Non-fluorescent quencher compounds and biomolecular assays

• US 8,637,658 B2
• Filed: 09/27/2012
• Issued: 01/28/2014
• Est. Priority Date: 08/27/2001
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Ewing, Gregory J., Mullah, Khairuzzaman Bashar, Graham, Ronald
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/629,021
• Publication Number:
  US 20130123475A1
• Time in Patent Office: 488 Days
• Field of Search: 546/75, 536/26.6, 530/300, 435/4
• US Class Current: 536/26.6
• 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: C07C 245/10 with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings being part of condensed ring systems
• CPC Class Codes: C07C 255/67 having cyano groups and azido groups bound to the same carbon skeleton
• CPC Class Codes: C07C 317/32 with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 241/16 Halogen atoms; Nitro radicals
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• CPC Class Codes: C07F 9/1411 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/2404 the ester moiety containing a substituent or a structure which is considered as characteristic
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

Bis-diazo, triaryl and aryldiazo-N-arylphenazonium quencher moieties, substituted with electron-withdrawing and electron-donating substituents which induce polarity in the delocalized aryl/diazo ring systems, are useful as labels when attached to biomolecules such as polynucleotides, nucleosides, nucleotides and polypeptides. The quencher moieties are non-fluorescent and accept energy transfer from fluorescent reporter labels by any energy-transfer mechanism, such as FRET.

Fluorescence quencher compositions are useful in preparing quencher labeled biomolecules for various molecular biology assays based on fluorescence detection.

Claims

1. A composition useful for synthesizing a quencher-labeled polynucleotide which has the structure (II):

2. The composition of claim 1, wherein the electron-withdrawing groups are each, independently of one another, selected from NO₂, CN, CF₃, CO₂H, CO₂R′

, C(O)NH₂, N(O)NHR′

, C(O)NR′

R′

, CHO, C(O)R′

, SO₂R′

, SO₂CF₃, SO₂OR′

, SO₃H, NO and C₅-C₁₄ aryl, wherein each R′

is independently H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

3. The composition of claim 1, wherein the electron-donating groups are each, independently of one another, selected from O⁻

, S⁻

, NR″

R″

, NHR″

, NH₂, NHC(O)R″

, OR″

, OH, OC(O)R″

, SR″

, SH, Br, I, Cl, F, R″ and

C₅-C₁₄ aryl, wherein each R″

is independently H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

4. The composition of claim 1, wherein R¹⁰ is NO₂.

5. The composition of claim 1, wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ is OCH₃.

6. The composition of claim 1, wherein the reactive linking group comprises an electrophile.

7. The composition of claim 6, wherein the electrophile of the reactive linking group is a carboxylic acid or an ester.

8. The composition of claim 1, wherein one R″

is substituted C₁-C₁₂ alkyl and the other R″

is unsubstituted C₁-C₁₂ alkyl.

9. The composition of claim 8, wherein the one R″

that is unsubstituted C₁-C₁₂ alkyl is CH₂CH₃.

12. The composition of claim 1, wherein A is selected from;

13. The composition of claim 1, wherein X is selected from DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.

14. The composition of claim 1, wherein the solid support is selected from polystyrene, controlled-pore glass, silica gel, silica, polyacrylamide, polyacrylate, hydroxyethylmethycrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such polymers.

15. A quencher-labeled conjugate, produced by reacting a phosphoramidite reagent of a nucleoside, a nucleotide or a polynucleotide with the alkoxydiyl L² moiety of the composition of claim 1, after removal of the acid labile X moiety of the composition.

16. The quencher-labeled conjugate of claim 15, wherein the conjugated substance is a polynucleotide comprising 2-100 nucleotides, and protected forms thereof.

17. The quencher-labeled conjugate of claim 16, further comprising a fluorescent moiety covalently attached to the polynucleotide, the fluorescence of which is capable of being quenched by the quencher moiety when the fluorescent and quencher moieties are in close proximity to one another.

18. A method of attaching a nucleotide to the composition of claim 1, comprising:

removing the acid labile protecting group X from the composition thereby forming a deprotected composition;

contacting the deprotected composition with a phosphoramidite nucleotide reagent, thereby forming a quencher labeled nucleotide.

10. A compound having a structure of one of the following formulae:

11. A compound having a structure of one of the following formulae:

19. A 3′

quencher polynucleotide bound to a solid support, having the structure;

20. The 3′

quencher polynucleotide of claim 19, wherein A is selected from;

21. The 3′

quencher polynucleotide of claim 19, wherein the electron-withdrawing groups are each, independently of one another, selected from NO₂, CN, CF₃, CO₂H, CO₂R′

, C(O)NH₂, N(O)NHR′

, C(O)NR′

R′

, CHO, C(O)R′

, SO₂R′

, SO₂CF₃, SO₂OR′

, SO₃H, NO and C₅-C₁₄ aryl, wherein each R′

is independently H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

22. The 3′

quencher polynucleotide of claim 19, wherein the electron-donating groups are each, independently of one another, selected from O⁻

, S⁻

, NR″

R″

, NHR″

, NH₂, NHC(O)R″

, OR″

, OH, OC(O)R″

, SR″

, SH, Br, I, Cl, F, R″ and

C₅-C₁₄ aryl, wherein each R″

is independently H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

23. The 3′

quencher polynucleotide of claim 19, wherein the reactive linking group comprises an electrophile.

24. The 3′

quencher polynucleotide of claim 19, further comprising a fluorescent dye label.

25. A 5′

quencher polynucleotide bound to a solid support, having the structure;

26. The 5′

quencher polynucleotide of claim 25, wherein A is selected from;

27. The 5′

quencher polynucleotide of claim 25, wherein the electron-withdrawing groups are each, independently of one another, selected from NO₂, CN, CF₃, CO₂H, CO₂R′

, C(O)NH₂, N(O)NHR′

, C(O)NR′

R′

, CHO, C(O)R′

, SO₂R′

, SO₂CF₃, SO₂OR′

, SO₃H, NO and C₅-C₁₄ aryl, wherein each R′

is independently H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

28. The 5′

quencher polynucleotide of claim 25, wherein the electron-donating groups are each, independently of one another, selected from O⁻

, S⁻

, NR″

R″

, NHR″

, NH₂, NHC(O)R″

, OR″

, OH, OC(O)R″

, SR″

, SH, Br, I, Cl, F, R″ and

C₅-C₁₄ aryl, wherein each R″

is independently H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

29. The 5′

quencher polynucleotide of claim 25, wherein the reactive linking group comprises an electrophile.