Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1990-11-16
1993-09-21
Griffin, Ronald W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435100, 435200, 435 74, C12P 1904, C12P 1912, C07H 306
Patent
active
052468402
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention relates to a method for enzymatic synthesis of an oligosaccharide compound, which either consists of or is a fragment or an analog of the carbohydrate part in a glycoconjugate. Furthermore, the invention relates to the use of the product prepared by this method.
It has been found that the oligosaccharide part of various glycoconjugates (especially glycolipids and glycoproteins) have a number of important functions in vivo (Biology of Carbohydrates, Vol. 2., Ginsburg et al., Wiley, New York (1984); The Glycoconjugates, Vol. 1-V, Academic Press, New York; S. Hakomori, Ann. Rev. Biochem. Vol. 50, pp. 733-64); Feizi, Nature, pp. 314 (1985); S. Hakomori, Chemistry and Physics of Lipids, Vol. 42, pp. 209-33). Among other things it was found that localisation, immunogenicity and degradation of glycoproteins; antigens); pathogens, proteins, hormones, toxins and during cell-cell interactions; have been found to be cancer-associated antigenic determinants; carbohydrate part of the glycoconjugate is required for full biological activity (e.g. receptor activity).
Universities and industry are at present working intensely on developing the use of biologically active oligosaccharides within a number of different fields, such as
Besides the above-mentioned fields, a considerable future market is envisaged for fine chemicals based on biologically active carbohydrates.
The organic chemical techniques used today for synthesis of these carbohydrate structures require an extensive protective group chemistry with many steps of synthesis and expensive catalysts. Low total yields are obtained in those complicated reaction schemes and the technique is not favorable, especially for larger scale work.
Enzymes are nature's own catalysts with many attractive characteristics, such as high stereo-, regio- and substrate selectivity as well as high catalytic efficiency under mild conditions. Today, great hopes are therefore placed in being able to utilize enzymes for large-scale selective synthesis of oligosaccharides with fewer reaction steps and consequently higher total yields than by organic chemical methodology.
Both hydrolases (glycosidases. EC 3.2) and glycosyltransferases (EC 2.4) can be used for synthesis (glycosidases: see Nisizawa et al. in The Carbohydrates, Chemistry and Biochemistry, 2nd Ed., Vol. IIA, pp. 242-290, Academic Press, New York (1970)). With glycosidases reversed hydrolysis (equilibrium reaction) or transglycosylation (kinetic reaction) are often used to obtain synthesis (see e.g. K.G.I. Nilsson, Carbohydrate Res., Vol. 167, pp. 95-103 (1987)). With transferases a nucleotide sugar (UDP-Gal, CMP-Sia, UDP-GalNAc, GDP-Fuc, etc), which is relatively expensive, is used as donor. Both types of enzymes have advantages. Glycosidases are abundant and can often be used directly without purification, glycosyltransferases show high regio- and acceptor-selectivity. However, both types of enzymes have disadvantages when used for synthesis. Glycosidases have a low or often wrong regioselectivity which may result in complicated product mixtures and thus purification problems. As a result glycosidases are often not suitable for synthesis of higher oligosaccharides. Glycosyltransferases are often present in small amounts in living cells and are thus often of low availability. Furthermore, as mentioned above, the transferases are cofactor dependent.
One of the objects of the present invention is to use the properties of glycosidases and glycosyltransferases in a favorable way for efficient synthesis of oligosaccharides. This is achieved according to the invention by combining glycosidase-catalysed synthesis of an oligosaccharide compound with glycosyltransferase-catalysed synthesis of the final, higher oligosaccharide. An easily available glycosidase is thus used for synthesis of the shorter oligosaccharide compound and a regiospecific enzyme (i.e. glycosyltransferase) is used when a higher regioselectivity is required, i.e., for synthesis of the final oligosaccharide. This is illustrated in the
REFERENCES:
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Thiem J., et al., "Synthesis of the Trisaccharide Neu-5-Ac-.alpha.(2.fwdarw.6) Gal-.beta.(1.fwdarw.4) G1cNAc by the Use of Immobilized Enzymes", Angew. Chem. Int. Ed. Engl. (1986), vol. 25, pp. 1096-1097.
Paulson, J. C., et al., "Use of Glycosyltransferases and Glycosidases in Structural Analysis of Oligosaccharides", Glycoconjugate Research (1979) vol. 1, pp. 247-250.
Griffin Ronald W.
Procur AB
Webber Pamela S.
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