Fluorescent quenching detecting reagents and methods

• US 7,897,736 B2
• Filed: 01/06/2010
• Issued: 03/01/2011
• Est. Priority Date: 12/08/1999
• Status: Expired due to Fees

• Current Assignee: ELITechGroup Inc. (Bruker Corporation)
• Original Assignee: Elitech Holding BV (Bruker Corporation)
• Inventors: Dempcy, Robert O., Lukhtanov, Eugeny Alexander, Vermeulen, Nicolaas M. J., Reed, Michael W., Gall, Alexander A.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/683,326
• Publication Number:
  US 20100174058A1
• Time in Patent Office: 419 Days
• Field of Search: 536/4.1, 536/23.1, 536/25.3, 536/26.6, 435/6
• US Class Current: 536/4.1
• 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: 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. A modified base comprising a quencher moiety and having the structure:

2. The modified base of claim 1 wherein one of Ar¹ and Ar² is a substituted aryl group.

3. The modified base of claim 1 wherein one of Ar¹ and Ar² is substituted with -halogen, —

OR′

, —

OC(O)R′

, —

NR′

R″

, —

SR′

, —

R′

, —

CN, —

NO₂, —

CO₂ R′

, —

CONR′

R″

, —

C(O)R′

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NRC″

C(O)₂ R′

, —

NR′

—

C(O)NR″

R′

″

, —

NH—

C(NH₂)NH, —

NR′

C(NH₂)NH, —

NH—

C(NH₂)═

NR′

, —

S(O)R′

, —

S(O)₂ R′

, —

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.

4. The modified base of claim 1 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 increase the wavelength absorbance maximum of the compound.

5. The modified base of claim 4 wherein Ar³ is substituted with -halogen, —

OR′

, —

OC(O)R′

, —

NR′

R″

, —

SR′

, —

R′

, —

CN, —

NO₂, —

CO₂ R′

, —

CONR′

R″

, —

C(O)R′

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NRC″

C(O)₂ R′

, —

NR′

—

C(O)NR″

R′

″

, —

NH—

C(NH₂)NH, —

NR′

C(NH₂)NH, —

NH—

C(NH₂)═

NR′

, —

S(O)R′

, —

S(O)₂ R′

, —

S(O)₂ NR′

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.

6. The modified base of claim 1 having the structure:

7. The modified base of claim 1 having the structure:

8. The modified base of claim 1 having the structure:

9. A method for synthesizing an oligonucleotide or oligonucleotide conjugate having one or more modified bases comprising synthesizing an oligonucleotide or oligonucleotide conjugate and incorporating into the oligonucleotide or oligonucleotide conjugate at least one modified base of claim 1.

10. A modified base comprising a quencher moiety and having the structure:

11. The modified base of claim 10 wherein one of Ar¹ and Ar² is a substituted aryl group.

12. The modified base of claim 10 wherein one of Ar¹ and Ar² is substituted with -halogen, —

OR′

, —

OC(O)R′

, —

NR′

R″

, —

SR′

, —

R′

, —

CN, —

NO₂, —

CO₂ R′

, —

CONR′

R″

, —

C(O)R′

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NRC″

C(O)₂ R′

, —

NR′

—

C(O)NR″

R′

″

, —

NH—

C(NH₂)NH, —

NR′

C(NH₂)NH, —

NH—

C(NH₂)═

NR′

, —

S(O)R′

, —

S(O)₂ R′

, —

S(O)₂ NR′

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.

13. The modified base of claim 10 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 increase the wavelength absorbance maximum of the compound.

14. The modified base of claim 13 wherein Ar³ is substituted with -halogen, —

OR′

, —

OC(O)R′

, —

NR′

R″

, —

SR′

, —

R′

, —

CN, —

NO₂, —

CO₂ R′

, —

CONR′

R″

, —

C(O)R′

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NRC″

C(O)₂ R′

, —

NR′

—

C(O)NR″

R′

″

, —

NH—

C(NH₂)NH, —

NR′

C(NH₂)NH, —

NH—

C(NH₂)═

NR′

, —

S(O)R′

, —

S(O)₂ R′

, —

S(O)₂ NR′

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.

15. A method for synthesizing an oligonucleotide or oligonucleotide conjugate having one or more modified bases comprising synthesizing an oligonucleotide or oligonucleotide conjugate and incorporating into the oligonucleotide or oligonucleotide conjugate at least one modified base of claim 10.

16. A modified base comprising a quencher moiety and having the structure:

17. A method for synthesizing an oligonucleotide or oligonucleotide conjugate having one or more modified bases comprising synthesizing an oligonucleotide or oligonucleotide conjugate and incorporating into the oligonucleotide or oligonucleotide conjugate at least one modified base of claim 16.