TECHNICAL FIELD

This invention relates to a process for preparing an enantiomer of an acid by enantioselective hydrolysis of a mixture of the corresponding R and S amide in the presence of an enantioselective amidase.

It also relates to a microorganism that produces amidase and to immobilized amidase for use in said process.

BACKGROUND ART

Many agrochemical intermediates and pharmaceutical products of the general formula I described below are currently marketed and used as racemic or diastereomeric mixtures. In many cases, the physiological effect specifically derives from only one enantiomer/diastereomer, whereas, the other enantiomer/diastereomers are inactive or even harmful. Chemical or enzymatic techniques for enan- tiodifferentiation are becoming increasingly important tools for production of chemicals of high optical purity. Enzymatic production of optically active acids such as 2-arylalkanoic acids by enantioselective hydrolysis of the corresponding racemic amides in the presence of a micro¬ organism or an enzyme is known from European patent applica¬ tion having publication No. 326,482. The microorganisms used belong to the genera Corynebacterium and Brevibacterium. The process was performed batchwise without organic solvent, and the enzymaticaliy active material was discarded after being used once. Data in the examples of this European patent ap¬ plication having publication No. 326,482 indicate that the conversion of S amide into acid ranged from about 40 to 65%, i.e. 35 - 60% of the S amide remained unconverted. The enan¬ tiomer excess of the S form in the acid produced was 92 -

97%. The microorganisms were cultivated on a fermentation substrate that included N-methylacetamide.

European patent application having publication No. 356,912 discloses microorganisms from the genera of Pseudo- monas. Fusarium, Rhodococcus, Brevibacterium , Micrococcus. Bacteridium and Bacillus capable of converting racemic ali¬ phatic 2-substituted nitriles into the optically active ali¬ phatic 2-substituted carboxylic acid. It is stated that the microorganisms are also active on aromatic 2-substituted ni- triles, but no supporting data are given. The enantiomer ex¬ cess of S acid resulting from the corresponding nitrile after completion of the reaction was maximum 84%. In the case of using Rhodococcus for converting a nitrile into an acid, the enantiomer excess was 35%. The fermentation substrate in- eluded nitrile.

European patent application having publication No. 348,901 relates to a process for producing an optically ac¬ tive α-substituted organic acid by treating a racemic α-sub- stituted nitrile or amide with a microorganism selected among the genera Alcalicrenes. Pseudomonas. Rhodopseudomonas, Corynebacterium, Acinetobacter. Bacillus, Mycobacterium, Rhodococcus and Candida. This 348,901 publication does not disclose any microorganism employed in the process of this invention. It is the object of this invention to provide a process with improved enantioselective conversion of amides, for example 2-arylalkanoic amides.

A work describing an examination of 809 strains isolated from 330 different soil samples to produce an S acid from the corresponding nitrile and amide appears in Appl.En¬ viron.Microbiol. 56 (1990) , 3125 et seq. , which was published after the priority date of this patent application.

It also is the object of this invention to provide a process by which a mixture of R and s amides being enantiomers is converted to the acid corresponding to one of the two amide enantiomers in a high yield and high purity.

It is also the object of this invention to provide a method of producing enantioselective amidase without the need for nitrile or amide as inducer.

STATEMENT OF THIS INVENTION

This invention provides a process for producing an enantiomer of an acid by enantioselective hydrolysis of a mixture of the corresponding S and R amide in the presence of an enzymaticaliy active biological material with enantio¬ selective amidase activity. By this process, two sorts of yields are important. Briefly, the two sorts of yields are, on one hand, the degree of conversion and, on the other hand, the purity of the resulting acid. The degree of conversion is the degree in which one of the two enantiomer amides is converted into the acid. The purity of the resulting acids has herein been designated enantiomer excess and is defined below. As mentioned below, this invention is superior as far as at least one of these two sorts of yields is concerned.

We have found that, surprisingly, it is possible to obtain a very high degree of conversion by the process of this invention. Under certain reaction conditions, it is even possible to obtain a degree of conversion of nearly 100%.

In addition and, surprisingly, a very high enantiomer excess is obtainable by the process of this invention. Under certain reaction conditions, it is even possible to obtain an enantiomer excess of nearly 100%.

Hence, after completion of the process of this invention and removal of the amide(s) , the resulting R or S acid has a very high purity, in some cases a purity of almost 100%. Thus, from a starting mixture of an R and S amide being enantiomers, optionally a racemic mixture of R and S amides, it is possible, by the process of this invention, either to produce directly a mixture of S acid and unconverted R amide essentially devoid of R acid and S amide or to produce a mixture of R acid and unconverted S amide essentially devoid

of S acid and R amide. This allows for simplified separation and higher yield.

Examples of suitable acids which can be prepared by the process of this invention are compounds of the general formula I wherein X represents a phenyl group or a naphthyl group which groups optionally are substituted with halogen, alkyl, alkoxy or benzoyl, R ¹ represents hydroxy, amino or alkyl, and R ² represents hydrogen or alkyl.

Examples of amides which can be used as starting material in the process of this invention are compounds of the general formula II

X-CR ¹ R ² -C0NH ₂ (II) wherein R ¹ , R ² and X each are as defined above.

Obviously, both the phenyl and naphthyl groups mentioned above may be substituted. The naphthyl group may be an α or β naphthyl group. Halogen is, preferably, chloro and fluoro. The alkyl and alkoxy groups are preferably lower alkyl and lower alkoxy groups. Hereinafter the term lower indicates that the group in question contains not more than 10 carbon atoms, preferably not more than 4 carbon atoms.

The amide used by the process of this invention may optionally have been produced in situ. As the enzymaticaliy active biological material used in the process of this invention has an enantioselective amidase activity, this material may herein be designated an amidase.

In one aspect of this invention, .the process is characterized in that said biological material is immobiliz¬ ed. In a second aspect, the process is characterized in that the hydrolysis is carried out in the presence of an organic solvent.

The enantioselectivity of the amidase used in the process of this invention may be determined by hydrolysis of a racemic amide, for example, an acid of formula II, for example, racemic 2-(4-chlorophenyl)-3-methylbutyramide.

The degree of conversion obtainable by the process of this invention is preferably above about 65%, more preferred above about 90%, even more preferred above about 95%, most preferred above about 99%. The preferred high degrees of conversion are obtainable by using especially preferred reaction conditions which are further illustrated in the examples below.

The enantiomer excess of the resulting enantiomer acid is preferably above about 85%, more preferred above about 90%, even more preferred above 95%. Using especially preferred reaction conditions which are further illustrated in the examples below, the enantiomer excess of the resulting enantiomer acid may be above 99%, more preferred above about 99.5%, most preferred above about 99.9%. The enantiomer excess is calculated from the concentration of R and S forms using the following equation:

([S] - [ ])/([S] + [RJ) x 100% wherein [R] and [S] is the concentration of the R and S form, respectively. This invention also provides a biologically pure culture of an enzymaticaliy active microorganism with enan¬ tioselective amidase activity. In fact, this invention describes a Rhodococcus strain that produces the amidase activity constitutively, i.e. without the need for an inducer such as an amide.

Further, this invention provides immobilized, en¬ zymaticaliy active biological material with enantioselective amidase activity for use in the above process. Furthermore, this invention also provides biological material having enan- tioselective amidase activity, characterized by being derived from a strain of Rhodococcus. and a method of preparing biological material having enantioselective amidase activity, characterized by comprising cultivation of an amidase produc¬ ing Rhodococcus strain in a medium that does not include nitrile or amide (unsubstituted or N-substituted) .

In addition, this invention also provides biologically pure cultures of enzymaticaliy active microorganisms with enantioselective amidase activity and a

method of preparing such biological material. Such microorganisms may be obtained from strains of Serratia, Moraxella or Pseudomonas.

DETAILED DESCRIPTION OF THIS INVENTION

The process of this invention is performed in a manner known per se by subjecting the mixture of the R and S amide being enantiomers to the action of the biological material in question in a pertinent reaction medium. The skilled art worker is able to determine convenient conditions for carrying out the process for this invention, due care being taken to this specification, including the examples below.

The biological material used in the process of this invention is prepared in a manner known per se. The biological material having amidase activity is preferably obtained in a manner known per se from a constitu¬ tive amidase producing strains of Rhodococcus. especially Rhodococcus erythropolis DP-10. This microbial strain was de¬ posited under the terms of the Budapest Treaty at DSM (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Braunschweig, Germany) on March 23, 1990 under the accession number DSM 5910. Other Rhodococcus erythropolis strains deposited on 21 February 1991 as described above include DSM 6374, DSM 6375 and DSM 6378 (Rhodococcus erythropolis Nos. DP-11, DP-26 and DP-25, respectively) .

Strain DSM 5910 is capable of hydrolyzing a wide range of aliphatic and aromatic amides into their correspond¬ ing acids. The strain, however, hydrolyses both the D and L form of amino acid amides such as phenylglycine amide and a number of aliphatic amino acid amides. It is highly surpris¬ ing that this strain (and other strains belonging to the genus Rhodococcus) possesses the ability to perform the enan¬ tioselective hydrolysis of racemic amides according to this invention. Strain DSM 5910 is advantageous in being constitu-

tive for production of amidase, i.e. no inducers are needed for maximum expression of amidase activity.

The Rhodococcus amidase preferably has an enantio- selectivity above 85%, more preferably above 90%, even more preferably above 95%, and most preferably above 99%.

The biological material having amidase activity is also obtainable in a mammer known per se from amidase producing strains of Serratia. Moraxella and Pseudomonas. These strains were deposited under the terms of the Budapest treaty at NRRL (Northern Regional Research Laboratories, Peoria, II, USA) and with accession numbers noted below. Pseudomonas putida NRRL-B-18669 and Moraxella sp. NRRL-B- 18671 were deposited on 8 July 1990 and Pseudomonas putida NRRL-B-18820. Pseudomonas sp. NRRL-B-18819 and Serratia liquefaciens NRRL-B-18821 were deposited on 9 May 1991.

According to this invention, the process is generally conducted in homogeneous or heterogeneous aqueous or aqueous-organic medium at temperature and pH conditions determined as a function of the immobilized cells, whole cells or cell extract from the microorganism and the mixture of amide and the resulting acid. The enzymatic reaction may be carried out using immobilized cells under batch or conti¬ nuous conditions.

The starting material for the process of this in- vention may be a previously prepared amide; this can be prepared, for example, by chemical or enzymatic hydrolysis of the corresponding nitrile. Alternatively, the amide may be produced JLn situ, for example by enzymatic hydrolysis of the corresponding nitrile. After completion of the reaction, the enantiomer acid can be recovered from the reaction mixture and purified by conventional methods. If a mixture of R and S amide (for example a racemic mixture) is used as starting material, the process results in a mixture of S acid and R amide or a mix- ture of R acid and S amide. After separating the amide from the acid, the amide can be racemized by known methods into racemic amide which can be recycled and hydrolyzed as already described. Racemization can be performed by refluxing the

amide with an anion exchange resin that comprises quaternary ammonium functionality, for example, Amberlite IRA-400 in OH form in toluene or an other non-aqueous solvent.

The enzymaticaliy active biological material to be used in this invention may, for example, be whole cells, cell paste, homogenized cells or a crude or purified enzyme solu¬ tion. Immobilization may be carried out by known methods, such as cross-linking, for example, with glutaraldehyde or polyazetidine according to US patent specification No. 4,892,825.

The immobilized material is preferably used in a continuous process, either in a fixed-bed column or a stirred tank reactor. If organic solvent is used, it is particularly preferred to use a stirred tank reactor, where the immobi- lized material and the organic solvent phase are retained by a hydrophilic membrane (cf. Example 8, Fig. 1)

Organic solvent to be used in the process of this invention may be water-miscible (for example, dimethyl sul- foxide) or water-immiscible (for example, toluene or octane) . The amount of solvent is generally 2 - 20% by weight of the reaction system.

The process of this invention may be used to pro¬ duce 2-arylalkanoic acid of the general formula I.

The process of this invention is particularly suited for production of acids where the aryl group X is phe¬ nyl, p-chlorαphenyl, p-isobutylphenyl, 3-benzoylphenyl, β- naphthyl or 6-methoxy-2-naphthyl, and where the group desig¬ nated R is hydroxy, methyl, ethyl or isopropyl.

Some specific examples of acids that may be pro- duced according to this invention are 2-(4-chlorophenyl)-3- methylbutyric acid (hereinafter designated CPIA) , (6-methoxy- 2-naphthyl) ydroxypropionic acid, 2-(6-methoxy-2-naphthyl)- propionic acid, 2-(4-isobutylphenyl)propionic acid, 2-phenyl- 2-hydroxypropionic acid and 2-(3-benzoylphenyl)propionic acid.

ABBREVIATIONS

CPIAm is 2-(4-chlorophenyl)-3-methylbutyramide, CPIA is 2-(4-chlorophenyl)-3-methylbutyric acid, IBAm is 2- (4-isobutylphenyl)propionamide, IBAC is 2-(4-isobutylphenyl)- propionic acid (ibuprofen) , NPAm is 2-(6-methoxy-2-naphthyl)- propionamide, NPAC is 2-(6-methoxy-2-naphthyl)propionic acid (naproxen) , ATAm is 2-phenyl-2-hydroxypropionamide, ATAC is 2-phenyl-2-hydroxypropionic acid and HPLC is High-Performance Liquid Chromatography.

ANALYTICAL PROCEDURE

In the examples that follow, the amide and acid products listed above were measured by reverse phase high performance chromatography. A Zorbax C18 column employing mobile phases of 70 - 75% methanol and 25 - 30% H ₂ 0 acidified with 0.1% H ₃ P0 ₄ or 67% acetonitrile and 33% H ₂ 0 acidified with 0.1% H ₃ P0 ₄ was used. Chromatographic identity and quantitation of acid products were determined by comparison with authentic standards.

Chiral HPLC for the separation of CPIAm/CPIA, NPAm/NPAC and IBAm/IBAC enantiomers was carried out with an ^α l-AGP column obtained from Chromtech (Sweden) . The mobile phases for separation of various enantiomers is summarized below.

Chiral HPLC Separation of Amide and Acid Enantiomers

Enantiomers Mobile Phase

CPIAm, CPIA 95% 0.01 M Phosphate Buffer

(pH 6.0) :5% Ethanol

NPAm, NPAC 95% 0.04 M Phosphate Buffer

(pH 5.6) :5% Etanol IBAm, IBAC 96% 0.02 M Phosphate Buffer

(pH 5.2) :4% Ethanol

Chiral HPLC for the separation of ATA and ATAC enan¬ tiomers was carried out with a Resolvosil BSA-7 column (Alltech, Inc.). The mobile phase employed for ATAm/ATAC enantiomer composition was 0.01 M phosphate buffer (pH 6.0). Enantiomeric composition, purity and chromatographic identity of the above amide and acids were determined by comparison with authentic standard enantiomers or racemic mixtures.

PRODUCTION EXAMPLE

Chemical hydrolysis of R,S-CPIN to R,S-CPIAm

A suspension containing 9.69 g of racemic 2-(4-chloro- phenyl)-3-methylbutyronitrile (50 mmoles), 75 ml of dioxane, 75 ml of water and 25.2 g of Amberlite IRA-400 (OH) resin was stirred and heated to reflux for 64 hours. After filtration of the resin _r - the filtrate was evaporated to dryness and dried in a vacuum oven at 50 - 55°C to give 9.8 g of crude racemic CPIAm. The composition of the recovered material was determined by HPLC.

The recovered material contained 3.6 mmoles of racemic 2-(4-chlorophenyl)-3-methylbutyronitrile and 46.4 mmoles of racemic CPIAm.

EXAMPLE 1

Preparation of biological material with enantioselective amidase activity

Microorganisms used are Rhodococcus erythropolis DSM 5910 (DP-10) , Rhodococcus erythropolis DSM 6374 (DP-11) ,

Rhodococcus erythropolis DSM 6378 (DP-25) , Rhodococcus erythropolis DSM 6375 (DP-26) , Pseudomonas putida NRRL-B-

18669 (13-5S-ACN-2a) , Moraxella sp. NRRL-B-18671 (3L-A-1-5-

la-1) , Pseudomonas putida NRRL-B-18820 (2D-ll-5-lb) , Pseudomonas sp. NRLL-B-18819 (2D-ll-5-lc) and Serratia lique- faciens NRRL-B-18821 (MOB IM/N3) .

The growth medium used for the cultivation of Pseudo- monas, Serratia and Moraxella strains was made up of the fol¬ lowing constituents.

SZI

KH ₂ P0 ₄ 8.85

Sodium citrate 2.25 MgS0 ₄ .7H ₂ 0 0.5

FeS0 ₄ .7H ₂ 0 0.05

Glucose ^a 10.0

Trace element solution ⁰ 1.0 ml

Vitamin solution ^d 1.0 ml Nitrile 2.7 g

^a Added after autoclaving. ^b Trace element solution: 10 ml of 25% HCl, 1.5 g of

FeCl ₂ .4H ₂ 0, 0.019 g of COC1 ₂ .6H ₂ 0, 0.1 g MnCl _2> 4H ₂ 0,

0.07 g of ZnCl ₂ , 0.062 g of H ₃ BO ₃ , 0.036 g of NaMo0 ₄ .2H ₂ 0, 0.024 g of NiCl ₂ .6H ₂ 0 and 0.017 g of

CuCl ₂ .2H ₂ 0. The FeCl ₂ .4H ₂ o is dissolved in HCl and distilled water is added to 1 liter. ^c Vitamin solution: 0.01 g of biotin, 0.01 g of folic acid, 0.05 g of pyridoxine.HCl, 0.025 g of riboflavin, 0.025 g of thiamine HCl, 0.025 g of nicotinic acid,

0.025 g of pantothenic acid, 0.0065 g of vitamin B12,

0.025 g of p-aminobenzoic acid and 0.025 g of thioacetic acid. ^d 1,4-dicyanobutane (Pseudomonas putida 13-5S-ACN-2a) , 2-methylglutaronitrile (Pseudomonas sp. 2D-ll-5-lc,

Pseudomonas putida 2D-ll-5-lb, Serratia liσuefaciens

MOB IM/N3, Moraxella sp. 3L-A-l-5-la-l) .

A 10 ml volume of the above medium (PR/glucose) was inoculated with 0.1 ml of frozen stock culture. Following

overnight growth at room temperature (22 - 25°C) on a shaker at 250 RPM, the 10 ml inoculum was added to 990 ml of fresh medium in a 2 liter flask. The cells were grown for 18 - 24 hours at room temperature with magnetic stirring at a rate high enough to cause bubble formation in the medium. Cells were harvested by centrifugation, washed once with 0.85% saline and the concentrated paste immediately placed in a -70°C freezer for storage.

EXAMPLE 2

Preparation of biological material with enantioselective amidase activity

The growth medium used for cultivation of Rhodococcus ervthropolis DP-10 was made up of the following constituents:

3/1 KH ₂ P0 ₄ 9.00

Na ₂ HP0 ₄ -2H ₂ 0 21.00

* added after autoclaving

** trace element solution SL-7 (cf. DSM, Catalogue of strains 1983, p. 296)

These constituents were dissolved in 900 ml of water, the pH value was adjusted to 7.5 by addition of phosphoric acid/sodium hydroxide, and the solution was made up to 1 liter by addition of water.

100 ml of the above growth medium was added to a 500 ml Erlenmeyer flasks and autoclaved. Glucose was added to the cooled medium from a sterile stock solution (20%, weight/ volume) . Erlenmeyer flasks inoculated with Rhodococcus erythropolis DSM 5910 were incubated at 30"C on a shaker for 28 hours.

Cells were harvested by centrifugation (at 20,000 rpm for 15 minutes on a Sorvall* centrifuge) , washed twice with 0.1 M phosphate buffer of pH value 7. The washed cell pellet (= cell paste) was stored at 4°C or frozen (-25°C) .

EXAMPLE 3

Immobilization of biological material with enantioselective amidase activity

Fresh cell paste obtained as described in Example 2 was immobilized as previously described (US patent specifica¬ tion No. 4,892,825, Example 11).

EXAMPLE 4

Hydrolysis of S-CPIAm to S-CPIA by Rhodococcus erythropolis DP-10

A 75 mg sample of dried, immobilized Rhodococcus erythropolis DP-10 was added to 3 ml of phosphate buffer (100 mM, pH value: 7.0) and incubated for 1 hour at 4 ^β C. The immobilized cell suspension was warmed to room temperature and 6.3 mg (29.8 μmoles) of S-CPIAm in 120 μl of dimethyl sulfoxide (hereinafter designated DMSO) was added. After incubation with agitation at 50°C for 48 hours, the reaction was acidified with 3M H ₂ S0 ₄ to a pH value of 3.0. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of

nitrogen gas and the residue was suspended in 3 ml of metha¬ nol. The composition of the methanol solution was determined by HPLC.

The extracted supernatant contained 0.5 μmole of CPIAm and 28.3 yxmoles of CPIA.

Measurement of the enantiomeric composition by chiral HPLC showed that the enantiomer excess of S-CPIA was 99%.

COMPARATIVE EXAMPLE

Hydrolysis of R-CPIAm by Rhodococcus erythropolis DP-10

A 75 mg sample of dried immobilized Rhodococcus erythropolis DP-10 was added to 3 ml of phosphate buffer (100 mM, pH value: 7.0) and incubated for 1 hour at 4°C. The immobilized cell suspension was warmed to room temperature and 6.3 mg (29.8 /xmoles) of R-CPIAm in 120 μl of DMSO was added. After incubation with agitation at 50°C for 48 hours, the reaction was acidified with 3M H ₂ S0 ₄ to a pH value of 3.0. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas. The composition of the methanol solu¬ tion was determined by HPLC.

The extracted supernatant contained 22.7 moles of CPIAm and less than 0.5 jumole of CPIA.

The amount of acid corresponded to the amount present as an impurity in the starting material.

Incomplete recovery of R-CPIAm was most likely due to experimental errors and/or adsorption of substrate to cells and/or the support material.

EXAMPLE 5

Hydrolysis of R,S-CPIAm to S-CPIA by Rhodococcus erythropolis DP-10

A 75 g sample of dried immobilized Rhodococcus erythropolis DP-10 was added to 3 ml of phosphate buffer (100 mM, pH value: 7.0) and incubated for 1 hour at 4 ^β C. The immobilized cell suspension was warmed to room temperature and 6.3 mg (29.8 moles) of R,S-CPIAm in 120 μl of DMSO was added. After incubation with agitation at 50 ^β C for 48 hours, the reaction was acidified with 3M H ₂ S0 ₄ to a pH value of

3.0. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas. The composition of the methanol solution was determined by HPLC.

The extracted supernatant contained 12.5 moles of CPIAm and 11.8 moles of CPIA.

Measurement of the enantiomeric composition by chiral HPLC showed that the enantiomer excess of R-CPIAm was 100% and that the enantiomer excess of S-CPIA was 100%.

EXAMPLE 6

Hydrolysis of R,S-CPIAm to S-CPIA by Rhodococcus erythropolis DP-10

The procedure of Example 5 was repeated with 59.8 moles of R,S-CPIAm and a 72 hour incubation. The composition of the methanol solution was determined by HPLC.

The extracted supernatant contained 31.9 μmoles of CPIAm and 21.8 moles of CPIA.

Measurement of the enantiomeric composition of the extracted supernatant by chiral HPLC showed 4.8 /xmoles of S- CPIAm, 27.1 moles of R-CPIAm and 21.8 μmoles of S-CPIA. The enantiomer excess of S-CPIA was 100%.

EXAMPLE 7

Hydrolysis of S-CPIAm to CPIA in toluene by Rhodococcus erythropolis DP-10

A 300 mg sample of dried immobilized Rhodococcus

5 erythropolis DP-10 was added to 12 ml of phosphate buffer

(100 mM, pH value: 7.0) and the mixture was incubated for 1 hour at 4°C. The immobilized cell suspension was warmed to room temperature and the phosphate buffer was removed by decantation. 12 ml of toluene saturated with 100 mM phosphate ιo buffer (pH value: 7.0) was added. Toluene saturated buffer was prepared by mixing equal volumes of toluene and 100 mM phosphate buffer (pH value: 7.0), shaking, allowing the phase to separate and removing the aqueous layer. After the addition of 118 μ-moles of S-CPIAm (19.7 μmoles/2 ml), the

15 toluene suspension was incubated with agitation at 30°C. A 2 ml sample was removed at 48 hours and 120 hours and each sample was evaporated to dryness under a stream of nitrogen gas. Each sample was resuspended in 2 ml of methanol and the composition of the methanol solution was determined by HPLC.

20 After 48 hours, the toluene supernatant samples con¬ tained 11.4 μmoles of CPIAm and 5.7 μmoles of CPIA and after 120 hours it contained 1.9 μ-moles of CPIAm and 9.4 μ-moles of CPIA.

EXAMPLE 8

25 S-CPIAm hydrolysis to S-CPIA in buffer/octane by Rhodococcus erythropolis DP-10

A 500 mg sample of dried immobilized Rhodococcus erythropolis DP-10 was added to a biphasic solution consisting of 16 ml of phosphate buffer (100 mM, pH value:

307.0) and 4 ml of n-octane. The immobilized cell suspension was incubated for 3 hours at 50°C. After the addition of 10

mg (47.4 μmoles) of S-CPIAm and incubation for 6 hours at 50°C and 600 rpm, 0.5 ml aliquots of each phase was removed. Each aliquot was adjusted to a pH value of 2.0 with ION HCl. Four volumes of methylene chloride were added to each aliquot and the suspensions were agitated for 15 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of air and each residue was resuspended in 0.5 ml of methanol. The composition of the methanol solution was determined by HPLC. The extracted supernatants contained:

Compound Buffer Octane phase phase

CPIAm 0.5 μmoles 0

CPIA 19.8 μmoles 1.9 μmoles

EXAMPLE 9

Continuous S-CPIAm hydrolysis to S-CPIA in buffer/octane by Rhodococcus erythropolis DP-10

A 3.74 g sample of dried immobilized Rhodococcus erythropolis DP-10 was added to a reactor consisting of a Millipore ultrafiltration cell with a hydrophilic membrane

(PTGC 043 10) installed at the bottom. The immobilized cells were incubated in 50 ml of phosphate buffer (100 mM, pH value: 7.0) plus 20 ml of n-octane at 50"C for 2 hours. After the addition of 64 mg of S-CPIAm (303.3 μmoles), the reactor was closed and the peristaltic pump which delivered phosphate buffer (100 mM, pH value: 7.0) pre-saturated with S-CPIAm was started. The liquid feed rate was adjusted to 20 ml/hour to give a 3.5 hour hydraulic retention time for the reacting system. The hydrophilic membrane selectively passes the buffer phase and retained the n-octane and immobilized Rhodococcus erythropolis DP-10. The reactor configuration is shown schematically in Figure 1.

The reactor effluent was collected at time intervals, was extracted as described in Example 7 and was analyzed by HPLC for CPIAm and CPIA.

The sample analysis translated into the following rates at the exit:

Elapsed time, CPIAm, CPIA,

Minutes μmoles/hour μmoles/hour

40 12.5 14.5

115 9.1 24.1 180 4.4 32.7

EXAMPLE 10

Resin racemization of S-CPIAm

A suspension containing 1.0 g of S-CPIAm (4.7 mmoles), 1.0 g of Amberlite IRA-400 (OH) resin and 25 ml of toluene was stirred and heated to reflux for 40 hours. After removal of the resin by filtration, the filtrate was evaporated to give 0.94 g of crystalline solid. The enantiomeric composition of the recovered material was measured by chiral HPLC and showed 54% S-CPIAm and 46% R-CPIAm.

EXAMPLE 11

Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-10

A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-10 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. Then 9.4 μmole of S-CPIAm or R,S-CPIAm in 40 μl of dimethylsulfoxide was added. After incubation at 50°C for 48 hours, the reactions were acidified with 3M H ₂ S0 ₄ to a pH value of 3. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 min. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas and the

residue was suspended in 1 ml of methanol. The composition of the methanol solution was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 1.

TABLE 1 S-CPIAm _/ R-CPIAm and R,S-CPIAm Hydrolysis by Rhodococcus erythropolis DP-10

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA

^a ND = None detected. ^b Data corrected for R-CPIA impurity in R-CPIAm starting material. ^c NT = Not tested.

TR = Trace amount detected.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 12

Hydrolysis of R,S-ATAm to R-ATAC by Rhodococcus erythropolis DP-10

A 50 g sample of frozen cell paste of Rhodococcus erythropolis DSM 5910 (DP-10) was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. Then 12.1 μmole of R,S-ATAm in 40 μl of dimethylsulfoxide was added. After incubation at 28°C with agitation for 48 hours, the reaction suspension was centrifuged to remove cell debris and the supernatant was passed through a 0.2 μ membrane filter. The composition of the clarified supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 2.

TABLE 2 R _/ S-ATAm Hydrolysis by Rhodococcus erythropolis DP-10

HPLC Analysis (u ole recovered)

Substrate Reverse-Phase Chiral

(μmole added) ATAm ATAC S-ATAm R-ATAm S-ATAC R-ATAC

R,S-ATAm 3.3 4.2 3.3 TR ^a 0.3 3.9 (12.1)

a TR = Trace amount detected.

Incomplete recovery of R,S-ATAm was most likely due to experimental errors and/or adsorption of substrate to cells. Enantiomer excess of R-ATAC recovered was 86%.

EXAMPLE 13

Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-11

A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-11 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 11, 9.4 μmole of S-CPIAm or R-CPIAm or R,S- CPIAm was added. Following the same incubation and extraction protocols as in Example 11, the composition of the extracted supernatant was determined by reverse-phase and chiral HPLC. The results are shown in Table 3.

TABLE 3 s,CPIAm, R-CPIAm and R,S-CPIAm Hydrolysis by Rhodococcus erythropolis DP-11

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA

^a TR = Trace amount detected. ND = None detected. ° Data corrected for R-CPIA impurity in R-CPIAm starting material.

^d NT = Not tested.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 14

Hydrolysis of R,S-ATAm to R-ATAC by Rhodococcus erythropolis DP-11

A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-11 was added to 1 ml of phosphate buffer (100 mM, pH value: 7.0) at room temperature. In the same manner as in Example 12, 12.1 μmole of R,S-ATAm was added. Following the same incubation, centrifugation and filtration protocols as in Example 12, the composition of the extracted superna¬ tant was determined by reverse-phase HPLC and chiral HPLC. The results are shown in Table 4.

TABLE 4 R,S-ATAm Hydrolysis by Rhodococcus ervthropolis DP-11

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) ATA ATAC S-ATAm R-ATAm S-ATAC R-ATAC

R,S-ATAm 4.8 1.2 3.4 1.4 ND 1.2 (12.1)

ND = Trace amount detected.

Incomplete recovery of R,S-ATAm was most likely due to experimental errors and/or adsorption of substrate to cells. Enantiomer excess of R-ATAC recovered was 100%.

EXAMPLE 15

Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-25

A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-25 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 11, 9.4 μmole of S-CPIAm or R-CPIAm or R,S- CPIAm was added. Following the same incubation and extraction protocols as in Example 11, the composition of the extracted supernatant was determined by reverse-phase and chiral HPLC. The results are shown in Table 5.

TABLE 5

S-CPIAm, R-CPIAm and R,S-CPIAm Hydrolysis by Rhodococcus erythropolis DP-25

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA

S-CPIAm (9.4) 6.0

R-CPIAm (9.4) 8.4 R,S-CPIAm 6.2 (9.4)

ND = None detected.

^b Corrected for R-CPIA impurity in R-CPIAm starting materi¬ al. ^c NT = Not tested.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 16

Hydrolysis of R,S-ATAm to R-ATAC by Rhodococcus erythropolis DP-25

A 50 mg sample of frozen cell paste of 24 Rhodococcus erythropolis was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 5, 12.1 μmole of R,S-ATAm was added. Following the same incubation, centrifugation and filtration protocols as in Example 12, the composition of the extractant supernatant was determined by reverse-phase HPLC and chiral HPLC. The results are shown in Table 6.

TABLE 6

R,S-ATAm Hydrolysis by Rhodococcus erythropolis DP-25

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) ATAm ATAC S-ATAm R-ATAm S-ATAC R-ATAC

R,S-ATAm 9.7 1.8 6.2 3.5 ND ^a 1.8 (12.1)

a ND = None detected.

Enantiomer excess of R-ATAC recovered was 100%.

EXAMPLE 17

Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-26

A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-26 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 11, 9.4 μmole of S-CPIAm or R-CPIAm or R,S- CPIAm was added. Following the same incubation and extraction protocols as in Example 11, the composition of the extracted supernatant was determined by reverse-phase and chiral HPLC. These results are shown in Table 7.

TABLE 7

Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-26

HPLC Analysis (ttmole recovered)

Substrate Reverse-Phase Chiral

(μmole added) CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA

- R = Trace detected. ^b ND = None detected.

^c Data corrected for R-CPIA impurity in R-CPIAm starting material.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 18

5 Hydrolysis of R,S-ATAm to R-ATAC by Rhodococcus erythropolis DP-26

A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-26 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same 10 manner as in Example 12, 12.1 μmole of R,S-ATAm was added. Following the same incubation, centrifugation and filtration protocols as in Example 12, the composition of the extracted supernatant was determined by reverse-phase and chiral HPLC. The results are shown in Table 8.

15 TABLE 8

R,S-ATAm Hydrolysis by Rhodococcus erythropolis DP-26

HPLC Analysis (ttmole recovered)

Substrate Reverse-Phase Chiral

20 (μmole added) ATA ATAC S-ATAm R-ATAm S-ATAC R-ATAC

R,S-ATAm 8.5 3.0 6.2 2.3 ND ^a 3.0 (12.1)

_Z m a ND = None detected.

Enantiomer excess of R-ATAC recovered was 100%.

EXAMPLE 19

Hydrolysis of R,S-NPAm to S-NPAC by Pseudomonas putida 13-5S- ACN-2a

A 25 mg sample of frozen cell paste of Pseudomonas putida i3-5S-ACN-2a was added to 1 ml of phosphate buffer (100 mM) , pH value: 7.2) at room temperature. Then 1 μmole of R,S-NPAm in 40 μl of dimethylsulfoxide was added. After in¬ cubation at 28"C with agitation for 48 hours, the reaction was acidified to a pH value of 3 with 3M H ₂ S0 ₄ . Four volumes of methylene chloride were added and the suspension was agi¬ tated for 30 min. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen. The residue was redissolved in 1 ml of methanol. The composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 9.

TABLE 9

R,s-NPAm Hydrolysis by Pseudomonas putida 13-5S-ACN-2a

HPLC Analysis (ttmole recovered)

Substrate Reverse-Phase Chiral

(μmole added) NPAm NPAC S-NPAm R-NPAm S-NPAC R-NPAC

R,S-NPAm 0.42 0.44 ND ^C 0.42 0.44 ND ^C (1.0)

a ND = None detected.

Enantiomer excess of S-NPAC recovered was 100%.

EXAMPLE 20

Hydrolysis of R,S-IBAm to S-IBAC by Pseudomonas putida 13-5S- ACN-2a

A 50 mg sample of frozen cell paste of Pseudomonas putida 13-5S-ACN-2a was added to 1 ml of phosphate buffer (100 M) , pH value: 7.0) at room temperature. Then 9.8 μmole R,S-IBAm in 40 μl of dimethylsulfoxide was added. After in¬ cubation at 28°C with agitation for 48 hours, the reaction was acidified with 3M H ₂ S0 ₄ to a pH value of 3. Four volumes of methylene chloride were added and the suspension was agi¬ tated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas and the residue was suspended in 1 ml of acetonitrile. The composition of the acetonitrile solution was determined by reverse phase HPLC and chiral HPLC and is shown in Table 10.

TABLE 10

R,s-IBAm Hydrolysis by Pseudomonas putida l3-5S-ACN-2a

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) IBAm IBAC S-IBAm R-IBAm S-IBAC R-IBAC

R,S-IBAm 4.8 1.3 1.4 3.4 1.3 ND ^a (9.8)

a ND = None detected.

Incomplete recovery of R,S-IBAm was most likely due to experimental errors and/or adsorption of substrate to cells. Enantiomer excess of S-IBAC recovered was 100%.

EXAMPLE 21

Hydrolysis of R,S-NPAm to S-NPAC by Pseudomonas putida 2D-11- 5-lb

A 20 mg sample of frozen cell paste of Pseudomonas putida 2D-ll-5-lb was added to 1 ml of phosphate buffer (100 mM) , pH value: 7.2) at room temperature. In the same manner as Example 19, 1 μmole of R,S-NPAm in 40 μl of dimethylsulfoxide was added. Following incubation for 24 hours and the same extraction protocol as in Example 19, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 11.

TABLE 11

R,S-NPAm Hydrolysis by Pseudomonas putida, 2D-ll-5-lb

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral (μmole added) NPAm NPAC S-NPAm R-NPAm S-NPAC R-NPAC

R,S-NPAm 0.60 0.40 0.05 0.55 0.39 0.01 (1.0)

Enantiomer excess of S-NPAC recovered was 95%.

EXAMPLE 22

Hydrolysis of R,S-IBAm to S-IBAC by Pseudomonas putida 2D-11- 5-lb

A 50 mg sample of frozen cell paste of Pseudomonas putida 2D-ll-5-lb was added to 1 ml of phosphate buffer (100 mM, pH value: 7.0) at room temperature. In the same manner as Example 20, 9.8 μmole of R,S-IBAm in 40 μl of dimethylsulfoxide was added. Following the same incubation and extraction protocols as in Example 20, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 12.

TABLE 12

R,S-NPAm Hydrolysis by Pseudomonas putida 2D-ll-5-lb

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) IBAm IBAC S-IBAm R-IBAm S-IBAC R-IBAC

R,S-IBAm 3.8 2.1 0.6 3.2 2.0 0.1 (9.8)

Incomplete recovery of R,S-IBAm was most likely due to experimental errors and/or adsorption of substrate to cells. Enantiomer excess of S-IBAC recovered was 90%.

EXAMPLE 23

Hydrolysis of R,s-NPAm to S-NPAC by Pseudomonas sp. 2D-11-5- lc

A 20 mg sample of frozen cell paste of Pseudomonas sp. strain 2D-11-5-1C, was added to 1 ml of phosphate buffer (100 mM, pH value: 7.2) at room temperature. In the same manner as Example 19, 1 μmole of R,S-NPAm in 40 μl of dimethylsulfoxide was added. Following the same incubation and extraction protocols as in Example 19, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 13.

TABLE 13

R,S-NPAm Hydrolysis by Pseudomonas sp. 2D-11-5-1C

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) NPAm NPAC S-NPAm R-NPAm S-NPAC R-NPAC

R,S-NPAm 0.54 0.48 ND ^a 0.54 0.47 0.01 (1.0)

^a ND = Not detected.

Enantiomer excess of S-NPAC recovered was 96%.

EXAMPLE 24

Hydrolysis of R,S-NPAm to NPAC by Serratia liσuefaciens MOB IM/N3

A 20 mg sample of frozen cell paste of Serratia liquβ- faciens MOB IM/N3 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.2) at room temperature. In the same manner as Example 19, 1 μmole of R,S-NPAm in 40 μl of di¬ methylsulfoxide was added. Following the same incubation and extraction protocols as in Example 19, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 14.

TABLE 14

R,S-NPAm Hydrolysis by Serratia liσuefaciens MOB IM/N3

HPLC Analysis (mmole recovered)

Substrate Reverse-Phase Chiral

(μmole added) NPAm NPAC S-NPAm R-NPAm S-NPAC R-NPAC

R,S-NPAm (1.0) 0.64 0.26 0.20 0.44 0.25 0.01

Enantiomer excess of S-NPAC recovered was 92%.

EXAMPLE 25

Hydrolysis of R,S-IBAm to S-IBAC by Serratia liquefaciens MOB IM/N3

A 50 mg sample of frozen cell paste of Serratia lique- faciens MOB IM/N3 was added to 1 ml of phosphate buffer (lOOmM) , pH value: 7.0) at room temperature. In the same manner as Example 20, 9.8 μmole of R,S-IBAm in 40 μl of di¬ methylsulfoxide was added. Following the same incubation and extraction protocols as in Example 20, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 15.

TABLE 15

R,S-IBAm Hydrolysis by Serratia liσuefaciens. MOB IM/N3

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) IBAm IBAC S-IBAm R-IBAm S-IBAC R-IBAC

R,S-IBAm (9.8) 4.9 0.7 2.0 2.9 0.7 NDa

^a ND = None detected.

Incomplete recovery of R,S-IBAm was most likely due to experimental errors and/or adsorption of substrate to cells. Entantiomer excess of S-IBAC recovered was 100%.

34

EXAMPLE 26

Hydrolysis of R,S-ATAm to R-ATAC by Moraxella sp. 3L-A-1-5- la-l

A 50 mg sample of frozen cell paste of Moraxella sp. 3L-A-l-5-la-l was added to 1 ml of phosphate buffer (100 mM, pH value: 7.0) at room temperature. Then 12.1 μmole of R,S- ATAm in 40 μl of dimethylsulfoxide was added. After incuba¬ tion at 28 ^β C with agitation for 48 hours, the reaction suspension was centrifuged to remove cell debris and the supernatant was passed through a 0.2 μ membrane filter. The composition of the supernatant was determined by reverse- phase HPLC and chiral HPLC and is shown in Table 16.

TABLE 16

R,S-ATAm Hydrolysis by Moraxella sp. 3L-A-l-5-la-l

HPLC Analysis (umole recovered)

Substrate Reverse-Phase Chiral

(μmole added) ATA ATAC S-ATAm R-ATAm S-ATAC R-ATAC

R,S-ATAm 7.6 1.5 4.9 2.7 ND ^C 1.5 (12.1)

ND = None detected.

Incomplete recovery of R,S-ATAm was most likely due to experimental errors and/or adsorption of substrate to cells. Entantio er excess of S-ATAC recovered was 100%.

EXAMPLE 27

Comparison of CPIAm Bioconversion by Rhodococcus erythropolis DP-10 and Brevibacterium sp. R312

Fifty mg samples of frozen cell paste of Rhodococcus erythropolis DP-10 and Brevibacterium sp. R312 were added to 1 ml volumes of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. Then 9.4 μmole of S-CPIAm of R,S-CPIAm in 40 μl of dimethylsulfoxide was added. After incubation at 50°C for 2, 5, 8, 16 and 24 hours, the reactions were acidified with 3M H ₂ S0 ₄ to a pH value of 3.0. Four volumes of methylene chloride were added and the residues were suspended in 1 ml of methanol. The composition of the methanol solu¬ tions were determined by reverse-phase HPLC. The results expressed as specific activity are shown in Table 17.

TABLE 17

S-CPIAm and R,S-CPAm Hydrolysis by Rhodococcus erythropolis DP-10 and Brevibacterium sp. R312

Bioconversion Specific Activity (μmoles

Strain Time CPIA/mα Cells ^a /hr x 10 ² ) hours S-CPIAm R,S-CPIAm

R. erythropolis 2 5.2 6.5 DP-10 5 5.6 6.5

8 5.2 4.4

16 4.1 2.1

24 2.9 1.6

Brevibacterium sp. 2 2.4 3.3 R312 5 3.0 3.6

8 3.0 3.1

16 2.8 2.2

24 2.8 1.7

Dry cell weight.

36

Higher specific activities were obtained for both S- CPIAm and R,S-CPIAm hydrolysis to S-CPIA with Rhodococcus erythropolis DP-10.

International Application No: PCT/

MICROORGANISMS

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