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Non-fluorescent quencher compounds and biomolecular assays
Inactive Publication Date: 2003-05-01
APPL BIOSYSTEMS INC
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0004] The present invention provides bis-diazo,triaryl I and aryldiazo-N-arylphenazonium II compounds which are useful as quen
Problems solved by technology
Fluorescent quenching acceptors, e.g. TAMRA (tetramethylrhodamine), have been employed in FRET assays, but have at least two significant limitations: background fluorescence and the preclusion of detection of reporter fluorescence at the acceptor fluorescence emission.
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example 1
Synthesis of Ethyl 4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4
[0146] 25
[0147] 2,5-Dimethoxyaniline 1 (7.7 gm, 0.050 moles) was dissolved in 40 ml tetrahydrofuran (THF) and chilled to 0.degree. C. under argon. Acetic anhydride (7.72 ml, 0.082 moles) was added. After stirring for 2 hours, thin-layer chromatography (TLC) indicated the reaction was complete. The mixture was evaporated under reduced pressure and kept under vacuum overnight.
[0148] Crude 2,5-dimethoxyacetanilide 2 was dissolved in 80 ml THF and stirred under argon. Borane-methylsulfide complex (62.5 ml, 2.0M in THF, 0.125 moles) was added by syringe over 10 minutes. After 10 minutes at ambient temperature, the mixture was heated to reflux for 3 hours. Analysis by TLC showed only a trace of 2. After cooling to room temperature, 42 ml water was cautiously added with stirring, followed by 4.5 ml conc. HCl. The reaction was heated for 1 hour. Next added sequentially with vigorous stirring were 125 ml water, 250 ml dichlorom...
example 2
Synthesis of Ethyl 4-(ethylphenylamino)butanoate 6
[0150] 26
[0151] N-ethylaniline 5 (2.0 gm, 0.016 moles), triethylamine (5.0 ml, 0.036 moles), and ethyl 4-bromobutyrate (3.45 gm, 0.018 moles) were heated at 110.degree. C. for 14 hours under argon. The mixture was cooled to room temperature, diluted with 50 ml ethyl acetate, washed with 50 ml water and 50 ml saturated NaCl, dried over MgSO.sub.4, filtered, and evaporated under reduced pressure to give crude ethyl 4-(ethylphenylamino)butanoate 6 (3.0 gm, 77% yield). .sup.1H NMR (CDCl.sub.3) .delta. 7.2, 2H, m; 6.6, 3H, m; 4.15, 2H, q; 3.3, 4H, m; 2.38, 2H, t; 1.9, 2H, m; 1.25, 3H, t; 1.10, 3H, t.
example 3
Synthesis of Methyl 2-(ethylphenylamino)acetate 7
[0152] 27
[0153] N-Ethylaniline 5 (2 g, 16.52 mmol) was added to a stirred suspension of sodiumhydride (436 mg, 18.17 mmol) in DMF (20 ml) at ambient temperature under argon. After 45 minutes, bromomethyl acetate (3.02 g, 18.17 mmol) was added and the reaction mixture was stirred for 22 hr. DMF was removed under reduced pressure and the residue was treated with ethyl acetate (60 ml) and water (50 ml). The organic extract was washed with saturated brine (50 ml), dried (Na.sub.2SO.sub.4), filtered and evaporated to give crude Methyl 2-(ethylphenylamino) acetate 7 as liquid (3.06 g, 96%).
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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 from fluorescent reporter labels by any energy-transfer mechanism, such as FRET. Fluorescence quencher compositions are useful in preparing quencher labelled biomolecules for various molecular biology assays based on fluorescence detection.
Description
I. FIELD OF THE INVENTION[0001] This invention relates generally to the field of molecular biology and more particularly, to methods and reagents of biomolecular detection and fluorescence-based assays.II. INTRODUCTION[0002] Researchers use fluorescencequenching biomolecular assays to detect the interaction, assembly, cleavage, dissociation and conformations of proteins, nucleic acids, and other biomolecules. Fluorescenceresonanceenergy transfer (FRET) is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity, conformation, and bond formation and cleavage. FRET has been applied in detection of labelled biomolecules to important areas of genomics: identification of single-nucleotide polymorphisms and other allelic variants, protein-protein interactions, and genome-wide analysis of regulatory sequences. Genetic information is being rapidly gleaned by automated sequencing of genomes from all kinds of organisms. As this ...
Claims
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