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Fluorescent dyes, energy transfer couples and methods

a technology of fluorescence and energy transfer, applied in the field of fluorescence dyes and fluorescence energy transfer, can solve the problems of imposing five severe constraints on selection, adding undesired time and expense, and difficult to find a set of dyes whose emission spectra are spectrally resolved

Inactive Publication Date: 2006-11-16
MILLIPORE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such purification steps add undesired time and expense to what should be a rapid automated process.
Multi-color fluorescent detection imposes five severe constraints on the selection of dye labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and / or treatment with enzymes, e.g., automated fluorescence-based DNA sequencing.
First, it is difficult to find a set of dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm) and the width of available spectrum is limited by the excitation light source.
Second, even if dyes with non-overlapping emission spectra are found, the set may still not be suitable if the respective fluorescent efficiencies are too low.
Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are widely separated.
One of the limitations of such presently available technologies arises from the available pool of restriction enzymes.
Another limitation pertains to the nature of information that is discoverable, i.e., whether the identified target is present, and its relative concentration.
It is not, however, readily possible to determine the relative proximity of target sites to each other under fixed or changing environmental conditions.

Method used

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  • Fluorescent dyes, energy transfer couples and methods
  • Fluorescent dyes, energy transfer couples and methods
  • Fluorescent dyes, energy transfer couples and methods

Examples

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Effect test

example 1

[0381] 2,3,4,5-Tetrachloro-6-(2-(4′-carboxyphenyl)-7-chloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid

[0382] 1A. 2,3,4,5-Tetrachloro-6-(5-chloro-2,4-dihydroxy-benzoyl)-benzoic acid (0.43 g, 1.0 mmol) and 2′,4′-dihydroxy-biphenyl-4-carboxylic acid (3, 0.23 g, 1.0 mmol) in methanesulfonic acid (10 mL) was placed in a pre-heated oil bath (T=130° C.) and stirred over a period of 2 h. The reaction mixture was allowed to cool to room temperature and quenched by pouring into ice cold water (200 mL). The solution was allowed to stand over a period of 2 h and the crude product was extracted with ethylacetate (EtOAc, 2×100 mL). The combined organic portions were washed with water (1×100 mL), and saturated sodium chloride (1×100 mL), dried (Na2SO4), and concentrated to give crude 1 as a dark brown solid. Purification via column chromatography (silica gel, 100 g, 1 / 1 EtOAc:hexanes 0.5% AcOH) gave the title compound (0.26 g, 42%) as an orange solid. Absorbance maximum (λmax 540 nm, 1×PBS), ...

example 2

[0388] 2,3,4,5-Tetrachloro-6-(2,7-bis(4′-carboxyphenyl)-6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid)

[0389] Tetrachloropthallic anhydride (31 mg, 0.11 mmol) and 2′,4′-dihydroxy-biphenyl-4-carboxylic acid (50 mg, 0.22 mmol) in methanesulfonic acid (2 mL) was placed in an pre-heated oil bath (T=130° C.) and stirred over a period of 0.75 h. The reaction mixture was allowed to cool to room temperature and quenched by pouring into ice cold water (50 mL). The solution was allowed to stand over a period of 2 h and the crude product was extracted with ethylacetate (EtOAc, 3×25 mL). The combined organic portions were washed with water (1×50 mL), and saturated sodium chloride (1×50 mL), dried (Na2SO4), and concentrated to give crude 1 as a dark brown solid. Purification via preparative layer chromatography (silica gel RP-18, 1 mm, 95 / 35 100 mM TEAB: AcCN, X 2) giving 4 as the major product. λmax 550 nm, MeOH; λem 575 nm, MeOH.

example 3

[0390] 2,3,4,5-Tetrachloro-6-(4-(4′-carboxyphenyl )-5-hydroxy-9-oxo-9H-benzo[a]xanthen-12-yl)-benzoic acid

[0391] Tetrachloropthallic anhydride (0.32 g, 1.1 mmol) and 2′,4′-dihydroxy-biphenyl-4-carboxylic acid (0.26 g, 1.1 mmol), 1,3-dihydroxynaphtalene (0.18 g, 1.1 mmol), in methanesulfonic acid (4 mL) was placed in an pre-heated oil bath (T=110° C.) and stirred over a period of 17 h. The reaction mixture was allowed to cool to room temperature and quenched by pouring into ice cold water (100 mL). The solution was allowed to stand over a period of 2 h and the crude product was extracted with ethylacetate (EtOAc, 3×25 mL). The combined organic portions were washed with water (1×50 mL), and saturated sodium chloride (1×50 mL), dried (Na2SO4), and concentrated to give crude 1 as a dark brown solid. Purification via column chromatography (silica gel 50 g, 4 / 1 EtOAc:hexanes, 1% AcOH) giving the title compound as the major product. ABS λmax 540 nm, 1×PBS; EM λem 570 nm, 1×PBS.

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Abstract

Fluorescent dyes, fluorescence energy transfer dye couples, multi-color dye sets, can be employed in art-recognized assays and certain novel methods, such as in proximity assays.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 612,297, filed Jul. 1, 2003, now U.S. Pat. No. ______, issued ______, 2006, and claims the benefit of provisional U.S. applications Ser. No. 60 / 393,338, filed Jul. 1, 2002 and Ser. No. 60 / 422,621, filed Oct. 30, 2002, all of which are incorporated herein by reference.FIELD OF THE INVENTION [0002] This invention relates to fluorescent dyes, fluorescence energy transfer, particularly to fluorescence energy transfer dye sets, donor dyes, acceptor / reporter dyes, linkers, combinations and methods of their use, e.g., in flow cytometry, DNA sequencing, protein arrays, DNA arrays, markers in microscopy. The compounds of the invention useful individually and particularly for multiplexing. BACKGROUND OF THE INVENTION [0003] The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modem analytical biotechnology. By eliminating ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C07D405/14C07D403/14A61P43/00C07D209/14C07D311/78C07D311/82C09B11/06C09B11/08C09B11/24C09B11/28C09B23/00C09B23/08C09B56/10C09K11/06G01N21/78G01N33/542
CPCC09B11/06C09B11/08G01N33/542C09B11/28C09B23/083C09B11/24A61P43/00
Inventor O'NEILL, ROGERFISHER, PETER V.
Owner MILLIPORE CORP
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