JPH03103185A - Production of isoolefin compound - Google Patents

Abstract

PURPOSE:To obtain the title compound useful as drug, agricultural chemical, other physiologically active substance and synthetic intermediate thereof by oxidizing squalene with a bacterium belonging to the genus Acinetobacter. CONSTITUTION:Squalene is converted with a bacterium (e.g. FERM 10,793) belonging to the genus Acinetobacter capable of oxidizing squalene into 4,9- dimethyl-12-hydroxy-4,8-dodecadienoic acid shown by the formula (R is hydroxy or carboxyl) or 4,9-dimethyl-4,8-dodecadienoic diacid, which is optionally converted into a salt thereof and the acid or the salt is collected. The culture condition, for example, is 25-40 deg.C for 2-5 days by shaking culture.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides an inolefin compound 4,9-dimethyl-12
-Hydroxy-4,8-dodecadienoic acid and 4,9-
The present invention relates to a novel method for producing dimethyl-4,8-dodecadienioic acid and salts thereof.

The inolefin compounds produced by the method of the present invention are useful as medicines, agricultural chemicals, other physiologically active substances, and their synthesis or intermediates.

[Conventional technology]

The compound produced by the method of the present invention is an inolefin compound having 14 carbon atoms, and is considered to be a type of terbenoid. Monoterpenes having 10 carbon atoms, such as citral, citronellal, linalool, and geraniol, are known as essential oils of higher plants and are used in fragrances and the like. Furthermore, seswaterpenes having 15 carbon atoms form a very interesting group of compounds that contain many physiologically active substances such as plant hormones and insect hormones.

Among the compounds produced by the method of the present invention, formula (I[I
4,9-dimethyl-12-hydroxy-4,8-dodecadienoic acid represented by formula (II) is a new compound created by the method of the present invention, and
, 9-dimethyl-12-hydroxy-4,8-dodecadienoic acid is a known compound (Literature Liebigs Anna
len der Chemie 639. 9-24
[Problems to be Solved by the Invention] The present invention provides isoolefin compounds useful as pharmaceuticals, agricultural chemicals, other physiologically active substances, and their synthesis or intermediates4.
．． The present invention aims to provide a novel method for producing 9-dimethyl-12-hydroxy-4,8-dodecadienoic acid and 4,9-dimethyl-4,8-dodecadienoic acid using microorganisms.

[Means to solve the problem]

The present inventors conducted various studies to develop a method for microbially producing isoolefin compounds. As a result, microorganisms belonging to Acinetobacter oxidize squalene to produce 4.9-dimethyl-12-hydroxy-4.8 The inventors obtained a completely new finding that 4,9-dimethyl-4,8-dodecadienoic acid and 4,9-dimethyl-4,8-dodecadienoic acid were produced, and completed the present invention based on this finding.

Therefore, the present invention provides 4.9-dimethyl-12-hydroxy-4.8-dodecadienoic acid and 4.9-dimethyl-12-hydroxy-4.8-dodecadienoic acid represented by the following formula (■): (wherein R represents a hydroxymethyl group or a carboxyl group) -dimethyl-4.8
- A method for producing dodecadienioic acid and its salts,
A microorganism belonging to Acinetobacter that can oxidize squalene converts squalene into 49-dimethyl-12 represented by formula (I).
-Hydroxy-4,8-dodecadienoic acid or 4,9-
A method characterized by converting dimethyl-4,8-dodecadienioic acid into dimethyl-4,8-dodecadienioic acid, optionally converting these into its salts, and collecting the acid or its salt: and the following formula (■)
In the method for producing 4.9-dimethyl-4,8-dodecadienioic acid and its salts represented by the following formula (■): 4.9 represented by the following formula (■): - Dimethyl-12-hydroxy-4,8-dodecadienoic acid is converted into 4,9-dimethyl-4,8-dodecadienoic acid represented by the above formula (II), and optionally converted into a salt, and the A method for collecting acids or salts is provided.

[Specific explanation]

A strain capable of oxidizing squalene that can be used in the method of the present invention can be obtained, for example, by the following method. That is, a naturally occurring isolation source is added to a medium containing squalene as the sole carbon source and cultured, and a strain capable of oxidizing squalene is isolated from the culture solution. The medium used here can be any commonly used medium, except for squalene as the only carbon source, and contains nitrogen sources, inorganic salts, and special requirements that the strain needs for growth. (e.g. vitamins, amino acids,
(nucleobases, etc.). The carbon source is squalene as the only carbon source and 0.00% per liter of culture medium. Add 1 to 100 g, preferably 1 to 10 g. As the nitrogen source, one or more of potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea, and the like can be used. The addition ratio varies depending on the type of nitrogen source, but the normal medium 1f! For 0
．． The amount is 1 to 10 g, preferably 1 to 3 g. As the inorganic salt, one or more of potassium phosphate, sodium phosphate, magnesium sulfate, iron sulfate, iron chloride, calcium chloride, etc. can be used. The addition ratio varies depending on the type of inorganic salt, but is usually 0.001 to 10 g per culture medium if, preferably 0.01 to 5 g. As the specially required substances, one or more of vitamins, yeast extract, peptone, corn steep liquor, etc. are used. The addition ratio varies depending on the type of substance, but it is 0.0% to normal medium LA.
0.05 to 10 g, preferably 0.1 to 5 g. The pH of the medium is usually adjusted to 2-11, preferably 5-10. Furthermore, it is preferable to add antibiotics, bactericidal agents, etc. to selectively propagate specific bacterial strains. For example, in order to selectively grow yeast and suppress bacterial growth, streptomycin was administered at 0.00%. 1 = 100mg/1
, preferably 10 to 50 mg/j! Can be added. Minutei! The source can be environmental substances in which microorganisms are thought to exist, such as soil, wastewater, rotten fruit, etc. Isolation source: 1-100 g for medium H
, preferably 30 to 50 g. Culture temperature is 15℃
It is carried out at ~80°C, preferably 25-40°C. The culture time is usually 1 to 30 days, preferably 4 to 7 days, then subcultured to a fresh medium, and similarly cultivated for 1 to 30 days.
Preferably, culturing is carried out for 4 to 7 days, and this is repeated 2 to 5 times. Culture is carried out under normal aerobic conditions. Growth of the bacterial strain is carried out by measuring the turbidity of the medium and observing it with a microscope. 00610 0. 1-10, preferably 0.2
It is preferable to perform a separation operation when the value reaches 1. That is, the strain is isolated from the culture solution. It is preferable to use a plate culture method, which is a conventional pure separation method. The plate medium may be any medium containing 0.1 to 10% agar; for example, a bouillon medium containing 2% agar may be used.

As an example of the microorganism obtained by the above method and which can be used for producing the compound of the present invention, the bacterium SQ75
- Two strains can be mentioned.

This microorganism strain SQ75-2 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbial Research Institute No. 10793 (FBRM P-1).
0793).

This microbial strain has the following mycological properties.

(a) Morphology Liquid medium ■ Cell shape and size Short rods, 0.8-1. 2 X 1. 0~1.5ha ■
・ Cell pleomorphism None ■ Motility None ■ Spore form or none ■ Durham staining Negative (b) Growth status in medium ■ Juicy agar plate culture Growth mode Colony color Colony shape Colony gloss Diffusive pigment (c ) Physiological properties Reduction of nitrate ■ Good growth of oisidase Milky white round (smooth) Presence or absence ■ Catalase + ■ Attitude towards oxygen Aerobic ■ 〇1 F test ■ Liquefaction of gelatin 10 ■ Assimilation (■ Adibate (2 L) - Aspartate (3 L-arabinose (4 D-xylose (5 DL-lactate)
ticus).

In the method of the present invention, in addition to the above-mentioned microorganisms, for example, Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) is used.
obacter calcoaceticus) IF
O 12552 (ATCC 14987), IFO
13006, ATCC 9036, ATCC23
055, ATCC 19606. ATCC 138
09, ATCC 17912, ATCC 17913
and ATCC 33304, and Acinetobacter
Ruu #7i (Acinetobacter lwoff)
ii) ATCC 9036 and the like can be used, and these strains are freely available from IF○ or ATCC.

Examples of the culture medium for producing the above compound using a strain capable of oxidizing squalene are as follows.

That is, it contains carbon sources, nitrogen sources, inorganic salts, and, if necessary, special required substances (eg, vitamins, amino acids, nucleobases, etc.) necessary for growth of the producing bacteria.

The starting material squalene as the sole carbon source or 4.
Although 9-dimethyl-12-hydroxy-4,8-dodecadienoic acid can be used, carbon sources include sugars such as glucose, sucrose and sorbitol, hydrocarbons such as n-baraffin, and alcohols such as ethanol and propanol. 1 to 100 g, preferably 2 to 1 og, of the same may be added per 11 media. As the nitrogen source, one or more of potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea, and the like can be used. The addition rate varies depending on the type of nitrogen source, but it is usually 0.00% per liter of culture medium.
The amount is 1 to 10 g, preferably 1 to 3 g. As the inorganic salt, one or more of potassium phosphate, sodium phosphate, magnesium sulfate, iron sulfate, iron chloride, calcium chloride, etc. can be used. The addition ratio varies depending on the type of inorganic salt, but is usually 0.01 to 5 g per liter of culture medium.

Specially required substances include one or two of vitamins, nucleic acids, yeast extract, pebtone, corn steep liquor, etc.
More than one species is used. Although the addition ratio varies depending on the type of substance, it is usually 0.05 to 10 g, preferably 0.1 to 5 g per medium 11. The pH of the medium is 2-11,
Preferably it is adjusted to 5-10. In addition to the above-mentioned media, there are known bouillon media for bacteria, yeast extract for molds and yeast,
A malt extract medium or the like can be used.

The above production medium contains squalene or
．． 9-dimethyl-12-hydroxy-4,8-dodecadienoic acid or a salt thereof is added. These starting materials may be present in the medium from the start of culture, or may be added intermittently or continuously during culture. The total amount of starting materials used per culture medium is, for example, 0. 1-100g
/A preferably 2-10 g/I! It is.

In order to produce a large amount of the target substance, it is necessary to perform large-scale culture, and for this purpose, it is preferable to perform a preculture before the main culture (production culture) to grow the production bacteria in advance. As a preculture medium for this purpose, the above-mentioned medium can be used, but squalene or 4.9-dimethyl-12-hydroxy-4.8-
It is not absolutely necessary to contain dodecadienoic acid.

The culture conditions for preculture and main culture (production culture) are 1.
The temperature is 5°C to 80°C, preferably 25 to 40t,
Shaking culture or aerated agitation culture is usually carried out for 1 to 20 days, preferably 2 to 5 days.

According to the present invention, the production microorganisms are once cultivated, and then the cultured microorganisms are brought into contact with the starting material in an aqueous medium under aerobic conditions, for example, under conditions similar to those for the above-mentioned cultivation. It is also possible to produce the target compound.

Finally, the culture solution or reaction solution is extracted with a solvent to obtain the compound of the present invention. At this time, it is suitable to adjust the pH of the culture solution to 1 to 6, preferably 2 to 3.

As the solvent used here, any solvent can be used as long as it is a compound in which the compound of the present invention can be dissolved. For example, carbon tetrachloride, trichloroethylene, toluene, benzene,
Organic solvents such as dichloromethane, chloroform, diethyl ether, ethyl acetate, and acetone are used, preferably dichloromethane, chloroform, or diethyl ether. Conventional methods such as adsorption, elution and dissolution, and distillation can be used for purification.

The compounds of the invention have a carboxyl group and can therefore form salts. These salts include, for example, alkali metal salts such as lithium, sodium, and potassium salts, alkaline earth metal salts such as potassium and magnesium salts, and ammonium salts. These salts can be obtained from the free compound according to a conventional method, for example, by neutralizing the free compound with a desired base.

The infrared absorption spectrum of the new compound 49-dimethyl-12-hydroxy-48-dodecadienoic acid produced by the method of the present invention is shown in Figure 1, and the 1H nuclear magnetic resonance spectrum is shown in Figure 2. The magnetic resonance spectrum is shown in FIG.

The infrared absorption spectrum of 4,9-dimethyl-4,8-dodecadiennic acid is shown in FIG. 4, the 'H nuclear magnetic resonance spectrum in FIG. 5, and the 13C nuclear magnetic resonance spectrum in FIG.

〔Effect of the invention〕

According to the present invention, 4,9-dimethyl-12-hydroxy-4 is extracted from squalene by a novel method using microorganisms.
．． 8-dodecadienoic acid and 4,9-dimethyl-4,8-
Dodecadienic acid and salts thereof can be produced, and 4,9-dimethyl-12-hydroxy-4.
4,9-dimethyl-4,8-dodecadienic acid and its salts can be produced from 8-dodecadienoic acid.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1: 50 ml of a culture medium consisting of the set shown in Table 1 was placed in a 500 rnl capacity Sakaguchi flask and steam sterilized at 121° C. for 20 minutes. Add 1 g of soil (Honmoku district, Yokohama City) to the same medium, culture with shaking at 30°C for 5 days, and use the culture limbs that have grown to grow the strain SQ on a bouillon plate medium containing 2% agar.
75-2 (M Koken Bacteria No. 10793) was isolated.

This strain was cultured in a bouillon liquid medium for 16 hours with shaking at 30°C, and the culture solution was inoculated at a ratio of 1% by volume into medium 50rd consisting of the composition shown in Table 1, and cultured with shaking at 30°C for 3 days. I did it. This culture solution was extracted with diethyl ether under hydrochloric acid acidity (pH 2), and after distilling off the solvent, preparative TLC
(Silca gel) was separated using a developing solvent of 100:100:-1, and the corresponding substance with an Rf value of 0.1 was scraped off and extracted with diethyl ether under acidic sulfuric acid.This compound is colorless. It was oily and the yield was 20 mg per 11 of the culture solution.This compound was 4.9-dimethyl-12-hydroxy-4.8-
Confirmation that it was dodecadienoic acid was performed by infrared absorption spectroscopy, IH nuclear magnetic resonance spectroscopy, 13C nuclear magnetic resonance spectroscopy, and mass spectrometry. These results are shown in FIGS. 1 to 3.

In addition, the spot at the Rf value of 0.15 was scraped off and extracted with diethyl ether under acidic sulfuric acid. After distilling off the solvent, a white solid compound was obtained. The yield of this compound was 15 mg per 11 culture fluids. This compound is 4,9-dimethyl-
The identification of 4.8-dodecadienic acid was carried out by infrared absorption spectroscopy, 'H nuclear magnetic resonance spectroscopy, +30 nuclear magnetic resonance spectroscopy, and mass spectrometry.

These results are shown in FIGS. 4 to 6.

Table 1 Added amount squalene 2.0 g/I! NH, NO
3 2.5 g#KH2PO,
1. 5 g/ANa2HPO4
1.5 g/j2 MgSO<・7H. 0 0.5 g/f
lFeSO.・7H20 0.01 g/
I! CaCl2・2H20 0. 01
g/l yeast extract 0.2 g/12
11H7. 2 Example 2. The method of Example 2 was repeated. However, in the culture medium composition shown in Table 1, 4,9-dimethyl-12-
Hydroxy-4,8-dodecadienoic acid 2 g/f! was used. This yielded 400 mg of raw material. It was confirmed that this compound was the known substance 4,9-dimethyl-4,8-dodecadienioic acid by infrared absorption spectroscopy, 1H nuclear magnetic resonance spectroscopy, 13C nuclear magnetic resonance spectroscopy, and mass spectrometry.

Example 3. The method of Example 1 was repeated. However, the strain is S Q752.
(Feikoken Bacterial Serial No. 10793) instead of Acinetobacter calcoaceticus (Acinetobalcoc
alcoaceticus) IPO 13006 was used. As a result, 4,9-dimethyl-
12-hydroxy-4,8-dodecadienoic acid 9.9
■, 8 4,9-dimethyl-4,8-dodecadienneic acid
．． I got 2 mg.

Example 4. The method of Example 1 was repeated. However, the bacterial strain is Acinetobacter calcoaceticus (Acinetobalco aceticus) instead of S Q75-2 (Feikoken Bacteria No. 10793).
calcoaceticus) IFO 12552 was used. As a result, 14 4.9-dimethyl-12-hydroxy-4,8-dodecadienoic acid was added per 11 culture fluid.
3.9 μg of 4.9-dimethyl-4,8-dodecadienneic acid were obtained.

[Brief explanation of drawings]

Figure 1 shows 4,9-dimethyl-12-hydroxy-4,
The infrared absorption spectrum of 8-dodecadienoic acid is shown. Figure 2 shows the nuclear magnetic resonance spectrum of the same substance. FIG. 3 shows the 13C nuclear magnetic resonance spectrum of the same substance. FIG. 4 shows an infrared absorption spectrum of 4,9-dimethyl-4,8-dodecadienioic acid. FIG. 5 shows the 1H nuclear magnetic resonance spectrum of the same substance. FIG. 6 shows the 13C nuclear magnetic resonance spectrum of the same substance. (ppm) Figure 2

Claims (1)

[Claims] 1. The following formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R represents a hydroxymethyl group or a carboxyl group) 4,9- dimethyl-12-hydroxy-
4,8-dodecadienoic acid and 4,9-dimethyl-4,8
- A method for producing dodecadiene dioic acid and its salts,
Acinetobacter (￥Acinetobacter￥)
Microorganisms that can oxidize squalene belong to A process characterized by converting them into acids, optionally converting them into their salts, and collecting the acids or their salts. 2. In the method for producing 4,9-dimethyl-4,8-dodecadiene dioic acid and its salts represented by the following formula (II): ▲Mathematical formulas, chemical formulas, tables, etc.▼(II), squalene, which belongs to Acinetobacter, Microorganisms that can oxidize 4,9-dimethyl-12-hydroxy-, which is expressed by the following formula (III): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III)
4,8-dodecadienoic acid represented by the above formula (II)
, 9-dimethyl-4,8-dodecadiene dioic acid,
A method characterized in that, if desired, the acid or salt is converted into a salt and the acid or salt is collected.