WO1997010355A1 - Processus de production d'amide d'acide actif sur le plan optique - Google Patents

Processus de production d'amide d'acide actif sur le plan optique Download PDF

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Publication number
WO1997010355A1
WO1997010355A1 PCT/JP1995/001823 JP9501823W WO9710355A1 WO 1997010355 A1 WO1997010355 A1 WO 1997010355A1 JP 9501823 W JP9501823 W JP 9501823W WO 9710355 A1 WO9710355 A1 WO 9710355A1
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ferm
group
rhodococcus
compound
nitrile
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PCT/JP1995/001823
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English (en)
Japanese (ja)
Inventor
Kazumasa Otsubo
Keizou Yamamoto
Wataru Takahashi
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Nagase & Company, Ltd.
Asahi Chemical Industry Co., Ltd.
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Priority to PCT/JP1995/001823 priority Critical patent/WO1997010355A1/fr
Publication of WO1997010355A1 publication Critical patent/WO1997010355A1/fr

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    • 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

Definitions

  • the present invention relates to a method for producing an acid amide compound having a high optical purity from a racemate or a corresponding nitrile compound having a low optical purity by using a living organism or a processed product thereof. More specifically, it is useful as an intermediate for producing a drug substance such as a drug for treating arrhythmia or a drug for treating arteriosclerosis, and as an intermediate for producing an amine having high optical purity which is important as an optical resolving agent.
  • Useful acid amide compounds with high optical purity use specific microorganisms that have nitrile hydratase activity, but have very low nitrilase and amidase activities and very low racemase activity, or processed products thereof. Accordingly, the present invention relates to a method for producing from a racemate or a corresponding nitrile compound having low optical purity. Further, the present invention relates to a novel microorganism useful for the above method.
  • Methods for producing an optically active acid amide include a method using an optically active resolving agent, a method using asymmetric synthesis, and a method for treating an organism or a processed product thereof (crushed bacterial cells, crude or purified enzyme active substances). Etc.) are known.
  • the method using an optically active resolving agent is relatively expensive since the optical resolving agent used is relatively expensive, and the method used several times to obtain an optical purity of 98% ee or more generally required for pharmaceuticals.
  • the asymmetric synthesis method is disadvantageous in that the steps are complicated and many steps must be performed, and the yield and optical purity are low.
  • Racemic 2-aryl alkane nitriles are acted on by microorganisms acting on Pseudomonas ⁇ , celiacia ⁇ or moraxella ⁇ to produce an optically active amide having a hydroxyl group or a lower alkyl at the ⁇ -position.
  • Production method PC International Publication W092 / 052755): This method has low amide generation activity of all microorganisms used, and the nitrile concentration used in the reaction is about 0. It is industrially disadvantageous in that it is as low as 2 to 0.8%.
  • the optical purity of the generated optically active amide is extremely low, about 30% e.e., and its industrial value as an optically active amide is low.
  • the present inventors have conducted intensive studies on a method for producing an acid amide using a microorganism or a processed product thereof, which has a high acid amide generation rate and high optical purity.
  • the racemic or low-purity L-butyryl compound represented by the following general formula (I) can be converted into the optically pure diacid amide compound represented by the following general formula (II)
  • the nitrilase activity to convert nitril to the corresponding carboxylic acid and the amidase activity to convert the generated acid amide to the corresponding carboxylic acid are extremely weak.
  • these microorganisms can convert even a high nitrile concentration into an acid amide, and can provide a high acid amide production rate and a high optical purity.
  • microorganisms convert only one optical isomer of the nitrile compound to the acid amide and not the other, but the recovery and re-racemization of the other optically active nitrile compound is not possible. It is industrially advantageous because it is easy and can be reused as a starting material.
  • a racemic or nitrile compound having a low optical purity represented by the following general formula (I) can be converted into an acid amide having a high optical purity represented by the following general formula ( ⁇ ) the compound was Kotogawa or ⁇ efficiently be produced '0
  • Corynebacterium sp. A68 (FERM BP— 5215), Rhodococcus sp KPO-2028 (FERM BP— 5216), Rhodococcus sp. PP-25 (FERM BP— 5217), Rhodococcus sp. PP-121 (FERM) BP-5218), Rhodococcus 'Maris BP-479-9 (FERM BP-5219), Bacillus' subtilis PP-116-15 (FERM BP-5220), Brevibacterium sp PP-133-7 (FERM BP- 52 21),
  • the present invention provides a novel microorganism, Rhodococcus maris BP-479-9 (FERM BP-5219), which is particularly useful in performing the above method.
  • R 2 represents water purple or a ⁇ ( ⁇ alkyl group.
  • R 2 is a CC * alkyl group, the group may be present in any of the ortho, meta and para positions.
  • the ⁇ C beta alkyl group represents a straight ⁇ or branched ⁇ alkyl group having 1 to 6 carbon atoms, e.g., methyl, Echiru, .eta. propyl, Isoburopiru, .eta. butyl, t- butyl group , Isobutyl, n-pentyl, neopentyl, and ⁇ -hexyl group.
  • a (: 4 alkyl group represents a directly-sold or branched alkyl group having 1 to 4 coal atoms.
  • 1-3 pieces of Fuyuniru groups S substitution has been C, and -C 3 alkyl group represents methyl substituted with 1-3 Fuyuniru group, Echiru, a n- propyl or isopropyl group, such as benzyl And benzhydrinole, trityl, phenethyl, and vinyl group.
  • the C 3 -C 8 cycloalkyl group a cycloalkyl group from 3 to eight carbon atoms, i.e., Shikurobu port pills, cyclobutyl, cyclopentyl, key sill cyclohexane, butyl or Shikurookuchiru group cyclohexylene.
  • the nitrile compound of the general formula (I) wherein R 2 is water purple is converted.
  • R 1 is ( ⁇ 4 alkyl group, methyl or ethyl group substituted with 1 or 2 phenyl groups, or C S -C 7 cycloalkyl group
  • R 2 is hydrogen
  • ( ⁇ to (: 4 alkyl groups are as defined above, and C 5 to C 7 cycloalkyl groups are cyclopentyl and cycloalkyl) Represents a hexyl or cycloheptyl group.
  • R 1 is ( ⁇ to (: 4 alkyl group, benzyl group, benzhydryl group, or C 5 to C 7 cycloalkyl group
  • R 2 is hydrogen
  • the nitrile compound of general formula (I) is converted to the corresponding acid amide according to the method of the present invention.
  • R 1 is The nitrile compound represented by the above general formula (I), which is an alkyl group and R 2 is hydrogen, is converted into a corresponding acid amide according to the method of the present invention.
  • the nitrile compound of the above general formula (I) wherein R 1 is an isopropyl or t-butyl group and R 2 is hydrogen is converted to the corresponding acid amide according to the method of the present invention. Convert to
  • the nitrile compound represented by the above general formula (I), which is a starting material in the method of the present invention, can be obtained, for example, from Journal of American Chemical Society (J. Am. Chen. So), Vol. 79 Pp. 3467-3469 (1957); JP-A-51-77044; JP-A-51-122636; Synthesis. (1986) and the like. In addition, starting materials available from commercial products may be purchased and used.
  • the optical purity is low in one of the (S) -form or the (R) -form. Mixtures can also be used.
  • the acid amide represented by the general formula (II) having high optical purity produced by the method of the present invention is an amine having high optical purity which is important as an intermediate for producing a pharmaceutical substance or as an optical resolving agent.
  • Useful as an intermediate for the production of Representative examples of these acid amides ( ⁇ ) include, for example, (R) -body or (S) -body, or (+)-body or (one) -body:
  • the present invention relates to a method for converting an optically inactive nitrile into a corresponding optically active acid amide by utilizing a microorganism or a processed product thereof. is important.
  • nitril is converted to an acid amide by using any of these strains or a processed product thereof.
  • the treated product is preferably used in the method of the present invention.
  • the processed product is used in the method of the present invention.
  • Maris BP-479-9 (FERM BP-5219), or
  • the processed product is used in the method of the present invention.
  • Rhodococcus' maris BP-479-9 (FERM BP-5219) or a processed product thereof is used in the method of the present invention.
  • mutants obtained by treating these strains with a known mutagen such as N-methyl- ⁇ '-nitronitrosoguanidine (NTG) or gene engineering can be used.
  • NTG N-methyl- ⁇ '-nitronitrosoguanidine
  • Strains modified by a genetic method can also be used in the method of the present invention.
  • Corynepacterium 'SP68 (FERM BP-5215) is a known strain described in JP-A-3-19695 (for the mycological K, refer to the same publication). This strain was introduced in 1988 On August 22, he was originally deposited with the National Institute of Biotechnology and Industrial Technology, and was transferred to the International Depositary on September 1, 1995, and was assigned the indicated accession number.
  • Rhodococcus sp. KP0-2028 (FERM BP-5216), Rhodococcus sp. PP-25 (FERM BP-5217), Rhodococcus sp.
  • PP-121 (FERM BP-5218), Rhodococcus maris BP-479-9 (FERM BP-5219), Bacillus subtilis PP-116-15 (FERM BP-5220), and Brevibacterium 'SP PP-133-7 (FERM BP-5221) are all new. This is a new strain isolated as nitrile bacteria from soil. All of these strains were originally deposited on November 2, 1993 at the Institute of Life Science and Industrial Technology, National Institute of Advanced Industrial Science and Technology, and transferred to an international deposit on September 1, 1995, and given the respective accession numbers. Have been.
  • Rhodococcus' SP KPO-2028 (FERM BP-5216) Cell shape
  • Rhodococcus' Maris BP-479-9 (FERM BP-5219) cell shape; rod-like
  • the microorganism used in the method of the present invention is cultured. Cultivation of the microorganism can be performed according to a known method.
  • the medium used may be a medium containing the usual nutrients for the usual microorganisms, ie coal, nitrogen, mineral nutrients and the like.
  • a carbon source glucose, glycerin, ethanol, sucrose, glutamic acid, acetic acid, citric acid, fumaric acid and the like can be used.
  • an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonia, urea, or an organic nitrogen source such as bran extract, malt extract, peptone, meat extract, etc. can be used. .
  • Phosphoric acid, magnesium, potassium, iron, cobalt, manganese, lanthanum and the like can be used as inorganic nutrients. These nutrient sources may be used in appropriate combination. Further, as a substance for increasing the reaction activity of the microorganism, a cyano compound such as acetonitrile and isobutyronitrile and an amide compound such as cabrolactam may be added.
  • the pH of the medium may be in the range of 5-10, preferably in the range of 6-8.
  • the culture temperature is from 18 to 50 ° C, preferably from 25 to 40 ° C.
  • the culturing time varies depending on the culturing conditions, but is usually 1 to 4 days, and culturing may be performed until the activity is maximized.
  • the above-mentioned processed microorganism is a nitrile hydratase activity capable of optically selectively hydrating a nitrile group of a racemic or nitrile compound (I) having a low optical purity. If it has the property, it means that it may be in any form. Its form differs depending on whether the enzyme activity is mainly present in the cells or extracellularly.
  • the enzyme activity is present in the cells, the culture of the above microorganism, the cells collected therefrom, the treated cells (eg, the crushed cells, the separation / extraction from these cells) Enzymatically active substances at various purification stages), the cells immobilized on a carrier by a known appropriate method, or the treated cells.
  • the activity is present outside the cells, the culture of the microorganism, the culture broth, the enzymatic active separated and extracted from the culture broth, or immobilized on a carrier by a known suitable method Refers to the activated enzyme and the like.
  • the enzyme active substance can be isolated and purified from the microorganism using a known enzyme purification means.
  • the enzyme is an intracellular enzyme
  • the cells are collected by, for example, centrifugation of a microorganism culture, and the cells are disrupted by mechanical means such as ultrasonic treatment or a dyno mill.
  • a crude enzyme solution is obtained by removing solid matter such as cell debris by centrifugation or the like, and the crude enzyme solution is subjected to ultracentrifugation separation, salting out, organic solvent precipitation, adsorption chromatography, Purify by ion-exchange chromatography, gel chromatography, reverse-phase chromatography, etc.
  • a stabilizer for preventing inactivation of the enzyme during the enzyme purification a commonly used stabilizer or about 1 to 10 O mM of isobutylamide may be added.
  • the microorganism obtained as described above or a treated product thereof is usually mixed with a suitable reaction medium in a suitable reaction medium. It may be brought into contact with racemate or nitrile (I) having low optical purity under the conditions.
  • Suitable reaction media include water, aqueous media such as buffers or hair fluids, water Mixed medium of a water-soluble organic solvent and a water-soluble organic solvent such as methanol, dimethyl sulfoxide or acetone, and a two-phase medium comprising an aqueous medium and a water-insoluble organic solvent such as hexane, ethyl acetate, ethyl ether or toluene (Including water-insoluble solvents saturated with water).
  • a suitable surfactant may be added to the reaction medium in an amount of about 0.01 to 2%.
  • Racemic or nitrile (I) of low optical purity may be added to the reaction medium in powder or liquid form or dissolved in a suitable solvent.
  • concentration of nitrile (I) added to the reaction solution is about 0.01 to 70% by weight, preferably about 1,0 to 50% by weight.
  • Nitril (I) need not be completely dissolved in the reaction medium.
  • the concentration of the microorganism or the processed product used in the above reaction varies depending on the degree of nitrile hydratase activity.
  • concentration of the cells in the reaction solution may be generally in the range of 0.05 to 20% by weight.
  • the treated product is added in such an amount that an activity equivalent to that in the case of using a microbial cell is obtained.
  • the reaction temperature may be about 5-60 ° (preferably, about 15-50 ° C.)
  • the reaction pH may be about 4-11, preferably about 7-10.
  • the reaction time varies depending on the reaction conditions, but is usually in the range of 100 to 100 hours.
  • the progress of the reaction is monitored by high performance liquid chromatography (HPLC), etc., and the optical activity generated
  • HPLC high performance liquid chromatography
  • the reaction is preferably terminated when the optical purity of the acid amide ( ⁇ ) is higher than the desired optical purity, preferably at least 80% ee, particularly preferably at least 90% ee.
  • a new nitrile (I) may be added to the reaction solution so that the nitrile (I) concentration is maintained within the above range
  • the optically active acid amide ( ⁇ ) can be isolated from the reaction mixture by various methods, for example, hexane or azo acid And a method of separation by chromatography on a silica gel column. If the generated optically active acid amide ( ⁇ ) has low solubility in the reaction medium, crystals precipitate during the reaction, and this can be recovered by centrifugation or the like.
  • the optical purity can be increased by the operation of preferential crystallization.
  • the enzyme nitrile hydratase of the microorganism used in the present method selectively acts on only one isomer of racemic nitrile (I) to generate an optically active acid amide ( ⁇ ). It is based on that.
  • these microorganisms have extremely low nitrilase activity of converting nitrile to acid and amidase activity of converting acid amide to acid, optically active acid amide ( ⁇ ) can be obtained without generation of acid.
  • the racemase activity of converting an optically active acid amide ( ⁇ ) into a racemate is extremely low, the acid amide ( ⁇ ) can be recovered without lowering the optical purity.
  • an optically active acid amide ( ⁇ ) When an optically active acid amide ( ⁇ ) is produced by the method of the present invention, an unconverted optically active nitrile compound remains. It can be recovered by simple operations such as extraction, centrifugation, and column separation. The recovered optically active nitrile compound itself can be converted into an optically active amine or optically active carboxylic acid which is useful as a pharmaceutical or an optical resolving agent and used. Further, the recovered optically active nitrile compound can be easily racemized by treating it with, for example, ammonia or sodium hydroxide in a solvent such as methanol. This racemic nitrile compound (I) can be reused in the method of the present invention. Therefore, if the purpose is to produce only the optically active acid amide ( ⁇ ), the desired optically active acid amide ( ⁇ ) can be produced in high yield. Can be
  • the first example is that an optically active acid amide (D) is hydrogenated under acidic conditions.
  • the desired optically active amine (III) is obtained by performing steric retention reduction with sodium borohydride.
  • This a reaction is carried out in an inert solvent.
  • an aprotic solvent such as dimethyl sulfoxide (DMSO), diglyme, ethylene glycol dimethyl ether, tetrahydrofuran (THF), or 1,4-dioxane is preferable.
  • Examples of the acid used in the primary reaction include, for example, inorganic acids such as hydrobromic acid, hydrochloric acid, sulfuric acid, and nitric acid; organic acids such as sulfuric acid, P-toluenesulfonic acid, and methanesulfonic acid; Ether complex, Al chloride Emit Lewis acids such as titanium tetrachloride.
  • chloroic acid which provides a high yield and is easy to handle, is a preferred solvent.
  • Use of acid :! Is about 1 to 20 molar equivalents, preferably about 3 to 10 molar equivalents, based on the optically active acid amide ( ⁇ ).
  • the amount of sodium borohydride used is about 1 to 20 mole equivalents, preferably about 3 to 10 mole equivalents, based on the acid amide ( ⁇ ).
  • the reaction is usually carried out at a temperature of about 20 to 200 ° C, preferably at a temperature of about 50 to 180 ° C to control side reactions.
  • the optically active amine (m) obtained by this general reaction can be recovered from the reaction solution, for example, as follows. That is, the reaction solution is made strongly acidic with an aqueous hydrochloric acid solution or the like, and this solution is washed with a water-immiscible organic solvent such as dichloromethane or chloroform. Next, the aqueous layer is made strongly alkaline, and extracted with a water-immiscible organic solvent such as dichloromethane and chloroform, and the solvent of the extract is distilled off to obtain an optically active amine (m).
  • the desired optical activity is obtained by dehydrating the optically active acid amide ( ⁇ ) while maintaining the steric state and hydrogenating the obtained optically active nitrile (IV) while maintaining the steric state.
  • This is a method to obtain a good amine (m).
  • the steric retention dehydration reaction of an optically active acid amide ( ⁇ ) is carried out by treating with a dehydrating agent in an inert solvent.
  • Examples of the dehydrating agent to be used include a dehydrating agent such as thionyl chloride, anhydrous dianhydride and diphosphorus pentoxide, and an azeotropic dehydrating agent such as sulfuric acid and P-toluenesulfonic acid.
  • thionyl chloride which provides a high yield and can control racemization, is a preferred dehydrating agent.
  • the amount of thionyl chloride to be used is about 1 to 20 equivalents, preferably about 1 to 3 equivalents, relative to the optically active acid amide ( ⁇ ). This reaction can be carried out at a temperature of about 50-150 ° C, In order to control the temperature, it is preferable to carry out at a temperature of about 70 to 100.
  • Examples of the inert solvent include ethyl acetate and toluene.
  • the optically active nitrile (IV) obtained by this dehydration reaction is recovered from the reaction solution by washing the reaction solution with a weak alkaline aqueous solution, water, or a saline solution, and then distilling off the solvent. be able to.
  • the steric retention hydrogenation of the thus obtained optically active nitrile (IV) is carried out by hydrogenation in an inert solvent in the presence of a catalyst.
  • a catalyst As the source catalyst, a homogeneous catalyst or a heterogeneous catalyst can be used.
  • Raney nickel is preferably used as a heterogeneous catalyst which can be added in a simple and high yield with water purple.
  • the amount of the catalyst used is about 1 to 50% (wtZwt ratio), preferably about 10 to 30% Ot / wt ratio, based on the optically active nitrile (IV).
  • inert solvent examples include alcohol solvents such as methanol, ethanol and isopropanol, and ether solvents such as diglyme, tetrahydrofuran (THF) and 1,4-dioxane.
  • alcohol solvents such as methanol, ethanol and isopropanol
  • ether solvents such as diglyme, tetrahydrofuran (THF) and 1,4-dioxane.
  • ethanol or isobrovanol are preferred solvents.
  • the hydrogenation is preferably carried out at room temperature to 120, preferably at room temperature to 80.
  • the hydrogen pressure may be between 1 and 100 kgZcm 2 'G, preferably between 5 and 800 kgZcm 2 ' G.
  • aqueous ammonia to the reaction solution in order to suppress the generation of by-products such as dialkylamine.
  • the amount of ammonia to be added may be about 0.01 to 1.0 molar equivalent relative to the optically active nitrile (IV), and about 0.1 to 0.5 to suppress racemization. It is preferably a molar equivalent.
  • the obtained optically active amine (II) can be recovered from the reaction solution in the same manner as described above.
  • the optical purity of the starting materials, intermediate products and target products is determined, for example, by Chiral Cell 0D-R, Chiral Cell 0J, Chiral AGP, Crown Pack (CBOfNPAK) CR (Daicel Chemical Industries) , Ceramosper Chiral RU-1 ( ⁇ seido), Sumichiral (SUMICHIBAL) 0 A (Sumitomo Chemical Analysis Center), Opti-pak TA (Waters), etc. It can be measured by the HP LC analysis used.
  • the percentages in the following examples represent weight Z capacity O / v)% unless otherwise specified.
  • the production rate and optical purity of the optically active acid amide were measured by high performance liquid chromatography (HPLC). The yield was calculated based on the total amount of nitrile (racemic) used in the reaction (therefore, the maximum value was 50%). Optical purity was indicated by the enantiomeric excess.
  • the culture was centrifuged to collect the cells, and the cells were dissolved in 40 ml of a 0.01 M phosphate solution (pH 7.5). Then, 20 g of oily 2-fu: L 2-loo 3-methylbutyronitrile was added, 35. The reaction was carried out for 40 hours while stirring with C. When a part of the reaction solution was analyzed under the following HPLC analysis conditions, the yield of 2-fluoro-3-methylbutylamide was 34%, and the optical purity of the S-isomer was 92.0% ee. . No production of 2-phenyl-3-methylbutyric acid was observed.
  • Retention time 3.9 minutes for S-form, 5.0 minutes for R-form.
  • the other organisms listed in Table 1 below were allowed to act on 2-fluoro-3-methylbutyronitrile in the same manner as described above.
  • Table 1 shows the production rate and optical purity of 2-phenyl-3-methylbutylamide measured by HP LC.
  • Rhodococcus * Maris BP-479-9 strain (FERM BP-5219) was cultured under the same culture conditions as in Example 1. Then, the cells were collected by centrifugation, washed, and suspended in 10 ml of distilled water. To this, 10 Otng of 2-cyclohexyl-2-phenylacetonitrile was added and reacted at 30 for 24 hours. Analysis by HPLC revealed that (S) -2-cyclohexyl-2-phenylacetamide was produced at a production rate of 18%, and the optical purity of the S-isomer was 8 l% e.e. Incidentally, 2-cyclohexyl-2-phenylacetic acid was not produced.
  • Retention time S body 9.5 minutes, R body 12.0 minutes
  • the cells were collected by centrifugation, and the cells were suspended in a mixed solution of 90 ml of 0.05 M phosphate buffer (pH 7.5) and 10 ml of dimethyl sulfoxide (DMSO). Next, 0.1 lg of 2,3,3-triphenylpropionitrile was added, and the mixture was reacted at 30 parts for 48 hours. After completion of the reaction, the reaction mixture was analyzed by HPLC under the following conditions. The title compound was produced at a production rate of 11%, and the optical purity was 82% ee. It turned out to be.
  • Retention time R body 7.7 minutes, S body 8.6 minutes
  • Rhodococcus maris BP-479-9 strain (FERM BP-5219) was cultured in a 2 L liquid medium in the same manner as in Example 1, and then the culture was centrifuged to collect 26 g of cells. This is washed with 30 mM potassium phosphate buffer containing 40mM Isobuchiruami de (P H 7.3), and suspended in the same buffer 60 ml. Then, the cells were sonicated at 9 KHz under ice-cooling for about 10 minutes to disrupt the cells. This cell lysate was centrifuged at 1800 Orpm for 20 minutes to remove cell debris and obtain a cell extract. The specific activity of nitrile hydratase in the extract was 0.126 U / mg.
  • the extract was dialyzed against 2 L of the same buffer as above, and the resulting dialysate was placed in a column of DEAE-cellulose, and a 5 OmM potassium phosphate buffer containing 0 to 0.5 M sodium chloride was added. Elution with linear (pH 7.0) gradient did. The fractions containing the enzymatic activity were collected, 15% ammonium sulfate was added, and the mixture was placed in a column of Phenyl Sepharose CL-14B, followed by 5 OmM potassium phosphate buffer containing 4 OmM isobutylamide (pH 7.3). Eluted. Fractions containing the enzyme activity were collected and subjected to humidification with 60% ammonium sulfate saturation. The obtained precipitate was dissolved in a small amount of 5 OmM potassium phosphate buffer (pH 7.3) and dialyzed with the same buffer to obtain purified nitrogen.
  • the method for measuring the sex and fermentation activities of the purified enzyme is as follows.
  • SDS-polyacrylamide gel electrophoresis detected two submissions with molecular weights of 24000 and 25100.
  • this enzyme is a nitrile rehydratase that converts a nitrile compound to an acid amide compound, and is a nitrile compound having 2-phenyl-3-methylbutyronitrile and its analogous structure. It is an enzyme that has an excellent effect of optically hydrating A.
  • Retention time R body 7.7 minutes, S body 8.6 minutes
  • an acid amide compound having a high optical purity which is useful as a pharmaceutical intermediate or an intermediate for an optical resolving agent, can be efficiently converted from an inexpensive raw material such as a racemate or a nitrile compound having a low optical purity.
  • an inexpensive raw material such as a racemate or a nitrile compound having a low optical purity.
  • it can be produced with high purity, and this method is extremely useful in industry.

Abstract

Cette invention concerne un procédé de production d'amide d'acide actif sur le plan optique à partir d'un composé de nitrile correspondant et inactif sur le plan optique, au moyen d'un micro-organisme spécifique qui peut avoir été préalablement traité de manière adéquate. Ce procédé permet d'obtenir un composé d'amide d'acide d'une grande pureté optique, lequel composé peut être utilisé en qualité de produit intermédiaire dans la fabrication de médicaments ou d'un agent de résolution optique devant être produits de manière efficace et avec une grande pureté à partir d'un composé de nitrile peu coûteux racémique ou d'une faible pureté optique.
PCT/JP1995/001823 1995-09-13 1995-09-13 Processus de production d'amide d'acide actif sur le plan optique WO1997010355A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001211892A (ja) * 2000-02-03 2001-08-07 Asahi Kasei Corp グリシンの微生物学的製造法
JP2001258586A (ja) * 2000-03-24 2001-09-25 Asahi Kasei Corp アンモニアの反応分離を利用したグリシンの微生物学的製造方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0319695A (ja) * 1989-06-16 1991-01-28 Asahi Chem Ind Co Ltd トランス―４―シアノシクロヘキサンカルボン酸アミドの製造法およびそれに用いる酵素
JPH04222591A (ja) * 1990-12-20 1992-08-12 Nitto Chem Ind Co Ltd Ｓ−（＋）−マンデルアミドおよびその誘導体の製造法
JPH05252990A (ja) * 1992-03-12 1993-10-05 Sumitomo Chem Co Ltd 光学活性なニトリルおよび光学活性なアミドの製造方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0319695A (ja) * 1989-06-16 1991-01-28 Asahi Chem Ind Co Ltd トランス―４―シアノシクロヘキサンカルボン酸アミドの製造法およびそれに用いる酵素
JPH04222591A (ja) * 1990-12-20 1992-08-12 Nitto Chem Ind Co Ltd Ｓ−（＋）−マンデルアミドおよびその誘導体の製造法
JPH05252990A (ja) * 1992-03-12 1993-10-05 Sumitomo Chem Co Ltd 光学活性なニトリルおよび光学活性なアミドの製造方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001211892A (ja) * 2000-02-03 2001-08-07 Asahi Kasei Corp グリシンの微生物学的製造法
JP4560164B2 (ja) * 2000-02-03 2010-10-13 旭化成ケミカルズ株式会社 グリシンの微生物学的製造法
JP2001258586A (ja) * 2000-03-24 2001-09-25 Asahi Kasei Corp アンモニアの反応分離を利用したグリシンの微生物学的製造方法
JP4497638B2 (ja) * 2000-03-24 2010-07-07 旭化成ケミカルズ株式会社 アンモニアの反応分離を利用したグリシンの微生物学的製造方法

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