Non-fluorescent asymmetric cyanine dye compounds useful for...

Details Non-fluorescent asymmetric cyanine dye compounds useful for... Non-fluorescent asymmetric cyanine dye compounds useful for...

1999-07-20

2002-02-19

Higel, Floyd D. (Department: 1626)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C546S087000, C546S165000, C546S174000, C546S270100, C546S284100, C435S006120, C536S022100, C536S025300

Reexamination Certificate

active

06348596

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to compounds useful as quenchers in a reporter-quencher energy transfer dye pair. Specifically, the present invention relates to non-radiative cyanine quencher compounds, reagents, such as nucleosides/tides and polynucleotides, incorporating such compounds and methods utilizing such compounds and/or reagents.
BACKGROUND
Nucleic acid hybridization assays comprise an important class of techniques in modern biology. Such assays have diverse applications, including the diagnosis of inherited disease, human identification, identification of microorganisms, paternity testing, virology, and DNA sequencing, e.g., sequencing by hybridization.
An important aspect of nucleic acid hybridization assays is the method used to facilitate detection of the hybridization event. A particularly important class of methods used in nucleic acid hybridization assays employs a reporter-quencher energy-transfer compound pair comprising a “reporter” compound and a “quencher” compound that interact through a fluorescence resonance energy transfer (FRET) process. In these methods, the reporter is a luminescent compound that can be excited either by chemical reaction, producing chemiluminescence, or by light absorption, producing fluorescence. The quencher can interact with the reporter to alter its light emission, usually resulting in the decreased emission efficiency of the reporter. This phenomenon is called quenching. The efficiency of quenching is strongly correlated with the distance between the reporter molecule and the quencher molecule. Thus, in a nucleic acid hybridization assay, detection of a hybridization event is accomplished by designing an energy transfer system in which the spacing between a reporter and a quencher is modulated as a result of the hybridization.
Quencher compounds that are presently used in FRET-based nucleic acid hybridization assays are themselves fluorescent. That is, in addition to quenching the fluorescence of the reporter, the quencher produces fluorescent emissions. This is problematic, particularly in assays employing multiple spectrally resolvable reporters. Because the quencher fluorescence can interfere with the fluorescent signal produced by one or more of the reporters, detection of a hybridization event can be problematic. Accordingly, there remains a continuing need for quencher compounds that are substantially non-fluorescent. In addition, there remains a need for reagents, such as nucleoside/tides and polynucleotides, that incorporate such quencher compounds in order to more reliably monitor, e.g., hybridization events.
SUMMARY OF THE INVENTION
The present invention is directed to Applicants' discovery of a class of non-fluorescent cyanine quencher compounds that are useful in the context of a reporter-quencher energy transfer compound pair. These quencher compounds find particular application in nucleic acid hybridization assays employing fluorescence energy transfer as a means of detection.
In one embodiment, the present invention relates to compounds of formula (I):
alone or in combination with a counterion thereof, wherein:
p is 0 or 1;
n is 0 or 1;
X is S, Se or O;
N
1
is nitrogen;
Z is selected from the group consisting of:
either:
(a) R
2
is A, Y
1
is H, and R
1
is a linking group, or
(b) R
2
is a linking group, and:
(i) R
1
is A, or
(ii) p is 1, and R
1
and Y
1
taken together are (CH
2
)
q
;
A is selected from the group consisting of alkyl, aryl, —CH
2
aryl, and —CH
2
)
m
N
+
(CH
3
)
3
;
q is an integer ranging from 2 to 4;
each m is independently an integer ranging from 2 to 12;
A or (CH
2
)
q
is unsubstituted or independently substituted with one or more of the same or different —NO
2
, —OH, alkoxy, —COOH, —COOC
1
-C
4
alkyl, —NHCHO, —NHCOC
1
-C
4
alkyl, —NHCOCH
3
, —NHCOCH
2
Cl, —NHCOCHCl
2
, —NHCOCCl
3
, —NHCOCF
3
, —NHCOCH
2
C
6
H
4
-o-NO
2
, —NHCOCH
2
OC
6
H
4
-o-NO
2
, —NHCOCH
2
COCH
3
, —NHCOCH
2
—N
+
C
5
H
5
Cl
−
, —NHCOCH
2
NHCS
2
CH
2
C
6
H
5
, —NHCOCH
2
CH
2
C
6
H
5
, —NHCOCH
2
CH
2
C
6
H
4
-p-OH, —NHCOCH
2
CH
2
C
6
H
4
-o-NO
2
, —NHCOC(CH
3
)
2
OC
6
H
4
-o-NO
2
, —NHCOC(CH
3
)
2
OC
6
H
4
-o-N═NC
6
H
5
, —NHCO(CH
2
)
3
Cl, —NHCOCH(CH
3
)
2
, —NHCOCH═CHC
6
H
4
-o-NO
2
, or —NHCO-2-pyridyl groups; either:
(a) R
3
, R
5
and R
6
are H and R
4
is —NO
2
; or
(b) R
3
and R
4
taken together form a benzo group substituted with one or two —NO
2
groups and R
5
and R
6
are hydrogen; or
(c) R
4
and R
5
taken together form a benzo group substituted with one or two —NO
2
groups and R
3
and R
6
are hydrogen; or
(d) R
5
and R
6
taken together form a benzo group substituted with one or two —NO
2
groups and R
3
and R
4
are hydrogen; and with the proviso that when R
1
in the compounds of formula (I) has an sp
3
hybridized carbon atom that is covalently attached to N
1
, then that carbon atom is methyl or, when substituted, primary,
and protected derivatives thereof.
The compounds of formula (I) are useful as non-fluorescent quenchers. In addition, they are useful in a composition further comprising a reporter dye, which have utility in determining how well matched are the reporter and quencher dyes with each other with respect to the ability of the quencher to quench the fluorescence of the reporter. The compounds of formula (I) are also useful: (i) when linked to a biomolecule, wherein the biomolecule is also linked to a reporter dye; and (ii) when linked to a biomolecule in a composition with a second biomolecule linked to a reporter dye in detecting the presence of a specific nucleotide sequence in a nucleic acid sample, in detecting the presence of contiguous sequences on a target nucleic acid, in detecting the presence of mutations within a target nucleic acid sequence, in monitoring the kinetics of nucleic acid hybridization, and in monitoring the progression of PCR reactions.
In a second embodiment, the present invention relates to compounds of formula (II) NUC—L′—R
41
—L—D alone or in combination with a counterion thereof, wherein: NUC is a nucleoside, a nucleotide, a nucleoside analog, or a nucleotide analog; L′ is a bond or a first spacer; R
41
is a covalent linkage; L is a bond or a second spacer; and D is the chromophore of a compound of formula (I), defined above, and protected derivatives thereof.
The compounds of formula (II) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a third embodiment, the present invention relates to compounds of the formula (III):
alone or in combination with a counterion thereof, wherein:
R
2
-R
6
, n, p, m, X, Z, NUC, L, L′ and R
41
are defined for formula (I); and
Y
1
is H, and protected derivatives thereof.
The compounds of formula (III) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a fourth embodiment, the present invention relates to compounds of formula (IV):
alone or in combination with a counterion thereof, wherein:
R
1
, R
3
-R
6
, n, p, X, L, L′, Y
1
, NUC and R
41
are defined as for formulas (I) and (II); and
Z is selected from the group consisting of:
and protected derivatives thereof.
The compounds of formula (IV) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a fifth embodiment, the present invention relates to compounds of formula (V)
NUC—C≡C—CH
2
—O—CH
2
—CH
2
—NR
57
—R
58
—R
41
—L—D  (V)
alone or in combination with a counterion thereof wherein:
NUC and D are defined as for formula (II);
R
41
is selected from a covalent linkage selected from the group consisting of carboxamides, esters, imines, hydrazones, oximes, alkyl amines, thioethers, ethers, thiophenols, aryl amines, boronate esters, hydrazides, N-acylureas or anhydrides, aminotriazines, triazinyl ethers, amidines, ureas, urethanes, thioureas, phosphite este

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