JPH01273595A - Production of gamma-halo-beta-hydroxybutyric acid ester - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a synthetic raw material for agricultural chemicals and medicines in high yield by reacting a gamma-haloacetic acid ester with a culture or fungal cells (treated substances) of a specific mold. CONSTITUTION:A gamma-haloacetic acid ester is reacted with a culture, fungal cells or their treated substance of a mold, belonging to the genus Cephalosporium, Paecilomyces, Verticillium, Fusarium, Ustilago or Gibberella [e.g., Cephalosporium charticola IFO 8952] and having the ability to convert the above-mentioned compound into a gamma-halo-beta-hydroxybutyric acid ester to afford the aimed compound. Furthermore, the culture is preferably normally carried out under aerobic conditions of pH3-8 and 10-40 deg.C for 1-10 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing γ-halo-β-hydroxybutyric acid ester by allowing filamentous fungi to act on T-haloacetoacetic ester. γ-halo-β-hydroxybutyric acid ester is useful as a raw material for agricultural and pharmaceutical synthesis.

[Problems to be solved by the prior art and the invention] Chemically reducing T-haloacetoacetate to obtain the corresponding γ-
When producing halo-β-hydroxybutyric acid ester, there are disadvantages in that side reactions are likely to occur and the yield of the target product is low. Therefore, in order to solve these problems, a method using the fermentation enzyme action of microorganisms that produce L-β-hydroxyacyl Co^ dehydrogenase (Japanese Patent Application Laid-Open No. 11809-1989) was proposed.
Public Book No. 3) was proposed. However, the reported microorganisms have practical problems such as enzyme activity, and there is a need to establish a method to utilize advantageous microorganisms.

[Means to solve the problem]

The present invention converts the γ-haloacetoacetate into the corresponding T-
Cephalosporium has the ability to convert to helo-β-hydroxybutyrate.
m) Genus, Paecilomyces (Paecilon+yce)
s), Verticillum spp., Fusarium spp., Ustilago spp.
sttlago) and Gihberella
a) γ-halo-β-, which is characterized in that one type of culture, bacterial cells, or treated bacterial cells selected from filamentous fungi of the genus is allowed to act on T-haloacetoacetic acid ester, and the product is collected. This is a method for producing hydroxybutyric acid ester.

The T-haloacetoacetic acid ester used in the present invention has the general formula 'R+-CH□CO・CH2C00RZ (in the formula, R1 is a halogen, and R2 is an arbitrary organic residue such as an alkyl group, a phenyl group, an aryl group, etc.) This is a compound represented by

The T-haloacetoacetate used in the present invention can be obtained, for example, by reacting a halogen and diketene in an organic solvent, but if necessary, it can also be produced from the T-haloacetoacetate by a conventional Grignard reaction.

The microorganism used in the present invention is a type of filamentous fungus that has the ability to convert T-haloacetoacetate to the corresponding γ-halo-β-hydroxybutyrate.
orium charticola) IFO8952
, Paecilomyces elegans
mycesele3ans) IFo 6987, Ustilago Zeae I
FO5346, Particillium niveostratosum (
VerticilliumniveosLratosu
m) IPO6625, Gibberella fujikuroi (Gi
bberella fujikuroi) IFO63
49, Fusarium merismoides (Fusariu
m merismoides) IFO30040 is preferably used.

Although the above-mentioned microorganisms can generally undergo mutation by natural or artificial means, all microorganisms that reduce T-haloacetoacetate and convert it into γ-halo-β-hydroxybutyrate can be used in the production method of the present invention. It can be used.

The microorganisms used in the present invention can be cultured according to conventional methods. The medium used for culture is a normal medium containing a urea source, a nitrogen source, inorganic substances, etc. necessary for growth. Furthermore, desirable results are often obtained by adding organic mW nutrients such as vitamins and amino acids.

The culture is carried out under aerobic conditions for 1 to 10 days while controlling the pH to 3 to 8 and the temperature to an appropriate range of 10 to 40°C. In the reaction, either a culture solution or cultured bacterial cells decomposed and collected from the culture solution can be used.

In addition, the bacterial cell treatment products include dried bacterial cells obtained by freeze-drying, acetone drying, etc., bacterial cell suspension obtained by grinding, autolysis, ultrasonication, etc., as well as T-haloacetate Enzyme protein fractions having enzymatic activity for converting acetate esters into the corresponding γ-halo-β-hydroxybutyric esters, immobilized products of these microbial cells or processed products of the microbial cells, and any others can be used.

T-haloacetoacetate corresponding to γ-halo-β-
The conversion to hydroxybutyric acid ester can be carried out by contacting T-haloacetoacetic ester with a culture solution, bacterial cells, treated bacterial cells, or a product obtained by immobilizing these by a known method in an aqueous medium. .

As the aqueous medium during such a reaction, water, a buffer solution, and a water-containing organic solvent can be used.

In order to allow the above microorganisms to act on γ-haloacetoacetate, the pH is usually 3 to 8 and the reaction temperature is 10 to 60"C.
Do this in a controlled manner.

The T-haloacetoacetate is added to the reaction system as it is, dissolved in a solvent, or dispersed.

The ester concentration in the reaction system is usually preferably in the range of 0.001 to 50% by weight. The T-haloacetoacetic acid ester can be added at any stage of the reaction, and can be carried out collectively, continuously, or in portions.

The reaction can also be carried out in the presence of sugars such as glucose, nutrients for microorganisms, surfactants, and the like. The reaction time can be adjusted depending on conditions such as concentration, but it is long (total
Over a period of time, the γ-haloaceto 11 ester is converted to the corresponding γ-halo-β-hydroxybutyric acid ester.

In order to collect the γ-halo-β-hydroxybutyric acid ester obtained in this way from the culture solution or reaction solution, the bacterial cells or the treated bacterial cells are removed by a conventional method such as centrifugation or ultrafiltration. Conventional methods such as extraction using an organic solvent such as ether, carbon tetrachloride, Hensen, ethyl acetate, etc. can be employed.

〔Example〕

Next, the method of the present invention will be explained in more detail by way of examples.

Example 1 Medium (pH 6.5) consisting of 5% glucose by weight and 5% corn steep liquor (pH 6.5) 5 m! ! was placed in a test tube, inoculated with the microorganisms shown in Table 1, and cultured with shaking at 28°C for 48 hours.

To this system, 25 μl of methyl γ-chloroacetoacetate was added, followed by shaking culture for 24 hours, followed by reaction.

After the obtained reaction solution was sterilized by centrifugation, reaction solution 2
ml was extracted with 4 ml of ethyl acetate, and subjected to gas chromatography (GC-9APF
m, 150℃, Nz30mj! /min). The results are shown in Table 1.

Example? A reaction was carried out and analyzed in the same manner as in Example 1 using ethyl γ-chloroacetoacetate as a substrate. The results are shown in Table 1.

Example 3 5 ml of a medium (ρl+6.5) consisting of 5% by weight of glucose and 5% by weight of corn staple liquor was placed in a test tube, inoculated with Paecilomyces elegans IF06987, and cultured with shaking at 28° C. for 24 hours. A culture solution was obtained.

Next, 100+nl of a medium with the same composition as above was placed in a 500me capacity Sakaro flask, and 5IIll of seed culture solution was added.
Shaking culture was performed at 8°C.

The obtained culture solution was centrifuged, washed with 0.9% NaC (! water, and then mixed with 100 ml of 0.1 M phosphate buffer (pH 6,0) containing l (w/v)% glucose.
! 1.0 g of ethyl T-chloroacetoacetate was added thereto, and the reaction was carried out for 18 hours with ventilation and shaking.

After sterilizing the resulting reaction solution by centrifugation, 300 ml of ethyl acetate was added. Extraction was performed using (100 ml x 3 times). Anhydrous magnesium sulfate was added to this ethyl acetate layer to dehydrate it, followed by concentration under reduced pressure to obtain 0.98 g of an oily product. This product was distilled under reduced pressure and IR (Island IIR-4
35), NMR (JEOL PMX60SI), gas chromatography (quality GC-9^PFXPEG 20
Mx 1m, 150℃, Nz 30ml/min
), it was confirmed that it was ethyl γ-chloroβ-hydroxybutyrate.

NMR δ (CDC1, medium): δ (ppm) 1.25 (30, t), 2.60 (2H, d
), 3.35 (L H,s, exchangea
ble, OH) 3.60 (2H, d), 4.
2 (2H,q)C R・T (min) 4.6 Example 4 Using octyl γ-chloroacetoacetate as a substrate, the reaction was carried out in the same manner as in Example 3. , 0V-1x 1m, 125°
c, Nz 30 ml/min), IR (quality IR-435
), NMR (JEOLGX-270) ``liI'' confirmed that it was octyl T-chloro-β-hydroxybutyrate.The reaction yield was 75%.

The substrate was 1 ml of 10% Tween 80 (KAO
-ATLAS) and added to the reaction system.

Example 5 10 g of wet bacterial cells obtained in the same manner as in Example 3 was added to 20 ml of
．． Suspend in 1 M phosphate buffer (pl + 6.5), perform 5-minute ultrasonic treatment 4 times while cooling with ice water,
A crude enzyme solution was obtained by removing insoluble matter by centrifugation.

Add 2 NADPI+ (Sigma) to 10ml of this crude enzyme solution.
00 When the reaction solution was analyzed in the same manner as in Example 3, T
The yield of ethyl -chloro-β-hydroxybutyrate was 95%.

〔Effect of the invention〕

According to the present invention, γ-halo-β-hydroxybutyric acid ester can be obtained from T-haloacetoacetic ester in high yield, which is industrially advantageous.

Patent applicant: Denki Kagaku Kogyo Co., Ltd.

Claims (1)

[Claims] (1) The genus Cephalosporium, the genus Paecilomyces, and the genus Verticillum (
Genus Verticillum, Fusarium
One type of culture, bacterial cells, or treated bacterial cells selected from filamentous fungi of the genus Ium, Ustilago, and Gibberella is allowed to act on γ-haloacetoacetic ester, and the product is collected. A method for producing γ-halo-β-hydroxybutyric acid ester.