Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-08-06
1999-03-30
Weber, Jon P.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 87, C12P 1930, C12P 1938
Patent
active
058887776
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for the preparation of nucleoside diphosphate and triphosphate sugars, wherein the hydroxyl protective groups are removed enzymatically, as well as to the preparation of these sugars which comprises coupling a nucleotide with a sugar-1-phosphate activated with a carbonyl bisazole and then removing the hydroxyl protective groups enzymatically.
Aside from their importance as energy carriers and structural units, carbohydrates have an important part to play as information carriers in intercellular communication and in intercellular recognition processes. They are naturally obtained as poly-, oligo-, di- or monosaccharides or as glycoconjugates, typically glycoproteins, glycolipids and glycosteroids. Oligosaccharides and glycoconjugates may be built most efficiently by a combination of chemical and enzymatic methods. The key enzymes which are, inter alia, used here are the so-called glycosyltransferases of the Leloir 202:215-238 (1992)!. Said enxymes are distinguished by high regiospecificity and stereospecificity with respect to the glycoside bond to be linked.
The enzymes transfer a sugar unit of so-called activated nucleoside monophophate and diphosphate sugars (donors) to a very wide range of different acceptor substrates which are, however, specific for the respective enzyme. The donors are considered as limiting for this type of glycoside synthesis, in particular because it has so far not been possible to prepare these compounds simply, on a large scale and in high yield. One possible chemical synthesis consists in reacting an unprotected sugar phosphate with a nucleotide activated, for example by a morpholine group Hindsgaul, O., Palcic, M. M., Can. J. Chem. 68:1063-1071 (1990)!. This linkage of a pyrophosphate bond is highly susceptible to water and very difficult to carry out with unprotected educts owing to the hygroscopic hydroxyl groups. Furthermore, as the unprotected educts are only sparingly soluble in the conventional coupling solvents (dimethyl formamide, Rieks-Everdiking, A., Klaffke, W., Synthesis 689-691 (1994)!. In addition to forming undesirable by-products, many unprotected sugar phosphates are instable. In consequence, the yield is usually low and never above 50% 59:2086-2095 (1981); Gokhale, U. B., Hindsgaul, O., Palcic, M. M., Can. J. Chem. 68:1063-1071 (1990)!. Protected reagents have not been used so far because with the known processes it did not seem possible to completely remove the protective groups after the coupling step while retaining the desired target structure. Thus a guanosine diphosphate glucose which is O-benzoyl-protected at the carbon atoms 2 and 3 can only be debenzoylated with substantial decomposition. The complete removal of protective groups without decomposition has only been possible in the case of uncharged Rev. 94:911-937 (1994)!. An exception are certain O-acyl-protected uridine diphosphate hexoses, all of which carry a base-stable NHAc substituent at the carbon atom 2 of the hexose and are .alpha.-configurated at the carbon atom 1. They may be chemically deacylated with 1M sodium hydroxide Res. 79:C9-C12 (1980); Yamazaki, T., Warren, C. D., Herscovics, A., Jeanloz, R. W., Can. J. Chem. 59:2247-2252 (1981); Thomas, R. L., Abbas, S. A., Matta, K. L., Carbohydr. Res. 184:77-85 (1988)!.
In addition to this possible chemical synthesis, specific naturally occuring donors can also be prepared using multienzyme systems. The great disadvantage of these enzyme systems is, however, that they are not easily Lees, W. J., Pale, P., Whitesides, G. M., J. Org. Chem. 57:146-151 (1992)!.
Surprisingly, it has now been found that nucleoside diphosphate and triphosphate sugars can be synthesised in high yield by using O-acyl protective groups in the sugar residue and, in the final reaction step, removing these protective groups under the action of specific enzymes. Accordingly it is also possible for the first time to carry out prior synthesis steps using reagents carrying protective groups, which is particularly useful for coupling t
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Lehning
Baisch Gabriele
Oehrlein Reinhold
Ferraro Gregory D.
Novartis AG
Weber Jon P.
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