WO1993004190A1 - Arylalkanoic acid resolution - Google Patents

Abstract

In a process for preparing predominantly one enantiomer of an optically-active 2-arylalkanoic acid by biotransformation, using an appropriate enzyme, of a mixture of enantiomers of an ester of the acid, the ester is derived from an alcohol of more than 4 carbon atoms, and the enzyme is in crude or only partially purified form. For example, S-ketoprofen can be thus prepared economically and in high ee, from racemic octyl ketoprofen.

Description

ARYLALKANOIC ACID RESOLUTION Field of the Invention

This invention relates to a process for resolving arylalkanoic acids, by a biotransformation according to the following reaction scheme:

wherein R¹ is an alkyl group, Ar is an aromatic residue and, for example, R is an aliphatic residue of 1 to 4 carbon atoms. Background of the Invention

In the prior art, a preference has often been made for carrying out such biotransformations with a small alkyl residue for R' . Thus, in EP-A-0196625, where 2-halopropionic acids are resolved by Candida cylindracea lipase (CCL) in a two-phase system, R¹ is defined as C,-C₄ alkyl. Moreover, later work with this enzyme, as described by Wu et al, J. Am. Chem. Soc. (1990) 112. 1990-5, concentrates on using the methyl ester for resolution of arylpropionic acids. The enzyme is purified, by addition of sodium deoxycholate to the enzyme solution and subsequent precipitation of the protein in 1:1 ethanol/diethyl ether.

This preference, for esters of ketoprofen at least, when using a crude preparation of CCL, is supported by the Table, below: enantiospecificity, as determined by the enantiomeric excess (ee) of the acid formed, declines with chain length from R'=methyl to R'=n-propyl.

Cambou and Klibanov, Appl. Biochem. Biotechnol. (1984) 9_.:255-60, report that CCL hydrolysis is selective for octyl 2-chloropropionate but not for the methyl ester. However, the failure of this methyl ester to exhibit

SUBSTITUTE SHEET enantiospecificity is considered to be due to its water- solubility, causing it not to form an interface. Summary of the Invention

As also shown in the Table, it has now surprisingly been found that when the esters are derived from increasingly longer chain alcohols, the ee subsequently increases to a higher figure than that obtainable with the methyl ester, of approximately 98% at R'=C₈ and C₁₀ n-alkyl. According to the present invention, the reaction illustrated above is conducted using crude (or partially- purified) enzyme, and R¹ has more than 4, e.g. 5, 6 or 7 to 15 to 20 C atoms. Subsequent work with purified enzyme showed that high ee was achieved whatever the chain length, but the purification adds to the overall cost. Description of the Invention

CCL has been identified as the most promising enzyme for enantiosspecific hydrolysis of racemic esters of ketoprofen. This enzyme gives the fastest rate and, unlike the majority of other active lipases, preferentially hydrolyses the S-ester, enabling simple purification of S-ketoprofen.

It appears that contaminating activities in crude CCL do not carry out the non-selective hydrolysis with esters of long-chain alcohols. Thus, there is advantage in using a long chain ester to enable the economy of using a crude or only partially pure enzyme. More particularly, the fact that, say, octyl ketoprofen can be used as a substrate with partially purified enzyme producing S-ketoprofen with ee of greater than 95% allows the development of a commercial process.

The following Examples illustrate the invention or are comparative. Examples

CCL is used as the biocatalyst to resolve racemic mixtures of various alkyl esters of ketoprofen; S- ketoprofen is formed. The same reaction conditions are used, i.e. 10 ml of 0.1 M potassium phosphate, pH 7.5 plus 2 ml cyclohexane, 100 mg alkyl ketoprofen, 50 mg enzyme, 30°C. Results are given in the Table.

The CCL is used in crude form, or after a three- step purification, comprising water extraction, filtra¬ tion and column separation, as follows: The enzyme was resuspended at 50 mg/ml in deionised water. After stirring at room temperature for 30 minutes, the inso¬ luble material was spun out. The supernatant was dia- filtered against 10 volumes of Mcllvaine's buffer (20 mM K₂HP0. + 10 mM citric acid, pH 3.7) at room tempera¬ ture. Following diafiltration, the enzyme solution was concentrated to 1/5 of its original volume. The enzyme solution was bonded onto an AP-sephadex C-50 column (20 ml enzyme solution: 20 ml of gel) equilibrated with Mcllvaine's buffer. The column was washed with Mcllvaine's buffer + 20 mM NaCl. After the first peak had eluted and the absorbance (at 280 nm) had returned to baseline, Mcllvaine's buffer + 200 mM NaCl was used to elute a second protein peak.

The final step of purification yielded two activi¬ ties. The protein eluted at a higher salt concentration was found to be highly enantiospecific.

The respective properties of the CCL biocatalysts were:

A - Assayed using olive oil as substrate, 37°C, pH 7.5. B - 100 ml 0.1 M ketoprofen: pH 6.5, 2 ml cyclohexane, 100 mg methyl ketoprofen, 30,000 U CCL, 30°C. Table: Enantiomeric excess values(%) of S-ketoprofen by Candida cylindracea lipase hydrolysis of esters from various alcohols.

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Claims

1. A process for preparing predominantly one enantiomer of an optically-active 2-arylalkanoic acid by biotransformation, using an appropriate enzyme, of a mixture of enantiomers of an ester of the acid, characterised in that the ester is derived from an alcohol of more than 4 carbon atoms, and that the enzyme is in crude or only partially purified form.

2. A process according to claim 1, wherein the enzyme is Candida cylindracea lipase.

3. A process according to claim 1 or claim 2, wherein the one enantiomer is the S-enantiomer.

4. A process according to claim 3, wherein the S- enantiomer is S-ketoprofen.

5. A process according to any preceding claim, wherein the alcohol has 8 to 10 carbon atoms.

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