Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-05-12
1999-03-16
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 73, 435 84, 435193, 536 111, 536 41, 536 187, C12P 1944, C12P 1926, C12P 1964
Patent
active
058829024
DESCRIPTION:
BRIEF SUMMARY
The present invention describes a new method to produce O-glycosylated amino acids, O-glycosylated peptides or derivatives of these. In a further aspect the present invention relates to products produced by the above method as well as uses of the resulting products.
Glycoconjugates contain oligosaccharide chains with up to twenty monosaccharide units and several sequences have been shown to have biological activity e.g. in the binding to different cells, pathogens, toxins, antibodies or other proteins on cell surfaces, in cancer metastasis, in inflammation processes (for example selectin-carbohydrate interactions), binding of white blood cells to the blood vessel wall, as a modifier of the activity, stability and biological activity of proteins, and as immunogenic substances which have potential for vaccination against different diseases. An extensive literature has been developed during the last few years in this field and there are several review articles on this type of biology, glycobiology, e.g. in Annual Review of Biochemistry and in current opinion in Structural Biology (see for example volume 3, 1993) incorporated herein by reference.
One type of glycoconjugate, the glycoproteins, contain carbohydrate-peptide sequences in which the carbohydrate unit is bound to the peptide or protein chain via mainly three different types of linkages, the O-glycosidic linkage represented by GalNAc.alpha.-OSer and GalNAc.alpha.-OThr, that is the linkage between N-acetyl-D-galactosamine and the hydroxyl group on a L-serine or a L-threonine residue in the peptide of protein chain (several such linkages can be found in a peptide or protein chain depending on the number of serine or threonine units in the molecule), the N-glycosidic linkage between N-acetyl-D-glucosamine and the amide function in asparagine, GlcNAc.beta.-N-Asn, and the O-glycosidic linkage between galactose or xylose and different hydroxyl group containing amino acids. Recently, also the .beta.-O-glycosidic linkage between N-acetyl-D-glucosamine and the hydroxyl group on serine or threonine, abbreviated GlcNAc.beta.-OSer and GlcNAc.beta.-OThr, respectively, has been of increased interest, e.g. because of its suggested importance in the DNA-replication in the nucleus of eucaryotic cells.
These types of linkage are of interest to produce by synthetic methods for fundamental studies and for synthesis of biologically or pharmaceutically active fragments of glycoproteins, for instance for use as vaccines or therapeutics. It is also important to be able to synthesize analogs or derivatives of the structures above for example with other types of sugar and/or configuration, as e.g. mannosamine and the .alpha.- or .beta.-configuration to modify or to increase the biological activity of the conjugate.
Several of these conjugates have earlier been synthesized by chemical or enzymatic methods. The disadvantages with chemical synthesis is that several reaction steps are required with extension protection group chemistry to obtain stereo- and regioselective synthesis of the conjugate and often, as a side reaction, .beta.-elimination and racemization of the amino acid residue is obtained due to the weakly acidic nature of the optically active C--H linkage. The stereospecific preparation of GalNAc.alpha.-OSer, GalNAc.alpha.-OThr and derivatives of these structures is especially difficult to achieve with chemical methods.
This invention describes a novel method based on enzymatic synthesis for the preparation of such GalNAc.alpha.-linked compounds.
Enzymatic synthesis of glycosidic linkages can be carried out with glycosyltransferases (EC 2.4) and with glycosidases (EC 3.2) (see e.g. K. Nilsson, Trends in Biotechnology, 1988, pages 256-264, incorporated herein). Glycosyltransferases are in general not available in quantity for large-scale synthesis, have a high acceptor specificity which limits the efficiency with unnatural or modified acceptors and are dependent on nucleotide sugars as glycosyl donors. Glycosidases are abundant and have been used for synthesis of non-m
REFERENCES:
patent: 5583042 (1996-12-01), Roth
APS Abstract JP409037790A (Feb. 10, 1997) Ajisaka et al.
Bioflexin AB
Lilling Herbert J.
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