Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-09-29
2001-09-04
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S100000, C435S200000, C435S201000, C435S209000
Reexamination Certificate
active
06284494
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to methods and compositions for synthesizing oligosaccharides, and to the oligosaccaharide products which can be obtained using such methods and compositions.
Oligosaccharide are copounds with considerable potential both as therapeutics and as reagents for clinical assays. The very nature of the saccharide subunits, however, makes the sythensis of oligosaccharide of potential interest a daunting task because of many posibilities for information of positional isomers in which different substituent groups on the sugars become involved in bond formation and potential for the fomation of different anomeric forms. Because of these factors, chemical synthezis of most oligosaccharides while possible is not generally feasible on a commercial scale because of poor yields of the desired product.
An alternative to chemical synthesis of oligosacaharides is enzymatic synthesis. In particular, enzymatic synthesis using glycosyl transferases, glycosidases or combinations thereof has been considered as a possible approach to the synthesis of oligosaccharides.
Glycosyl transferases catalyse the reaction
Glycosyl transferases can be very effective for producing specific products with good stereochemical and regiochemical control, if a transferase with the desired specificity is available. The enzymes can be expensive and hard to handle since they are often membrane-associated and unstable, however, and the required nucleotide sugar substrates can be quite expensive. Furthermore, glycosyl transferases possessing the desired specificity to make many interesting oligosaccharides are not available.
Glycosidases catalyze the reaction
and synthesize oligosaccharides when the reaction is run in reverse from the normal direction. In addition, oligosaccharide synthesis can be achieved by adding a second sugar to the reaction mixture which competes with water and reacts in its place with the first sugar in a transglycosylation reaction. Glycosidases are generally available and easy to handle and have the potential to make many different products using inexpensive substrates. Unfortunately, it is difficult to control the reverse hydrolysis reaction which leads to poor product yields. In addition, while the stereochemical control (i.e., the formation of only one anomer) is generally good, it is hard to predict or control the regiochemistry (i.e., the formation of 1-2 vs 1-3 vs 1-4 vs 1-6 bonds).
To realize the potential of enzymatic oligosaccharide synthesis, there is therefore a need for a synthetic approach which avoids the drawbacks of the known techniques. 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, and to provide enzymes suitable for practicing these techniques.
SUMMARY OF THE INVENTION
These and other objects of the invention can be achieved through the use of mutant glycosidase enzymes, which cannot hydrolyze disaccharide products, but can still form them. Thus, a first aspect of the present invention is a method for forming an oligosaccharide. In this method a mixture of a glycosyl donor and a glycoside acceptor molecule is prepared. The glycosyl donor is selected from among molecules having substituents at the 1-position which are good leaving groups. The glycosyl donor is then enzymatically coupled to the glycoside acceptor molecule to form a glycosyl glycoside product using a mutant glycosidase enzyme in which one of two key amino acids has been changed, and the glycosyl glycoside product is recovered. In the case of a “retaining” glycosidase, the mutant enzyme is one in which the normal nucleophilic amino acid within the active site had been changed to a non-nucleophilic amino acid. In the case of an “inverting” glycosidase, the mutant enzyme in one in which the amino acid which normally functions as a base has been replaced by a non-ionizable amino acid. In both cases, the glycosyl donor is selected to have the opposite anomeric configuration from the desired product.
A further aspect of the present invention is a mutant glycosidase enzyme of the retaining type, in which the normal nucleophilic amino acid within the active site has been changed to an amino acid other than glutamic acid or aspartic acid. One such enzyme is a mutant form of Agrobacterium &bgr;-Glucosidase in which the normal glutamic acid residue at position 358 is replaced with an alanine residue.
A further aspect of the present invention is a mutant glycosidase enzyme of the inverting type, in which the normal amino acid that functions as a base within the active site has been changed to a non-ionizable amino acid.
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MacKenzie Lloyd
Wang Qingping
Withers Stephen G.
Oppedahl & Larson LLP
Slobodyansky Elizabeth
The University of British Columbia
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