Electron-deficient nitrogen heterocycle-substituted...

Details Electron-deficient nitrogen heterocycle-substituted... Electron-deficient nitrogen heterocycle-substituted...

2000-02-07

2001-04-24

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S546000, C436S800000, C530S402000, C536S024300, C544S235000, C544S238000, C544S283000, C544S333000, C544S353000, C544S405000, C546S119000, C546S144000, C546S173000, C546S283100, C548S146000, C548S159000, C548S215000, C548S217000, C548S250000, C548S256000, C548S266200, C548S305100, C548S311400, C548S364400

Reexamination Certificate

active

06221604

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to the field of fluorescent dye compounds useful as labelling reagents to prepare molecular probes. More specifically, this invention relates to fluorescein dyes with a xanthene ring structure and electron deficient nitrogen heterocycle substituents.
BACKGROUND
The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modern analytical biotechnology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact and costs associated with reagent disposal are greatly reduced. Examples of methods utilizing such fluorescent detection methods include automated DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
In many important applications, it is advantageous to employ multiple spectrally distinguishable fluorescent labels in order to achieve independent detection of a plurality of spatially overlapping analytes, i.e. multiplex fluorescent detection. Examples of methods utilizing multiplex fluorescent detection include single-tube multiplex DNA probe assays, PCR, single nucleotide polymorphisms, immunoassays, and multi-color automated DNA sequencing. The number of reaction vessels may be reduced thereby simplifying experimental protocols and facilitating the production of application-specific reagent kits. In the case of multi-color automated DNA sequencing, multiplex fluorescent detection allows for the analysis of multiple nucleotides in a single electrophoresis lane thereby increasing throughput over single-color methods and reducing uncertainties associated with inter-lane electrophoretic mobility variations. Automated four-color Sanger-type DNA sequencing has enabled entire genome characterization at the molecular level.
Assembling a set of multiple spectrally distinguishable fluorescent labels useful for multiplex fluorescent detection is problematic. Multiplex fluorescent detection imposes at least six severe constraints on the selection of component fluorescent dye labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and/or treatment with enzymes, e.g., automated DNA sequencing. First, it is difficult to find a set of structurally similar dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm). Second, even if dyes with non-overlapping emission spectra are identified, the set may still not be suitable if the respective fluorescent quantum efficiencies are too low. Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are usually widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobility of the analyte. Fifth, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the analyte, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like. Sixth, the dye must have sufficient photostability to withstand laser excitation.
Currently available multiple dye sets suitable for use in four-color automated DNA sequencing applications require blue or blue-green laser light to adequately excite fluorescence emissions from all of the dyes making up the set, e.g., argon-ion lasers. As lower cost red lasers become available, a need develops for fluorescent dye compounds and their conjugates which satisfy the above constraints and are excitable by laser light having a wavelength above about 500 nm.
SUMMARY
The present invention relates to dye compounds suitable for the creation of sets of spectrally-resolvable fluorescent labels useful for multi-color fluorescent detection.
Generally the dyes of the invention comprise a fluorescein-type, xanthene ring structure I:
substituted with at least one electron-deficient nitrogen heterocycle linked to the fluorescein ring system at R
1
, R
2
, R
3
, R
4
, R
5
, or R
7
.
R
1
, when taken alone, is H, F, Cl, (C
1
-C
6
) alkyl, (C
1
-C
6
) substituted alkyl, (C
1
-C
6
) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R
7
is benzo or heterocycle.
R
2
, when taken alone, is H, F, Cl, (C
1
-C
6
) alkyl, (C
1
-C
6
) substituted alkyl, (C
1
-C
6
) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle.
R
3
, when taken alone, is H, F, Cl, (C
1
-C
6
) alkyl, (C
1
-C
6
) substituted alkyl, (C
1
-C
6
) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle.
R
4
, when taken alone, is H, F, Cl, (C
1
-C
6
) alkyl, (C
1
-C
6
) substituted alkyl, (C
1
-C
6
) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R
5
is benzo or heterocycle.
R
5
, when taken alone, is H, F, Cl, (C
1
-C
6
) alkyl, (C
1
-C
6
) substituted alkyl, (C
1
-C
6
) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R
4
is benzo or heterocycle.
R
7
, when taken alone, is H, F, Cl, (C
1
-C
6
) alkyl, (C
1
-C
6
) substituted alkyl, (C
1
-C
6
) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R
4
is benzo or heterocycle.
R
6
can be (C
1
-C
6
) alkyl, (C
2
-C
6
) alkene, (C
2
-C
6
) alkyne, cyano, heterocyclic aromatic, phenyl, and substituted phenyl having the structure II:
wherein X
1
, X
2
, X
3
, X
4
and X
5
taken separately are H, Cl, F, (C
1
-C
6
) alkyl, (C
2
-C
6
) alkene, (C
2
-C
6
) alkyne, CO
2
H, SO
3
H, CH
2
OH, or reactive linking group.
In one aspect, the invention provides a method of labelling a substrate with a fluorescein dye of structure I, where the substrate reacts with the reactive linking group of the dye and a substrate-dye conjugate is formed. Substrate dye-labelled conjugates comprise the electron deficient nitrogen heterocycle-substituted fluorescein dye, according to I, and a substrate, i.e. another molecule or substance. The substrate may be labelled with one or more dyes of the invention, which may be the same or different. Fluorescence from the dyes provides detectable signals across a spectral range, enabling differentiation of differently labelled substrates in a single sample or mixture.
In one embodiment, the electron deficient nitrogen heterocycle-substituted fluorescein dye is covalently conjugated to another dye compound to form an energy-transfer dye compound.
In another embodiment, the electron deficient nitrogen heterocycle-substituted fluorescein dye is covalently conjugated to a nucleoside, nucleotide, nucleoside and nucleotide analog, polynucleotide or polynucleotide analog to form labelled conjugates therewith.
In yet another aspect, the invention provides phosphoramidite reagents including the electron deficient nitrogen heterocycle-substituted fluorescein dyes of the invention.
In another aspect, the invention provides various methods for synthesizing oligonucleotides labelled with electron-deficient nitrogen heterocycle-substituted fluorescein dyes, and employing the dyes for detection of fluorescent labelled polynucleotides.
In another aspect, the invention provides kits comprising electron-deficient nitrogen heterocycle-substituted fluorescein dyes and reagents useful for labelling molecules and/or for performing assays such as DNA sequencing and amplification, e.g. polymerase chain reaction.
The electron-deficient nitrogen hetero

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