IE63329B1 - Process for preparing optically active 2-(aryloxy) - or 2-(arylthio) alkanoic acids - Google Patents

Abstract

Process for preparing optically active 2-aryloxy- or 2-arylthioalkanoic acids by enantioselective hydrolysis of the corresponding racemic amides in the presence of a microorganism or of an enzyme selected as a function of its ability to selectively hydrolyse racemic alpha -phenylpropionamide to S alpha -phenylpropionic acid.

Description

The present invention relates to the preparation of optically active 2-(aryloxy)- or 2-(arylthio)alkanoic acids of general formula: (I) Ar in which Ar denotes an aromatic radical, optionally substituted by at least one alkyl, alkoxy, halo or hydroxyl group, the alkyl or alkoxy groups preferably having 1 to 4 carbon atoms each; A denotes an oxygen or sulphur atom; R denotes an alkyl radical having 1 to 6 carbon atoms .

More especially, the present invention provides a process for the preparation of an R-(+) enantiomer of a 2(aryloxy)- or 2-(arylthio)alkanoic acid of general formula (I), and more particularly an R-(+) enantiomer of a 2phenoxy- or 2-(phenylthio)propionic acid of general formula: Ar CO,H (II) in which Ar has the same meaning as in formula I and, most preferably, (R)- ( + )-2-(4-hydroxyphenoxy)propionic acid.

According to the present invention, the 2-(aryloxy)or 2-(arylthio)alkanoic acids of formula (I) in the R form * are obtained by the enantioselective hydrolysis of the corresponding racemic amides optionally prepared in situ in the presence of a microorganism which is a Brevibacterium or Corynebacterium strain, or an enzyme derived from a said microorganism, having nitrilase activity and able to hydrolyse racemic α-phenylpropionamide enantioselectively to (S)- α-phenylpropionic acid with an enantiomeric excess of more than 65%, and then separating the (R)-2-(aryloxy)or -2-(arylthio)alkanoic acid from the unhydrolysed amide of the (S)-2-(aryloxy)- or -2-(arylthio)alkanoic acid.

The selection of the agent which brings about the enantioselective hydrolysis of the racemic amide of the 2(aryloxy)- or (2-(arylthio)alkanoic acid of general formula (I) is accomplished by bringing the agent into contact with racemic α-phenylpropionamide in a suitable medium until 20% of the product is converted, and then measuring the enantiomeric excess.

The microorganisms which are especially suitable are chosen from Brevibacterium R312 (CBS 717.73) and Corynebacterium N711 (FERM P 4445) or Corynebacterium N774 u (FERM P 4446), which enable (S)-2-arylalkanoic acids to be obtained with an enantiomeric excess of S isomer generally of more than 90% from the corresponding amide. -4It is surprising to note that, among these microorganisms, Brevibacterium R312, which is described in French Patent Specification FR 7,901,803/2,447,359 and in Advances in Biochemical Engineering, vol. 14, p. 1-32 (1980) as possessing non-stereospecific general amidase, hydrolyses the racemic amides of 2-arylalkanoic acids stereospecifically, whereas its A4 mutant, which possesses a stereospecific L-amidase, does not hydrolyse the racemic amides of 2-arylalkanoic acids.

It is appropriate to note that Corynebacterium N771 and Corynebacterium N774, which are described in European Patent Specification EP 0,187,680, hydrolyse, for example, lactonitrile to DL-lactic acid and a-aminophenylpropionitrile to DL-phenylalanine without stereoselectivity being observed.

The process of the present invention is generally carried out in an aqueous or homogeneous or heterogeneous aqueous-organic medium, under temperature and pH conditions determined in accordance with the nature of the microorganisms or the enzyme used, by stirring a suspension of cells or cell extracts of the microorganism and the amide of the racemic 2-(aryloxy)- or 2-(arylthio)alkanoic acid.

The process leads to a mixture of the (R)-2(aryloxy) - or -2-(arylthio)alkanoic acid and the amide of the (S)-2-(aryloxy)- or -2-(arylthio)alkanoic acid. At the end of the process the (R)-form acid and the (S)-form amide are separated.

When the microorganism used possesses the property of hydrolysing nitriles, combined with the property of enantioselectively hydrolysing a-phenylpropionamide, it is possible to obtain an (R)-2-phenoxy- or -2(phenylthio)alkanoic acid either from the nitrile of the racemic 2-phenoxy- or 2-(phenylthio)alkanoic acid or from the amide of the racemic 2-phenoxy- or 2-(-phenylthio) alkanoic acid.

The examples which follow show how the invention may be put into practice.

EXAMPLE 1 a) Brevibacterium strain R 312 (CBS cultured in an agitated flask at 28°C for 14 medium whose composition is as follows: Glucose (NH4)2SO4 kh,po4 Na2HPO4.12H2O K2HPO4 g g 1-01 g 1.64 g 717.73) is hours in a ΙΟ.82 g CaCl2.2H20 0.012 g ZnCl2 0.0012 g FeSO4.7H2O 0.0012 g MnS04. H20 0.0012 g MgSO4.7H2O 0.5 g Thiamine hydrochloride 0.002 g Water q.s. 1000 cm3 This preculture is used for inoculating a culture medium having the same composition but containing Nmethylacetamide at a concentration of 20 mM. Culturing is performed in an agitated flask for 24 hours at 28°C. The biomass obtained is separated by centrifugation and then washed twice with sodium chloride solution at a concentration of 9 g/litre. b) A centrifugation pellet containing 13 mg of Brevibacterium R 312 cells, expressed as dry matter, is suspended in 2 cm3 of phosphate buffer (50 mM) at pH 7.25 mg of racemic 2-phenylpropionitrile (190 μΐηοΐ) are added. After 24 hours' agitation at 25°C, the reaction mixture is diluted by adding 23cm3 of an acetonitrile/Nhydrochloric acid (90:10 by volume) mixture. The bacteria are removed by centrifugation. The composition of the supernatant is determined by high performance liquid chromatography (HPLC).

The supernatant contains: 119 μ mol of 2-phenylpropionamide 62 μΐηοΐ of 2-phenylpropionic acid.

Sodium chloride is added to promote separation of the aqueous and organic phases. After evaporation of the organic phase to dryness, the residue is taken up with 20 cm3 of a chloroform/0.1 N sodium hydroxide (1:1 by volume) mixture.

The basic phase is acidified and extracted with chloroform.

Measurement of the optical rotation shows that the enantiomeric excess of S-( + ) isomer is 100%.

After derivatization of the 2-phenylpropionic acid with (R)-(+)-a-methylbenzylamine, analysis by HPLC shows that the enantiomeric excess is 96.4%.

EXAMPLE 2 A centrifugation pellet containing 1.33 mg of Brevibacterium R 312 cells, expressed as dry matter, is suspended in 2 ml of phosphate buffer (50 mM) at pH 7.0. 32.6 mg of racemic 2-(4-hydroxyphenoxy)propionamide (0.180 mmol) are added. After 24 hours' agitation at 25°C, the reaction mixture is diluted by adding 23 cm3 of an acetonitrile/N hydrochloric acid (90:10 by volume) mixture. The bacteria are removed by centrifugation. The composition of the supernatant is determined by high performance liquid chromatography (HPLC): 0.0884 mmol of 2-(4-hydroxyphenoxy)propionamide 0.0864 mmol of 2-(4-hydroxyphenoxy)propionic acid.

Sodium chloride is added to promote separation of the organic and agueous phases. After evaporation of the organic phase to dryness, the residue is taken up with 10 ml of ethyl acetate plus 6 ml of a 10% w/v solution of sodium hydrogen carbonate in water. After settling has taken place, the agueous solution is separated and extracted with twice 10 ml of ethyl acetate. The agueous phase is acidified to pH 1.0 with N hydrochloric acid solution and then extracted with three times with 10 ml ethyl acetate. These organic phases containing the acid are dried over sodium sulphate and evaporated to dryness. The residue is taken up in acetone. Measurement of the optical rotation of this acetone solution shows that the enantiomeric excess of R-(+) isomer is 99%.

After derivatization of the 2-(4-hydroxyphenoxy)propionic acid with (R)-(+)-a-methylbenzylamine, HPLC analysis shows that the enantiomeric excess of R-(+) isomer is 94%.

EXAMPLE 3 a) Corynebacterium strain N 771 (FERM P 4445) is cultured in an agitated flask at 28°C for 14 hours in a medium having the following composition: yeast extract 3 g malt extract 3 g lactopeptone 5 g glucose 10 g FeSO4.7H2O water q.s. 1 litre and the pH of which is adjusted to 7.5 by adding sodium hydroxide before sterilization.

This preculture is used for inoculating, at a 1:40 ratio, a medium of the same composition.

After incubation for 24 hours at 28°C in an agitated flask, the biomass is separated by centrifugation and then washed twice with aqueous sodium chloride solution at a concentration of 9 g/litre. b) A centrifugation pellet containing 11.3 mg of Corynebacterium N 771 cells, expressed as dry matter, is suspended in 2 cm3 of phosphate buffer (50 mM) pH 7 . 34 mg of racemic 2-(4-hydroxyphenoxy)propionamide (0.187 mmol) are added, and the mixture is then stirred for 24 hours at 25°C. The reaction mixture is treated under the conditions of Example 2.

The supernatant contains: 0.083 mmol of 2-(4-hydroxyphenoxy)propionamide 0.079 mmol of 2-(4-hydroxyphenoxy)propionic acid.

The enantiomeric excesses of 2-(4-hydroxyphenoxy)propionic acid, measured by the optical rotation and after derivatization with (R)-(+)-a-methylbenzylamine, are, respectively, 95% and 91% in respect of the R-( + ) isomer.

EXAMPLE 4 Corynebacterium N774 cells are prepared under the conditions described in Example 3a) for the preparation of a cell pellet of Corynebacterium N771. ϊ A centrifugation pellet containing 10.8 mg of Corynebacterium N774 cells, expressed as dry matter, is suspended in 2 cm3 of phosphate buffer (50 mM) pH 7. 34.5 mg of racemic 2-(4-hydroxyphenoxy)propionamide (0.190 mmol) are added and the mixture is then stirred for 24 hours at 25°C. The reaction mixture is treated under the conditions of Example 1.

The supernatant contains: 0.078 mmol of 2-(4-hydroxyphenoxy)propionamide 0.080 mmol of 2-(4-hydroxyphenoxy)propionic acid. The enantiomeric excesses of 2-(4-hydroxyphenoxy) propionic acid, measured by the optical rotation and after derivatization with (R)-(+)-a-methylbenzylamine, are, respectively, 95% and 92% in respect of the R-( + ) isomer.

Claims (7)

1. Process for preparing an R-(+) enantiomer of a 2. -(aryloxy)- or 2-(arylthio)alkanoic acid of formula: H c (I) in which Ar denotes an optionally substituted aromatic radical, R denotes an alkyl radical having 1 to 6 carbon atoms and A denotes an oxygen or sulphur atom, which comprises enantioselectively hydrolysing an amide of the corresponding racemic 2-(aryloxy)- or 2-(arylthio)alkanoic acid, optionally prepared in situ, in the presence of a microorganism which is a Brevibacterium or Corynebacterium strain, or an enzyme derived from a said microorganism, having nitrilase activity and able to hydrolyse racemic aphenylpropionamide enantioselectively to (S) - aphenylpropionic acid with an enantiomeric excess of more than 65%, and then separating the (R)-2-(aryloxy)- or -2(arylthio)alkanoic acid from the unhydrolysed amide of the (S)-2-(aryloxy)- or -2-(arylthio)alkanoic acid.

2. Process according to claim 1, in which the said microorganism is Brevibacterium R 312 (CBS 717.73), Corynebacterium N 771 (FERM P 4445) or Corynebacterium N 774 (FERM P 4446) .

3. Process according to claim 1, in which the starting amide is obtained in situ by hydrolysis of the nitrile of the racemic 2-(aryloxy)- or 2-(arylthio)alkanoic acid in the presence of the microorganism used or of the enzyme derived from this microorganism.

4. Process according to any one of claims 1 to 3 in which the acid produced has the formula: (ϋ) Ar^ in which Ar has the same meaning as in claim 1.

5. Process according to claim 4, in which the compound of formula (II) is 2-(4-hydroxyphenoxy)propionic acid in the R-(+) form.

6. Process according to claim 1 substantially as described in any one of Examples 1 to 4.

7. An enantiomer of a 2-(aryloxy)- or 2(arylthio)alkanoic acid as defined in claim 1 when produced by the process claimed in any of claims 1 to 6.