Title of Invention

"A PROCESS FOR THE PREPARATION OF ENANTIONMERICALLY PURE D-(-)-3-ACETYLTHIO-2-METHYLPROPANOIC ACID"

Abstract

A Process for the preparation of enantiomerically pure D-(-) -3- acetylthio -2-methylpropanoic acid, which comprises: contacting DL-(+)-methyl 3-acetylthio-2- methylpropanoate. under agitation at pH 6.8 with a halotolerent strain of pseudomonas fluorescens having characteristics as described herein grown in a known manner in a conventional nutrient medium at pH between 5.0 to 11.0. at temperature between 15 to 37°C for at least a period of 10 min., separating the biomass by conventional methods and recovering D-(-)-3-acetylthio-2- methylpropanoic acid by conventional solvent extraction methods.

Full Text

The present invention relates to a process for the preparation of enantiomerically pure D-(-)-3-acetylthio-2-methylpropanoic acid.of formula (1) shown in drawing accompanying this specification is prepared by chemoselective and enantioselective hydrolysis of DL-(+)-methyl 3-acetylthio-2-methylpropanoate of formula (2). More particularly the present invention relates to production of D-(-)-3-acetylthio-2-methyipropanoic acid (1) by using a strain of Pseudomonas fluorescens as biocatalyst. D-(-)-3-acetylthio-2-methylpropanoic acid is thus obtained in optically pure form. The optically pure D-(-)-3-acetylthio-2-methylpropanoic acid is a crucial intermediate in the production of Angiotensin Converting Enzyme (ACE) inhibitors, such as Captopril and Alacepril which are used for the treatment of hypertension and congestive heart failure. This biocatalyst also hydrolyses methyl 3-chloro-2-methylpropanoate, methyl 2-phenylpropanoate and ethyl 2-chloropropanoate of formulae (3), (4) and (5) respectively, which are useful as intermediates for Pharmaceuticals and agro - chemicals. DL-(±)-3-acetylthio-2-methylpropanoic acid can be prepared from methyl methacrylic acid and thioacetic acid. This racemic material can be chemically resolved to D-(-)-3-acetylthio-2-methylpropanoic acid by using varoius optically active amines such as 1,2-diphenylethyl amine or 2-amino-1-butanol (Houbiers, Johannes Paulus Marie, Japan Kokai Tokyo Koho, 80-38386,1980; N. Ohashi, S. Nagata, S. Katsube, Japan Kokai Tokkyo Koho, 81 - 7756, 1981; J. Iwao, M. Oya, E. Kato, T. Watanabe, Japan Kokai Tokkyo Koho, 78 - 151912, 1978). Alternately D-(-)-3-acetylthio-2- methylpropanoic acid can be prepared by chemical synthesis using D-3- hydroxy-2-methylpropanoic acid as starting material (M. Shimazaki, J. Hasegawa, K. Kan, K. Nomura, Y. Nose, H. Kondo, T. Ohashi, K.Watanabe, Chem. Pharm. Bull., 30, 3139, 1982). However these methods consumed large amount of energy and expenstve agents, which had tendency to remain as impurities in D-(-)-3-acetylthio-2- methylpropanoic acid. Moreover, conversion of D-3-hydroxy-2- methylpropanoic acid to D-(-)-3-acetylthio-2-methylpropanoic acid required several steps of chemical reactions. The enzymatic methods are economically and environmentally friendly and provide much cleaner products. D-(-)-3-Acetylthio-2-methylpropanoic acid has also been produced by the resolution of DL-(±)-methyl 3-acetylthio-2-methylpropanoic acid by Pseudomonas fluorescens, IFO 3081 (A. Sakimae, A. Hosoi, E. Kobayashi, N. Ohsuga, R. Numazawa, I. Watanabe, H. Ohnishi, Biosci. Biotech. Biochem., 56, 1252, 1992). The organism was isolated by screening of organisms from stock cultures obtained from Institute of Fermentation, Osaka (IFO), the Institute of Applied Microbiology, University of Tokyo (IAM) and American Type Culture Collection (ATCC). Several organisms, which were capable of producing D- (-) -3-Acetylthio-2-methylpropanoic acid, were isolated, but Pseudomonas fluorescens, IFO 3081 was found to be the best and was used for further studies. To obtain D-(-)-3-Acetylthio-2-methylpropanoic acid with Pseudomonas fluorescens, IFO 3081, the reaction was done at substrate concentration of 0.225 moles per liter at pH 7.0 at 30°C. The cells had an activity of 558 units/g-cells. The D-(-)-3-acetylthio-2-methylpropanoic acid was produced in 97.2% enantiomeric excess. Another strain of Pseudomonas putida MR2068 has also been described (A. Sakimae, E. Ozaki, H. Toyama, N. Ohsuga, R. Numazawa, I. Muraoka, E. Hamada, H. Ohnishi, Biosci. Biotech. Biochem., 56, 1341, 1992) which produced D-(-)-3-acetylthio-2-methylpropanoic acid in 98.6 % enantiomeric excess. The product obtained by these methods was contaminated with the undesired L form of the acid owing to chemical degradation or non-specific enzymatic reaction or both. The main objective of the present invention is to provide a process for the preparation of D-(-)-3-acetylthio-2-methylpropanoic acid from DL-(±)-methyl 3-acetylthio-2-methylpropanoate as a single enantiomer, using a strain of Pseudomonas fluorescens as biocatalyst. Another objective is to use a strain of Pseudomonas fluorescens, deposited in Microbial Type Culture Collection (a constituent laboratory of applicant), Institute of Microbial Technology, Chandigarh, India, having accession number MTCC B0015, where B denotes a Bacterium, as biocatalyst. The strain designated MTCC B0015 was found Gram's negative, aerobic, rod shaped, fluorescent, green pigment producing and non-endospore forming. The characteristics usually associated with genus Pseudomonas (Sergey's Manual of Systematic Bacteriology, Vol. 1, Williams and Wilkins, Baltimore, USA, 1994). The strain of Pseudomonas fluorescens MTCC BO015 used as biocatalyst is different from those reported earlier in that: (i) it has activity of about 2400 units/g-cells, (ii) it produces D-(-)-3-acetylthio-2-methylpropanoic acid as a single enantiomer and (iii) it can be used at substrate concentration of up to 3 moles per liter. Accordingly, the present invention provides a process for the preparation of enantiomerically pure D-(-)-3-acetylthio-2-methylpropanoic acid, which comprises: contacting DL-(±)-methyl 3-acetylthio-2-methylpropanoate, under agitation at pH 6.8 with a halotolerent strain of Pseudomonas fluorescens having characteristics as described herein grown in a known manner in a conventional nutrient medium at pH between 5.0 to 11.0, at temperature between 15 to 37°C for at least a period of 10 min., separating the biomass by conventional methods followed by recovering D-(-)-3-acetylthio-2-methylpropanoic acid by conventional solvent extraction methods. In an embodiment of the present invention, the conventional nutrient medium used may be such as peptone and beef extract or a medium consisting of glucose, ammonium sulphate, ammonium phosphate, yeast extract and trace amounts of mineral salts such as ferrous sulphate, magnesium sulphate and copper sulphate In another embodiment of the present invention contacting of DL-(±)-methyl 3-acetylthio-2-methylpropanoate with a halotolerent strain of Pseudomonas fluorescens may be effected for a period of 10 min. to 6 hr. In yet another embodiment of the present invention the contacting of DL-(±)-methyl 3-acetylthid-2-methylpropanoate with a halotolerent strain of Pseudomonas fluorescens may be effected at substrate concentration of 0.01 moles to 3 moles per liter of buffer. In yet another embodiment of the present invention, the separation of biomass may be effected by filteration or centrifugation. In yet another embodiment of the present invention, the solvents used for the extraction of D-(-)-3-acetylthio-2-methylpropanoic acid may be such as diethyl ether, ethyl acetate, chloroform, dichloromethane or similar water immissible solvents. The biocatalyst used for the production of optically pure D-(-)-3- acetylthio-2-methylpropanoic acid should have the following properties : (i) it should chemoselectively hydrolyze the methoxycarbonyl group. The acetylthio group which is also present and prone to undergo hydrolysis should remain unaffected, (ii) it should enantioselectively hydrolyze the S - enantiomer, the R - enantiomer should remain unaffected, (iii) it should work under neutral conditions of pH, as both the substrate and product are prone to undergo degradation at acidic as well as basic pH and (iv) from a commercial point of view it should be able to tolerate high substrate concentration. The organism is isolated in the following manner. The soil and water samples from Lothal, Gujarat, were collected, as they were expected to yield halotolerant strains -of microorganisms. Lothal is an area in Western India, which is in close proximity with seawater. These samples were plated after dilution with sterile distilled water on nutrient agar plates containing tributyrin. The plates were incubated at a temperature between 15 and 30°C for a period between 24 and 48 hr. The microbial colonies producing a clearing zone, underneath or around the growth on the plates were picked up and purified by repeated streaking on nutrient agar plates, -The purified microorganisms were tested for their ability to produce D-(-)-3-acetylthio-2-methylpropanoic acid from DL-(±)-methyl 3-acetylthio-2-methylpropanoate. The organism, which produced maximum yield of optically pure D- (-) -3-acetylthio-2-methylpropanoic acid, was selected. The selected isolate was deposited in Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India and has been given an accession number MTCC B0015, where B denotes a bacterium. The isolated strain was found to be Pseudomonas fluorescens, based on its morphological and physiological characteristics. It has been observed that the strain designated as MTCC B0015 produces D-(-)-3-acetylthio-2-methylpropanoic acid from DL-(±)-methyl 3-acetylthio 2-methylpropanoate at a rate of about 2.4 g per hr. per g of cells (dry cell weight). It has also been observed that the strain designated as MTCC B0015 remains effective for the production of D-(-)-3-acetylthio-2-methylpropanoic acid from DL-(±)-methyl 3-acetylthio-2-methylpropanoate at a substrate concentration of up to three moles per liter. Details of the process are given below: The soil and water samples were collected from Lothal, Gujarat, India. The samples were diluted with sterile distilled water and plated on nutrient agar - tributyrin plates. The plates were incubated at a temperature ranging from 15 to 37°C for a period of between 24 and 48 hr. The microbes that produced a clearing zone underneath or around the growth on the plates were purified by repeated restreaking of individual colonies on nutrient agar plates. A few cells from the purified colonies were transferred to a nutrient agar plate containing DL-(±)-methyl 3-acetylthio-2-methylproponate and alizarin red dye. The colonies of microorganisms, which produced yellow zone underneath or around the growth, were selected for further screening. A loopful of each of these selected microorganisms was inoculated in a medium consisting of peptone and beef extract or the like, pH adjusted to neutral and incubated in an orbital shaker at 200 rpm. at a temperature between 15 and 37°C. After a period between 24 and 48 hr., cells from these cultures were harvested by centrifugation and washed with phosphate buffer. The washed cells were used for reaction with DL-(±)-methyl 3-acetylthio-2-methylpropanoate in phosphate buffer at pH 7.0 and production of D-(-)-3-acetylthio-2-methylpropanoic acid was monitored by thin layer chromatography or high performance liquid chromatography. The following tests were conducted on the strain designated as MTCC B0015: Growth on MacConkey agar, methyl red test, Voges Proskauer test, citrate and acetate utilization, casein hydrolysis, starch hydrolysis, urea hydrolysis, aesculin hydrolysis, ONPG hydrolysis, nitrate reduction, hydrogen sulphide production, oxidase test, gelatin liquefaction, arginine dihydrolase and production of acid from carbohydrates. These tests were conducted according to Sergey's Manual of Systematic Bacteriology, Vol. 1, Williams and Wilkinson, Baltimore, USA, 1994. Based upon the morphological features and the results of the tests described above the strain designated as MTCC B0015 was found to be Pseudomonas fluorescens. A further description of the invention is given below in the examples, which should however not be construed to limit the scope of the present invention. EXAMPLE 1 The soil and water samples were collected from Lothal, Gujarat, India. The samples were screened for esterase activity by diluting the samples with sterile distilled water and plating on tributyrin containing agar plates (1.5ml tributyrin, 0.2 g calcium carbonate and 3.0 g agar in 200 ml of water). The plates were incubated at a temperature of 30°C for 24 hr. The microbial colonies producing a clearing zone underneath or around the growth on the plates were picked up and purified by repeated streaking on the nutrient agar plates. The purified microorganisms were plated on nutrient agar plates containing DL-(+)-methyl 3-acetylthio-2-methylpropanoate and alizarin red at pH of 7.0. The microbial colonies that produced a yellow zone underneath or around were screened for their ability to produce D-(-)-3-acetylthio-2-methylpropanoic acid from DL-(±)-methyl 3-acetylthio-2-methylpropanoate by individual culture of pure microbes in minimal medium broth in 100 ml flasks at 30°C. The cells were harvested by centrifugation of culture after 24 hr. and 48 hr. and washed with phosphate buffer at pH 7.0. The washed cells were suspended in phosphate buffer at pH 7.0, DL-(±)-methyl 3-acetylthio-2-methylpropanoate added to it and the contents incubated at 30°C for 12 hr. The cells were removed by centrifugation and the pH of the supernatant was adjusted "to 2.0 with 1 N hydrochloric acid. The supernatant was extracted with diethyl ether. The ethereal layer was separated and shaken with water containing 2% (w/v) sodium bicarbonate. The aqueous layer was separated, its pH adjusted to 2.0 with 12 N hydrochloric acid and extracted with diethyl ether. The ether layer was dried over anhydrous sodium sulfate and evaporated to leave on oily residue, which was dissolved in chloroform. The optical rotation of chloroform solution was recorded on a polarimeter. Two isolates were found, which gave positive direction of optical rotation. The selected isolates were grown on a medium containing (per liter) of peptone 10 g, beef extract 5 g, pH 7.0 at 30°C at 200 rpm on an orbital shaker." After 24 hr. D-(-)-3-acetylthio-2-methylpropanoic acid producing enzyme was assayed as follows The cells were isolated by centrifugation and washed with phosphate buffer. 10.5 g of these cells were suspended in 100 ml of 0.05M phosphate buffer, pH 7.0 and 10 g of DL-(±)-methyl 3-acetylthio-2-methylpropanoate added to it and the volume made up to 200 ml with phosphate buffer. The reaction was done at 30°C for one hr., maintaining the pH of the reaction mixture at 7.0 by the addition of 1 N sodium hydroxide. The hydrolytic activity was defined as one unit when 1/162.2 mmol sodium hydroxide was added to the reaction mixture in one hr. The specific activity was defined as number of units per gram of cells (dry cell weight). One of the isolates, which produced about 2400 units per gram of cells (dry cell weight) was selected and deposited with Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh and has been given accession number MTCCB0015. EXAMPLE 2 The strain designated as MTCC B0015 as obtained in example 1 was subjected to the following tests: growth on MacConkey agar, indole test, methyl red test, Voges Proskauer test, citrate and acetate utilization test, casein hydrolysis, starch hydrolysis, urea hydrolysis, aesculin hydrolysis, ONPG hydrolysis, nitrate reduction, hydrogen sulphide production, oxidase test, catalase test, oxidation/fermentation, gelatin liquefaction, and production of acid from carbohydrates. These tests were conducted according to Bergey's manual of systematic bacteriology, vol. 1, Williams and Wilkinson, Baltimore, USA, 1994. The results of these tests are given in Table 1. 1. Growth on MacConkey agar: The organism was streaked on the plates containing (per liter) of 17g peptone, 3g polypeptone, 10g lactose, 1.5g bile salts, 13.5g agar, 0.03g neutral red, O.OOIg crystal violet at pH of 7.1. The growth of red shaded colonies indicated the presence of a lactose fermentor. 2. Indole test: After incubation in 5 ml indole production medium (1% tryptone broth), added 2 ml of the reagent (5g p- dimethylaminobenzaldehyde, 75 ml iso-amyl alcohol and 25 ml cone, hydrochloric acid). The contents of the tube were shaken vigorously and examined for the appearance of any pink colour in alcohol layer. 3. Methyl red test: Two glass tubes with 1.5 ml sterile medium containing (per liter) 5g peptone, 5g dipotassium hydrogen phosphate, 5g glucose at pH 7.5 were inoculated with the organism. One was incubated at 37°C for 2 days and other at room temp, for 5 days. The contents of the tubes were shaken vigorously with 2 drops of methyl red solution and observed for any change in colour. 4. Voges - Proskauer test: The organism was grown in 5 ml of VP broth (per liter; 7g proteose peptone, 5g glucose; pH 6.5). 3 ml of 40% (W/v) sodium hydroxide and 0.5-1.0 mg of creatine was added to the culture broth. It was kept at room temperature for 30-60 min. and observed for the appearance of red colour. 5. Citrate/Acetate utilization test: Slants of the citrate/acetate medium were made which contained (per liter) of 1g ammonium dihydrogen phosphate, 1g dipotassium phosphate, 5g sodium chloride, 2g sodium citrate (or 1g sodium acetate) 15g agar, 0.08g bromothymol blue at pH of 7.0. A well- isolated colony from medium was picked and inoculated as a single streak on the slant surface of citrate/acetate agar plate. The plate was incubated at 35°C for 24-28 hr. and observed for the appearance of deep blue colour. 6. Casein hydrolysis: The organism was grown on milk agar plates. Hydrolysis of casein was indicated by appearance of clear zone underneath and around the growth. The milk agar was made by mixing autoclaved skim milk solution (10%) with equal volume of 2X nutrient agar. 7. Starch hydrolysis: The organism was grown on nutrient agar containing 1% soluble starch. The plates were flooded with 95% ethanol. Hydrolysis of starch is indicated by the appearance of a clear zone underneath or around the growth. 8. Urea hydrolysis: Sterile agar plates containing (per liter) 1g peptone, 5g sodium chloride, 2g potassium dihydrogen phosphate, 10g agar no. 1 (oxoid), 1g glucose, 6 ml phenol red (0.2% aqueous solution), 100 ml urea (20% aqueous solution) were inoculated with a light suspension of the organism, incubated and observed for any colour change. 9. Aesculin hydrolysis: Sterile agar plates containing (per liter) 10g peptone, 5g sodium chloride, 0.5g ferric chloride, 20g agar at pH 7.3 were inoculated with the organism and examined for up to 5 days. The blackening around the growth indicated a positive test. 10. ONPG hydrolysis: 0-Nitrophenyl- a -D-galactopyranoside (ONPG) 6.0 g/liter solution was made & pH was adjusted to 7.6 with disodium hydrogen phosphate. It was mixed with peptone water (per liter; 10g peptone, 5g sodium chloride, pH 7.3) in ratio of 1:3, inoculated with a drop of suspension of the organism and incubated for 48 hr. The appearance of yellow colour was observed. 11. Nitrate reduction: The organism was inoculated in nitrite free broth (1 g potassium nitrate per liter of nutrient broth). It was observed for gas in the Durham's tubes. 1 ml of reagent A (0.8% sulphanilic acid in 5 N acetic acid) followed by 1 ml of reagent B (0.6% - - naphthylamine in 5 N acetic acid or 0.5% - naphthylamine in 5 N acetic acid) was added. Appearance of red colour indicated the presence of nitrite, which has been formed by reduction of nitrate. To the tubes not showing red colour, powdered zinc (5 mg per ml) was added and the appearance of red colour on standing was observed. 12. Production of catalase: The growth of organism on nutrient agar was flooded with 10% hydrogen peroxide. Production of gas bubbles indicated the presence of catalase. 13. Hydrogen sulphide production test: Triple Sugar Iron (TSI) agar (per liter; 3g beef extract, 3g yeast extract, 20g peptone, 5g sodium chloride, 10g sucrose, 1g dextrose, 0.3g ferric citrate, phenol red q.s, 12g oxoid sugar no 3) was sterilized by autoclaving at 15°C for 20 min and allowed to settle in slope form with a butt about 3 cm long. It was inoculated by stabbing the butt and streaking the slope and observed for blackening in the butt only for up to 5 days. 14. Oxidative/Fermentative test: 10 ml of sterile medium (per liter : 2g peptone, 5g sodium chloride, 0.3g dipotassium hydrogen phosphate, 3g agar, 6 ml of 1% aqueous bromothymol blue (and 10g glucose) was distributed to the tubes with 16 mm diameter. The organism was inoculated in duplicate tubes. Soft paraffin (melted) was added to one tube to the height of about 3.0 mm above the medium. It was incubated and examined for up to 5 days. The yellow colour in open tubes and green colour in sealed tubes is indicative of positive oxidative and negative fermentative test, respectively. 15. Gelatin liquefaction: Growth on the nutrient agar gelatin (nutrient agar with 0.4% gelatin) was flooded with 10 ml of sulfuric acid pre saturated with sodium sulphate. Gelatin hydrolysis was indicated by a clear zone underneath or around the growth. 16. Oxidase test: The organism were grown on nutrient agar, cells were removed with a toothpick and applied on a filter paper. 2-3 drops of 1% aqueous solution of tetramethyl-p-phenylenediamine dihydrochloride were put on the cells. A positive reaction was shown by the development of a purple colour within ten seconds. 17. Acid from carbohydrates: The organism was inoculated into tubes of acid producing medium. Charge in colour from blue to yellow indicated the production of acid. The composition of acid production medium was (per liter) 1g diammonium hydrogen phosphate, 0.2g potassium chloride, 0.2g magnesium sulphate, 0.2g yeast extract, 15g agar, 15 ml 0.04% (w/v) solution of bromocresol purple and sugars at final concentration of 0.05%. Thus, the strain designated at MTCC B0015 was identified as Pseudomonas fluorescens based on the morphological characteristics and the results of the above tests. TABLE 1 : SUMMARY OF THE RESULTS OF THE MORPHOLOGICAL, BIOCHEMICAL AND PHYSIOLOGICAL TESTS CONDUCTED ON STRAIN DESIGNATED AS MTCC B0015 Morphological Characteristics: Rods, Gram's negative, Motile, Translucent colonies with green pigments, Fluorescent, No Endospore forming BIOCHEMICAL AND PHYSIOLOGICAL TESTS: (Table Removed) Example 3 This example pertains to the preparation of biocatalyst. The strain of Pseudomonas fluorescens isolated as described in example 1 was maintained on nutrient agar. The slants were incubated at 37°C for 24 hr. A loopful of this actively growing culture was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of sterile medium consisting of (per liter) 10g peptone and 5g beef extract. The culture flask was incubated at 37°C in an orbital shaker at 200 rpm for 12 hr. A 2ml portion of this culture was used to inoculate 200 ml of sterile medium of above composition in each of the several one-liter flasks. These flasks were incubated at 37°C at 200 rpm on an orbital shaker. The growth was monitored spectrophotometrically every 2 hr. The enzyme activity in cells was monitored by withdrawing an aliquot of 2 ml every 2 hr. and reacting with DL-(+)-raethyl 3-acetylthio-2- methylpropanoate at 30°C for one hr. maintaining the pH at 7.0 with 0.05 N sodium hydroxide. The fermentation was terminated when there was no further increase in activity. The fermentation broth was centrifuged at 10,000 g for 20 min. to give 14.0 g cells per liter of broth. The dry cell weight was obtained by sequentially heating the cells to a constant weight at 50°C for 12 hr., 80°C for 2hr. and 120°C for 1hr. Thus, 10.5 grams of wet cells, as obtained above, when dried in such away gave rise to approximatly 1.6g of dry cells of the microbial strain designated as MTCC B0015. EXAMPLE 4: For the production of D-(-)-3-acetylthio-2-methylpropanoic acid, 6 g of DL-(±)-methyl 3-acetylthio-2-methylpropanoate was added to 100 ml of 0.05 M phosphate buffer of pH 7.0, containing a suspension of 45g of the cells (wet cell weight) of Pseudomonas fluorescens obtained as in Example 3. The reaction was started at 30°C, maintaining the pH of the reaction mixture at 7.0 by the addition of 10% (w/v) aqueous sodium hydroxide. The progress of the reaction was monitored by High Performance Liquid Chromatography. After 2 hr. the reaction mixture was cooled to 4°C in an ice bath and centrifuged at 10000 g for 20 min. The pH of the supernatant was adjusted to 2.0 with 12 N hydrochloric acid. The supernatant was extracted with diethyl ether. The ethereal layer was shaken with 2% (w/v) aqueous sodium bicarbonate. The aqueous layer was cooled in an ice bath, its pH adjusted to 2.0 with 12 N hydrochloric acid and extracted with diethyl ether. The evaporation of diethyl ether from ethereal layer gave D-(-)-3-acetylthio-2-methylpropanoic acid as colorless oil; Yield 49.1%, [α]D 25°, -57.8° (c=2, CHCI3); 1H NMR (CDCI3): 1.29 (doublet, J=7.2 Hz), 2.35 (singlet), 2.73 (multiple!), 3.05 (doublet of doublet, J=6.1 & 7.5 Hz), and 3.13 (doublet of doublet, J=7.1 & 7.5 Hz). D-(-)-3-Acetylthio-2-methylpropanoic acid, so produced was found to have ee of 99.7% based on High Performance Liquid Chromatography and 1H NMR spectroscopic analysis of a diastereomer produced by its reaction with R-(+)-1-(1-naphthyl)ethylamine in dichloromethane in presence of dicyclohexyl carbodiimide by standard protocol. EXAMPLE 5 For the production of D-(-)-3-acetylthio-2-methylpropanoic acid, 17.6 g of DL-(±)-methyl 3-acetylthio-2-methylpropanoate was added to 100 ml of 0.05 M phosphate buffer of pH 7.0, containing a suspension of 40g of the cells (wet cell weight) of Pseudomonas fluorescens obtained as in Example 3. The reaction was started at 30°C, maintaining the pH of the reaction mixture at 7.0 by the addition of 10% (w/v) aqueous sodium hydroxide. The progress of the reaction was monitored by High Performance Liquid Chromatography. After 6 hr. the reaction mixture was cooled to 4°C in an ice bath and centrifuged at 10000 g for 20 min. The pH of the supernatant was adjusted to 2.0 with 12 N hydrochloric acid. The supernatant was extracted with ethyl acetate. The ethyl acetate layer was shaken with 2% (w/v) aqueous sodium bicarbonate. The aqueous layer was cooled in an ice bath, its pH adjusted to 2.0 with 12 N hydrochloric acid and extracted with ethyl acetate. The evaporation of ethyl acetate gave D-(-)-3-acetylthio-2- methylpropanoic acid as colorless oil; Yield 48.8%, [α]D 25°, -57.7° (c=2, CHCI3); 1H NMR (CDCI3): 1.29 (doublet, J=7.2 Hz), 2.35 (singlet), 2.73 (multi- plet), 3.05 (doublet of doublet, J=6.1 & 7.5 Hz), and 3.13 (doublet of doublet, J=7.1 & 7.5 Hz). D-(-)-3-Acetylthio-2-methylpropanoic acid, so produced was found to be a single enantiomer based on High Performance Liquid Chromatography and 1H NMR spectroscopic analysis of a diastereomer produced by its reaction with R-(+)-1-(1-naphthyl)ethylamine in dichloromethane in presence of dicyclohexyl carbodiimide by standard protocol. EXAMPLE 6 For the production of D-(-)-3-acetylthio-2-methylpropanoic acid, 0.176 g of DL-(±)-methyl 3-acetylthio-2-methylpropanoate was added to 100 ml of 0.05 M phosphate buffer of pH 7.0, containing a suspension of 15g of the cells (wet cell weight) of Pseudomonas fluorescens obtained as in Example 3. The reaction was started at 30°C, maintaining the pH of the reaction mixture at 7.0 by the addition of 10% (w/v) aqueous sodium hydroxide. The progress of the reaction was monitored by High Performance Liquid Chromatography. After 10 min. the reaction mixture was cooled to 4°C in an ice bath and centrifuged at 10000 g for 20 min. The pH of the supernatant was adjusted to 2.0 with 12 N hydrochloric acid. The supernatant was extracted with diethyl ether. The ethereal layer was shaken with 2% (w/v) aqueous sodium bicarbonate. The aqueous layer was cooled in an ice bath, its pH adjusted to 2.0 with 12 N hydrochloric acid and extracted with diethyl ether. The evaporation of diethyl ether from ethereal layer gave D-(-)-3-acetylthio-2-methylpropanoic acid as colorless oil; Yield 48.2%, [α]D 25°, -58° (c=2, CHCI3); 1H NMR (CDCI3): 1.29 (doublet, J=7.2 Hz), 2.35 (singlet), 2.73 (multiple!), 3.05 (doublet of doublet, J=6.1 & 7.5 Hz), and 3.13 (doublet of doublet, J=7.1 & 7.5 Hz). D-(-)-3-Acetylthio-2-methylpropanoic acid, so produced was found to be a single enantiomer based on High Performance Liquid Chromatography and 1H NMR spectroscopic analysis of a diastereomer produced by its reaction with R-(+)-1-(1-naphthyl)ethylamine in dichloromethane in presence of dicyclohexyl carbodiimide by standard protocol. Advantages of the present invention are: 1. The strain of Pseudomonas fluorescens used as biocatalyst hydrolyses DL-(+)-methyl 3-acetylthio-2-methylpropanoate to D-(-)-3-acetylthio-2-methylpropanoic acid under very mild reaction conditions of temperature (30°C) and pH (7.0) and at substrate concentration of up to 3 moles per liter. The conversion rate is high; approximately 2.4 g of acid is produced in one hr. by 1 g of cells (dry cell weight), so that the reaction can be completed in shorter time. These features lead to (i) reduced energy requirements and (ii) high quality of the product as substrate as well as product do not undergo chemical degradation under these conditions, thus leading to an eco-friendly and economic process. 2. The biocatalyst used in the process is (i) highly chemoselective for methoxycarbonyl group.The acetylthio group, prone to hydrolysis and present in the molecule remains unaffected and (ii) highly enantioselective for S enantiomer. These features lead to high chemical and optical purity for the product, D-(-)-3-acetylthio-2-methylpropanoic acid. 3. D-(-)-3-Acetylthio-2-methylpropanoic acid was produced as a single enantiomer. We claim: 1. A process for the preparation of enantiomerically pure D-(-)-3-acetylthio- 2-methylpropanoic acid, which comprises: contacting DL-(±)-methyl 3- acetylthio-2-methylpropanoate, under agitation at pH 6.8 with a halotolerent strain of Pseudomonas fluorescens having characteristics as described herein grown in a known manner in a conventional nutrient medium at pH between 5.0 to 11.0, at temperature between 15 to 37°C for at least a period of 10 min., separating the biomass by conventional methods and recovering D-(-)-3-acetylthio-2-methylpropanoic acid by conventional solvent extraction methods. 2. A process as claimed in claim 1 where in the conventional nutrient medium used has composition such as peptone and beef extract or glucose, ammonium sulphate, ammonium phosphate, yeast extract and trace amounts of mineral salts such as ferrous sulphate, magnesium sulphate and copper sulphate 3. A process as claimed in claim 1 and 2 where in contacting of DL-(+)- methyl 3-acetylthio-2-methylpropanoate with a halotolerent strain of Pseudomonas fluorescens may be effected for a period of 10 min. to 6 hr. 4. A process as claimed in claim 1 to 3 where in contacting of DL-(±)-methyl 3-acetylthio-2-methylpropanoate with a halotolerent strain of Pseudomonas fluorescens may be effected at substrate concentration of 0.01 moles to 3 moles per liter of buffer. 5. A process as claimed in claim 1 to 4 where in the separation of biomass may be effected by filtration or centrifugation. 6. A process as claimed in claim 1 to 5 where in the solvents used for the extraction of D-(-)-3-acetylthio-2-methylpropanoic acid may be such as diethyl ether, ethyl acetate, chloroform, dichloromethane or similar water immissible solvents. 7. A process for the preparation of enantiomerically pure D-(-)-3-acetylthio- 2-methylpropanoic acid substantially as herein described with reference to the examples.

Documents:

1287-del-1999-abstract.pdf

1287-del-1999-claims.pdf

1287-del-1999-complete specification (granted).pdf

1287-del-1999-correspondence-others.pdf

1287-del-1999-correspondence-po.pdf

1287-del-1999-description (complete).pdf

1287-del-1999-drawings.pdf

1287-del-1999-form-1.pdf

1287-del-1999-form-2.pdf

1287-del-1999-form-4.pdf

1287-del-1999-form-9.pdf

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