Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Patent
1993-07-12
1998-04-14
Cintins, Marianne M.
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
540122, 540131, 540132, 540139, 540140, 544102, 544103, 544106, 544109, 544110, 562 7, C09B 4704, C09B 6200, C09B 6700
Patent
active
057393184
DESCRIPTION:
BRIEF SUMMARY
This case is a 371 of PCT/EP91/02160, filed on Nov. 13, 1991.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel compounds useful as labelling agents, more particularly to novel dyes exhibiting absorption maxima at or beyond the red extreme of the visible spectrum.
2. Description of the Prior Art
Labelling agents are frequently required in studies of blood samples, e.g. in measurements of glycosylated haemoglobin as part of an evaluation of blood glucose levels in patients suffering from diabetes mellitus. Such agents are described in, for example, DE-A-3720736 and U.S. Pat. No. 4,861,728 and typically comprise a boronic acid reagent which is capable of reacting with the cis-diol grouping of the glycosyl moiety and which is bonded to a fluorescent and/or coloured dye, e.g. a diazo conjugate or fluorescein, rhodamine or phycobiliprotein. Assessment of quantification of the fluorescence or absorbance, e.g. by reflectometry, affords a measure of the glycosylated haemoglobin levels in the blood samples.
A disadvantage of the use of phycobiliprotein conjugates of boronic acids is that in most glycosylated haemoglobins the glycosyl moiety, which is principally attached to the N-terminal valine amino acid of the beta-chain, is effectively located in a narrow "pocket" of the haemoglobin molecule and is therefore not readily accessible to high molecular weight reagents.
Other previously proposed coloured labels such as fluorescein and rhodamine suffer the disadvantage that their absorption spectra overlap significantly with the absorption spectrum of haemoglobin; this can prevent or seriously inhibit effective spectroscopic quantification of the label.
SUMMARY OF THE INVENTION
The present invention is based on our discovery of new classes of labelling compounds which exhibit an absorption maximum at not less than 600 nm, at which wavelengths haemoglobin has minimal absorption.
According to one aspect of the invention we provide non-proteinaceous boronic acid conjugate compounds having absorption maxima at not less than 600 nm. Such compounds may, for example, be represented by the formula (I) absorption and/or emission maximum at not less than 600 nm and W is a linking organic group.
It will be appreciated that the dihydroxyboryl residue may also exist in the anionic form employed and that such compounds are to be regarded as within the scope of formula (I).
The boronic acid conjugates of the invention are useful in spectroscopic studies relating to estimation or quantification of cis-diols, particularly of glycosylated moieties, e.g. proteins, in the presence of haemoglobin since there is virtually no overlap between the absorption spectra of haemoglobin and the chromophore.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The boron atom of the boronic acid residue is advantageously attached to a phenyl group, e.g. an aminophenyl group, such as a m-aminophenyl group. Depending on the pKa value desired for the boronic acid, the phenyl group may optionally be further substituted, e.g. by one or more substituents which influence the pKa value without sterically interfering with bonding between the boronic acid residue and the target cis-diol. Examples of electron-withdrawing substituents which may be employed to enhance the degree of ionisation and thus the association constant of the boronic acid to target cis-diols such as glycosylated moieties include nitro, lower alkoxy such as methoxy or ethoxy, and acyl groups such as formyl or lower alkanoyl. Representative boronic acids of this type include 2-nitro-5-aminophenyl boronic acid and the corresponding 3-nitro and 4-nitro isomers. In general any such phenyl or substituted phenyl groups may be linked directly to the chromophoric and/or fluorophoric moiety V, so that the phenyl group constitutes the group W in formula (I), or via linking or spacing groups, e.g. as known in the art.
One useful category of compounds according to the invention comprises compounds of formula (II) ##STR1## wherein X represents an oxygen or sulphur atom;
REFERENCES:
patent: 4496722 (1985-01-01), Gallop et al.
patent: 4830786 (1989-05-01), Pease et al.
patent: 4861728 (1989-08-01), Wagner
patent: 5242842 (1993-09-01), Sundrehagen
Fabian et al., "Light Absorption of Organic Colorants," Springer-Verlag, 1980, p. 157.
Koyama, Chemical Abstracts, 114:118100h, 1990.
Frantzen Frank
Sundrehagen Erling
Axis Research AS
Cintins Marianne M.
Jones Dwayne C.
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