Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-04-09
2001-03-20
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S100000, C435S101000, C435S174000, C435S179000, C435S193000
Reexamination Certificate
active
06204029
ABSTRACT:
FIELD OF THE INVENTION
In one aspect, this invention relates to the synthesis of oligosaccharides. In particular, it relates to improved syntheses of oligosaccharides using non-naturally occurring glycoside derivatives. In addition, this invention relates to methods of generating nucleotide phosphate sugars in situ and analogs thereof.
BACKGROUND OF THE INVENTION
Oligosaccharides are compounds with considerable potential both as therapeutics and as reagents for clinical assays. Synthesis of many oligosaccharides of potential interest is difficult because of the very nature of the saccharide subunits. A multitude of positional isomers in which different substituent groups on the sugars become involved in bond formation, along with the potential formation of different anomeric forms, are possible. As a result of these problems, large scale chemical synthesis of most oligosaccharides is not possible due to economic considerations arising from the poor yields of desired products.
Enzymatic synthesis provides an alternative to chemical synthesis of oligosaccharides. Enzymatic synthesis using glycosidases, glycosyl transferases, or combinations thereof, have been considered as a possible approach to the synthesis of oligosaccharides.
Glycosidases catalyze the reaction:
and synthesize oligosaccharides when the natural reaction is run in reverse. Oligosaccharides may also be synthesized by adding a second sugar to the reaction mixture which competes with water, reacting in its place with the first sugar in a transglycosylation reaction. While glycosidases are generally available and easy to handle, difficulties controlling the reverse reaction result in poor yields of product. Additionally, although stereochemical control (i.e., the formation of only one anomer) is good, it is difficult to predict or control the regiochemistry (i.e., the formation of 1-2 versus 1-3 versus 1-4 versus 1-6 bonds).
Glycosyl transferases catalyze the reaction:
Glycosyl transferases naturally function to synthesize oligosaccharides. They produce specific products with excellent stereochemical and regiochemical control. This reaction proceeds with substantial yield because the reverse reaction does not occur. Unfortunately, because they are often membrane-associated, these enzymes tend to be unstable in solution and expensive to buy. In addition, the sugar nucleotide substrates required by these enzymes are quite expensive. Furthermore, glycosyl transferases possessing the desired specificity to make many interesting oligosaccharides are not commercially available. Recent progress in cloning techniques, however, have made several glycosyl transferases available in sufficient quality and quantity, making enzymatic oligosaccharide synthesis more practical (see, for example, Paulson, et al.,
J. Biol. Chem
. 264:17615 (1989)).
To realize the potential of enzymatic oligosaccharide synthesis, there is therefore a need for a synthetic approach which avoids the principal drawbacks of the known techniques (i.e., the cost of the sugar nucleotide substrates). It is an object of this invention to provide such a technique which permits the synthesis of a wide variety of oligosaccharides in good yield.
SUMMARY OF THE INVENTION
The present invention provides methods of preparing oligosaccharides using activated glycoside derivatives. In one aspect, this invention relates to a process for making a glycosylated acceptor, by mixing in an aqueous medium an activated glycoside derivative, an acceptor substrate, a glycosyl transferase, and a nucleotide phosphate or a nucleotide phosphate analog to form a glycosylated acceptor.
In another aspect, this invention relates to a process of making a nucleotide phosphate glycoside, by mixing in an aqueous medium an activated glycoside derivative, a glycosyl transferase and a nucleotide phosphate or a nucleotide phosphate analog, to form a nucleotide phosphate glycoside.
In yet another aspect, this invention relates to a process of making saccharides, oligosaccharides or carbohydrate containing moieties using a nucleotide phosphate glycoside coupled with a nucleotide phosphate sugar dependent enzyme.
The products of the foregoing methods will typically be isolated or recovered following enzymatic synthesis, although in some embodiments, multiple glycosylation reactions will be carried out either in a single reaction vessel or multiple vessels.
In other aspects, the present invention provides compositions which are useful for the formation of glycosidic linkages and compositions which are formed by the methods described herein.
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Lougheed Brenda
Withers Stephen G.
Naff David M.
The University of British Columbia
Townsend and Townsend / and Crew LLP
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