Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Patent
1996-03-19
1998-05-05
Soderquist, Arlen
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
436 56, 436 95, 436161, 436172, 536 111, 536 172, 536104, 5361231, 53612313, G01N 3300, C07H 100, C08B 3500
Patent
active
057473471
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to carbohydrates and in particular to (oligo)saccharides conjugated with a label which facilitates their identification and analysis.
BACKGROUND OF THE INVENTION
Oligosaccharide analysis may be performed by a variety of standard techniques including, but not limited to, gel filtration, ion exchange, hydrophobic interaction and hydrophilic interaction chromatography, mass spectrometry, gel electrophoresis and capillary electrophoresis. Tritium-labelling is often used. There are also several reports detailing methods, especially by reductive amination, for labelling the reducing terminus of an oligosaccharide with, for example, UV-absorbing, fluorescent, or electro-chemically active labels.
Many laboratories would prefer to avoid the use of radio-isotopes. For example, WO-A-9105256 discloses labelling carbohydrates with a fluorescent naphthalene ring structure which includes a charge-carrying substituent, for the purpose of separating and analysing the carbohydrates. The preferred labelling reagents are aminonaphthalenesulphonic acids.
Such labels for oligosaccharides are generally relatively large, charged molecules, making them unsuitable, for instance, for the gel filtration chromatography of uncharged (oligo) saccharides,e.g. using Bio-Rad P4 gels.
Smaller labels, e.g. p-aminobenzoates, have been used in mass spectrometry. However, they are liable to undergo hydrolysis at elevated pH.
Relatively small fluorescent labels are also known. For example, Hase et al, J. Biochem. 85:989-994 (1979), disclose 2-aminopyridine for use as a fluorescent label for oligosaccharides and their separation by paper electrophoresis. Yalpani et al, Can. J. Chem. 59:2934-2939 (1981), disclose bicyclic, e.g. naphthalenic, labels and also p-fluoroaniline as a starting material for fluorescent labelling, via p-fluorochlororoacetamidoaniline (i.e. Cl--CH.sub.2 --CO--NH--Ph--F wherein Ph is 1,4-phenylene), of 1,2:3,4-di-O-isopropylidene-.alpha.-D-galactopyranose.
Bisswanger et al, Biochemistry 18(26):5946-5953 (1979), report investigation of tryptophan biosynthesis, specifically of the multifunctional enzyme (phosphoribosyl)anthranilate isomerase-indoleglycerol phosphate synthase. N-(5-phosphoribosyl)anthranilate was prepared and identified as a substrate of greater stability and purity than the natural substrates. This synthetic substrate was reported as having different absorption and fluorescence spectra on binding to each of the enzyme's two binding sites.
O'Brien et al, J. Het. Chem. 7:99-105 (1970), disclose the synthesis of antiriboflavin compounds using as starting materials dimethylanthranilic acid and anthranilic acid. However, the anthranilic compounds were used here only as synthesic precursors and not as fluorescent labels.
SUMMARY OF THE INVENTION
According to the present invention, a saccharide is labelled with a fluorescent group of the formula
The labelled compounds are stable, soluble in water and also organic solvents such as DMSO. For example when the group is --NR--Ph--CONR'--, they are uncharged but run satisfactorily on gels. R and R' may independently be H, C.sub.1-6 alkyl, e.g. methyl, or any substituent provided that the fluorescent property, and adequate hydrophilicity, are retained. Ph is phenylene.
DESCRIPTION OF THE INVENTION
The label may be attached by reaction with reducing sugars under a variety of conditions, in the presence of a reducing agent (may be added sequentially) such as sodium cyanoborohydride, or a borane-amine complex, such as the borane-dimethylamine complex. After reaction, the derivatised saccharide may be purified by various methods, or directly analysed on, for instance, P4 gel filtration chromatography.
The novel compounds may be readily prepared from readily-available starting materials, by reductive amination of the reducing terminal of a saccharide. For example, the saccharide (represented as oligo--CHO) may be reacted with an anthranilamide, e.g. 2-aminobenzamide, as shown in Chart A. Either or each N atom may be substit
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Charles Stephen Alexander
Goulding Paul
Hawke David Harry
Parekh Rajesh Bhikhu
Oxford Glycosystems Ltd.
Soderquist Arlen
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