 , and R 3 are members independently selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted unsaturated alkyl, with the proviso that at least two of R 1 , R 2 and R 3 are members selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and combinations thereof.

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, —NH 2 , 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 C 1 , C 2 , C 3 , C 4 , C 5 , or C 6 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:

wherein

R 5 , R 6 and R 7 are members independently selected from halogen, —NR′R″, substituted or unsubstituted aryl, nitro, substituted or unsubstituted C 1 -C 6 alkyl, and substituted or unsubstituted C 1 -C 6 alkoxy,

wherein R′ and R″ are independently selected from H and substituted or unsubstituted C 1 -C 6 alkyl;

X is a substituted alkyl amine comprising 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, —NH 2 , and phosphoramidite; or X is a substituted alkyl amine comprising a covalent bond to a solid support or to a nucleic acid;

Y is selected from the group consisting of reactive functional groups;

n is a number from 0 to 1, inclusive;

a is a number from 0 to 4, inclusive, such that when a is greater than 1, the R 5 groups are the same or different;

b is a number from 0 to 4, inclusive, such that when (v×b) is greater than 1, the R 6 groups are the same or different;

c is a number from 0 to 5, inclusive, such that when c is greater than 1, the R 7 groups are the same or different; and

v is a number from 1 to 10, inclusive, such that when v is greater than 1, the value of b on each of the v phenyl rings is the same or different.

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, —NH 2 , 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:

wherein X 5 is —NR′R″,

wherein R′ and R″ are members independently selected from unsubstituted alkyl and substituted alkyl,

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 C 1 , C 2 , C 3 , C 4 , C 5 , or C 6 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:

wherein

X and Y are members independently selected from reactive functional groups;

wherein at least one of X and Y is —NR′R″, wherein R′ is unsubstituted alkyl; and R″ is substituted C 1 , C 3 , C 4 , C 5 , or C 6 alkyl bearing a reactive functional group;

m is a number selected from 0 to 4, inclusive, such that when m is greater than 1, the X groups are the same or different;

v is a number from 1 to 10 and when v is greater than 1, the R 2 groups are the same or different; and

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, —NH 2 , and phosphoramidite.

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

wherein

wherein R′ and R″ are independently selected from H and substituted or unsubstituted C 1 -C 6 alkyl;

X is —NR′R″, wherein, for X, R′ is unsubstituted alkyl; and R″ is substituted C 1 , C 3 , C 4 , C 5 , or C 6 alkyl bearing a reactive functional group;

Y is selected from the group consisting of reactive functional groups;

n is a number from 0 to 1, inclusive;

a is a number from 0 to 4, inclusive, such that when a is greater than 1, the R 5 groups are the same or different;

b is a number from 0 to 4, inclusive, such that when (v×b) is greater than 1, the R 6 groups are the same or different;

c is a number from 0 to 5, inclusive, such that when c is greater than 1, the R 7 groups are the same or different; and

v is a number from 1 to 10, inclusive, such that when v is greater than 1, the value of b on each of the v phenyl rings is the same or different.

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

wherein X 5 is —NR′R″,

wherein

R′ is unsubstituted alkyl; and

R″ is substituted C 1 , C 3 , C 4 , C 5 , or C 6 alkyl bearing a reactive functional group.

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, —NH 2 , and phosphoramidite.

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

wherein

X and Y are members independently selected from reactive functional groups;

wherein at least one of X and Y is

wherein

R 9 is unsubstituted alkyl;

R 10 is substituted alkyl;

X 1 is —CH 3 ; and

X 2 is a member selected from —OH, —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

m is a number selected from 0 to 4, inclusive, such that when m is greater than 1, the X groups are the same or different;

v is a number from 1 to 10 and when v is greater than 1, the R 2 groups are the same or different; and

22. The quencher of excited state energy according to claim 21 , wherein X 3 is cyanoethyl; and X 4 is isopropyl.

23. The quencher of excited state energy according to claim 21 , wherein R 10 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:

wherein

wherein R′ and R″ are independently selected from H and substituted or unsubstituted C 1 -C 6 alkyl;

X is

wherein

R 9 is unsubstituted alkyl;

R 10 is substituted alkyl;

X 1 is —CH 3 ; and

X 2 is a member selected from —OH, —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

Y is selected from the group consisting of reactive functional groups;

n is a number from 0 to 1, inclusive;

a is a number from 0 to 4, inclusive, such that when a is greater than 1, the R 5 groups are the same or different;

b is a number from 0 to 4, inclusive, such that when (v×b) is greater than 1, the R 6 groups are the same or different;

c is a number from 0 to 5, inclusive, such that when c is greater than 1, the R 7 groups are the same or different; and

v is a number from 1 to 10, inclusive, such that when v is greater than 1, the value of b on each of the v phenyl rings is the same or different.

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

wherein X 5 is

wherein

R 9 is unsubstituted alkyl;

R 10 is substituted alkyl;

X 1 is —CH 3 ; and

X 2 is a member selected from —OH, —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl.

27. The quencher of excited state energy according to claim 25 , wherein X 3 is cyanoethyl; and X 4 is isopropyl.

28. The quencher of excited state energy according to claim 25 , wherein R 10 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:

wherein

X and Y are members independently selected from reactive functional groups;

wherein at least one of X and Y is

wherein

X 1 is —CH 3 ;

X 2 is a member selected from —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

p is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

q is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

m is a number selected from 0 to 4, inclusive, such that when m is greater than 1, the X groups are the same or different;

v is a number from 1 to 10 and when v is greater than 1, the R 2 groups are the same or different; and

31. The quencher of excited state energy according to claim 30 , wherein X 3 is cyanoethyl; and X 4 is isopropyl.

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

wherein

wherein R′ and R″ are independently selected from H and substituted or unsubstituted C 1 -C 6 alkyl;

X is

wherein

X 1 is —CH 3 ;

X 2 is a member selected from —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

p is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

q is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

Y is selected from the group consisting of reactive functional groups;

n is a number from 0 to 1, inclusive;

a is a number from 0 to 4, inclusive, such that when a is greater than 1, the R 5 groups are the same or different;

b is a number from 0 to 4, inclusive, such that when (v×b) is greater than 1, the R 6 groups are the same or different;

c is a number from 0 to 5, inclusive, such that when c is greater than 1, the R 7 groups are the same or different; and

v is a number from 1 to 10, inclusive, such that when v is greater than 1, the value of b on each of the v phenyl rings is the same or different.

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

wherein X 5 is

wherein

X 1 is —CH 3 ;

X 2 is a member selected from —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

p is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

q is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

34. The quencher of excited state energy according to claim 32 , wherein X 3 is cyanoethyl; and X 4 is isopropyl.

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

wherein

X and Y are members independently selected from reactive functional groups;

wherein at least one of X and Y is

wherein

X 1 is —CH 3 ;

X 2 is a member selected from —OH, —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

p is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

q is a number selected from 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

m is a number selected from 0 to 4, inclusive, such that when m is greater than 1, the X groups are the same or different;

v is a number from 1 to 10 and when v is greater than 1, the R 2 groups are the same or different; and

36. The quencher of excited state energy according to claim 35 , wherein X 3 is cyanoethyl; and X 4 is isopropyl.

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

wherein

wherein R′ and R″ are independently selected from H and substituted or unsubstituted C 1 -C 6 alkyl;

X is

wherein

X 1 is —CH 3 ;

X 2 is a member selected from —OH, —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

p is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

q is a number selected from 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

Y is selected from the group consisting of reactive functional groups;

n is a number from 0 to 1, inclusive;

a is a number from 0 to 4, inclusive, such that when a is greater than 1, the R 5 groups are the same or different;

b is a number from 0 to 4, inclusive, such that when (v×b) is greater than 1, the R 6 groups are the same or different;

c is a number from 0 to 5, inclusive, such that when c is greater than 1, the R 7 groups are the same or different; and

v is a number from 1 to 10, inclusive, such that when v is greater than 1, the value of b on each of the v phenyl rings is the same or different.

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

wherein X 5 is

wherein

X 1 is —CH 3 ;

X 2 is a member selected from —OH, —COOH, —NH 2 , —SH, and —OP(OX 3 )N(X 4 ) 2 ,

wherein X 3 and X 4 are members independently selected from alkyl and substituted alkyl;

p is a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

q is a number selected from 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

39. The quencher of excited state energy according to claim 37 , wherein X 3 is cyanoethyl; and X 4 is isopropyl.

Assignments (4)

RELEASE OF SECURITY INTEREST IN PATENTS PREVIOUSLY RECORDED AT REEL/FRAME (035957/0976) Recorded Mar 8, 2016

From: HSBC CORPORATE TRUSTEE COMPANY (UK) LIMITED

To: BIOSEARCH TECHNOLOGIES, INC.

Reel/Frame 038037/0669 →

CORRECTIVE ASSIGNMENT TO CORRECT THE INCORRECT APPL. NO. 61/996,902 PREVIOUSLY RECORDED AT REEL: 035957 FRAME: 0976. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Aug 11, 2015

From: BIOSEARCH TECHNOLOGIES, INC.

To: HSBC CORPORATE TRUSTEE COMPANY (UK) LIMITED, AS SECURITY AGENT

Reel/Frame 036335/0516 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 30, 2015

From: BIOSEARCH TECHNOLOGIES, INC.

To: HSB CORPORATE TRUST COMPANY (UK) LIMITED, AS SECURITY AGENT

Reel/Frame 035957/0976 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 15, 2014

From: COOK, RONALD M; LYTTLE, MATT; DICK, DAREN

To: BIOSEARCH TECHNOLOGIES, INC.

Reel/Frame 033738/0104 →

Continuity (6)

Continuation 12765844 · Apr 22, 2010

Continuation 12546927 · Aug 25, 2009

Continuation 11437991 · May 19, 2006

Division 11192705 · Jul 29, 2005

Continuation 09567863 · May 9, 2000

Related Publication 20140323703A1 · Oct 30, 2014