Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1995-08-08
1997-11-04
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 72, 435100, 435177, 435183, 435190, 435232, C12P 900, C12P 1900, C12P 1902, C12N 988
Patent
active
056838972
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a process for the production of dihydroxyacetone phosphate (DHAP) by enzymatic oxidation of glycerophosphate as well as its use in situ by conversion in a coupled enzymatic aldol addition.
The stereoselective asymetric synthesis of polyhydroxylated compounds is of current interest due to their importance as components or precursors of active pharmaceutical substances such as antibiotics or glycosidase inhibitors. Enzymatic aldol additions are a valuable method since these can be used to synthesize all four possible diastereomeric ketoses and certain derivatives under mild reaction conditions in a highly diastereoselective manner and thus in high chemical and optical purity (DE 41 11 971). While the corresponding enzymes, the DHAP aldolases, accept a variety of aldehydes as electrophilic components, these enzymes additionally require dihydroxyacetone phosphate (DHAP) as an essential aldol donor substrate.
Commercial DHAP or direct acetal precursors do not come into consideration as a starting material for technical syntheses for economic reasons. Moreover, free DHAP is relatively unstable and decomposes into methyl glyoxal, which irreversibly inactivates the enzyme and into inorganic phosphate which inhibits aldolases. Although various processes are known for the chemical or enzymatic synthesis of DHAP, each of these has serious disadvantages with regard to complexity and product quality: acetonitrile/pyridine yields a product in poor yield (60%, non-reproducible according to several reports) which is highly contaminated with inorganic phosphate (C.-H. Wong and G. M. Whitesides, J. Org. Chem. 1983, 48, 3199). time-consuming synthesis from 3-chloro-1,2-propanediol (8 steps in the synthesis, total yield ca. 17%; C. E. Ballou and H. O. L. Fischer, J. Am. Chem. Soc. 1956, 78, 1659). considerable expenditure of material and time (3-5 steps in the synthesis) by three variants e.g. with diphenylchlorophosphate (R. L. Colbran et al., Carbohydr. Res. 1967, 4, 355), phosphoryl chloride (F. Effenberger and Straub, Tetrahedron Lett. 1987, 28, 1641) or dibenzyl-N,N-diethylphosphoramidite (R. L. Pederson et al. Tetrahedron, 1991, 47, 2643). DHAP, which is produced from this precursor by acid hydrolysis, is contaminated with ca. 20% inorganic phosphate and is only obtained in a total yield of 50%. Crans and G. M. Whitesides, J. Am. Chem. Soc. 1985, 107, 7019) yields a DHAP quality which is characterized by a high degree of contamination with acetate and phosphate since the acetyl phosphate used for cofactor regeneration is hydrolytically unstable. The enzyme is irreversibly inactivated to a considerable extent by the strongly electrophilic DHAP and can only be stabilized to a limited extent even by immobilization. fructose-1,6-diphosphate-aldolase uses a comparatively elaborate starting material, yields only a low equilibrium concentration of DHAP and is limited to synthetic reactions with the same aldolase (O. Meyerhof and K. Lohmann, Biochem. Z. 1934, 271, 89; W.-D. Fessner and C. Walter, Angew. Chem. 1992, 104, 643). glycerophosphate oxidase only leads to DHAP in non-isolatable amounts on a nanomolar to micromolar scale (JP 03/228 699, FR 2 620 732, U.S. Pat. No. 4,784,945, DE 3 731 876, EP 0 255 334, EP 0 354 551).
The object of the invention is therefore to produce dihydroxyacetone phosphate in high chemical purity and yield from readily available starting materials and in doing so to work under such mild reaction conditions by means of enzymatic catalysis that the labile product can be converted in situ if possible by all types of DHAP aldolases in a coupled reaction.
The object is achieved by a process for the production of DHAP which is characterized in that glycerol monophosphate is oxidized by air or hydrogen peroxide and excess amounts of a H.sub.2 O.sub.2 specific enzymatic activity of the enzyme preparation used in each case and due to limitations of enzyme solubility, the enzymes are preferably used in the following concentration ranges: GPO 1-1000 U/ml and catalase 5-1,000,000 U/ml an
REFERENCES:
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Boehringer Mannheim GmbH
Lilling Herbert J.
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