EP0662149A1 - A process for the preparation of enantiomeric 2-alkanoic acids - Google Patents

A process for the preparation of enantiomeric 2-alkanoic acids

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Publication number
EP0662149A1
EP0662149A1 EP93917294A EP93917294A EP0662149A1 EP 0662149 A1 EP0662149 A1 EP 0662149A1 EP 93917294 A EP93917294 A EP 93917294A EP 93917294 A EP93917294 A EP 93917294A EP 0662149 A1 EP0662149 A1 EP 0662149A1
Authority
EP
European Patent Office
Prior art keywords
nitrile
amide
enantiomeric
alkyl
mixture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93917294A
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German (de)
French (fr)
Inventor
Robert Donald Fallon
Barry Stieglitz
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EIDP Inc
Original Assignee
EI Du Pont de Nemours and Co
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Publication date
Application filed by EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of EP0662149A1 publication Critical patent/EP0662149A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

  • X-CHR-COOH are currently marketed as racemic or diastereomeric mixtures.
  • the physiological effect derives from only one enantiomer/diastereomer while the other enantiomer/diastereomer is inactive or even harmful.
  • Certain chemical and enzymatic techniques for separating enantiomers are known.
  • WO92/05275 discloses an enantioselective process for producing (S)-2-alkanoic acids from the corresponding racemic nitriles via enantiomeric amide intermediates in a 2-step, biologically-catalyzed sequence.
  • This invention pertains to a method for converting an (S)-amide, or stereospecifically converting a mixture of (R)- and (S)-amides, of Formula I
  • A is selected from the group consisting of:
  • A-l A-2 R 1 is C]-C 4 alkyl
  • R 3 is H; F; Cl; Br; OH; C,-C 3 alkyl; or C r C 3 alkoxy;
  • the solvent for the transformation may be an aqueous buffer or a biphasic mixture of aqueous buffer and organic solvent.
  • This invention also pertains to a method for converting (S)-nitrile of
  • R 1 and R 2 are defined as for compounds of Formula I, to the corresponding enantiomeric (S)-carboxylic acid comprising contacting said nitrile with a mixture of Pseudomonas chlororaphis B23 and Pseudomonas putida 5B-MGN-2p in a solvent and recovering the product.
  • Preferred is the method wherein R 1 is CH(CH3)2, R 2 is Cl and the nitrile is racemic.
  • the solvent for the transformation can be an aqueous buffer or a biphasic mixture of aqueous buffer and organic solvent.
  • this invention pertains to a method for converting (S)-nitrile of
  • Preferred is a method for converting (S)-2-(6-methoxy-2- naphthyl)propionitrile, or stereospecifically converting a mixture of (R)- and (S)-2-(6-methoxy-2-naphthyl)propionitrile, to the enantiomeric (S)-carboxylic acid, through the corresponding amide, comprising contacting said nitrile with Pseudomonas chlororaphis B23 in a solvent and recovering the product.
  • microorganisms used in the present invention are Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23.
  • the methods of this invention should be understood to include use of these microorganisms and/or the amidase and nitrile hydratase enzymes from said microorganisms.
  • stereospecifically refers to a chemical reaction which is stereospecific.
  • enantiomeric refers to a compound which is a single enantiomer and substantially free of the other enantiomer. Whether a reaction is “stereospecific” or “stereoselective” is determined by the enantiomeric ratio (E) for the product (R)- and (S)-enantiomers. E corresponds to the ratio of the rate of formation of the two enantiomers. When E is greater than 7, the reaction is stereospecific and when E is 7 or less, the reaction is stereoselective. Preferred reactions are those wherein E is above 8.5 and most preferred reactions are those wherein E is 10 or above.
  • the hydrolysis of the nitrile to the amide is accomplished by the action of a stereospecific nitrile hydratase enzyme originating in the P. putida 5B-MGN-2p obtained by culturing the microorganism in the presence of a medium suitable for production of the stereospecific nitrile hydratase.
  • This medium need include only an appropriate source of nitrogen for growth (e.g., ammonium chloride) because P. putida 5B-MGN-2p produces the enzyme constitutively in the absence of an inducer.
  • the nitrile hydratase thus obtained is added to the mixture of (R)- and (S)-nitrile to yield the corresponding (S)-amide.
  • the amide intermediate is hydrolyzed by a stereospecific amidase enzyme originating in P. chlororaphis B23 to yield the corresponding (S)-acid.
  • One method for performing the nitrile-to-acid transformation is to collect the nitrile hydratase enzyme from P. putida 5B-MGN-2p and the amidase enzyme from P. chlororaphis B23 and use the enzymes together as an enzyme preparation in a biologically-recognized manner.
  • the stereospecific conversion to the corresponding (S)-acid may be accomplished using P. chlororaphis B23 without the presence of P. putida 5B-MGN-2p.
  • the intermediate in the conversion is the corresponding amide.
  • the conversion of the nitrile to the amide is not stereospecific, but the conversion of the racemic or enantiomerically-enriched amide to (S)-acid is stereospecifc.
  • the most common solvent used in microbial conversions is aqueous buffer.
  • most organic substrates are only sparingly soluble in water, and therefore the microbial reaction concentrations are significantly lower than conventional organic chemical transformations.
  • Large reaction vessels must be utilized, or numerous reactions must be performed, to produce large amounts of product.
  • the methods of the present invention can employ a biphasic solvent which contains an aqueous buffer and an organic solvent.
  • the biphasic solvents allow the reactions to be run at higher concentration which improves the practicality of using microbial conversions on a preparative scale.
  • Suitable aqueous buffers include phosphate and pyrophosphate.
  • the "organic solvent” is defined as a single non-aqueous liquid selected from the group comprising Cg-Cjg hydrocarbons, toluene, and dibutyl phthalate, and monophasic mixtures thereof.
  • the ratio of aqueous buffer to organic solvent can range from about 95:5 to 50:50.
  • the recycle process can be configured such that the (S)-acid is continually removed from the medium, and the by-product (R)-nitrile can be racemized and recycled in a continuous process in which it is combined with additional alkyl nitrile and contacted with the enzyme mixture to form the alkyl acid (see WO92/05275).
  • the present invention is particularly characterized by the biological material, P. chlororaphis B23 which is employed alone in some of the conversions, and by the combination of biological materials, P. chlororaphis B23 and P.
  • putida 5B-MGN-2p used to convert nitrile to (S)-acid.
  • the microorganism P. putida 5B-MGN-2P was deposited under the terms of the Budapest Treaty at NRRL (Northern Regional Research Center, U.S. Department of Agriculture, 1815 North University St., Peoria, IL) and bears the accession number NRRL-B- 18668.
  • the strain was isolated from soil collected in Orange, Texas. Standard enrichment procedures were used with the following modified medium (PR Basal Medium). Isolates of P.
  • putida 5-B-MGN-2P were purified by repeated passing on Bacto Brain Heart Infusion Agar followed by screening for ammonia production from the enrichment nitrile, and identified by methods disclosed in WO92/05275.
  • Pseudomonas chlororaphis B23 accession number FERM BP-187 is a Nitto Chemical patent strain obtained from the Fermentation Research Institute, Agency of Industrial Science and Technology, 1-1-3 Higashi, Tsukuba, Ibaraki 305, Japan.
  • the P. chlororaphis B23 and P. putida 5B-MGN-2p were propagated on PR basal medium as described below.
  • Pseudomonas putida 5B-MGN-2p was also grown in the absence of a nitrile or amide inducer with 25 mM NH C1 or 25 mM (NH 4 ) 2 SO 4 replacing the nitrile or amide.
  • a 10 mL volume of complete PR basal medium was inoculated with 0.1 mL of frozen stock culture of P. chlororaphis B23 or P. putida 5B-MGN-2P. 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-L flask. The cells were grown overnight at room temperature with 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 cell paste was immediately placed in a -70°C freezer for storage. Thawed cell pastes containing approximately 80% water were used in all bioconversions.
  • concentrations of nitrile, amide and acid products derived via microbial hydrolysis were measured by reverse-phase HPLC. Detection was by ultraviolet light absorbtion.
  • a Du Pont Zorbax® C18 column employing a mobile phase of 70-75% methanol and 25-30% H 2 O acidified with 0.1 % H 3 PO 4 was used.
  • Examples 1 to 3 describe P. chlororaphis B23 hydrolysis of (S)-CPIAm at 28°C, 35°C and 50°C. Comparisons are provided for (R)-CPIAm hydrolysis at 28°C and 35°C (Comparison Tests 1 and 2).
  • Examples 4 to 7 describe the stereoselective hydrolysis of (S)-CPIN and (R,S)-CPIN at 28°C and 35°C, i.e., both (S)-CPIN and (R)-CPIN are hydrolyzed but more (S)-CPIN is converted to (S)-CPIAm. Again the stereospecific bioconversion of (S)-CPIAm to (S)-CPIA is demonstrated as only (S)-CPIA is generated. Comparisons are provided for (R)-CPIN hydrolysis at 28°C and 35°C (Comparison Tests 3 and 4).
  • Example 8 describes the stereospecific hydrolysis of (R,S)-NPAm to (S)- NPAC.
  • Example 10 describes Pseudomonas putida 5B-MGN-2P and P. chlororaphis B23 bioconversion of (R,S)-CPIN to (S)-CPIA. In this
  • Example 11 describes a biphasic (buffer/ organic solvent) bioconversion of (R,S)-CPIN to (S)-CPIA with both P. putida 5B-MGN-2P and P. chlororaphis B23.
  • the major product is (S)-CPIA but a small amount (0.5 ⁇ mole) of (R)-CPIAm was also generated.
  • Examples 1 to 3 and Comparison Tests 1 and 2 Hydrolysis of (S)-CPIAm and (R)-CPIAm Pseudomonas chlororaphis B23 at
  • 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.
  • Incomplete recovery of (S)-CPIN or (R)-CPIN may be attributable to experimental error or adsorption of substrate to cells.
  • the methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen and the residue was redissolved 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 3.
  • Example 9 Hydrolysis of (R,S)-NPCN by Pseudomonas chlororaphis B23
  • a 12.5 mg sample of frozen cell paste of Pseudomonas chlororaphis B23 was added to 1 mL of phosphate buffer (100 mM, pH value: 7.2) at room temperature. Then, 1 ⁇ mole of (R,S)-NPCN in 40 ⁇ L of dimethylsulfoxide was added. Following the incubation and extraction protocol in Example 8, the composition of the extracted supernatant was determined by reverse phase HPLC and chiral HPLC. The results are shown in Table 4.
  • Pseudomonas chlororaphis B23 Fifty milligram samples of frozen cell pastes of Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23 were added to 1 mL of pyrophosphate buffer (5 mM, pH value: 7.5) at room temperature. This was transferred to a vial containing 20.7 ⁇ mole (R,S)-CPIN. After incubation at 35°C for 48 h and following the extraction protocol in Examples 1 to 3, the composition of the extracted supernatant was determined by reverse -phase HPLC and chiral HPLC. The results are shown in Table 5.

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Abstract

Converting 2-alkyl nitriles of formula (1), wherein A and R1 are as defined in the text, to the corresponding 2-alkanoic acids; 2-alkyl nitriles to the corresponding amides; and 2-alkyl amides to the corresponding acids, all employing one or both of Pseudomonas putida 5B-MGN-2p and Pseudomonas chlororaphis B23.

Description

TITLE A PROCESS FOR THE PREPARATION OF ENANTIOMERIC 2-ALKANOIC ACIDS Many agrichemicals and pharmaceuticals of the general formula,
X-CHR-COOH, are currently marketed as racemic or diastereomeric mixtures. In many cases, the physiological effect derives from only one enantiomer/diastereomer while the other enantiomer/diastereomer is inactive or even harmful. Certain chemical and enzymatic techniques for separating enantiomers are known. For instance, WO92/05275 discloses an enantioselective process for producing (S)-2-alkanoic acids from the corresponding racemic nitriles via enantiomeric amide intermediates in a 2-step, biologically-catalyzed sequence.
Nothing in the art suggests the method of this invention for stereospecifϊcally converting amides of Formula I containing either the A- 1 or A-2 moieties to the corresponding acids, nor the stereospecific conversion of the defined nitriles to the corresponding amides by the microorganisms defined in the disclosed solvent systems, nor the one-step, two-microorganism method for sterospecifically converting nitriles directly to the corresponding acids. SUMMARY OF THE INVENTION
This invention pertains to a method for converting an (S)-amide, or stereospecifically converting a mixture of (R)- and (S)-amides, of Formula I
RJ
wherein:
A is selected from the group consisting of:
A-l A-2 R1 is C]-C4 alkyl;
R2 is H; F; Cl; Br; OH; C,-C3 alkyl; OCF2H; or H2C=C(CH3)CH2NH; and
R3 is H; F; Cl; Br; OH; C,-C3 alkyl; or CrC3 alkoxy;
to the corresponding enantiomeric (S)-carboxylic acid comprising contacting said amide with Pseudomonas chlororaphis B23 in a solvent. Preferred is the above method wherein A is A-l, R1 is CHCGH^, R2 is Cl and the amide is (S)-amide. Also preferred is the method wherein A is A-2, R1 is methyl, R3 is methoxy and the amide is racemic. The solvent for the transformation may be an aqueous buffer or a biphasic mixture of aqueous buffer and organic solvent.
This invention also pertains to a method for converting (S)-nitrile of
Formula II, or stereospecifically converting a mixture of (R)- and (S)-nitriles of
Formula II,
II
wherein R1 and R2 are defined as for compounds of Formula I, to the corresponding enantiomeric (S)-carboxylic acid comprising contacting said nitrile with a mixture of Pseudomonas chlororaphis B23 and Pseudomonas putida 5B-MGN-2p in a solvent and recovering the product. Preferred is the method wherein R1 is CH(CH3)2, R2 is Cl and the nitrile is racemic. Again, the solvent for the transformation can be an aqueous buffer or a biphasic mixture of aqueous buffer and organic solvent. In addition, this invention pertains to a method for converting (S)-nitrile of
Formula II, or stereospecifically converting a mixture of (R)- and (S)-nitriles of Formula II, to the corresponding enantiomeric (S)-amide comprising contacting said nitrile with a Pseudomonas putida 5B-MGN-2p in a biphasic mixture of aqueous buffer and organic solvent and recovering the product. This invention also pertains to a method for converting an (S)-nitrile of
Formula III, or stereospecifically converting a mixture of (R)- and (S)-nitriles of Formula III, wherein R ' and R3 are defined as for compounds of Formula I, to the enantiomeric (S)-carboxylic acid, through the corresponding amide, comprising contacting said nitrile with Pseudomonas chlororaphis B23 in a solvent and recovering the product. If desired, the amide intermediate can be isolated.
III
Preferred is a method for converting (S)-2-(6-methoxy-2- naphthyl)propionitrile, or stereospecifically converting a mixture of (R)- and (S)-2-(6-methoxy-2-naphthyl)propionitrile, to the enantiomeric (S)-carboxylic acid, through the corresponding amide, comprising contacting said nitrile with Pseudomonas chlororaphis B23 in a solvent and recovering the product.
DETAILS OF THE INVENTION The (S)-2-alkanoic carboxylic acids made by a method of this invention have the formula:
IV
The microorganisms used in the present invention are Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23. The methods of this invention should be understood to include use of these microorganisms and/or the amidase and nitrile hydratase enzymes from said microorganisms.
The term "stereospecifically" refers to a chemical reaction which is stereospecific. The term "enantiomeric" refers to a compound which is a single enantiomer and substantially free of the other enantiomer. Whether a reaction is "stereospecific" or "stereoselective" is determined by the enantiomeric ratio (E) for the product (R)- and (S)-enantiomers. E corresponds to the ratio of the rate of formation of the two enantiomers. When E is greater than 7, the reaction is stereospecific and when E is 7 or less, the reaction is stereoselective. Preferred reactions are those wherein E is above 8.5 and most preferred reactions are those wherein E is 10 or above. In the method for converting a mixture of (R)- and (S)-nitrile of Formula II wherein A is A-l to the corresponding enantiomeric (S)-acid, the hydrolysis of the nitrile to the amide is accomplished by the action of a stereospecific nitrile hydratase enzyme originating in the P. putida 5B-MGN-2p obtained by culturing the microorganism in the presence of a medium suitable for production of the stereospecific nitrile hydratase. This medium need include only an appropriate source of nitrogen for growth (e.g., ammonium chloride) because P. putida 5B-MGN-2p produces the enzyme constitutively in the absence of an inducer. The nitrile hydratase thus obtained is added to the mixture of (R)- and (S)-nitrile to yield the corresponding (S)-amide.
The amide intermediate is hydrolyzed by a stereospecific amidase enzyme originating in P. chlororaphis B23 to yield the corresponding (S)-acid. One method for performing the nitrile-to-acid transformation is to collect the nitrile hydratase enzyme from P. putida 5B-MGN-2p and the amidase enzyme from P. chlororaphis B23 and use the enzymes together as an enzyme preparation in a biologically-recognized manner.
When the nitrile is of Formula II and A is A-2, the stereospecific conversion to the corresponding (S)-acid may be accomplished using P. chlororaphis B23 without the presence of P. putida 5B-MGN-2p. The intermediate in the conversion is the corresponding amide. The conversion of the nitrile to the amide is not stereospecific, but the conversion of the racemic or enantiomerically-enriched amide to (S)-acid is stereospecifc.
The most common solvent used in microbial conversions is aqueous buffer. However, most organic substrates are only sparingly soluble in water, and therefore the microbial reaction concentrations are significantly lower than conventional organic chemical transformations. Large reaction vessels must be utilized, or numerous reactions must be performed, to produce large amounts of product. The methods of the present invention can employ a biphasic solvent which contains an aqueous buffer and an organic solvent. The biphasic solvents allow the reactions to be run at higher concentration which improves the practicality of using microbial conversions on a preparative scale.
Suitable aqueous buffers include phosphate and pyrophosphate. The "organic solvent" is defined as a single non-aqueous liquid selected from the group comprising Cg-Cjg hydrocarbons, toluene, and dibutyl phthalate, and monophasic mixtures thereof. The ratio of aqueous buffer to organic solvent can range from about 95:5 to 50:50.
The treatment of a mixture of (R)- and (S)-amide using P. chlororaphis B23 yields a mixture of (S)-acid and unconverted (R)-amide. After separating the amide from the acid, the amide can be racemized by known methods to 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 (OH) in toluene or another non-aqueous solvent (see WO92/01062).
The methods described herein which start with a mixture of an (R)- and (S)- nitrile to produce an (S)-acid also produce an (R)-nitrile as the major by-product. Following separation of the nitrile from the desired acid, e.g., by base neutralization and solvent extraction of the nitrile, recycling of the (R)-nitrile also requires racemization. Enantiomeric or enantiomerically-enriched nitriles can be converted to racemic nitriles using a strongly basic ion exchange resin such as Amberlite® IRA-400 (OH), Amberlyst® A-26, or Dowex® 1X8 (after exchange with hydroxide ion) in an organic solvent. This procedure results in high racemic nitrile yields with no substantial hydrolysis of the nitrile to the corresponding amide or acid. The recycle process can be configured such that the (S)-acid is continually removed from the medium, and the by-product (R)-nitrile can be racemized and recycled in a continuous process in which it is combined with additional alkyl nitrile and contacted with the enzyme mixture to form the alkyl acid (see WO92/05275). The present invention is particularly characterized by the biological material, P. chlororaphis B23 which is employed alone in some of the conversions, and by the combination of biological materials, P. chlororaphis B23 and P. putida 5B-MGN-2p, used to convert nitrile to (S)-acid. The microorganism P. putida 5B-MGN-2P was deposited under the terms of the Budapest Treaty at NRRL (Northern Regional Research Center, U.S. Department of Agriculture, 1815 North University St., Peoria, IL) and bears the accession number NRRL-B- 18668. The strain was isolated from soil collected in Orange, Texas. Standard enrichment procedures were used with the following modified medium (PR Basal Medium). Isolates of P. putida 5-B-MGN-2P were purified by repeated passing on Bacto Brain Heart Infusion Agar followed by screening for ammonia production from the enrichment nitrile, and identified by methods disclosed in WO92/05275. Pseudomonas chlororaphis B23 (accession number FERM BP-187) is a Nitto Chemical patent strain obtained from the Fermentation Research Institute, Agency of Industrial Science and Technology, 1-1-3 Higashi, Tsukuba, Ibaraki 305, Japan.
The P. chlororaphis B23 and P. putida 5B-MGN-2p were propagated on PR basal medium as described below.
The following additions and or modifications were made to the PR basal media:
Pseudomonas putida 5B-MGN-2p was also grown in the absence of a nitrile or amide inducer with 25 mM NH C1 or 25 mM (NH4)2SO4 replacing the nitrile or amide.
For testing enzyme activity, a 10 mL volume of complete PR basal medium was inoculated with 0.1 mL of frozen stock culture of P. chlororaphis B23 or P. putida 5B-MGN-2P. 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-L flask. The cells were grown overnight at room temperature with 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 cell paste was immediately placed in a -70°C freezer for storage. Thawed cell pastes containing approximately 80% water were used in all bioconversions.
The abbreviations hereafter are used in the Examples which follow: CPIN - 2-(4-chlorophenyl)-3-methylbutyronitrile
CPIAm - 2-(4-chlorophenyl)-3-methylbutyramide CPIA - 2-(4-chlorophenyl)-3-methylbutyric acid
NPCN - 2-(6-methoxy-2-naphthyl)-propionitrile
NPAm - 2-(6-methoxy-2-naphthyl)-propionamide NPAC - 2-(6-methoxy-2-naphthyl)-propionic acid
HPLC - High-Performance Liquid Chromatography
The following enantiomers and mixtures were examined: (S)-CPIN, (R)- CPIN, (R,S)-CPIN, (S)-CPIAm, (R)-CPIAm, (R,S)-NPCN and (R,S)-NPAm. The (R,S)-designation indicates a racemic mixture of the indicated compound. One milliliter assays containing substrate and buffer were incubated at temperatures and for times indicated hereafter. Bioconversion broths were extracted and analyzed first by reverse-phase HPLC and then chiral HPLC as described hereafter. Analytical Procedures
The concentrations of nitrile, amide and acid products derived via microbial hydrolysis were measured by reverse-phase HPLC. Detection was by ultraviolet light absorbtion. A Du Pont Zorbax® C18 column employing a mobile phase of 70-75% methanol and 25-30% H2O acidified with 0.1 % H3PO4 was used.
Chromatographic identity and quantitation of nitriles and the resulting amide and acid products were determined by comparison with authentic standards.
Chiral HPLC for the separation of enantiomers was carried out with an α acid glycoprotein column obtained from Chromtech (Sweden). The mobile phases for separation of various enantiomers is summarized below.
Chiral HPLC Separation of Nitrile, Amide and Acid Enantiomers
Enantiomers Mobile Phase
CPIN, CPIAm, CPIA 95% 0.01 M Phosphate Buffer (pH 6.0):5% Ethanol NPCN, NPAm, NPAC 95% 0.01 M Phosphate Buffer (pH 5.6):5% Ethanol
The processes of this invention are illustrated by the following Examples. Examples 1 to 3 describe P. chlororaphis B23 hydrolysis of (S)-CPIAm at 28°C, 35°C and 50°C. Comparisons are provided for (R)-CPIAm hydrolysis at 28°C and 35°C (Comparison Tests 1 and 2).
Examples 4 to 7 describe the stereoselective hydrolysis of (S)-CPIN and (R,S)-CPIN at 28°C and 35°C, i.e., both (S)-CPIN and (R)-CPIN are hydrolyzed but more (S)-CPIN is converted to (S)-CPIAm. Again the stereospecific bioconversion of (S)-CPIAm to (S)-CPIA is demonstrated as only (S)-CPIA is generated. Comparisons are provided for (R)-CPIN hydrolysis at 28°C and 35°C (Comparison Tests 3 and 4).
Example 8 describes the stereospecific hydrolysis of (R,S)-NPAm to (S)- NPAC.
Example 10 describes Pseudomonas putida 5B-MGN-2P and P. chlororaphis B23 bioconversion of (R,S)-CPIN to (S)-CPIA. In this
2 microbe/1 step (R,S)-CPIN bioconversion only (S)-CPIA and (S)-CPIAm were detected as products.
Example 11 describes a biphasic (buffer/ organic solvent) bioconversion of (R,S)-CPIN to (S)-CPIA with both P. putida 5B-MGN-2P and P. chlororaphis B23. In this Example, the major product is (S)-CPIA but a small amount (0.5 μmole) of (R)-CPIAm was also generated. Examples 1 to 3 and Comparison Tests 1 and 2 Hydrolysis of (S)-CPIAm and (R)-CPIAm Pseudomonas chlororaphis B23 at
28°C, 35°C, and 50°C A 50 mg sample of frozen cell paste of Pseudomonas chlororaphis B23 was added to 1 mL of phosphate buffer (100 mM, pH value: 7.0) at room temperature. This was transferred to a vial containing 9.4 μmole of (S)-CPIAm or (R)-CPIAm. After incubation at 28°C or 35°C or 50°C for 24 or 48 h, the reactions were acidified with 3M H SO4 to pH 3. Six 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.
Incomplete recovery of (S)-CPIAm or (R)-CPIAm may be attributable to experimental error or adsorption of substrate to cells.
Tahle 1. S-CPIAm and R-CP1 Am Hvdrolvsis hv Pseudomonas chlororaphis B23 at 28°C. 35°C and 50°C
HPLC Analysis (μmole Recovered)
Reverse-Phase Chiral CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA
4.0 4.7 4.0 NDa 4.7 NDa
8.4 NDa-b NT0 NT^ NT0 NTC
4.0 3.0 NTC NTc Nτc NTc
4.6 4.1 4.6 NDa 4.1 NDa
.2 NDa'b NDa 8.2 NDa NDa>b
a ND - None detected.
" Data corrected for R-CPIA impurity in R-CPIAm starting material. c NT - Not tested.
Examples 4 to 7 and Comparison Tests 3 and 4 Hydrolysis of (S)-CPIN, (R)-CPIN and (R,S)-CPIN by Pseudomonas chlororaphis B23 at 28°C, 35°C A 50 mg sample of frozen cell paste of Pseudomonas chlororaphis B23 was added to 1 mL of phosphate buffer (100 mM, pH value: 7.0) or pyrophosphate buffer (5 mM, pH value: 7.5) at room temperature. This was transferred to a vial containing 10.4 μmole of either (S)-CPIN or (R)-CPIN or 20.7 μmole of (R,S)- CPIN. After incubation of 28°C and 35°C for 48 h and following the extraction protocol as in Examples 1 to 3, the composition of the extracted supernatant was determined by reverse phase HPLC and chiral HPLC. The results are shown in Table 2.
Incomplete recovery of (S)-CPIN or (R)-CPIN may be attributable to experimental error or adsorption of substrate to cells.
c Amount of product detected (21.6 μmole) slightly larger than amount of substrate (20.7 μmole) due to experimental error.
Example 8
Hydrolysis of (R,S)-NPAm by Pseudomonas chlororaphis B23 A 12.5 mg sample of frozen cell paste of Pseudomonas chlororaphis B23 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 incubation at 22°C for 48 h, the reaction was acidified with 3M H SO4 to pH 3. Four volumes of methylene chloride were added and the suspension was agitated for 30 min. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen and the residue was redissolved 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 3.
Incomplete recovery of (R,S)-NPAm may be attributable to experimental error or adsorption of substrate to cells.
Table 3. R.S-NPAm Hydrolysis by Pseudomonas chlororaphis B23
HPLC Analysis (μmole Recovered)
Example Substrate Reverse-Phase Chiral
No. (μmole Added) NPAm NPAC S-NPAm R-NPAm S-NPAC R-NPAC 8 R,S-NPAm
(1.0) 0.33 0.30 0.01 0.32 0.30 Trace
Example 9 Hydrolysis of (R,S)-NPCN by Pseudomonas chlororaphis B23 A 12.5 mg sample of frozen cell paste of Pseudomonas chlororaphis B23 was added to 1 mL of phosphate buffer (100 mM, pH value: 7.2) at room temperature. Then, 1 μmole of (R,S)-NPCN in 40 μL of dimethylsulfoxide was added. Following the incubation and extraction protocol in Example 8, the composition of the extracted supernatant was determined by reverse phase HPLC and chiral HPLC. The results are shown in Table 4.
Incomplete recovery of (R,S)-NPAm may be attributable to experimental error or adsorption of substrate to cells. Tahle 4. R.S-NPCN Hydrolysis by Pseudomonas chlororaphis B23
HPLC Analysis (μmole Recovered)
Example Substrate Reverse Phase Chiral No. (μmole Added) NPCN NPAm NPAC S-NPCN R-NPCN S-NPAm R-NPAm S-NPAC R-NPAC
9 R,S-NPCN (1.0) 0.05 0.14 0.21 ND3 0.05 0.04 0.10 0.21 NDa a ND - None detected.
Example 10
Hydrolysis of (R,S)-CPIN by Pseudomonas putida 5B-MGN-2P and
Pseudomonas chlororaphis B23 Fifty milligram samples of frozen cell pastes of Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23 were added to 1 mL of pyrophosphate buffer (5 mM, pH value: 7.5) at room temperature. This was transferred to a vial containing 20.7 μmole (R,S)-CPIN. After incubation at 35°C for 48 h and following the extraction protocol in Examples 1 to 3, the composition of the extracted supernatant was determined by reverse -phase HPLC and chiral HPLC. The results are shown in Table 5.
Table 5. R.S-CPIN Hydrolysis by Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23
HPLC Analysis (μmole Recovered)
Example Substrate Reverse Phase Chiral No. (μmole Added) CPIN CPIAm CPIA S-CPIN R-CPIN S-CPIAm R-CPIAm S-CPIA R-CPIA
10 R.S-CPIN (20.7) 13.6 3.4 0.9 5.2 8.4 3.4 NDa 0.9 NDa
a ND - None detected.
Example 1 1
Hydrolysis of (R,S)-CPIN by Pseudomonas putida 5B-MGN-2P and
Pseudomonas chlororaphis B23 in an Aqueous Buffer/Hydrocarbon Mixture
Fifty milligram samples of frozen cell pastes of Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23 were added to 0.8 mL of pyrophosphate buffer (5 mM, pH value: 7.5) at room temperature. Then,
20.7 μmole of (R,S)-CPIN in Soltrol® 220, a mixture of C13-C17 hydrocarbons
(Phillips 66 Co.) was added. After incubation at 35°C for 48 h and following the extraction protocol in Examples 1 to 3, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC. The results are shown in Table 6.
Table 6. R.S-CPIN Hydrolysis by Pseudomonas putida 5B-MGN-2P and Pseudomonas chlororaphis B23 in Buffer/Soltrol®
HPLC Analysis (μmole Recovered)
Example Substrate Reverse Phase Chiral No. (μmole Added) CPIN CPIAm CPIA S-CPIN R-CPIN S-CPIAm R-CPIAm S-CPIA R-CPIA
1 1 R.S-CPIN (20.7) 15.5 0.5 4.5 5.3 10.2 0.2 0.3 4.5 NDa
a ND - None detected.

Claims

3019CLAIMS What is claimed is:
1. A method for converting an (S)-amide, or stereospecifically converting a mixture of (R)- and (S)-amides, of Formula I
R1
I
A— CH— C— NH
II 2 o
I
wherein: A is selected from the group consisting of:
A-l A-2
R1 is CrC4 alkyl;
R2 is H; F; Cl; Br; OH; CrC3 alkyl; OCF2H; or H2C=C(CH3)CH2NH; and
R3 is H; F; Cl; Br; OH; CrC3 alkyl; or CrC3 alkoxy;
to the corresponding enantiomeric (S)-carboxylic acid comprising contacting said amide with Pseudomonas chlororaphis B23 in a solvent and recovering the enantiomeric (S)-carboxylic acid.
2. A method according to Claim 1 wherein A is A-l, R1 is CH(CH3) , R2 is Cl, and the amide is (S)-amide.
3. A method according to Claim 1 wherein A is A-2, R1 is methyl, R- is methoxy and the amide is racemic.
4. A method according to Claim 1 wherein the solvent is a biphas mixture of aqueous buffer and organic solvent.
5. A method for converting (S)-nitrile, or stereospecifically converting a mixture of (R)- and (S)-nitriles, of the formula R1 I ■CH- ■CN
wherein: A is selected from the group consisting of:
A-l A-2
R1 is CrC4 alkyl;
R2 is H; F; Cl; Br; OH; C,-C3 alkyl; OCF2H; or H2C=C(CH3)CH2NH; and
R3 is H; F; Cl; Br; OH; CrC3 alkyl; or CrC3 alkoxy;
to the enantiomeric (S)-carboxylic acid, through the corresponding amide, comprising contacting said nitrile with Pseudomonas chlororaphia B23 in a solvent and recovering the enantiomeric (S)-carboxylic acid.
6. A method according to Claim 5 wherein the nitrile is racemic 2-(6- methoxy-2-naphthyl)propionitrile or racemic 2-(4-chlorophenyl)-3- methylbutyronitrile.
7. A method according to Claim 5 comprising isolation of the intermediate amide.
8. A method for converting (S)-nitrile, or stereospecifically converting a mixture of (R)- and (S)-nitriles, of Formula II,
II
wherein
R1 is C,-C4 alkyl,; and
R2 is H; F; Cl; Br; OH; CrC3 alkyl; OCF2H; or H2C=C(CH3)CH2NH; to the corresponding enantiomeric (S)-carboxylic acid comprising contacting said nitrile with a mixture of Pseudomonas chlororaphis B23 and Pseudomonas putida
5B-MGN-2p in a solvent and recovering the enantiomeric (S)-carboxylic acid.
9. A method according to Claim 8 wherein R1 is CH(CH3)2, R2 is Cl and the nitrile is racemic.
10. A method according to Claim 8 wherein the solvent is a biphasic mixture of aqueous buffer and organic solvent.
11. A method for converting (S)-nitrile, or stereospecifically converting a mixture of (R)- and (S)-nitriles, of Formula II
II
wherein
R1 is CrC4 alkyl; and R2 is H; F; Cl; Br; OH; C,-C3 alkyl; OCF2H; or H2C=C(CH3)CH2NH;
to the corresponding enantiomeric (S)-amide comprising contacting said nitrile with a Pseudomonas putida 5B-MGN-2p in a biphasic mixture of aqueous buffer and organic solvent and recovering the enantiomeric (S)-amide.
EP93917294A 1992-09-21 1993-07-23 A process for the preparation of enantiomeric 2-alkanoic acids Withdrawn EP0662149A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US94818592A 1992-09-21 1992-09-21
US948185 1992-09-21
PCT/US1993/006821 WO1994006930A1 (en) 1992-09-21 1993-07-23 A process for the preparation of enantiomeric 2-alkanoic acids

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5991879A (en) * 1982-11-16 1984-05-26 Hideaki Yamada Method for cultivating bacterium of genus pseudomonas
JP2907479B2 (en) * 1990-02-28 1999-06-21 輝彦 別府 Genetic DNA encoding a polypeptide having nitrile hydratase activity, transformant containing the same, and method for producing amides
DK161690D0 (en) * 1990-07-05 1990-07-05 Novo Nordisk As PROCEDURE FOR PREPARING ENANTIOMERIC COMPOUNDS
ES2085487T3 (en) * 1990-09-20 1996-06-01 Du Pont A PROCEDURE FOR THE PREPARATION OF 2-ALCANOIC ENANTIOMERIC ACIDS.

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* Cited by examiner, † Cited by third party
Title
See references of WO9406930A1 *

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WO1994006930A1 (en) 1994-03-31

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