Fluorescent quenching detection reagents and methods

• US 6,790,945 B2
• Filed: 06/06/2001
• Issued: 09/14/2004
• Est. Priority Date: 12/08/1999
• Status: Expired due to Term

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Lukhtanov, Eugeny A., Gall, Alexander A., Dempcy, Robert O., Vermeulen, Nicolaas M. J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/876,830
• Publication Number:
  US 20020034754A1
• Time in Patent Office: 1,196 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.33, 536/25.34, 536/26.22, 536/25.32, 435/6, 534/727
• US Class Current: 536/23.1
• 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 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/572 Five-membered rings
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• 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 29/0813 substituted by OH, O-C(=X)-R, O-C(=X)-X-R, O-R (X being O,S,NR; R being hydrocarbonyl)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Oligonucleotide probes containing two labels are provided and are useful in hybridization assays. The probes can also contain a minor groove binding group.

Claims

1. A compound having the formula:

wherein

Ar¹ and Ar² are each independently a substituted or unsubstituted aryl group, where at least one of Ar¹ and Ar² is a substituted aryl;

X₁ is selected from the group consisting of OH, O-dimethoxytrityl, O-methoxytrityl, O-trityl and an oxygen atom having an acid labile blocking group;

X₂ is a phosphoramidite; and

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof.

12. A compound of claim 1 wherein W is acylic.

13. A compound of claim 1 wherein W comprises a cyclic group.

2. A compound having the formula:

wherein

Ar¹ and Ar² are each independently a substituted or unsubstituted aryl group, where at least one of Ar¹ and Ar² is a substituted aryl;

X₁ is selected from the group consisting of H, (C₁-C_1-C12)alkyl, aryl, heteroaryl, and protected or unprotected functional group;

X₂ is selected from the group consisting of a phosphorous coupling moiety, a pentafluorophenoxy moiety and a succinimidyl moiety; and

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof.

14. A compound of claim 2 wherein W is a cyclic.

15. A compound of claim 2 wherein W comprises a cyclic group.

3. A compound having the formula:

wherein

Ar¹ and Ar² are each independently a substituted or unsubstituted aryl group,where at least one of Ar¹ and Ar² is a substituted aryl;

X₁, is selected from the group consisting of H, (C₁-C₁₂)alkyl, aryl, heteroaryl and protected or unprotected functional group;

X₂ is a phosphoramidite; and

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof.

16. A compound of claim 3 wherein W is acyclic.

17. A compound of claim 3 wherein W comprises a cyclic group.

4. A compound having the formula:

wherein

Ar¹ and Ar² are each independently a substituted or unsubstituted aryl group;

X₁ is selected from the group consisting of H, (C₁-C₁₂)alkyl, aryl, heteroaryl, and protected or unprotected functional group;

X₂ is a moiety reactive towards nucleophiles;

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof; and

wherein one of Ar¹ and A² is directly or indirectly substituted with a substituted aryl group (A³), where Ar³ extends the resonance ability of the Ar¹—

N═

N—

A² aromatic system and thereby increases the wavelength absorbance maximum of the compound.

5. A compound of claim 4 wherein Ar¹ is directly substituted with Ar³.

6. A compound of claim 4 wherein Ar¹ is indirectly substituted with Ar³.

7. A compound of claim 4 wherein A² is directly substituted with Ar³.

8. A compound of claim 4 wherein Ar² is indirectly substituted with Ar³.

18. A compound of claim 4 wherein W is acyclic.

19. A compound of claim 4 wherein W comprises a cyclic group.

20. A compound of claim 5 wherein W is acyclic.

21. A compound of claim 5 wherein W comprises a cyclic group.

22. A compound of claim 6 wherein W is a cyclic.

23. A compound of claim 6 wherein W comprises a cyclic group.

24. A compound of claim 7 wherein W is acyclic.

25. A compound of claim 7 wherein W comprises a cyclic group.

26. A compound of claim 8 wherein W is acyclic.

27. A compound of claim 8 wherein W comprises a cyclic group.

34. A compound of claims 2, 4-8 or 18-27 wherein X₂ is a phosphoramidite.

9. A compound having the formula:

wherein

Ar¹ and A² are each independently a substituted or unsubstituted aryl group;

X₁, is selected from the, group consisting of H, (C₁-C₁₂)alkyl, aryl, heteroaryl, and protected or unprotected functional group;

X₂ is a phosphoramidite;

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof; and

wherein Ar¹ or Ar² is indirectly substituted with an aryl group through a group selected from —

(C═

C)_n— and

—

(CR′

CR′

)_n—

where n is 0 to 5 and R′

is independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (u nsubstituted aryl)oxy-(C₁-C₄)alkyl.

28. A compound of claim 9 wherein W is acyclic.

29. A compound of claim 9 wherein W comprises a cyclic group.

10. A compound having the formula:

wherein

Ar¹ and Ar² are each independently a substituted or unsubstituted aryl group;

X₁ is selected from the group consisting of H, (C₁-C₁₂)alkyl, aryl, heteroaryl, and protected or unprotected functional group;

X₂ is a phosphoramidite;

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof; and

Ar¹ or Ar² is indirectly substituted with an aryl group (Ar³) through a double bond selected from carbon—

carbon and nitrogen—

nitrogen double bonds.

30. A compound of claim 10 wherein W is cyclic.

31. A compound of claim 10 wherein W comprises a cyclic group.

11. A compound having the formula:

wherein

Ar¹ and A² are each independently a substituted or unsubstituted aryl group;

X₁ is selected from the group consisting of H, (C₁-C₁₂)alkyl, aryl, heteroaryl, and protected or unprotected functional group;

X₂ is a phosphoramidite;

W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof;

wherein one of Ar¹ and Ar² is directly or indirectly substituted with a substituted aryl group (Ar³), where Ar³ extends the resonance ability of the Ar¹—

N═

N—

Ar² aromatic system and thereby increases the wavelength absorbance maximum of the compound; and

at least one of Ar¹, A²and Ar³ is substituted with -halogen, —

OR′

, —

OC(O)R′

, —

NR′

R″

, —

SR′

, —

R′

, —

CN, —

NO₂, —

CO₂R′

, —

CONR′

R″

, —

C(O)R′

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NRC″

C(O)₂R′

, —

NR′

—

C(O)NR″

R′

″

, —

NH—

C(NH₂)NH, —

NR′

C(NH₂)NH, —

NH—

C(NH₂)═

NR′

, —

S(O)R′

, —

S(O)₂R′

, —

S(O)₂NR′

R″

, —

N3, —

CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and

where R′

, R″ and

R′

″

are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

32. A compound of claim 11 wherein W is acyclic.

33. A compound of claim 11 wherein W comprises a cyclic group.