Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1996-09-23
1998-11-03
Knode, Marian C.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
530395, 536 172, 536 179, 536 182, 536 185, 536 187, 536 291, 536 2912, 536 53, 536 54, 536 55, 536124, C07H 100, C07H 506
Patent
active
058310770
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/AU 94/00764 filed on 9 Dec., 1994.
TECHNICAL FIELD
The present invention relates to methods for the production of sugar derivatives, the elimination of sugars from glycoproteins, binding carbohydrates to solid supports and analysis of glycoamino acids using solid phase Edman degradation. All of the disclosures can be applied to monosaccharide or oligosaccharide moieties.
BACKGROUND ART
In view of the developing interest in the importance of the glycosylation of proteins, it has become essential to develop new methods for the study of the detailed structure of complex glycans.
The glycans of glycoproteins are typically either N-linked (attached to carry out structural analysis of glycoproteins, it is desirable to release the glycans from the protein backbone. To this end, several enzymatic and chemical methods are available. Enzymatic release of N-glycans can be glycans of both classes, and has been adapted, using milder conditions, release of O-glycans however is by digestion of the glycoprotein in mild disadvantage of this method is a reduction of the glycans to alditols and that the reaction is not truly specific for O-glycans as there is significant release of N-glycans due to the inclusion of borohydride.
To date, the most common method for immobilising reducing sugars has been polymer-bound primary amino groups by a labile glycosyl amine linkage which is then stabilised by reduction. This reduction is slow and the efficiency of binding is rather low when limited amounts of sugars are available. In recent variations on this approach, the glycosyl amine
There is a need for site specific identification and characterisation of protein associated glycosylation. Edman degradation is the ideal chemical method for identifying single sites of glycosylation in proteins. The predominant technology of absorption-phase Edman degradation, however, fails to extract the glycosylated amino acid for subsequent analysis. In contrast solid-phase Edman degradation extracts the N- and O-glycosylated amino acids in anhydrous trifluoroacetic acid and each glycosylated amino acid can be collected and subjected to carbohydrate analysis and/or modification (10). The harsh conditions of Edman degradation, however, result in the hydrolysis of sialic acid from the oligosaccharides (FIG. 1). In addition, unless the sialic acid is removed the sialylated oligosaccharide is attached to the solid-support during the immobilisation procedures (FIG. 2). Hence, there is a need to develop a method of modifying sialic acid so as to prevent its immobilisation during the covalent-coupling chemistry and to impart stability during the acid conversion of the sialylated thiazilinone to the thiohydantoin (FIG. 3).
There is a strong need to have simple experimental conditions for the specific release of O-linked glycans from glycoproteins, and for their isolation in an unreduced form suitable inter alia for chemical derivatisation, reductive incorporation of labels or the attachment of polymer supports.
The present inventors have developed chemical modification procedures for the analysis and manipulation of sugars (FIG. 4). It will be appreciated by one skilled in the art that the following disclosures can be applied to monosaccharide or oligosaccharide moieties.
DISCLOSURE OF THE INVENTION
Accordingly, in a first aspect the present invention consists in a method for the production of a sugar hydrazone comprising reacting aqueous hydrazine hydrate with a reducing sugar in the presence of a base, optionally followed by the removal of the hydrazine hydrate and the base.
In one embodiment of the first aspect, the base is sodium hydroxide, preferably at a concentration of 0.1M.
In another embodiment of the first aspect the base is triethylamine, preferably at a concentration of 0.2M.
Preferably, the removal of the hydrazine hydrate from the reaction mixture is by evaporation under reduced pressure or in a stream of inert gas, e.g. N.sub.2. When an inorganic base is used, it is removed from the reaction mixture
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Batley Michael
Cooper Catherine Anne
Gooley Andrew Arthur
Kerr Warren Charles
Packer Nicolle Hannah
Knode Marian C.
Lee Howard C.
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