Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Reexamination Certificate
2001-10-26
2004-12-07
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
C435S006120, C436S527000, C436S529000, C436S530000, C436S531000, C530S391300, C530S409000, C546S056000, C546S077000, C546S049000, C548S418000, C548S426000, C568S326000, C568S632000, C564S272000, C564S274000
Reexamination Certificate
active
06828159
ABSTRACT:
The invention relates to the use of carbopyronine compounds of the general formula (I) as labeling groups in procedures for the detection of analytes, to novel carbopyronine compounds and to a process for the preparation of these compounds.
In chemical, medical and biological analysis, dyes are used as labeling or detection groups. In particular, fluorescent dyes have gained importance in recent years and displaced other often cost-intensive procedures, which use, for example, radioisotopes for labeling.
In particular in the field of DNA sequencing, fluorometric procedures have gained acceptance in recent years and almost completely replaced the procedures customary up till then, which use radioactive isotopes.
In spite of the availability of various fluorescent dyes, such as, for example, FITC (fluorescein isothiocyanate), FLUOS (fluorescein N-hydroxysuccinimide ester), rhodamine derivatives etc., it was previously not possible to solve the problems due to background fluorescence, honspecific binding phenomena and the need for cost-intensive measuring equipment in a satisfactory manner.
As a result of background fluorescence and nonspecific binding, the sensitivity and accuracy of the measurements is reduced. In addition, in the case of available fluorescent dyes the absorption maximum lies in regions which do not make possible the use of light sources which are less expensive and which can be of small dimensions, such as, for example, He/Ne lasers and laser diodes.
An object of the present invention was thus to make available fluorescent dyes which can be employed as labeling groups in procedures for the detection of analytes and at least partially avoid the disadvantages of the prior art.
This object has been achieved by the use of compounds of the general formula (I)
as labeling groups in a procedure for the detection of an analyte, where
R
1
, R
2
, R
3
, R
4
, R
5
, R
6
and R
7
are in each case independently hydrogen, halogen, a hydroxyl, amino, sulfo or carboxyl or aldehyde group or a saturated or unsaturated, straight-chain, branched or cyclic hydrocarbon group having up to 20 C atoms, where the hydrocarbon groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, in particular phenyl, or/and heteroaryl radicals and optionally heteroatoms such as oxygen, sulfur or nitrogen atoms or/and two or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde, C
1
-C
4
-alkoxy or/and C
1
-C
4
-alkoxycarbonyl groups, or one or more of the radicals R
1
-R
7
, in each case with adjacent substituents, form a ring system which can contain one or more multiple bonds,
R
8
and R
8a
in each case independently are a saturated or unsaturated, straight-chain, branched or cyclic hydrocarbon group having up to 20 carbon atoms, e.g. a C
1
-C
6
-alkyl group, in particular methyl, ethyl, propyl or/and butyl, or an aryl or heteroaryl group, in particular phenyl, which optionally contain heteroatoms such as oxygen, sulfur or nitrogen atoms or/and one or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde, C
1
-C
4
-alkoxy or/and C
1
-C
4
-alkoxycarbonyl groups, or R
8
and R
8a
can form a ring system,
R
9
, R
10
, R
11
and R
12
in each case independently are hydrogen or a saturated or unsaturated, straight-chain, branched or cyclic hydrocarbon group having up to 20 C atoms, e.g. polyether, phenyl, phenylalkyl having 1-3 C atoms in the chain, where the hydrocarbon groups can optionally contain heteroatoms such as oxygen, sulfur or nitrogen atoms or/and one or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, phospho, carboxyl, carbonyl, alkoxy or/and alkoxycarbonyl groups,
or one or more of the radicals R
9
-R
12
, in each case with adjacent substituents, form a ring system which can contain one or more multiple bonds,
where —N(R
11
)(R
12
) or/and ═(R
9
)(R
10
) can be replaced by —OR
9
or/and ═O,
and X is optionally anions present for charge equalization.
The compounds of the general formula (I) can be employed as labeling groups in procedures for the qualitative or/and quantitative determination of an analyte. This determination can be carried out in aqueous liquids, e.g. samples of body fluids such as, for example, blood, serum, plasma or urine, wastewater samples or foodstuffs. The procedure can also be carried out as a wet test, e.g. in a cuvette, or as a dry test in an appropriate reagent carrier. The determination of the analyte can be carried out here by means of a single reaction or by means of a sequence of reactions. Surprisingly, the use of compounds of the general formula (I) showed very good results in chemical and in particular in medical and biological detection procedures for the determination of an analyte, especially in nucleic acid sequencing procedures and in protein analysis.
The compounds of the general formula (I) can be used in all chemical, medical and biological detection procedures known to the person skilled in the art in which fluorescent dyes are suitable as labeling groups. For this, the compounds of the general formula (I) are in general coupled covalently to a receptor which is specific for the analyte to be detected. This takes place using generally known procedures. The specific receptor can be any suitable compound or any suitable molecule, preferably it is a peptide, a polypeptide or a nucleic acid. The compounds or conjugates of these compounds can be used, for example, in nucleic acid, hybridization procedures, in particular for the sequencing of nucleic acids or immunochemical procedures. Procedures of this type are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor.
A further object of the present invention was to make available novel carbopyronine compounds which are suitable in particular for use as labeling groups in analyte detection procedures, can be prepared using simple and inexpensive processes, can be handled without problems and at least partially avoid the disadvantages of the prior art.
This object has been achieved by a compound of the general formula (I)
where
R
1
-R
12
and X have the meanings indicated above,
with the proviso that if R
1
-R
3
and R
5
-R
7
are hydrogen and R
8
, R
8a
and R
9
-R
12
are methyl,
R
4
is not hydrogen, methyl, isopropyl, phenyl, 2,6-dimethylphenyl or 2-isopropenylphenyl.
An advantage of the compounds (I) is that owing to an almost arbitrary substituent variation the properties of individual compounds, e.g. the spectroscopic properties, the position of the absorption and fluorescence maxima, the solubility properties, the fluorescence quantum yield and decay time, vary strongly and thus can be selected as desired. In this way, interferences with interfering substances in samples such as serum, blood or plasma etc. can be reduced or even avoided completely. The preparation of some compounds of the formula (I) can be carried out by processes known per se. Preferably, the synthesis is carried out, however, according to a novel process described below, which is particularly simple and inexpensive.
In a preferred class of the compounds (I), R
6
is bridged with R
11
or/and R
7
with R
12
, R
1
with R
10
or/and R
2
with R
9
and form a ring system which can contain one or more multiple bonds. The ring system preferably contains one or more 5- or 6-membered rings.
R
4
is preferably hydrogen, C
1
-C
6
-alkyl or a radical containing an aromatic ring system, e.g. a radical containing a carboxyl or/and halogen group, such as 2-carboxyphenyl, 2-carboxytetrachlorophenyl or pentafluorophenyl. R
8
and R
8a
are preferably in each case independently methyl, ethyl or/and optionally substituted phenyl.
Examples of particularly preferred classes of compound are shown in the general formulae IVa to IVe:
in which the dashed lines are optionally double bonds, in whose presence the radicals R bonded via a dashed line are absent,
R
1
, R
3
, R
4
, R
5
, R
6
, R
7
, R
8
, R
8a
, R
9
, R
11
, R
12
and X are as defined abo
Arden-Jacob Jutta
Drexhage Karl-Heinz
Frantzeskos Jörg
Zilles Alexander
Ceperley Mary E.
Day Ursula B.
Drexhage Karl-Heinz
Feiereisen Henry M.
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