Dark quenchers for donor-acceptor energy transfer

a technology of donor-acceptor energy and dark quenchers, which is applied in the direction of instruments, group 5/15 element organic compounds, organic chemistry, etc., can solve the problems of significant complicating restricting so as to reduce the fluorescence of the probe, restrict the nucleic acid sequence, and simplify the design of the probe.

Inactive Publication Date: 2006-02-16
HSBC TRUSTEE COMPANY UK LIMITED AS SECURITY AGENT
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0018] Many of the nucleic acid probes presently used rely on an interaction between the fluorophore and the quencher in order to minimize the fluorescence of the probe in the absence of its hybridization to a complementary nucleic acid. The interaction between the fluorophore and the quencher is typically brought about by using a nucleic acid probe sequence that forms a secondary structure (e.g., hairpin, loop, etc.). Requiring that a probe adopt a secondary structure significantly complicates the design of the probe and greatly restricts the nucleic acid sequences that can be used as components of the probes. In contrast, nucleic acid probes using BHQs of the present invention are found to facilitate the interaction between the quencher and the fluorophore without requiring concomitant formation of nucleic acid secondary structure, thereby allowing a much greater diversity of nucleic acid sequences to be used as components of fluorescent probes.
[0019] Moreover, by varying the number and identity of the members of the conjugated system of the BHQs, the spectral properties (e.g., absorbance) of a BHQ can be “tuned” to match the spectral characteristics (e.g., emission) of one or more fluorophores. For example, as the BHQs of the present invention can be selected to have a broad range of absorbance maxima, these quenchers are uniquely suited for use in multiplexing applications. Furthermore, the ability to select a BHQ having a particular spectral characteristic allows the use of BHQs in multiplexing applications using one or more distinct fluorophore in combination with one or more distinct BHQ, thereby expanding the choices of donor-acceptor pairs that can be incorporated into probes.

Problems solved by technology

Requiring that a probe adopt a secondary structure significantly complicates the design of the probe and greatly restricts the nucleic acid sequences that can be used as components of the probes.

Method used

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  • Dark quenchers for donor-acceptor energy transfer
  • Dark quenchers for donor-acceptor energy transfer

Examples

Experimental program
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example 1

[0240] This Example sets forth the synthesis and characterization of BH1 and derivatives thereof.

1.1 Synthesis of 4-methyl-2-nitrobenzylazo-2′-methyl-5′-nitrobenzylazo-4″-N,N-di(2-hydroxyethyl) azobenzene, (BH1 diol), 6

[0241] To a rapidly stirred suspension of 25 g (60 mmol) Fast Corinth V salt (Aldrich 22,736-5) in 400 mL of chilled water (ice bath) was added 50 g (276 mmol) N-phenyldiethanolamine dissolved in 400 mL methanol and 300 mL sat'd NaHCO3 over 20 min. The mixture changed color from yellow to deep red during the course of the addition. The mixture was chilled for an additional 1 hr after addition, then filtered through a medium glass frit. The dark red solid was washed with 300 ml of ice cold water and air dried for 3 days. The yield of 6 was 27 g, (91%). TLC rf 0.15 (Silica plate, 5% MeOH in CH2Cl2). MALDI M / e 493.11 (Calc'd 492.5). 1H NMR (CDCl3, δ) 7.9 (d, 2H), 7.65 (m, 2H), 7.6 (s, 1H), 7.45(d, 1H), 7.4(s, 1H), 6.8(d, 2H), 4.0(s, 3H), 3.95(t, 2H), 3.85(t, 2H), 3.7(t...

example 2

[0245] This Example sets forth the synthesis and characterization of BH2 and derivatives thereof.

2.1 Synthesis of 4-nitrobenzylazo-2′,5′-dimethoxybenzylazo-4″-N,N-di(2-hydroxyethyl)azobenzene, (BH2 diol), 10

[0246] To a rapidly stirred suspension of 25 g (60 mmol) Fast Black K salt (Aldrich 20,151-0) in 400 mL of chilled water (ice bath) was added 50 g (276 mmol) N-phenyldiethanolamine dissolved in 400 mL methanol and 300 mL sat'd NaHCO3 over 20 min. The mixture changed color from brown to deep blue during the course of the addition. The mixture was chilled for an additional 1 hr after addition, then filtered through a medium glass frit. The dark blue solid was washed with 300 ml of ice cold water and air dried for 3 days. The yield of 10 was 22 g, (74%). TLC rf 0.2 (Silica plate, 5% MeOH in CH2Cl2). MALDI M / e 493.13 (Calc'd 494.4). 1H NMR (CDCl3, δ) 8.3 (d, 2H), 8.0(d, 2H), 7.85(d, 2H), 7.4(d, 2H), 6.7(m, 2H), 4.1(s, 3H), 3.95(s, 3H), 3.9(t, 2H), 3.8(t, 2H), 3.7(t, 2H), 3.5(t, 3H)...

example 3

[0250] This Example sets forth the synthesis and characterization of BH3 and derivatives thereof.

3.1 Synthesis of 3-diethylamino-5-phenylphenazium-7-(4′-N,N-di(2-hydroxyethyl)azobenzene)chloride, (BH3 diol), 14

[0251] 3-amino-7-(diethylamino)-5-phenylphenazium chloride (methylene violet 3RAX, Aldrich 30,750-5), 10 g (26 mmol) was stirred in 200 mL 1 N HCl while in an ice bath. A solution of 2 g NaNO2 in 20 mL of cold water was added dropwise over 20 min. The solution was stirred for 30 min. N-phenyldiethanolamine, 4.7 g (27 mmol), was dissolved in 100 mL of methanol and added to the methylene violet solution, after the pH was adjusted to 6 with NaOH solution. The solution changed color from violet to dark green. The solution was stirred for 1 hr, then extracted three times with 200 mL of CH2Cl2. The aqueous layer was evaporated, and the dark green solid was triturated with 3 200 mL portions of pyridine. The pyridine was evaporated to give 2.9 g (21% yield) of 14 as a dark green sol...

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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.

Description

BACKGROUND OF THE INVENTION [0001] There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, antibodies, and enzymes and nucleic acids, particularly those implicated in disease states. [0002] The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity, which characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C07B61/00C07D241/46C07F9/09C07F9/24C07H21/00C09B31/043C09B56/00
CPCC07B2200/11C07D241/46C07F9/091C07F9/2408C07H21/00G01N33/582C09B56/00C40B40/00G01N33/542C07C245/08C12Q1/6818C09B31/043C09B56/12
Inventor COOK, RONALDLYTTLE, MATTDICK, DAREN
Owner HSBC TRUSTEE COMPANY UK LIMITED AS SECURITY AGENT
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