JPS6328591B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention is based on the following formula () (In the formula, R is a C _1-4 straight or branched alkyl group,
CF ₃ −CH ₂ −CH ₂ − group or

Regarding the method for producing 2-(substituted phenyl)propionic acid represented by the above formula (), in detail, 2-(substituted phenyl)propionic acid represented by the above formula () is converted into 2-(substituted phenyl)propionic acid represented by the following formula (). (In the formula, R is a C _1-4 straight or branched alkyl group,
CF ₃ −CH ₂ −CH ₂ − group or

The present invention relates to a method for microbially producing 1-(substituted phenyl)-1-phenylethane represented by the following formula: [Prior art] 2-(Substituted phenyl)propionic acid has anti-inflammatory and analgesic effects on humans and animals, and 2-(4-isobutylphenyl)propionic acid is particularly effective as an excellent anti-inflammatory agent. It is commonly used. Conventionally,
The only known method for producing this substance is chemical synthesis. As a chemical synthesis method, for example, Japanese Patent Publication No. 7491/1973, Japanese Patent Publication No. 18105/1973
Although various methods are known in addition to those disclosed in Japanese Patent Application Laid-Open No. 50-4040, these methods generally require long steps and complicated operations, and are not necessarily satisfactory. For example, JP-A-1988-1, which has a relatively short process.
The method of No. 4040 is based on the following steps. [Problems to be Solved by the Invention] A feature of the present invention is to propose a method that does not rely on chemical synthesis methods in order to improve the complexity of steps in conventional chemical synthesis methods. Structure of the Invention [Means for Solving Problems] The present invention is a method that utilizes a specific bacterium as an alternative to chemical synthesis, and is a method that utilizes 1-(substituted phenyl)-1- represented by the above formula (). A bacterium that assimilates phenylethane and produces 2-(substituted phenyl)propionic acid represented by the above formula () is used.
That is, the present invention provides 1-
Assimilate (substituted phenyl)-1-phenylethane,
2-(substituted phenyl) represented by the above formula ()
A bacterium belonging to the genus Pseudomonas that has the ability to produce propionic acid is inoculated into a medium supplemented with 1-(substituted phenyl)-1-phenylethane having the same substituted phenyl group as the target product, and cultured. ) 2-(substituted phenyl)
This is a method for collecting propionic acid. The present inventors have discovered that 1- expressed by the above formula ()
As a result of a search for bacteria that can assimilate (substituted phenyl)-1-phenylethane, bacteria newly isolated from soil in Iwaki City, Fukushima Prefecture oxidized the compound and produced 2-(substituted phenyl ethane) expressed by the formula (). ) was discovered to produce propionic acid, and by utilizing this microbial conversion, 2-(substituted phenyl) was produced with fewer steps than conventional chemical synthesis methods.
This is a new method for producing propionic acid. The newly isolated bacterium was analyzed in Bergy's Manual of Determination based on its mycological properties, which will be described later.
-minative Bacteriology, 8th edition and The
As a result of searching for Prokary-otes, it was found that it belongs to the genus Pseudomonas. When compared with known bacterial species, this species differs in that it assimilates 1-(substituted phenyl)-1-phenylethane, and was determined to be a new bacterial species, Pseudomonas cepacia . It was named KTB-03. This bacterium was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on May 25, 1980, and its accession number is FERM BP-534.
It is. The mycological properties of this bacterium are as follows.

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[Table] The above-mentioned strains can be cited as preferred examples of the bacterial strains used in the present invention, and of course, artificial and natural mutant strains thereof belong to the genus Pseudomonas and are represented by the formula () 1-
All bacteria that produce 2-(substituted phenyl)propionic acid represented by the above formula () from (substituted phenyl)-1-phenylethane can be used. For culturing the microorganisms used in the present invention, carbon sources that are commonly used as culture components can be used, but 2-(substituted phenyl)propionic acid represented by the above formula () can be produced in high yield. For this purpose, 1-(substituted phenyl)-1-phenylethane represented by the above formula () is used as the sole or main carbon source. In this case, the diphenylethane derivative is selected depending on the target 2-(substituted phenyl)propionic acid. That is, one having the same substituted phenyl group as the target propionic acid derivative is used. 1-(Substituted phenyl)-1-phenylethane as a carbon source is added to the medium at a concentration of 0.01 to 0.5% by weight, but the entire prescribed amount may be added at once or divided into several portions. It may also be added. 1-(substituted phenyl) represented by the above formula ()
More specific examples of preferred compounds as -1-phenylethane include 1-(4-methylphenyl)-1-phenylethane 1-(4-ethylphenyl)-1-phenylethane 1-(4-propylphenyl)-1-phenylethane 1-(4-isobutylphenyl)-1-phenylethane 1-[4-(3,3,3-trifluoropropyl)
-Phenyl]-1-phenylethane 1-[4-(2-trifluoromethylpropyl)phenyl]-1-phenylethane When culturing the bacteria used in the present invention, the nitrogen source of the medium used is particularly limited. However, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphorus, polypeptone, etc. can be used. In addition, as inorganic salts, disodium phosphate, monopotassium phosphate, dipotassium phosphate, etc. are used, and as trace metals, magnesium sulfate, calcium chloride,
Ferric chloride or the like can be used. Other additives include yeast extract, cornstarch liquor, meat extract, etc. to improve the growth of the bacterial strain. Cultivation methods include shaking culture, agitation culture, deep aeration culture, etc., and culture methods that are a mixture of these, such as deep aeration agitation culture, etc., can be used. Further, the appropriate culture temperature is 25 to 35°C, the culture pH is around neutral (6 to 8), and the number of culture days is 3 to 35 days. 2-(substituted phenyl) accumulated after completion of culture
Propionic acid can be obtained by operations such as filtering and centrifuging the culture solution. Purified 2-(substituted phenyl)propionic acid can be obtained by extraction and purification from the sterilized culture solution. Purification can be carried out by means such as solvent extraction, dehydration, decolorization, fractional distillation, column chromatography, etc. alone or in appropriate combinations. For example, after completing the culture, centrifuge the culture solution, remove the resulting supernatant, and use it as needed.
After adjusting the pH, the target substance is extracted using an organic solvent such as dichloromethane. Next, after dehydration and concentration, the mixture is subjected to silica gel chromatography, eluted and fractionated using benzene-ethyl acetate (7:1) to obtain an eluted fraction containing the target substance. By concentrating this fraction,
A crude target substance is obtained, but if necessary, it can be purified by operations such as recrystallization. Examples will be described below, but the present invention is not limited to the following examples. Example 1 K ₂ HPO ₄ 0.065g, KH ₂ PO ₄ 0.026g,
_{Na2HPO4 ・ 12H2O0.134g} , _NH4Cl0.005g ,
_MgSO4・_7H2O0.068g , _CaCl2 0.083g, _FeCl3・
6H ₂ O 0.0008g, polypeptone 0.2g, yeast extract
0.3 g, 100 ml of a liquid medium (PH7.2) having the composition shown in distilled water 1, and 1-[4-(3,3,3-trifluoropropyl)- synthesized according to JP-A-58-29725. phenyl]-1-phenylethane 0.05g
After putting it in a 300ml Erlenmeyer flask and sterilizing it under high pressure at 121℃ for 15 minutes, Pseudomonas cepersia KTB-
03 strain (Feikoken Joyori No. 534) was inoculated and incubated at 30℃ for 28 hours.
Rotary shaking culture was performed for days. The culture solution is centrifuged to remove bacterial cells, the supernatant is adjusted to pH 2 or less with 6N hydrochloric acid, and extracted with 100 ml of chloroform. This extract was transferred to 1N caustic soda, made acidic again, extracted with chloroform, and the chloroform layer was concentrated under reduced pressure to completely remove the solvent to obtain a slightly viscous reddish-brown liquid. This was purified by silica gel chromatography (benzene-ethyl acetate) and further recrystallized from n-hexane.
27 mg (yield: 61%, based on 1-[4-(3,3,3-trifluoropropyl)phenyl]-1-phenylethane) of white crystals was obtained. The melting point of the obtained crystal was 75 to 76.5℃, and the results of NMR, infrared absorption spectrum, and mass spectrometry were 2.
-[4-(3,3,3-trifluoropropyl)phenyl]-propionic acid (JP-A-58-65237). Example 2 100 ml of the liquid medium shown in Example 1 was
Put 0.05 g of 1-[4-2-trifluoromethylpropyl)phenyl]-1-phenylethane synthesized in the same manner as No. 29725 into a 300 ml Erlenmeyer flask,
After autoclaving at 121°C for 15 minutes, Pseudomonas cepersia KTB-03 strain was inoculated and cultured with rotary shaking at 30°C for 28 days. By treating the culture solution in the same manner as in Example 1, 12 mg of purified white crystals (yield 27%, based on 1-[4-(2-trifluoromethylpropyl)phenyl]-1-phenylethane) was obtained. . Example 3 Add 0.1 g of 1-(4-isobutylphenyl)-1-phenylethane to 100 ml of the liquid medium shown in Example 1.
After putting it in a 300ml Erlenmeyer flask and sterilizing it under high pressure at 121℃ for 15 minutes, Pseudomonas cepersia KTB-
03 strain was inoculated and cultured with rotational shaking at 30°C for 28 days. By treating the culture solution in the same manner as in Example 1, 61 mg of purified white crystals (yield 70.5%, however, 1-
4(-isobutylphenyl)-1-phenylethane) was obtained. The melting point of this substance is 75-77℃.
The results of NMR, infrared absorption spectrum, and mass spectrum were consistent with that of 2-(4-isobutylphenyl)propionic acid. Example 4 Short-term culture data 0.01 g of 1-(4-isobutylphenyl)-1-phenylethane in 50 ml of the liquid medium shown in Example 1.
was placed in a 300 ml Erlenmeyer flask, sterilized under high pressure at 121℃ for 15 minutes, and then Pseudomonas cepersia KTB
-03 strain was inoculated and cultured with rotary shaking at 30°C for 3 days. The culture solution is centrifuged to remove bacterial cells, the supernatant is adjusted to pH 2 or less with 6N hydrochloric acid, and extracted with 50 ml of chloroform. After concentrating the chloroform layer under reduced pressure, it was poured into a volume of 10 ml and determined by high performance liquid chromatography to yield 4 mg (yield 46%, based on 1-(4-isobutylphenyl)-1-phenylethane).
It was found that 2-(4-isobutylphenyl)propionic acid was produced. Example 5 Substrate 1 represented by the above formula in the same manner as in Example 1
-isopropylphenyl-1-phenylethane
0.2 g was assimilated by Pseudomonas cepersia KTB-03 strain. The yield of the product 2-isopropylphenylpropionic acid obtained after 21 days of cultivation was 49 mg (yield
28.6% (based on 1-isopropylphenyl-1-phenylethane).

Claims (1)

[Claims] 1 A bacterium belonging to the genus Pseudomonas, which has the following formula () (wherein, R is a C _1-4 straight or branched alkyl group,
The following formula () is obtained by oxidizing 1-(substituted phenyl)-1-phenylethane represented by CF ₃ -CH ₂ -CH ₂ - group or [Formula] group. (wherein, R is the same as R in formula ()) A bacterium capable of producing 2-(substituted phenyl)propionic acid represented by 1. A method for producing 2-(substituted phenyl) propionic acid, which comprises inoculating and culturing a medium to which phenylethane has been added, and collecting 2-(substituted phenyl) propionic acid represented by the above formula () from the culture.