GB2218985A - Producing optically active 2-aryloxypropionic acids - Google Patents

Abstract

A process for the stereospecific inversion of the S enantiomer to the corresponding R enantiomer of a 2-aryloxy-propionic acid of formula I <IMAGE> or a salt or ester thereof, wherein X represents a halogen atom and Y represents a halogen atom or an alkyl group to 1 to 4 carbon atoms, comprises supplying the S enantiomer or a mixture of the R and S enantiomers of a compound of formula (I), or a salt or ester thereof, to a microorganism having a stereospecific inverting enzyme system, or an extract of the microorganism containing said enzyme system, capable of converting the S-enantiomer to the corresponding R enantiomer. <IMAGE>

Description

PROCESS FOR PRODUCING OPTICALLY ACTIVE 2-ARYLOXYPROPIONIC ACIDS This invention relates to the preparation of optically active 2-aryloxypropionic acids and is particularly, though not exclusively, concerned with the preparation of optically active forms of herbicidal aryloxypropionic acids such as mecoprop (2- (4-chloro-2-methylphenoxy) propionic acid) and dichlorprop (2-(2,4-dichlorophenoxy)propionic acid).

Herbicidal 2-aryloxypropionic acids such as mecoprop and dichlorprop have been available for many years. The commercially available products are the racemates, although it is recognised that it is the R-enantiomers which are active as herbicides. While the active R-enantiomers can be manufactured chemically, for example from the appropriate optically active lactic acid, such a process is expensive. There is thus a need for economically viable methods of producing products predominantly in the R-form which do not involve stereospecific chemical syntheses.

EP-A-133033 indicates that certain phenoxypropionic acids having a substituent of formula -OR1 or NHR2 at the p-position (where R1 is hydrogen or a protecting group and R is hydrogen or methyl) can be microbially stereospecifically inverted. However these compounds are intended merely as intermediates which allow coupling of the p-substituent with a further aromatic ring to yield enantiomers of well known aryloxyphenoxypropionic acid and arylaminophenoxypropionic acid herbicides such as fluazifop (2- [4- (5-trifluoromethyl-2-pyridyl- oxy)phenoxy)propionic acid). Furthermore, the only examples concern /S 2-(p-hydroxyphenoxy)propionic acid and only a limited enrichment in R-enantiomer is obtained.Surprisingly, we have found that S enantiomers of certain aryloxypropionic acids which are themselves herbicides, and which have halogen substitution at the p-position can be microbially inverted to the R enantiomer in high yields, thus giving a means of obtaining a product which is highly enriched in R-enantiomer from the /S starting material.

According to this invention we provide a process for the stereospecific inversion of the S enantiomer to the corresponding R enantiomer of a 2-aryloxyprop ionic acid of formula I

or a salt or ester thereof, wherein X represents a halogen atom and Y represents a halogen atom or an alkyl group of 1 to 4 carbon atoms, comprising supplying the S enantiomer or a mixture of the R and S enantiomers of a compound of formula (I), or a salt or ester thereof, to a microorganism having a stereospecific inverting enzyme system, or an extract of the microorganism containing said enzyme system, capable of converting the S-enantiomer to the corresponding R enantiomer.

Preferably X represents a chlorine atom.

Preferably Y represents a chlorine atom or a methyl group. The substrate for the stereospecific inversion is therefore preferably racemic 2-(4-chloro-2-methylphenoxy)propionic acid or 2-(2,4-dichlorophenoxy)propionic acid.

The microorganism preferably is capable of stereospecifically inverting at least 90% of the S enantiomer present to the R enantiomer, more preferably at least 95%.

Suitable microorganisms for carrying out the stereospecific inversion include fungal and bacterial microorganisms belonging to the genera Rhodococcus, Arthrobacter, Bacillus, Leuconostoc, Proteus, Streptomyces, Penicillium, Botrytis and Nocardia.

Preferred microorganisms are those of the genus Rhodococcus, for example the species Rhodococcus rhodochrous. A specific example is Rhodococcus rhodochrous NCIB 12566 deposited with the National Collections of Industrial and Marine Bacteria, U.K.

on 16 October 1987, and variants or mutants thereof.

While the stereospecific inversion is usually carried out in the presence of the whole cells, the stereospecific inverting enzyme system may have been completely or partly extracted from the microorganism prior to carrying out the inversion. To avoid unnecessary separation, enzyme purification and enzyme immobilisation procedures, the enzyme is usually present with at least some of the cell components. When in association with the cells, these may be live or dead, intact, treated in some way or themselves immobilised and optionally homogenised, so long as the enzyme component itself is retained in active and stable form to allow the inversion to proceed. Immobilisation of the enzyme or the cells may be by any of the methods known in the art so long as the enzyme inverting activity is retained intact.

The microorganisms are preferably cultured prior to use for the stereospecific inversion for about 1 to 10 days, whereafter the cells are suspended in a liquid nutrient medium, preferably a minimal liquid nutrient medium, and compound (I) is subjected to the action of the cells. After the abovementioned cultivation the cells may be isolated from the culturing medium before suspending the cells in the minimal liquid nutrient medium.To grow the micro-organisms used for the stereospecific inversion of compound (I), ordinary culture media containing an assimilable carbon source (for example n-heptane vapour or glucose, lactate, sucrose, etc.), a nitrogen source (for example ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), with optionally an agent for an organic nutrient source (for example yeast extract, malt extract peptone, meat extract, etc.) and an inorganic nutrient source (for example phosphate, magnesium, potassium, zinc, iron and other metals in trace amounts) may be used.

A temperature between 0 and 450C and a pH between 3.5 and 8 is maintained during the growth of the microorganisms.

Preferably the microorganisms are grown at a temperature between 20 and 37'C and at a pH between 4 and 7.

The aerobic conditions required during the growth of the microorganisms can be provided according to any of the well-established procedures, provided that the supply of oxygen is sufficient to meet the metabolic requirement of the microorganisms.

This is most conveniently achieved by supplying gaseous oxygen, preferably in the form of air.

During the stereospecific inversion the microorganisms might be in a growing stage using an abovementioned ordinary culture medium.

Preferably during the stereospecific inversion, the microorganisms can be held in a substantially non-growing stage using a minimal culture medium. As minimal culture medium, an ordinary culture medium may be used containing an assimilable carbon source when required (for example glucose, lactate, sucrose, etc.), a nitrogen source when required (for example ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), with an agent for an organic nutrient source when required (for example yeast extract, salt extract, peptone, meat extract, etc.) and an inorganic nutrient source when required (for example phosphate, magnesium, potassium, zinc, iron and other metals in trace amounts). The microorganisms can be kept in the non-growing stage for example under exclusion of the assimilable carbon source or under exclusion of the nitrogen source. A temperature between 0-and 45"C and a pH between 3.5 and 8 is preferably maintained during this stage.

Preferably the microorganisms are kept at a temperature between 20 and 37'C and a pH between 4 and 7. The aerobic conditions required during this stage can be provided according to the abovementioned procedures, provided that the supply of oxygen is sufficient to meet the metabolic requirement of the microorganisms but also to convert compound (II) into compound (I). The compound (I) produced by the microorganisms as mentioned above, can be recovered and purified according to any of the procedures known per se for such products.

The invention is illustrated in the following examples. The media used were as follows: Ammonium salts medium (ASM) Composition g/litre Ammonium chloride 0.535 Potassium dihydrogenphosphate 0.531 Disodium hydrogenphosphate 0.866 Potassium sulphate 0.174 Magnesium sulphate.7H2O 0.037 Calcium chloride.2H20 0.00733 TK3 trace element solution 0.1 ml Ferrous sulphate.7H2 0 0.0189 (added after autoclaving) pH adjusted to 7.0 Phosphate medium (PSX) composition g/litre Potassium dihydrogen phosphate 8.92 Disodium hydrogen phosphate 2.84 Diammonium hydrogen phosphate 1.0 Ammonium sulphate 0.2 Potassium chloride 0.2 Trisodium citrate 0.294 Calcium sulphate.2H20 0.005 Magnesium sulphate.7H2O 0.2 PS2 T/E 10 ml TK3 trace element solution ZnSO4. 7H2O 0.288g MnSO4.4H2O 0.224g H3BO3 0.0618g CuSO4 .5H2O 0.l248g Na2MoO4.2HO 0.0484g CoC12.6H2O 0.0476g KI 0.083g 1N H2SO4 1 ml Dissolved in 1 litre distilled water PS2 T/E (NH4)2 SO4.FeSO4.6H2O 0.25g ZnSO4. 7H2O 0.05g MnC12.4H20 0.03g CuSO4.5H2O 0.015g CoCl2. 6H20 0.015g H3BO3 0.005g Na2MoO4.H2O 0.0055g KI 0.01g Dissolved in 1 litre distilled water Example 1 Conversion of JRS 2- (4-chloro-2-methylphenoxy) - propionic acid into R-2-(4-chloro-2-methylphenoxy)- propionic acid Rhodococcus rhodochrous NCIB 12566 was kept at 4 C on nutrient agar slopes and used for loop inoculation of 300 ml of PSX medium.The cells were grown, using n-heptane vapour as the sole carbon source, on a rotary shaker at 100 rpm at 300C for 5 days. The cells were collected by centrifugation and resuspended in 30 ml ASM medium to give a dry weight concentration of cells of 9 to 11 mg.ml -1 Powdered JRS 2-(4-chloro-2-methylphenoxy)propionic acid (15mg) was added to 30 ml of ASM cell suspension followed by incubation on a rotary shaker at 100 rpm at 30iC for 24 hours. The cells were then acidified with 5N sulphuric acid (1 mlj and extracted with dichloromethane (3 x 30 ml). The combined extracts were blown to dryness under a stream of nitrogen.The quantity of acid recovered was determined by HPLC by suspending the extracts in 70 parts by volume acetonitrile and 30 parts by volume 2% acetic acid and injecting onto a Lichrosorb RP-18 (250 x 4 mm) column using a 5 x 10 3 ml loop.

The recovered acid was purified by preparative HPLC using the column as described above and a 200 x 10 3 injection loop, followed by combination of the acid containing fractions and evaporation to dryness.

Derivatisation and chiral HPLC analysis were carried out as follows: An aliquot (0.5 ml) of the recovered acid in 100% ethanol was dispersed into a capped vial and the ethanol evaporated. To the solid (5-7 mg) was added dimethylformamide -dimethylacetal (0.5 ml; 2 mEq/ml in pyridine) and the mixture heated at 60*C for 10 minutes. The pyridine was evaporated and n-hexane (1 ml) added.

Chromatography was performed on a column of immobilised D-phenyl glycine (250 x 4.9 mm column containing CHI-PGC-250A (D-3 , 5-dinitrobenzoylphenyl- glycine bonded silica) supplied by Hichrom Ltd., Reading). The mobile phase was n-hexane at a flow rate of 1 ml mien 1. Detection was at 280 nm (u.v.).

Optical rotation of the acid was accomplished by dissolving the acids in 100% ethanol (4 ml) and measurement at the sodium D line in a Perkin Elmer polarimeter (Model No. 241; 589.3 nm; 1 decimeter light path).

HPLC analysis showed a recovery of 2-(4-chloro2-methylphenoxy)propionic acid of 91%. Optical rotation [&alpha;]20 D = + 17.1 (C = 0.276, 100% ethanol).

Chiral HPLC analysis showed 97.7% R-enantiomer.

Example 2 Conversion of JRS 2-(2,4-dichlorophenoxy)propionic acid into R-2-(2,4-dichlorophenoxy)propionic acid Example 1 was repeated using EJS 2-(2,4-dichlorophenoxy)propionic acid as substrate for the microorganism.

HPLC analysis showed a recovery of 2-(2,4-dichlorophenoxy)propionic acid of 94.9%.

Optical rotation 20D = + 17.75 (C = 0.304, 100% ethanol). The correlation of the R-enantiomer with dextrorotatory characteristics is shown by Fredga and Aberg (1965) Ann. Rev. P1. Physiol. 16, 53-72.

Chiral HPLC analysis was carried out by preparation of the ethyl esters and chromatography on a Nucleosil chiral 2 column, supplied by Machery Nagel. The mobile phase was n-heptane containing 0.2% isopropanol and 0.05% trifluoroacetic acid at a flow rate of 1.5 ml mien 1. Detection was at 280 nm. The enantiomeric purity was shown to be 94.1% R-enantiomer.

Claims (10)

1. PROCESS FOR PRODUCING OPTICALLY ACTIVE

   2-ARYLOXYPROPIONIC ACIDS 1. A process for the stereospecific inversion of the S enantiomer to the corresponding R enantiomer of a 2-aryloxypropionic acid of formula I

   or a salt or ester thereof, wherein X represents a halogen atom and Y represents a halogen atom or an alkyl group of 1 to 4 carbon atoms, comprising supplying the S enantiomer or a mixture of the R and S enantiomers of a compound of formula (I), or a salt or ester thereof, to a microorganism having a stereospecific inverting enzyme system, or an extract of the microorganism containing said enzyme system, capable of converting the S-enantiomer to the corresponding R enantiomer.
2. 2. A process according to claim 1 wherein X in formula I represents a chlorine atom.
3. 3. A process according to claim 1 or 2 wherein Y in formula I represents a chlorine atom or a methyl group.
4. 4. A process according to claim 1 wherein the substrate for the stereospecific inversion is racemic 2-(4-chloro-2-methylphenoxy)propionic acid or 2-(2,4-dichlorophenoxy)propionic acid.
5. 5. A process according to any one of claims 1 to 4 wherein the microorganism is capable of stereospecifically inverting at least 90% of the S enantiomer present to the R enantiomer.
6. 6. A process according to any one of the preceding claims wherein the microorganism belongs to the genus Rhodococcus, Arthrobacter, Bacillus, Leuconostoc, Proteus, Streptomyces, Penicillium, Botrytis or Nocardia.
7. 7. A process according to claim 6 wherein the microorganism belongs to the species Rhodococcus rhodochrous.
8. 8. A process according to claim 7 wherein the microorganism is Rhodococcus rhodochrous NCIB 12566 or a variant or mutant thereof.
9. 9. A process according to any one of the preceding claims wherein the product of the stereospecific inversion is in the form of a free acid which is subsequently converted to a salt or ester.
10. 10. A compound of formula I or a salt or ester thereof containing predominantly the R-enantiomer when obtained by the inversion process of any one of the preceding claims.