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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• 1993
  • 1993-07-23 EP EP93917294A patent/EP0662149A1/de not_active Withdrawn
  • 1993-07-23 JP JP6508066A patent/JPH08501219A/ja active Pending
  • 1993-07-23 WO PCT/US1993/006821 patent/WO1994006930A1/en not_active Application Discontinuation

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