Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Resolution of optical isomers or purification of organic...
Patent
1994-06-17
1996-05-14
Knode, Marian C.
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
435136, C12P 4100, C12P 740
Patent
active
055166903
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT EP92/01892 filed Aug. 19, 1992.
FIELD OF THE INVENTION
This invention relates to arylalkanoic acid resolution.
BACKGROUND OF THE INVENTION
Many pharmaceutically-active compounds produced by chemical synthesis are obtained and sold as mixtures of stereoisomers. However, it is often the case that only one these stereoisomers is pharmaceutically active. The contaminating enantiomer often has very poor, if any, activity or, in some instances, has unwanted physiological side-effects and shows toxicity.
Work by a number of researchers has shown that the anti-inflammatory activity of the 2-arylpropionic acids naproxen and ibuprofen is found with the (S) enantiomer. The same is true for ketoprofen, currently manufactured and sold as a racemate.
Arylalkanoic acids may be resolved by biotransformation, according to the following reaction scheme: ##STR1## wherein R' is an alkyl group, Ar is an aromatic residue and, for example, R is an aliphatic residue of 1 to 4 carbon atoms.
One particular object behind the present invention is to provide an economic route to the production of optically pure (S)-ketoprofen.
SUMMARY OF THE INVENTION
The present invention provides, inter alia, a method for the resolution of (S)-ketoprofen from a racemic mixture. This method involves biocatalytic hydrolysis of an ester of racemic ketoprofen, to yield a biotransformation broth containing ketoprofen acid substantially enriched in one enantiomer and ketoprofen ester substantially enriched in the other enantiomer. Provided that the acid product of the biotransformation is sufficiently enriched with the desired enantiomer, further improvement of the optical purity can readily and economically be achieved using standard chemical procedures by forming a salt with a chiral amine of high optical purity, followed by crystallisation from solution. The residual ketoprofen ester from the biotransformation can readily be purified, chemically racemised and recycled for use in further biotransformations, to minimise raw material costs.
DESCRIPTION OF THE INVENTION
A preferred aspect of the present invention is based on the discovery of a biocatalyst suitable for use in the biotransformation described above: a microbe (ENZA-I3) has been identified that is extremely useful in carrying out the required resolution. This organism was originally isolated by screening samples from the sewage works for growth on ethanol as a sole source of carbon. Subsequent screening of the organism on plates containing ethyl ketoprofen showed that, following growth, there was extensive clearance of the non-water-soluble ethyl ketoprofen around ENZA-I3 colonies (indicative of potential ester hydrolysis). Subsequent screening in liquid media showed ENZA-I3 to be considerably more active at hydrolysing ethyl ketoprofen than other isolates during the liquid screen.
This strain has proved to have a number of characteristics advantageous for the resolution of (S)-ketoprofen from the racemic ketoprofen ester, as follows: high enantioselectivity such that, at low conversions, greater than 95% purity (S)-ketoprofen can be obtained. At conversions approaching 50% (40-50%) this reduces to 90% purity. This selectivity is obtained without the need to purify out contaminating activities (which can be expensive) or over-express the activity of interest by cloning. possible to change the selectivity of the biocatalyst such that (R)-ketoprofen acid is preferentially accumulated instead of the (S) enantiomer. Thus, by taking the biotransformation to greater than 50%, (S)-ketoprofen can be produced as the methyl ester. of 1.5 to 2 hours, enabling biocatalyst to be easily and economically prepared.
The isolated strain has been identified by Centralbureau Voor Schimmelcultures (CBS) in the Netherlands as Trichosporon laibacchii (Windisch) which is also classified as Endomyces laibacchi. Several alternative strains of this species are publicly available from the CBS. These strains, for instance CBS 5791, 5790, 5381 and 2495, have been obtained and te
REFERENCES:
patent: 5322791 (1994-06-01), Sih
ATCC Catalogue of Yeasts pp. 95-96 (1990).
Carganico Germano
Evans Chriatopher T.
Stabler Peter J.
Wisdom Richard A.
Knode Marian C.
Laborabotios Menarini, S.A.
Saucier S.
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