Fluorescent quenching detection reagents and methods

• US 20030096254A1
• Filed: 03/29/2002
• Published: 05/22/2003
• Est. Priority Date: 12/08/1999
• Status: Abandoned Application

• Current Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Vermeulen, Nicolaas M.J., Gall, Alexander A., Dempcy, Robert O., Lukhtanov, Eugeny Alexander, Reed, Michael W.
• Application Number:
  US10/113,445
• Publication Number:
  US 20030096254A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• 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: C09B 69/106 containing an azo dye
• 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. An oligonucleotide probe compound having the formula:

6. A compound of claims 1 or 2 wherein Ar² is substituted with Ar³.

7. A compound of claim 6 wherein Ar² is indirectly substituted with Ar³.

8. A compound of claim 7 wherein Ar² is indirectly substituted with Ar³ through aromatic ring(s) and/or double bond(s).

9. A compound of claim 8 wherein Ar² is indirectly substituted with Ar³ through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.

10. A compound of claim 9 wherein Ar² is indirectly substituted with Ar³ through a group selected from —

(CH↓

C)_t— and

—

(CR′

═

CR′

)_t—

where t 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 (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

11. A compound of claims 1 or 2 wherein at least one of Ar¹, Ar² and 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′

, —

NR″

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.

12. A compound of claim 11 wherein Ar² is indirectly substituted with Ar³ through a double bond.

13. A compound of claim 12, wherein Ar²—

N═

N—

Ar¹—

has the structure;

14. A compound of claim 11 wherein Ar² is directly substituted with Ar³.

15. A compound of claim 14 wherein Ar²—

N═

N—

Ar¹—

has the structure;

2. A quencher-phosphoramidite reagent compound having the formula:

3. A compound of claim 2 wherein X₂ is selected from the group consisting of a phosphorous coupling moiety, a pentafluorophenoxy moiety and a succinimidyl moiety.

4. A compound of claim 2 wherein X₁ is selected from the group consisting of OH, O-dimethoxytrityl, O-methoxytrityl, O-trityl or an oxygen atom having an acid labile blocking group.

5. A compound of claim 2 wherein X₂ is a phosphoramidite of the formula —

O—

P(N(iPr)₂)(OCH₂CH₂CN).

16. An oligonucleotide probe compound having the formula:

19. A compound of claims 16 or 17 wherein W comprises a substituted aryl group (Ar³) which is directly or indirectly attached to Ar¹ and extends the resonance ability of the Ar¹—

N═

N—

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

20. A compound of claim 19 wherein Ar¹ is indirectly substituted with Ar³ through aromatic ring(s) and/or double bond(s).

21. A compound of claim 20 wherein Ar¹ is indirectly substituted with Ar³ through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.

22. A compound of claim 21 wherein Ar¹ is indirectly substituted with Ar³ through a group selected from —

(CH↓

C)_t— and

—

(CR′

═

CR′

)_t—

where t 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 (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

17. A quencher-phosphoramidite reagent compound having the formula:

18. A compound of claim 17 wherein X₂ is selected from the group consisting of a phosphorous coupling moiety, a pentafluorophenoxy moiety and a succinimidyl moiety.

23. A probe compound-for a hybridization assay comprising the formula FL-ODN-(W)_d-Q wherein

FL is a fluorophore with an emission wavelength in the range of about 300 to about 800 nm;

ODN is an oligonucleotide;

W is a linker group that has from 3 to 100 atoms other than hydrogen atoms, selected from C, N, O, S, P and Si, and is cyclic, acyclic, aromatic or a combination thereof, and d is 0 or 1; and

Q is a quencher moiety comprising the structure —

Ar¹—

N═

N—

Ar² wherein Ar¹ is joined to either ODN or W, and Q has a broader absorbance range than dabcyl (4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl, absorbance max=453 nm), wherein Ar¹ and Ar² are each independently a substituted or unsubstituted aryl group.

24. A compound of claim 23 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.

26. A compound of claims 24 or 25 wherein Ar² is indirectly substituted with Ar³ through aromatic ring(s) and/or double bond(s).

27. A compound of claim 26 wherein Ar² is indirectly substituted with Ar³ through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.

28. A compound of claims 24 or 25 wherein W comprises Ar³, and Ar¹ is indirectly substituted with Ar³ through aromatic ring(s) and/or double bond(s).

29. A compound of claim 28 wherein Ar¹ is indirectly substituted with Ar³ through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.

25. A compound of the formula

30. A compound of claim 25 wherein q is 0.

31. A compound of claim 25 wherein the solid support is selected from glass, controlled pore glass, polymeric materials, polystyrene, beads, coated glass or support that is compatible with oligonucleotide synthesis.