Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Patent
1988-06-17
1990-09-11
Lee, Mary C.
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
530360, 530409, 536 182, C07D31102, C07H 1500
Patent
active
049564803
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to new and novel compounds for use as fluorescent labelling agents and the invention also includes fluorescent conjugates of such new compounds.
Use of derivatives of 7-hydroxy coumarin for fluorometric assay of biologically active substances, such as enzymes is known. The labelling of such biologically active substances usually takes place by covalent coupling of the labelling agent to the substance involved. The use of 7-hydroxy coumarin derivatives in fluorometric assays is, however, subject to serious drawbacks, in view of the fact that the fluoroescence is developed only in the alkaline pH-range, usually above about pH 8. This is due to the fact that in order to develop fluorescence the hydroxyl group at the 7-position must be ionized. Since sensitive biological materials are involved in the assay the need to make the solutions alkaline before reading the fluorescence can constitute a factor that causes loss of sensitivity due to the necessary dilution with the alkaline reagents.
Another drawback of using known fluorescent labelling agents, such as fluorescein isothiocyanate (FITC) is that they emit radiation in a wavelength region that makes it difficult to distinguish from radiation emitted by autofluorescence of the biological material, such as cells or tissues. This autofluorescence of biological materials is often in the same yellow or green region, and the experimentator must make a subjective assessment as to whether he is examining true labelling characteristics or characteristics due to autofluorescence.
Yet another drawback of using 7-hydroxy coumarin derivatives is the fact that covalent coupling of for example 7-hydroxy coumarin-3-acetic acid to a polypeptide results in blocking of an amino group of the polypeptide with concomitant change of its acid-base balance. Such change may well cause alteration of the biological characteristics of the protein.
The invention has for its purpose to provide new coumarin derivatives, the use of which does not involve the implications of the known techniques as indicated above.
Another object of the invention is to provide fluorescent conjugates containing such new coumarin derivatives.
Still another object is to provide fluorescent labelling compounds emitting in a different region than the region of autofluorescence of biological material, such as cells and tissues.
In the instant disclosure the coumarin nucleus will be numbered as shown below, the numbering being given to facilitate the understanding of the following disclosure: ##STR3##
To attain these other and objects the invention provides for new and novel compounds having the formula (I). ##STR4##
In this structural formula it is preferred that m is 1 or 2, and particularly 1 corresponding to the 3-acetic acid derivative. For practical reasons reactive derivatives involving the carboxyl function of compounds (I) are preferred, such as esters, for example N-hydroxysuccinimidyl esters, the reason being to facilitate covalent coupling to the biologically active material of interest.
Accordingly, the compounds of the invention preferably have the following formula (II): ##STR5## wherein R.sub.3 is an N-oxysubstituent, such as an N-oxysuccinimide substituent. R.sub.3 may also, of course, be hydroxy.
The invention also provides for new fluorescent conjugates having the formula (III): ##STR6##
In this formula (III) R.sub.4 may be an organic residue, such as a protein residue or carbohydrate residue.
In the above the formulas I-III, R.sub.1 and R.sub.2 may be the same or different and are selected from hydrogen and lower alkyl. By teh expression "lower" is meant groups containing 1 to 4 carbon atoms. Compounds I and II, wherein R.sub.1 and R.sub.2 both are different from hydrogen are preferred in that substitution on the nitrogen atom prevents polymerization between identical molecules to form amide bonds.
In regard to compounds of the formula (II) R.sub.3 may be such as to result in reactive acyl derivatives, such as halides particularly acyl chloride.
In regard to fluores
REFERENCES:
patent: 3008969 (1961-11-01), Pretka
CA 97:49239b Goya et al., New Fluoresence Probes for Drug-Albumin Interaction Studies, 1982, Chem. Pharm Bull.
Biocarb AB
Kilby Scalzo Catherine S.
Lee Mary C.
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