Title of Invention	A PROCESS FOR HYDROLYZING A RACEMIC R- ALPHA-HYDROXY ARENE ACETONITRILE INTO R ISOMER OF CORRESPONDING ACID.
Abstract	A process for hydrolyzing a racemic RS-a-hydroxy arene acetonitrile into (R) isomer of the corresponding acid, comprising the steps: (a) Obtaining a supernatant containing nitilase by incubating a substrate culture of Pseudomonas fluorescens bearing accession No. MTCC 5110 in nutrient broth medium at 25-300C for 20-24 hours, (b) hydrolysing a-hydroxy arene acetonitrile such as herein described with the supernatant obtained in step (a) in aqueous medium, optionally in the presence of a buffer such as hereindescribed at pH of 5 and a temperature of 10 to 4500, and (c) recovering the corresponding (R)-carboxylic acid in high enantiomeric excess.

Title of Invention

A PROCESS FOR HYDROLYZING A RACEMIC R- ALPHA-HYDROXY ARENE ACETONITRILE INTO R ISOMER OF CORRESPONDING ACID.

Abstract

A process for hydrolyzing a racemic RS-a-hydroxy arene acetonitrile into (R) isomer of the corresponding acid, comprising the steps: (a) Obtaining a supernatant containing nitilase by incubating a substrate culture of Pseudomonas fluorescens bearing accession No. MTCC 5110 in nutrient broth medium at 25-300C for 20-24 hours, (b) hydrolysing a-hydroxy arene acetonitrile such as herein described with the supernatant obtained in step (a) in aqueous medium, optionally in the presence of a buffer such as hereindescribed at pH of 5 and a temperature of 10 to 4500, and (c) recovering the corresponding (R)-carboxylic acid in high enantiomeric excess.

Full Text	Field of the Invention The present invention relates to a process for hydrolyzing a racemic RS-cihydroxy arene acetonitrile into (R) isomer of the corresponding acid. The process of the invention is effected using a nitrilase produced by a novel mesophilic microorganism Pseudomonas fluorescens MTCC 5110. Background of the invention R-α-hydroxy arene acetic acids find numerous applications as chiral synthons and resolving agents. For example there are numerous uses of R-(-)-mandelic acid, a hydroxy acid in this class. (R)-(-)-mandelic acid is a useful resolving agent (Kinbara et. al., Design of resolving reagents: p-substituted mandelic acids as resolving reagents for 1-arylalkylamines. Tetrahedron: Asymmetry. 7:1539-1542. 1996; Drabowics et al., New procedures for the resolution of chiral tedbutylphenylphosphine oxide and some of its reactions. Tetrahedron: Asymmetry. 10: 2757-2763. 1999; Han et al.; First practical synthesis of enantiomerically pure (R)- and (S)- desmethyl sibutramine (DMS) and unambiguous determination of their absolute configuration by single-crystal X-ray analysis. Tetrahedron: Asymmetry. 13: 107-109. 2002) and a chiral synthon for the production of various pharmaceuticals such as semi-synthetic penicillin (Furlenmeier et al., 6-Acyl derivatives of aminopenicillanic acid. U.S. Patent 3957758. 1976), cephalosporins (Terreni et al., Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of cephamandole from cephalosporin C. Bioorg. Med. Chem. Lett. 11: 2429-2432. 2001), anti obesity agents (Mills et al. Phenethanolamines, compositions containing the same and method for effecting weight control. U.S. Patent 4391826. 1983), antiarrhythmic agents (Aav et al., Preparation of highly enantiopure stereoisomers of 1 -(2,6-dimethylphenoxy)-2-aminopropane (Mexiletine). Tetrahedron: Asymmetry. 10: 3033-3038. 1999), anti-tumour agents (Kotora and Negishi, Efficient and diastereoselective synthesis of (+)-goniobutenolide-A via palladium-catalysed ene-yne cross coupling-lactonization cascade. Tetrahedron Lett. 37: 9041-9042. 1996; Surivet et al.; Easy access to an enantiopure precursor of (+)-goniodiol. T-etrahedron: Asymmetry. 7: 3305-3308. 1999; Surivet et al., 1996) and the molecule itself also exhibits anti fungal properties (Kope et al., pHydroxybenzoylformic acid and R-(-)--p-hydroxymandelic acid, two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolilithus arhizus. Canadian J. Microbiol. 37: 258-264. 1991). Nitrilases (nitrile metabolising enzymes) catalyse the hydrolysis of nitriles to corresponding acid and ammonia. Based on their substrate specificity, microbial nitrilases are differentiated into three different categories, namely aliphatic nitrilases, aromatic nitrilases and arylacetonitrilases. Some nitrilases hydrolyze aromatic or heterocyclic nitriles to corresponding acids and ammonia. Others are known to hydrolyze preferentially either aliphatic nitriles or acylacetonitriles to respective carboxylic acids. Nitrilases are generally inducible enzymes, composed of one or two types of subunits of different sizes and numbers. Nitrilase do not show the presence of any cofactor or prosthetic group unlike other nitrile metabolising enzymes. All nitrilases are known to be sulphydryl enzymes wherein elimination of the -SH groups by chemical modification leads to enzyme inactivation. They are reported to have a catalytically essential cysteine residue at or near their active site (Kobayashi et al., Nitrilase from Rhodococcus rhodochrous Ji: sequencing and overexpression of the gene and identification of an essential cysteine residue. J. Biol. Chem. 267: 20746 — 20751, 1992). A possible mechanism for nitrilase-catalysed hydrolysis is a nucleophilic attack by thiol group on carbon atom of nitrile with concomitant protonation of nitrogen to form a tetrahedral thiomidate intermediate. Subsequent steps involved are, attack by two water molecules and protonation of nitrogen atom, which is lost as ammonia. The tetrahedral intermediate formed can break down anomalously to produce amide in some cases instead of normal acid product (Stevenson et al., Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnol. AppI. Biochem. 15: 283 — 302, '1992). While amides are reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous JI was surprisingly found to catalyse hydrolysis of amide to acid and ammonia stoichiometrically. This suggests the existence of a common tetrahedral intermediate in the reaction involving nitriles or amides as substrates (Kobayshi et al., The catalytic mechanism of amidase also involves nitrile hydrolysis. FEBS Left. 439: 325—328, 1998). Objects of the invention The main objective of the present invention is to provide a novel microorganism Pseudomonas fluorescens MTCC 5110 capable of producing nitrilase that preferentially hydrolyses R-stereo-isomer of (RS) —a-hydroxy arene acetonitrile. Another object is to provide a process for hydrolysing ci-hydroxy arene acetonitrile into corresponding acid, wherein R-isomer is obtained in a high enantiomeric excess. Description of Drawings: Fig. 1 is a schematic representation of the reaction of the invention. Statement of Invention: Accordingly, the invention provides a process for hydrolyzing a racemic RS-cihydroxy arene acetonitrile into (R) isomer of the corresponding acid, comprising the steps: (a) Obtaining a supernatant containing nitilase by incubating a substrate culture of Pseudomonas fluorescens bearing accession No. MTCC 5110 in nutrient broth medium at 25-300C for 20-24 hours, (b) hydrolysing ci-hydroxy arene acetonitrile such as herein described with the supernatant obtained in step (a) in aqueous medium, optionally in the presence of a buffer such as hereindescribed at pH of 5 and a temperature of 10 to 450C, and (c) recovering the corresponding (R)-carboxylic acid in high enantiomeric excess. Detailed Description of the invention: Accordingly, the invention provides a process for hydrolysing cx-hydroxy arene acetonitrile into its corresponding acid and producing R-isomer in high enantiomeric excess, the process comprising the steps of: (a) hydrolysing a racemic nitrile with a nitrilase produced by Pseudomonas fluorescens MTCC 5110 in aqueous medium optionally in the presence of a buffer, and (b) recovering a mixture of acids, wherein (R)-isomer is obtained in high enantiomeric excess. The present invention employs a novel microorganism Pseudomonas fluorescens MTCC 5110 which has been isolated from the soil at NIPER, Mohali, India. This organism has been deposited at Microbial Type Culture Collection (MTCC), IMTECH, India on 9th July, 2003 at accession No. 5110. This organism is unique in that it produces nitrilase, which can preferentially hydrolyse the ahydroxy arene acetonitrile as substrate and produce R-isomer in high enantiomeric excess. Such selectivity is very useful, especially in cases where the S-isomer happens to be an unwanted product. The process of the invention may preferably be affected in aqueous medium, with a buffer such as phosphate buffer. Ambient conditions may be employed for hydrolysis such as temperature in the range of 100C to 450C. If desired, the culture may be incubated for 4 to 6 hours and the pH may be adjusted from 5 to 10, preferably at 7.0. At the end of the reaction, the product accumulates in the reaction system and may be recovered by any known method such as concentration, extraction, crystallization, dialysis etc. The arene portion of cx-hydroxy arene acetonitrile may be a phenyl, substituted phenyl, furan, thiophene, pyrrole or other substituted or unsubstituted heterocycles. The substrate may be a nitrile compound such as ahydroxy arene acetonitrile for example mandelonitrile. The concentration of the substrate may be 0.1 to 0.2% (gm/100 ml) bywt, preferably 0.16% bywt. The amount of Pseudomonas fluorescens cells in the medium may be 0.6 to 2 % (gm/I 00 ml) by wt, on dry basis, preferably 2% by wt. Thus, hydrolytic activity of Pseudomonas fluorescens MTCC 5110 assists in the transformation of one of the enantiomers of racemic nitrile in to the corresponding acid. The R-isomer obtained may be in the range of 37-39% with enantioselectivity upto 99%. The invention is now illustrated in detail by the following examples, which are not to be construed as limitations on the scope of the invention. Example 1. Isolation and characterization of Pseudomonas fluorescens MTCC 5110. Various soil samples, obtained from NIPER, Mohali, India sprayed with varying concentration of the RS-mandelonitrile were extensively screened for the isolation of nitrilase producing strains. The screening was carried out by enriching the soil samples in the laboratory condition using minimal salt medium (MSM) which consisted of a few salts and 2-5 mM racemic mandelonitrile. The enriched samples were plated on MSM agar plates and incubated at 300C. The positive isolates were screened for on the basis of growth on MSM agar plate and were further purified by re-streaking individual colonies on MSM agar plates. A number of positive isolates were obtained from various soil samples on MSM agar plates and their production pattern was checked in liquid medium. Nutrient broth and nutrient broth plus isobutyronitirle (used as an inducer), was used as the inoculum generation and production medium respectively. The flasks were incubated at 300C for the production of the desired nitrilase. The nitrilase activity was determined at regular intervals of time using fluorescence method (Banerjee et al; 2003). The process was repeated a number of times to select the most potent nitrilase producer. The selected isolate was found to be Pseudomonas t7uorescens on the basis of morphological and biochemical characteristics listed below in Table 1. Table 1. (Table Removed) Example 1(a): Purification of Pseudomonas fluorescens Different bacterial cultures were isolated by enrichment technique in shake flasks starting with soil samples, which were acclimatized with RS-mandelonitrile. Treated soil samples were collected from different places and screened for isolation of nitrile hydrolysing strains. One gram soil sample was suspended in 5 ml demineralised water to prepare the inoculum. To the minimal salt medium (MSM), containing different nitriles (2 mM) as a sole carbon and nitrogen source, 2% (v/v) inoculum was added and incubated at both 30 C at 200 rpm for 1 to 2 weeks. Growth in the selective medium was checked by streaking samples onto the selective plates, which consisted of MSM, substrate and agar as gelling agent. Positive isolates were selected on the basis of growth on the selective plates, cultures were purified and maintained on nutrient agar plates. Composition of MSM (g/l) Na2HPO4 2 KH2PO4 1 NH4CI 0.4 MgCI2 0.4 Primary screen was designed based on the substrate tolerance of the isolates and was done by checking the growth of different isolates on selective plates of 2, 5,10 and 20 mM RS-mandelonitrile. Example 1(b) Characterization of Pseudomonas fluorescens. Isolation and characterization of selected nitrilase producing microorganism were carried out wherein the bacterial culture was subjected to different tests as per the standard identification protocols. Mandelonitrile hydrolysing organism i.e. P. fluorescens as obtained above is maintained on nutrient agar plates of following composition. Composition of nutrient agar (g/l) Peptic digest of animal tissue 5 NaCI 5 Beef extract 1.5 Yeast extract 1.5 Agar 1.5 The plates were incubated at 300C for 24 h. The culture was maintained by periodic transfer on nutrient plates and stored at 40C. The microorganism was inoculated in nutrient broth of following composition and cultured at 30 C for 12 h and 2% (vlv) inoculum was subsequently transferred to induction medium of the same composition containing isobutyronitrile as the inducer (1 (g/l). Composition of nutrient broth (g/l) Peptic digest of animal tissue 5 NaCI 5 Beef extract 1.5 Yeast extract 1.5 Microbial cells were harvested from the culture broth at intervals of 12, 18, 24, 36 and 48 h to determine the enzyme activity. Cells were washed with a 0.1 M phosphate buffer (pH 7) and suspended in the same buffer solution to prepare a resting cell suspension (200 mg/mI). The enzyme activity was determined using the fluorescence method. The reaction mixture containing 1.75 ml sodium phosphate buffer (0.1 M, pH 7), 200 p1 resting cell suspension (final cell conc. 20 mg/mi), and 50 p.1 mandelonitrile (500 mM) to obtain a final substrate concentration of 12.5 mM and was incubated at 30 C for 20 mm in a water bath. The reaction was stopped by adding 2 ml 0.1 N HCI. In control samples, substrate was added after stopping the reaction with HCI. The reaction mixture was centrifuged at 5000 x g and clear supernatant was used for ammonia estimation. Unknown concentrations of ammonia were determined from a standard curve prepared with NH4CI in a concentration range of 2.5- 1000 pM. Reagents used: 0.2 M phosphate buffer, pH 7.4 0.75 M alcoholic o-phthaldialdehyde solution 0.072 M alcoholic 2-mercaptoethanol solution Working solution was prepared by mixing 4.5 ml of alcoholic o-phthaldialdehyde solution and 4.5 ml of alcoholic 2-mercaptoethanol solution with 91 ml of phohosphate buffer (0.2 M, pH 7.2). To 0.1 ml of the reaction supernatant, 3.0 ml of working solution was added, thoroughly mixed and kept for 20 mm at room temperature. The fluorescence intensity was recorded at Xes 412 nm and Xem 467 nm using a spectrofluorimeter (Perkin Elmer, model LS5OB). One unit of nitrile hydrolyzing activity is defined as the amount of enzyme capable of releasing 1 pmole of ammonia under standard conditions of assay. Exam pIe 2 To determine the yield and enantiomeric excess (ee) of mandelic acid RP-HPLC and chiral HPLC were performed respectively. Cells of the Pseudomonas fluorescens MTCC 5110 were harvested at the time of maximum enzyme accumulation and washed with 0.1 M phosphate buffer (pH 7) as discussed above and suspended in the same buffer to obtain a final cell concentration of 20 mg/mI. To this cell suspension, mandelonitrile was added to a final concentration of 12.5 mM. The reaction mixture was incubated at 30 C with constant shaking (200 rpm) up to 18 h with intermittent withdrawal of samples at 0.5, 1, 2, 3, 4, 5, 6, and 12 h. The reaction mixture was centrifuged at 5000 x g and pH of the clear supernatant was brought down to 1.5 using 6 N HOl. After adjusting the pH, the supernatant was extracted with equal volume of ether and the extracted material was concentrated under reduced pressure and suspended in 2 ml methanol (enzyme and substrate blanks were also processed in similar way). Yield was determined from a standard curve prepared by using RS-mandelic acid in a concentration range of 50-1 000 pg/mI. Using 10 mM phosphate buffer (pH 4.8) and methanol (65:35, v/v) as mobile phase at a flow rate of 0.8 mI/mm the reaction components were resolved on a LiChroCART® RP-18 column (250 x 4 mm, 5 p.m) (MERCK, Germany). The retention time for mandelic acid, mandelamide and mandelonitrile were 2.6, 4.2 and 18.3 mm respectively. Absorbance of the reaction components was measured at 254 nm. The optical purity of mandelic acid was determined by direct analysis of the enantiomer on a JHIRALCEL-OD-H column (250 x 0.46 mm, 5 p.m) (DAICEL Chemical Industries, U.S.A.). The pH of the reaction mixture was adjusted to 8.4 with 2 N NaOH after which unreacted nitrile was extracted with equal volume of ether. After adjusting pH of the aqueous layer to 1.5 with 6 N HCI, the desired product was extracted again with equal volume of ether. The extract obtained was concentrated under reduced pressure and suspended in mobile phase to obtain a final concentration of 100 p.M and analysed on the chiral column at a flow rate of 0.5 mI/mm using hexane, isopropyl alcohol and tri-fluoro acetic acid (90: 10: 0.2 v/v) as mobile phase. The retention times for S and R enantiomer were 15.5 and 17.5 mm respectively. Absorbance of the desired product was measured at 254 nm. The main advantages of the present invention are: 1. The nitrilase-mediated hydrolysis of ci-hydroxy arene acetonitrile is always advantageous over synthetic chemical route, since the reaction can be performed at ambient temperature and pressure. 2. The time period of microbial hydrolysis of ci-hydroxy arene acetonitrile to corresponding acid is 6 h. 3. The yield and enantioselectivity of the product (acid) is also very high. We claim: 1. A process for hydrolyzing a racemic RS-cz-hydroxy arene acetonitrile into (R) isomer of the corresponding acid, comprising the steps: (a) Obtaining a supernatant containing nitilase by incubating a substrate culture of Pseudomonas fluorescens bearing accession No. MTCC 5110 in nutrient broth medium at 25-300C for 20-24 hours, (b) hydrolysing α-hydroxy arene acetonitrile such as herein described with the supernatant obtained in step (a) in aqueous medium, optionally in the presence of a buffer such as hereindescribed at pH of 5 and a temperature of 10 to 45c)C, and (c) recovering the corresponding (R)-carboxylic acid in high enantiomeric excess. 2. A process as claimed in claim 1 wherein arene portion of ci-hydroxy arene acetonitrile is a phenyl, substituted phenyl, furan, thiophene, pyrrole or other substituted or u nsu bstituted heterocycles. 3. A process as claimed in claim 1 wherein the α-hydroxy arene acetonitrile is mandelonitrile. 4. A process as claimed in claim 1 wherein the concentration of the substrate culture is 0.1 to 0.2% by wt. 6. A process as claimed in claim I wherein the preferred amount of Pseudomonas fluorescens MTCC 5110 cells is 0.6 to 2% by wt. 7. A process as claimed in claim 6 wherein the amount of Pseudomonas fluorescens MTCC 5110 cells is 2% by wt. 8. A process as claimed in claim I wherein the buffer is phosphate buffer. 9. A process as claimed in claim 1 wherein the reaction is effected at 30 0C. 10. A process for preparing R-α-hydroxy arene acetic acid substantiaily as hereindescribed with reference to the examples and drawings.

Full Text

Field of the Invention
The present invention relates to a process for hydrolyzing a racemic RS-cihydroxy arene acetonitrile into (R) isomer of the corresponding acid. The process of the invention is effected using a nitrilase produced by a novel mesophilic microorganism Pseudomonas fluorescens MTCC 5110.

Background of the invention

R-α-hydroxy arene acetic acids find numerous applications as chiral synthons and resolving agents. For example there are numerous uses of R-(-)-mandelic acid, a hydroxy acid in this class. (R)-(-)-mandelic acid is a useful resolving agent (Kinbara et. al., Design of resolving reagents: p-substituted mandelic acids as resolving reagents for 1-arylalkylamines. Tetrahedron: Asymmetry. 7:1539-1542. 1996; Drabowics et al., New procedures for the resolution of chiral tedbutylphenylphosphine oxide and some of its reactions. Tetrahedron: Asymmetry. 10: 2757-2763. 1999; Han et al.; First practical synthesis of enantiomerically pure (R)- and (S)- desmethyl sibutramine (DMS) and unambiguous determination of their absolute configuration by single-crystal X-ray analysis. Tetrahedron:
Asymmetry. 13: 107-109. 2002) and a chiral synthon for the production of various pharmaceuticals such as semi-synthetic penicillin (Furlenmeier et al., 6-Acyl derivatives of aminopenicillanic acid. U.S. Patent 3957758. 1976), cephalosporins (Terreni et al., Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of cephamandole from cephalosporin C. Bioorg. Med. Chem. Lett. 11: 2429-2432. 2001), anti obesity agents (Mills et al. Phenethanolamines, compositions containing the same and method for effecting weight control. U.S. Patent 4391826. 1983), antiarrhythmic agents (Aav et al., Preparation of highly enantiopure stereoisomers of 1 -(2,6-dimethylphenoxy)-2-aminopropane (Mexiletine). Tetrahedron: Asymmetry.
10: 3033-3038. 1999), anti-tumour agents (Kotora and Negishi, Efficient and diastereoselective synthesis of (+)-goniobutenolide-A via palladium-catalysed ene-yne cross coupling-lactonization cascade. Tetrahedron Lett. 37: 9041-9042. 1996; Surivet et al.; Easy access to an enantiopure precursor of (+)-goniodiol.

T-etrahedron: Asymmetry. 7: 3305-3308. 1999; Surivet et al., 1996) and the molecule itself also exhibits anti fungal properties (Kope et al., pHydroxybenzoylformic acid and R-(-)--p-hydroxymandelic acid, two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolilithus arhizus. Canadian J. Microbiol. 37: 258-264. 1991).
Nitrilases (nitrile metabolising enzymes) catalyse the hydrolysis of nitriles to corresponding acid and ammonia. Based on their substrate specificity, microbial nitrilases are differentiated into three different categories, namely aliphatic nitrilases, aromatic nitrilases and arylacetonitrilases. Some nitrilases hydrolyze aromatic or heterocyclic nitriles to corresponding acids and ammonia. Others are known to hydrolyze preferentially either aliphatic nitriles or acylacetonitriles to respective carboxylic acids.

Nitrilases are generally inducible enzymes, composed of one or two types of subunits of different sizes and numbers. Nitrilase do not show the presence of any cofactor or prosthetic group unlike other nitrile metabolising enzymes. All nitrilases are known to be sulphydryl enzymes wherein elimination of the -SH groups by chemical modification leads to enzyme inactivation. They are reported to have a catalytically essential cysteine residue at or near their active site (Kobayashi et al., Nitrilase from Rhodococcus rhodochrous Ji: sequencing and overexpression of the gene and identification of an essential cysteine residue. J. Biol. Chem. 267: 20746 — 20751, 1992).

A possible mechanism for nitrilase-catalysed hydrolysis is a nucleophilic attack by thiol group on carbon atom of nitrile with concomitant protonation of nitrogen to form a tetrahedral thiomidate intermediate. Subsequent steps involved are, attack by two water molecules and protonation of nitrogen atom, which is lost as ammonia. The tetrahedral intermediate formed can break down anomalously to produce amide in some cases instead of normal acid product (Stevenson et al., Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnol. AppI. Biochem. 15: 283 — 302, '1992). While amides are reported to
be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous JI was surprisingly found to catalyse hydrolysis of amide to acid and ammonia stoichiometrically. This suggests the existence of a common tetrahedral intermediate in the reaction involving nitriles or amides as substrates (Kobayshi et al., The catalytic mechanism of amidase also involves nitrile hydrolysis. FEBS Left. 439: 325—328, 1998).

Objects of the invention

The main objective of the present invention is to provide a novel microorganism
Pseudomonas fluorescens MTCC 5110 capable of producing nitrilase that
preferentially hydrolyses R-stereo-isomer of (RS) —a-hydroxy arene acetonitrile.

Another object is to provide a process for hydrolysing ci-hydroxy arene acetonitrile into corresponding acid, wherein R-isomer is obtained in a high enantiomeric excess.

Description of Drawings:

Fig. 1 is a schematic representation of the reaction of the invention.
Statement of Invention:

Accordingly, the invention provides a process for hydrolyzing a racemic RS-cihydroxy arene acetonitrile into (R) isomer of the corresponding acid, comprising the steps:
(a) Obtaining a supernatant containing nitilase by incubating a substrate culture of Pseudomonas fluorescens bearing accession No. MTCC 5110 in nutrient broth medium at 25-300C for 20-24 hours,
(b) hydrolysing ci-hydroxy arene acetonitrile such as herein described with the supernatant obtained in step (a) in aqueous medium, optionally in the presence of a buffer such as hereindescribed at pH of 5 and a temperature of 10 to 450C, and
(c) recovering the corresponding (R)-carboxylic acid in high enantiomeric excess.

Detailed Description of the invention:
Accordingly, the invention provides a process for hydrolysing cx-hydroxy arene acetonitrile into its corresponding acid and producing R-isomer in high enantiomeric excess, the process comprising the steps of:
(a) hydrolysing a racemic nitrile with a nitrilase produced by Pseudomonas fluorescens MTCC 5110 in aqueous medium optionally in the presence of a buffer, and
(b) recovering a mixture of acids, wherein (R)-isomer is obtained in high enantiomeric excess.

The present invention employs a novel microorganism Pseudomonas fluorescens MTCC 5110 which has been isolated from the soil at NIPER, Mohali, India. This organism has been deposited at Microbial Type Culture Collection (MTCC), IMTECH, India on 9th July, 2003 at accession No. 5110. This organism is unique in that it produces nitrilase, which can preferentially hydrolyse the ahydroxy arene acetonitrile as substrate and produce R-isomer in high enantiomeric excess. Such selectivity is very useful, especially in cases where the S-isomer happens to be an unwanted product.

The process of the invention may preferably be affected in aqueous medium, with a buffer such as phosphate buffer. Ambient conditions may be employed for hydrolysis such as temperature in the range of 100C to 450C. If desired, the culture may be incubated for 4 to 6 hours and the pH may be adjusted from 5 to 10, preferably at 7.0.

At the end of the reaction, the product accumulates in the reaction system and may be recovered by any known method such as concentration, extraction, crystallization, dialysis etc. The arene portion of cx-hydroxy arene acetonitrile may be a phenyl, substituted phenyl, furan, thiophene, pyrrole or other substituted or unsubstituted heterocycles. The substrate may be a nitrile compound such as ahydroxy arene acetonitrile for example mandelonitrile. The concentration of the substrate may be 0.1 to 0.2% (gm/100 ml) bywt, preferably 0.16% bywt.
The amount of Pseudomonas fluorescens cells in the medium may be 0.6 to 2 % (gm/I 00 ml) by wt, on dry basis, preferably 2% by wt.

Thus, hydrolytic activity of Pseudomonas fluorescens MTCC 5110 assists in the transformation of one of the enantiomers of racemic nitrile in to the corresponding acid. The R-isomer obtained may be in the range of 37-39% with enantioselectivity upto 99%.

The invention is now illustrated in detail by the following examples, which are not to be construed as limitations on the scope of the invention.

Example 1. Isolation and characterization of Pseudomonas fluorescens MTCC 5110.

Various soil samples, obtained from NIPER, Mohali, India sprayed with varying concentration of the RS-mandelonitrile were extensively screened for the isolation of nitrilase producing strains. The screening was carried out by enriching the soil samples in the laboratory condition using minimal salt medium (MSM) which consisted of a few salts and 2-5 mM racemic mandelonitrile. The enriched samples were plated on MSM agar plates and incubated at 300C. The positive isolates were screened for on the basis of growth on MSM agar plate and were further purified by re-streaking individual colonies on MSM agar plates. A number of positive isolates were obtained from various soil samples on MSM agar plates and their production pattern was checked in liquid medium. Nutrient broth and nutrient broth plus isobutyronitirle (used as an inducer), was used as the inoculum generation and production medium respectively. The flasks were incubated at 300C for the production of the desired nitrilase. The nitrilase activity was determined at regular intervals of time using fluorescence method (Banerjee et al; 2003). The process was repeated a number of times to select the most potent nitrilase producer. The selected isolate was found to be Pseudomonas
t7uorescens on the basis of morphological and biochemical characteristics listed below in Table 1.

Table 1.
(Table Removed)

Example 1(a): Purification of Pseudomonas fluorescens

Different bacterial cultures were isolated by enrichment technique in shake flasks starting with soil samples, which were acclimatized with RS-mandelonitrile.
Treated soil samples were collected from different places and screened for isolation of nitrile hydrolysing strains. One gram soil sample was suspended in 5 ml demineralised water to prepare the inoculum. To the minimal salt medium (MSM), containing different nitriles (2 mM) as a sole carbon and nitrogen source, 2% (v/v) inoculum was added and incubated at both 30 C at 200 rpm for 1 to 2 weeks. Growth in the selective medium was checked by streaking samples onto the selective plates, which consisted of MSM, substrate and agar as gelling agent. Positive isolates were selected on the basis of growth on the selective plates, cultures were purified and maintained on nutrient agar plates.

Composition of MSM (g/l)
Na2HPO4 2
KH2PO4 1
NH4CI 0.4
MgCI2 0.4
Primary screen was designed based on the substrate tolerance of the isolates and was done by checking the growth of different isolates on selective plates of 2, 5,10 and 20 mM RS-mandelonitrile.

Example 1(b) Characterization of Pseudomonas fluorescens.

Isolation and characterization of selected nitrilase producing microorganism were carried out wherein the bacterial culture was subjected to different tests as per the standard identification protocols.
Mandelonitrile hydrolysing organism i.e. P. fluorescens as obtained above is maintained on nutrient agar plates of following composition.
Composition of nutrient agar (g/l)
Peptic digest of animal tissue 5
NaCI 5
Beef extract 1.5
Yeast extract 1.5
Agar 1.5
The plates were incubated at 300C for 24 h. The culture was maintained by periodic transfer on nutrient plates and stored at 40C. The microorganism was inoculated in nutrient broth of following composition and cultured at 30 C for 12 h and 2% (vlv) inoculum was subsequently transferred to induction medium of the same composition containing isobutyronitrile as the inducer (1 (g/l).
Composition of nutrient broth (g/l)
Peptic digest of animal tissue 5
NaCI 5
Beef extract 1.5
Yeast extract 1.5
Microbial cells were harvested from the culture broth at intervals of 12, 18, 24, 36 and 48 h to determine the enzyme activity. Cells were washed with a 0.1 M phosphate buffer (pH 7) and suspended in the same buffer solution to prepare a resting cell suspension (200 mg/mI). The enzyme activity was determined using the fluorescence method. The reaction mixture containing 1.75 ml sodium phosphate buffer (0.1 M, pH 7), 200 p1 resting cell suspension (final cell conc. 20 mg/mi), and 50 p.1 mandelonitrile (500 mM) to obtain a final substrate concentration of 12.5 mM and was incubated at 30 C for 20 mm in a water bath. The reaction was stopped by adding 2 ml 0.1 N HCI. In control samples, substrate was added after stopping the reaction with HCI. The reaction mixture was centrifuged at 5000 x g and clear supernatant was used for ammonia estimation. Unknown concentrations of ammonia were determined from a standard curve prepared with NH4CI in a concentration range of 2.5- 1000 pM.
Reagents used:

0.2 M phosphate buffer, pH 7.4

0.75 M alcoholic o-phthaldialdehyde solution

0.072 M alcoholic 2-mercaptoethanol solution
Working solution was prepared by mixing 4.5 ml of alcoholic o-phthaldialdehyde solution and 4.5 ml of alcoholic 2-mercaptoethanol solution with 91 ml of
phohosphate buffer (0.2 M, pH 7.2). To 0.1 ml of the reaction supernatant, 3.0 ml of working solution was added, thoroughly mixed and kept for 20 mm at room temperature. The fluorescence intensity was recorded at
Xes 412 nm and Xem 467 nm using a spectrofluorimeter (Perkin Elmer, model LS5OB). One unit of nitrile hydrolyzing activity is defined as the amount of enzyme capable of releasing 1 pmole of ammonia under standard conditions of assay.

Exam pIe 2

To determine the yield and enantiomeric excess (ee) of mandelic acid RP-HPLC and chiral HPLC were performed respectively. Cells of the Pseudomonas fluorescens MTCC 5110 were harvested at the time of maximum enzyme accumulation and washed with 0.1 M phosphate buffer (pH 7) as discussed above and suspended in the same buffer to obtain a final cell concentration of 20 mg/mI. To this cell suspension, mandelonitrile was added to a final concentration of 12.5 mM. The reaction mixture was incubated at 30 C with constant shaking (200 rpm) up to 18 h with intermittent withdrawal of samples at 0.5, 1, 2, 3, 4, 5, 6, and 12 h. The reaction mixture was centrifuged at 5000 x g and pH of the clear supernatant was brought down to 1.5 using 6 N HOl. After adjusting the pH, the supernatant was extracted with equal volume of ether and the extracted material was concentrated under reduced pressure and suspended in 2 ml methanol (enzyme and substrate blanks were also processed in similar way).
Yield was determined from a standard curve prepared by using RS-mandelic acid in a concentration range of 50-1 000 pg/mI. Using 10 mM phosphate buffer (pH 4.8) and methanol (65:35, v/v) as mobile phase at a flow rate of 0.8 mI/mm the reaction components were resolved on a LiChroCART® RP-18 column (250 x 4 mm, 5 p.m) (MERCK, Germany). The retention time for mandelic acid, mandelamide and mandelonitrile were 2.6, 4.2 and 18.3 mm respectively. Absorbance of the reaction components was measured at 254 nm. The optical purity of mandelic acid was determined by direct analysis of the enantiomer on a
JHIRALCEL-OD-H column (250 x 0.46 mm, 5 p.m) (DAICEL Chemical Industries, U.S.A.).
The pH of the reaction mixture was adjusted to 8.4 with 2 N NaOH after which unreacted nitrile was extracted with equal volume of ether. After adjusting pH of the aqueous layer to 1.5 with 6 N HCI, the desired product was extracted again with equal volume of ether. The extract obtained was concentrated under reduced pressure and suspended in mobile phase to obtain a final concentration of 100 p.M and analysed on the chiral column at a flow rate of 0.5 mI/mm using hexane, isopropyl alcohol and tri-fluoro acetic acid (90: 10: 0.2 v/v) as mobile phase. The retention times for S and R enantiomer were 15.5 and 17.5 mm respectively. Absorbance of the desired product was measured at 254 nm.

The main advantages of the present invention are:
1. The nitrilase-mediated hydrolysis of ci-hydroxy arene acetonitrile is always advantageous over synthetic chemical route, since the reaction can be performed at ambient temperature and pressure.
2. The time period of microbial hydrolysis of ci-hydroxy arene acetonitrile to corresponding acid is 6 h.
3. The yield and enantioselectivity of the product (acid) is also very high.

We claim:

1. A process for hydrolyzing a racemic RS-cz-hydroxy arene acetonitrile into (R) isomer of the corresponding acid, comprising the steps:
(a) Obtaining a supernatant containing nitilase by incubating a substrate culture of Pseudomonas fluorescens bearing accession No. MTCC 5110 in nutrient broth medium at 25-300C for 20-24 hours,
(b) hydrolysing α-hydroxy arene acetonitrile such as herein described with the supernatant obtained in step (a) in aqueous medium, optionally in the presence of a buffer such as hereindescribed at pH of 5 and a temperature of 10 to 45c)C, and
(c) recovering the corresponding (R)-carboxylic acid in high enantiomeric excess.

2. A process as claimed in claim 1 wherein arene portion of ci-hydroxy arene acetonitrile is a phenyl, substituted phenyl, furan, thiophene, pyrrole or other substituted or u nsu bstituted heterocycles.

3. A process as claimed in claim 1 wherein the α-hydroxy arene acetonitrile is mandelonitrile.

4. A process as claimed in claim 1 wherein the concentration of the substrate culture is 0.1 to 0.2% by wt.

6. A process as claimed in claim I wherein the preferred amount of Pseudomonas fluorescens MTCC 5110 cells is 0.6 to 2% by wt.

7. A process as claimed in claim 6 wherein the amount of Pseudomonas fluorescens MTCC 5110 cells is 2% by wt.

8. A process as claimed in claim I wherein the buffer is phosphate buffer.

9. A process as claimed in claim 1 wherein the reaction is effected at 30 0C.

10. A process for preparing R-α-hydroxy arene acetic acid substantiaily as hereindescribed with reference to the examples and drawings.

Documents:

975-del-2003-abstract.pdf

975-del-2003-claims.pdf

975-del-2003-complete specification (granted).pdf

975-del-2003-correspondence-others.pdf

975-del-2003-description (complete).pdf

975-del-2003-drawings.pdf

975-del-2003-form-1.pdf

975-del-2003-form-19.pdf

975-del-2003-form-2.pdf

975-del-2003-form-26.pdf

975-del-2003-form-3.pdf

975-del-2003-form-5.pdf

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Patent Number

197592

Indian Patent Application Number

975/DEL/2003

PG Journal Number

29/2008

Publication Date

26-Sep-2008

Grant Date

02-Mar-2007

Date of Filing

07-Aug-2003

Name of Patentee

NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH (NIPER)

Applicant Address

SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ANIRBAN BANERJEE	SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.
2	PRAVEEN KAUL	SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.
3	ROHIT SHARMA	SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.
4	HARMANDER PAL SINGH CHAWLA	SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.
5	CHAMAN LAL KAUL	SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.
6	UTTAM CHAND BANERJEE	SECTOR 67, PHASE X, SAS NAGAR, MOHALI, DISTRICT ROPER, PANJAB 160 062, INDIA.

PCT International Classification Number

C07C 250/34

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA