FR2566425A1 - PROCESS FOR THE PRODUCTION OF 2- (PHENYL SUBSTITUTED) PROPIONIC ACID BY MICROORGANISM - Google Patents

Abstract

A PROCESS FOR THE PRODUCTION OF 2- (PHENYL SUBSTITUTE) PROPIONIC ACID, INCLUDING THE CULTURE OF A MICRO-ORGANISM BELONGING TO THE GENUS OF PSEUDOMONAS AND HAVING TRANSFORMATION ACTIVITY OF 1- (PHENYL SUBSTITUE) -1- (PHENYL SUBSTITUE) -1- (PHENYL SUBSTITUE) -1- IN 2- (SUBSTITUTE PHENYL) PROPIONIC ACID IN A CULTURE MEDIA CONTAINING 1- (SUBSTITUTE PHENYL) -1-PHENYLETHANE AND THE RECOVERY OF 2- (SUBSTITUTE PHENYL) PROPIONIC ACID FORMED IN THIS CULTIVATION MATERIAL.

Description

The present invention relates to a process for the production of 2- (phenyl

  substituted) propionic acid and in more detail a process for the microbiological production of 2- (substituted phenyl) propionic acid from 1- (phenyl)

substituted) 1-phenylethane.

  2- (substituted phenyl) propionic acid has anti-phlogistic activity and

  analgesic activity for humans and animals, and in particular 2- (4-

  isobutylphenyl) propionic acid has been generally used as an excellent anti-inflammatory agent. For its production, only the chemical synthesis process is known. As synthetic synthetic processes, those disclosed for example in Japanese Patent Publication Nos. 40-7491 (1965) and 47-18105 (1972), Japanese Patent Application No. 504040 (1975) and the like are known, however known method generally comprises many steps and the operations are complex and inconvenient, that is to say that the traditional methods are not

necessarily satisfactory.

  For example, the process disclosed in Japanese Patent Application Serial No. 504040 (1975) comprises the following steps: ## STR1 ## ## STR2 ## for 3 hours Cu (OCOC) .33 2 in c ns 6 - H- = c Pb (OCOCH) 2C C.3

i.

  KM nO Cf4 (2- (4-isobutylphenyl) propionic acid -2. The object of the present invention is to provide a process for producing 2- (substituted phenyl) propionic acid which is not based on a chemical synthetic method so to eliminate the complexity mentioned above and the problems of

  traditional chemical synthetic processes.

  After studying a process for producing 2- (substituted phenyl) propionic acid using a specified species of bacteria, the inventors of the present invention found that a specified bacterial species recently isolated from a soil at IWAKI City FUK USHIMA- Prefecture, Japan oxidizes 1- (substituted phenyl) -1-phenylethane to 2- (substituted phenyl) propionic acid, and based 1D on microbiological transformation, the present inventors have developed. the new method of producing 2- (substituted phenyl) propionic acid comprising

  fewer steps than the traditional chemical synthetic process.

  In one aspect of the present invention there is provided the production of 2 (substituted phenyl) propionic acid, comprising the steps of inoculating a microorganism belonging to the genus Pseudomonas and having a conversion activity of 1 ( substituted phenyl) -1-phenylethane in 2- (phenyl)

  substituted) propionic acid in a culture medium containing 1 (substituted phenyl) -

1-phenylethane.

  the culture of the micro-organism in this environment, thus producing micro-organisms

  2- (substituted phenyl) propionic acid organism, and recovery of 2 (phenyl

  substituted) propionic acid thus produced from this culture material.

  The present invention relates to a process for producing 2- (substituted phenyl) propionic acid, comprising the steps of inoculating a bacterium belonging to the genus Pseudomonas and having a 1- (substituted phenyl) -transforming activity. phenylethane in 2- (substituted phenyl) propionic acid o the substituent is the same as the substituent in 1- (substituted phenyl) -1-phenylethane, in a culture medium containing 1- (substituted phenyl) -1-phenylethane, the culture

  of the bacterium thus inoculated into this medium, thereby oxidizing the 1- (substituted phenyl) -1-

  phenylethane in 2- (substituted phenyl) -propionic acid and recovery of 2- (phenyl)

  substituted) propionic acid thus formed.

  The bacterium newly isolated by the present inventors and used in the present invention to convert 1- (substituted phenyl) 1-phenylethane to 2- (substituted phenyl) propionic acid belongs, this has been found, to the genus Pseudomonas as a result of the bacteriological properties of the latter, referring to the Manual of Bacteriological Determinants of Bergy 8th Edition, and the Prokaryotes, and after having found that the present bacterium differs from the known species of Pseudomonas for the use of 1 - (substituted phenyl) -1-phenylethane, the present inventors have decided that the newly isolated bacterium species is a novelty,

  and named the new species: Pseudomonas cepacia. A type of Pseudomonas.

  cepacia has been deposited at Fermentation Research Institute, Japan Agency for Industrial Science and Technology, under the filing number FERM-BP 534, filing date

May 25, 1984.

  The bacteriological properties of Pseudomonas cepacia are as follows (1) Morphological properties Shape of a cell: in rods Size of a cell: 1.0 to 1.5 μm wide and 1.5 to 2.5 μm long Pléomorphisme of a cell: none

Mobility: positive.

  Number of flagella: more than one Spore formation: none Gram stain: negative Acid resistance: negative (2) Developmental state (color, brightness, appearance, andC ooo) Test (culture conditions) Test results Culture broth-agar plate (1) pigment productivity: (+) pale yellow pigment (2) brightness: - (+) colony surfaces

were smooth.

  Slant agar broth culture (1) pigment productivity: (+) pale yellow pigment (2) luster (+) colony surfaces

were smooth.

  Culture broth-liquid (1) is not developed.

  Culture broth-gelatin (1) only develops on the surface of the medium (2) liquefaction of gelatin: (-) Sunflower-milk culture (1) the appearance of the medium did not change, coagulation: (-) - liquefaction: (-) (3) Effect of temperature on development: Temperature (OC) Devropp__

4 (-)

(-)

(+]

37 (+)

  -5 - (4) Effect of pH on development: Ph Development

4.0 (-)

, 0 (-)

6.0 (+)

8.0 (+)

9.0 C-)

, 0 (-)

  (5) Production of acid and / or gas from carbohydrate.

  1l Carbohydrate Acid production Gas production L-arabinose (+) (-) D-xylose (+) (-) D-glucose (+) (-) D-mannose (+) (-) D-fructose (+) C-) D-galactose (+) -) Maltose (+) (-) Sucrose '+] (-) Lactose (+) _ C-) Trehalose (+) D-sorbitol - (-) D- mannitol (+) -) Inositol (+) -) Glycerol -) (-) -25 Starch -) C-) -6 (6) Other types: Test Test result Development under aerobic conditions (+) Development in anaerobic conditions (-) Starch hydrolysis (-) Nitrate reduction (-H Denitrification (-H MR test () VP test (-) Indole formation (-) Hydrogen sulphide formation (-) Use of citric acid in Koser culture medium (-)

  in the Christensen culture medium -) -

  Use of a nitrate (-) Use of an ammonium salt (+) Formation of a pigment (+) yellow pigment

Urease formation (-.

  Oxidase Formation (+) Catalase Formation (+) O / F Test (Hugh-Leifson Method) - Oxidant As a species of bacteria to be used for the present invention Pseudomonas cepacia can be cited as a suitable example, however any artificial or natural mutant species thereof and any other microorganisms belonging to the genus Pseudomonas and producing 2- (substituted phenyl) propionic acid from 1- (substituted phenyl) -1-phenylethane may be used in

the present invention.

  In the culture of bacteria in the present invention, any carbon source that is usually used as a nutrient element may be used, however, such that the bacterium produces 2- (substituted phenyl) propionic acid in high yield, 1- ( substituted phenyl) -1-phenylethane is used as the sole source of carbon or an element of the carbon source. In such cases, 1- (substituted phenyl) -1-phenylethane is selected according to the desired 2- (substituted phenyl) propionic acid. That is, the substituent (group) in 1- (substituted phenyl) -1-phenylethane is the same as the substituent (group) in 2- (phenyl)

  substituted) objective propionic acid.

  The concentration of 1- (substituted phenyl) -1-phenylethane as the carbon source to be added is 0.01 to 0.5 wt., And the total amount of the carbon source can be added at a time or can be added several times after dividing into parts. As 1- (substituted phenyl) -1-phenylethane, it is soluble. R-represented by the formula (1) may be mentioned: ## STR1 ## wherein R 1 is a member selected from the group consisting of straight chain alkyl groups of 1 to 4 carbon atoms, groups branched chain alkyls of 3 and 4 carbon atoms, CP-CH 2 CH 2 - and CF 3 -C] HCH 2 - and as preferable starting material, the following compounds are given CH 3 as examples: 1- (4-methylphenyl) -1-phenylethane, 1- (4-Pre-thopylphenyl) -1-phenylethane, 1- (1 {4-propylphenyl) -1-phenylethane, 1- (4-isobutylphenyl) -1-phenylethane, 1 [4- (3,3,3 trifluoropropyl) phenyl-1-phenylethane and 1- [4- (2-trifluoromethylpropyl) phenyl-1-phenylethane As a nitrogen source used for the culture of bacteria according to the present invention, potassium nitrate, ammonium nitrate , ammonium sulphate, ammonium chloride, ammonium phosphate and polypeptone may be

used without restriction.

  In addition, as sources of inorganic phosphorus and potassium used in the culture medium, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the like are used. Magnesium sulphate, calcium chloride, ferric chloride and others are used as

  minor nutrient metal elements for bacteria.

  The other additives for the culture medium, yeast extract, macerated maize liquor, meat extract and others are used for favorable development

bacteria.

  As a culture method of the bacteria, there can be mentioned the shaking culture, the shaking culture or the submerged aeration culture and a method combining the above techniques, for example, culture with aeration.

  submerged with stirring and others can be used.

  The culture is suitably carried out at a temperature of 25 to 35 ° C.

  and at a pH of 6 to 8 for 3 to 35 days.

  After finishing the culture, by subjecting the cultured material to filtration or centrifugation to remove the bodies of bacteria, the supernatant containing 2- (substituted phenyl) propionic acid is obtained. The purified acid can be obtained by subjecting the supernatant element to the process comprising the steps of solvent extraction, dehydration, decolorization, fractionation, column chromatography

  and by a single method or the combination thereof.

  For example, after finishing the cultivation, the cultivated material is subjected to centrifugation to obtain the supernatant fraction, and after acidification of the fraction by adding an acid, if necessary, the object substance of the patent is extracted with a organic solvent such as dichloromethane and others. After subjecting the extract to dehydration and condensation, the condensate is subjected to silica gel chromatography for the elution and fractionation of the condensate using a mixture of 7/1 benzene and ethyl acetate. as an eluent. The fraction thus obtained containing the substance that is the subject of the patent is condensed, which makes it possible to obtain the product subject of the patent in the form of crude crystals, which are

  purified as needed by recrystallization, etc.

  The present invention will be explained in more detail with reference to the examples

following non-limiting

EXAMPLE 1

  In a 300 ml conical flask, 100 ml of an aqueous liquid culture medium (pH 7.2) of a composition comprising 0.065 g of K2HPO4, 0.026 g of KH2PO4, 0.134 g of Na2HPO4.12H2O, 0.005 g of NH4Cl. 0.068 g of MgSO4. 7H20, 0.083 g of CaCl2, 0.0008 g of FeCl3O6H2O, 0.2 g of polypepone, 0.3 g of yeast extract and 1 liter of distilled water and 0.05 g of 1-4 (3, 3,3-trifluoropropyl) phenyl! Synthetic -1-phenylethane according to the Japanese Patent Application No. 58-29725 (1983) were introduced, and after sterilization of the flask for 15 minutes at 121 ° C. under pressure, Pseudomonas cepacia (species No. KTB-03, deposited at Fermentation Research Institute,

  Agency for Industrial Science and Technology, Japan under deposit n FERM-

  BP 534) were inoculated into the culture medium thus prepared and cultured during

  28 days at 30 C turning and shaking the ball.

  After removing the bacterium bodies from the culture medium thus grown by centrifugation, the pH of the supernatant was adjusted to less than 2 by adding 6N aqueous hydrochloric acid and from the supernatant thus treated. The subject substance of the patent was extracted with 100 ml of chloroform. After dissolving the extract in 1N sodium hydroxide solution, the aqueous solution was acidified by addition of hydrochloric acid and the solution was again extracted with chloroform. By condensing the chloroform layer under reduced pressure to completely remove the solvent, a liquid was obtained.

slightly viscous red-brown.

- 1 0-

  The liquid thus obtained was subjected to silica gel chromatography using the mixture of benzene and ethyl acetate as eluent, thus purifying the liquid, and the fraction thus obtained was recrystallized from n-hexane to obtain 27 mg of white crystals in 61% yield based on the starting material, 1- [4- (3,3,3, trifluoropropyl) phenyl] -1-phenylethane. The melting point of the crystals thus obtained was 75 to 76.5 ° C. and the crystals thus obtained were identified as 2- (4- (3,3,3-trifluoropropyl) phenyl] propionic acid as a result of the examination. of NMR, infrared absorption spectrum, and mass spectrum with those of the autogenous specimen (see Japanese patent application

58-65237 (1983)).

  The conversion of the starting material to the substance subject of the patent is represented as follows: ## STR2 ##

CH3 CH5

EXAMPLE 2

  By culturing the bacteria in the same manner as in Example 1 except for the use of synthetic 1- [4- (2-trifluoromethyl-propyl) phenyl-1-phenylethane according to Japanese Patent Application 58-29725 (1983) instead of 1- [4- (3,3,3-trifluoropropyl) phenyl] -1-phenylethane in Example 1 as the starting material, 12 mg of purified white crystals were obtained in a yield of 27% in relation to the material

  starting material, 1- [4- (2-trifluoromethylpropyl) phenyl] phenylethane.

  The transformation of the starting material into the substance of the patent is represented as follows

CHB CH

CFl3 / Li3, H CHC142 CH-COOH

CF3 CH3 CF3 CH3

EXAMPLE 3

  By culturing the bacteria in the same manner as in Example 1 except for the use of 0.1 g of 1- (4-isobutylphenyl) -1-phenylethane instead of 0.05 g of (3,3,3-trifluoropropyl) phenyl] -1-phenylethane in Example 1, 61 mg of purified white crystals were obtained in a yield of 70.5% relative to the material.

  starting material, 1- (4-isobutylphenyl) -1-phenylethane.

  The melting point of the thus obtained cristal was 75 to 77 ° C., and the NMR spectrum, the infrared absorption spectrum and the mass spectrum were the same as those of the authentic specimen of 2- (4isobutylphenyl) propionic acid. The transformation of the starting material into the subject-matter of the patent is represented as follows

CH3 CH3

  / C 2 <tH <)> CHCH1 Q CH-COOH 2c ci CH 3 eH - 3cs l3

EXAMPLE 4

  Example of culture in short period, After introduction of 50 ml of the same culture medium as in Example 1 and 0.01 g of 1- (4-isobutylphenyl) -1-phenylethane in a conical flask of 300 ml, and after having subjected the medium to sterilization under high pressure for 15 minutes at 121 ° C., the same type of Pseudomonas cepacia as in Example 1 was inoculated into the culture medium thus sterilized, and the material to be inoculated was cultured during

  3 days b 30 C while turning and shaking the balloon.

  After subjecting the material thus cultivated to the. centrifugation, thereby removing the bodies of bacteria, the supernatant element was acidified to a pH below 2 by adding 6N hydrochloric acid and thereafter from the element

  supernatant, the substance subject of the patent was extracted with 50 ml of chloroform.

  After condensing the chloroform layer under reduced pressure, 10 ml of precisely measured condensate were subjected to high speed liquid chromatographic analysis, by which it was. found that 4 mg of 2- (4-isobutylphenyl) propionic acid was formed in 46% yield

  -1- (4-isobutylphenyl) -1-phenylethane starting material.

EXAMPLE 5

  In the same way as in example 1, 0.2 g of 1- (4-isopropylphenyl) -1-phenylethane was used in the culture of the same type of Pseudomonas cepacia as in Example 1 and converted into 2 - (4-isopropylphenyl) -propionic acid. The amount of product was 49 mg corresponding to a yield

  28.6% relative to the starting material, 1- (4-isopropylphenyl) -1-phenylethane.

  The transformation of the starting material into the subject-matter of the patent is represented as follows:

____ C.3 /. X _C-

chF

5 .. 0CH-. CH-COO3

C43 C3 3 C

3I. - 13-

Claims (4)

CLAIMS.   A process for the production of 2- (substituted phenyl) propionic acid, comprising the steps of inoculating a microorganism belonging to the genus Pseudomonas and having a 1- (substituted phenyl) -transforming activity phenylethane to 2- (substituted phenyl) propionic acid in a culture medium containing 1- (phenyl) substituted) -1-phenylethane.   the culture of the micro-organism in it, thus producing at the micro-   2- (substituted phenyl) propionic acid organism, and recovery of 2 (phenyl   substituted) -propionic acid thus produced from the material thus cultivated.   2. A process according to claim 1, characterized in that   (substituted phenyl) 1-phenylethane is a compound represented by the formula (1): wherein R denotes a member selected from the group consisting of straight-chain alkyl groups of 1 to 4 carbon atoms, alkylene groups having chains   branched 3 and 4 carbon atoms, CF -CH12CH1- and CF3-CHCH2-.   CH3 3. A process according to claim 1, characterized in that the culture of the microorganism is carried out at a temperature of 25 to 35 C and at a pH of 6-8.   4. A process according to claim 1, characterized in that the concentration of this 1- (substituted phenyl) -1-phenylethane in this culture medium is 0.01 to 0.5% by weight.