JPH03180196A - Production of optically active epichlorohydrin - Google Patents

Abstract

PURPOSE:To obtain optically active epichlorohydrin by reacting a specific bacterium of the genus Alcaligenes with racemic 2,3-dichloro-1-propanol in a culture medium and further reacting the resultant R-(+)-2,3-dichloro-1-propanol with an alkali agent. CONSTITUTION:A bacterium, having the ability to assimilate S-(-)-2,3-dichloro-1- propanol and belonging to the genus Alcaligenes or cultured bacterial cells thereof are reacted with racemic 2,3-dichloro-1-propanol (the aforementioned compound is hereinafter referred to as beta-DCH) in a culture medium and the resultant R-(+)-2,3-dichloro-1-propanol is the then reacted with an alkali agent to afford S-(+)-epichlorohydrin. The contact time of the bacterium with the racemic beta-DCH in the culture medium is normally about half a day to 10 days and the concentration of the beta-DCH may be about 0.1-0.6vol.% in the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to racemic 2,3-dichloro-1-propanol (hereinafter, this compound will be abbreviated as β-DC).

This invention relates to a method for producing optically active epichlorohydrin using as a raw material optically active β-DC obtained by microbial treatment of .

(Prior Art) Optically active epichlorohydrin is an important raw material for the synthesis of various pharmaceuticals. However, the method for producing this optically active epichlorohydrin is described in Baldwin, Journal, Orr. Organic Chemistry (J, O.
rg, Chem) Volume 43, 1978.

Page 4876 or Ems, Journal, Chemical,
Society, ,Chemical Communication, ,
(J,Cl1E)f,sOc,,CHEM,CO)I
MUN, 1984, p. 1600, but all of them require advanced synthesis techniques, and no simple manufacturing method is known.

(Problems to be Solved by the Invention) The present inventor has already discovered racemic β-DC and R-(10)-β-
Highly purified optically active S-(
-) -Method for obtaining β-DC port
6) and a method for obtaining R-(-)-epichlorohydrin by reacting this product with an alkaline agent (JP-A No. 6)
2-6697@publication), but a simple method for producing the opposite optical isomer, ie, S-(+)-epichlorohydrin, is not known. The present invention has solved this problem.

(Means for Solving the Problems) The present inventors have completed the present invention by discovering that the above optically active epichlorohydrin can be easily produced with high purity by microbial treatment.

That is, the present invention provides R-
(10)-β-DC eyes are reacted with an alkaline agent and S-(+
)-A method for producing optically active epichlorohydrin, which is characterized by obtaining epichlorohydrin.

The mycological properties of the microorganisms isolated and collected from soil by the present inventor and used in the present invention are shown in Table 1.

table a, form ■Cell shape and size ■Cell pleomorphism ■Presence or absence of motility ■Presence or absence of spores ■Durham stainability ■Anti-acidity Growth status in each medium ■Meat juice agar plate culture (30℃).

b) Slow colony shape 0〉Colony shape C) Shape of colony surface 2) Prominence of colony e) Colony periphery To〉Contents of the colony g) Colony color tone H) Colony transparency ri〉Colony gloss n) Generation of soluble pigments ■Meat juice agar slope culture l! (30℃.

b) Quality of growth Mouth) Colony shape C) Uplifted state of colony cross section 2> Colony gloss E〉Colony surface shape To> Colony transparency g) Colony color ■ Broth liquid culture (30℃).

b) Growth characteristics: Turbidity: 3-day culture) Normal, diameter: 3-4IIIm, circular, smooth, convex, circular, gold-rimmed, homogeneous, milky, translucent, 3-day culture: 11) Good growth, filamentous, smooth, flat, smooth, translucent, milky white 3 Daily cultivation! Bacillus, 0.4 to 0.6 Coloring■ Meat juice gelatin puncture culture■ Lidomus milk C6 Physiological test 1r Reduction of IR salts 2 MR test 3 VP test 4 Indole production 5 @Hydrogen production 6 Starch hydrolysis 7 Denitrification reaction 8 Citric acid utilization 9 Inorganic Utilization of nitrogen source 10 Pigment production 11 Urease 12 Oxidase +13 Catalase +14 Growth range
pH5.0-9.0. Temperature 20-4
5℃ 15m! S degree for elemental aerobic 16
0-F test (by Hugh Leirson method) 1
7 Presence or absence of acid and gas production from m-classes Sugars (1>D-glucose (2>D-galactose (3) sucrose <4) trehalose (5) starch (6) glycerin 18 arginine dihydrolase 19 PHB Accumulated gas + Acid + + + Based on the results of no formation, purge aids manual of
Systematic Bacteriology (Beraey's)
s Manual of Systematic Ba
cterioloc+y) Based on the description in Volume 1, the origin is identified as having characteristics of the genus Alcaligenes. The present inventor has identified his home country as Alcaligenes (Alcaligenes).
s) sp, DS- was named -338 (hereinafter referred to as
The home country is DS-338). In its home country, it has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Institute No. 11114 ("ERM P-11114>").

In the present invention, not only the above-mentioned DS-1338 strain, its variants and mutants, but also the 5-(
-) All bacteria capable of assimilating -2,3-dichloro-1-propanol can be used.

The gist of the present invention is to optically activate the racemic β-DC using the bacteria described above, and then react it with an alkaline agent by a conventional method to obtain optically active epichlorohydrin. In the present invention, the above-mentioned bacteria or their cultured cells may be used, or they may be immobilized. However, the methods for culturing and immobilizing the above-mentioned bacteria may be commonly used methods. That is, the culture method involves culturing the above-mentioned bacteria in a nutrient medium containing a carbon source, a nitrogen source, an organic nutrient source, and an inorganic nutrient source, such as a bouillon medium or a sweetened bouillon medium, allowing them to grow well, and culturing the resulting culture. Alternatively, cultured bacterial cells may be used. Carbon sources include carbohydrates such as glycerin, citric acid, maleic acid,
Organic acids such as malic acid and their salts, and ammonium sulfate, ammonium chloride, and ammonium nitrate as nitrogen sources.

Inorganic nitrogen such as ammonium phosphate, and organic nitrogen such as peptone, casein, yeast extract, meat extract, etc. can be used. Other inorganic salts include phosphates, magnesium salts, potassium salts, and iron salts.

Zinc salts, copper salts, etc. are used. The culture conditions are usually a temperature of about 20 to 45°C, preferably 25 to 37°C, and a pH of about 5.
The reaction is carried out aerobically at a temperature of ~9, preferably pns, o~7.5 by shaking or aerated stirring.

Further, the immobilization method may be, for example, a method of enclosing the living bacterial cells using acrylamide, carrageenan, agar, gelatin, sodium alginate, etc. After immobilization, the cells may be crushed into an appropriate size and shape for use. .

The reaction between the above bacteria and racemic β-DC is
The above-mentioned bacteria, their cultured cells, or their immobilized products may be stirred and brought into good contact with each other in a medium containing DCH, such as a synthetic medium, and the contact time is usually half a day to 10 days, and β-DC days. The concentration is approximately 0.1-0.6% by volume of the medium width.
It is sufficient as long as it is of a certain extent.

After the reaction is completed, the reaction solution is taken out and filtered to separate the cultured cells and the supernatant, or the immobilized material and the supernatant, and the R-(+)-β-DC port remaining in the supernatant is removed. The activated carbon column treatment,
It is fractionated by operations such as ether extraction and vacuum distillation. This fraction is treated with an alkaline agent, preferably a caustic alkali such as caustic soda or caustic potash, to produce epichlorohydrin.

Furthermore, if immobilized bacterial cells are used in the method of the present invention, operations such as centrifugation become easy, and the immobilized product can be used repeatedly.

(Example) The present invention will be specifically explained below using examples. In the examples, percentages are by weight unless otherwise specified.

Example 1 Yeast extract 1.0%, glycerin 2.0%, polypeptone 1.0%, l) H7.0 medium 20j at 30%. Place it in a Q-sized jar fermenter and heat sterilize it as usual.
-338 strain was inoculated into DS- and cultured for 24 hours under the following conditions.

Temperature 30℃ pH Initial pH 7.0 Airflow rate 2ON/min Stirring rotation speed: 300 r, p, m.

After the culture was completed, the microbial cells and the culture filtrate were separated using a centrifuge to obtain 600 g of viable cells. Next, the viable bacterial cells are
Soak it in the synthetic medium shown below 10.1! After adjusting the volume, it was fixed with acrylamide in a conventional manner. The immobilized product was crushed into pieces of 0.5 to 1 mm square using a mixer and thoroughly washed with a synthetic medium.

Components of synthetic medium Ammonium sulfate 0.05% by weight Ammonium nitrate 0.05 Potassium hydrogen phosphate
0.1 Monosodium phosphate 0.2 Sodium phosphate 0.1 Magnesium sulfate 0.05 N iron sulfate, copper sulfate, manganese sulfate Trace amount pH initial pH 6.8 Next, prepare in this way The immobilized product was placed in a 1,001-volume di-fermenter and added with a synthetic medium for 80 min.
Set to 0. Furthermore, 320 racemic β-DC eyes
rn1. 160 g of calcium carbonate was added and stirred under the following conditions.

Temperature 30℃ Airflow rate 40.11/min Rotation speed: 30 Or, p, m.

72 hours after the start of the reaction, the supernatant liquid and the immobilized product were separated by filtration,
The remaining β-DC was collected from this liquid using an activated carbon column, ether extraction, and distillation under reduced pressure, and 152 g was collected.

The substance was identified using the following method.

1) Identification column carrier PE by gas chromatography
As a result of comparison with commercially available β-DC using G-20MP, 5%, 60-80 mesh, the retention time was exactly the same. Purity 98.2% or higher.

2) Identification by IR (infrared absorption spectrum) As shown in the chart shown in Figure 1, the absorption pattern is that of commercially available β-D.
It was exactly the same as day C.

From the above, this substance was clearly found to be β-DC. The following method was used to confirm that this substance was R-(10)-β-DC.

1) Measurement of optical rotation The specific optical rotation of commercially available β-DC and this substance is as follows.

Commercially available β-DC [α] 萱 = 0.0" C = 1. Dichloromethane main substance (α) 萱 = 1,000 10.4°C = 1. Dichloromethane 2
) Preparation of R-(+)-α-methoxy-α-trifluoromethylphenylacetate ester and analysis by high-performance liquid chromatography R-(+)-α-methoxy-α-trifluoromethylphenylacetate chloride was prepared from commercially available β- After reacting with DCH and this substance to prepare its ester derivative, the analysis results by liquid chromatography were as follows.

Analysis conditions Column carrier ZORBAXODS 4.6 mm x 25 cm (manufactured by Ou Pont) Methanol: Water = 65: 35 (V/V) 1 m/man Absorbance at 260 nm Eluate Elution amount Detection method Analysis results Commercially available β-DC Japanese substance Retention time 50. Two peaks with the same area were given at 5 minutes and 52.0 minutes.

Peak only at retention time 52.0 minutes gives a peak at 50.5 minutes. I couldn't.

3> Preparation of dichloropropyl-N-phenyl carbamate and its optical rotation Add 1 g of this substance and 0.9 (l) of phenyl incyanate to 30 d of dry acetone and 0.3 ml of triethylamine, and heat for about 3 hours. After refluxing and preparing the dichloropropyl-N-7enyl carbamate, its specific rotation was measured.

Commercially available β-DC day (α) tube = 0.0'C = 1. Methanol main substance (α) tube = + 16.4° C = 1. From the results obtained with methanol, it was found that this substance was R-(10)-β-DC, and its optical purity was 99% or more.

Next, 100 g of this R-(+)-β-DC was mixed with 650 g of a 1.4 N aqueous sodium chloride solution in a 1000 flask and stirred vigorously at room temperature for 80 minutes. Furthermore, 200 m of ether was added and after stirring, ether The layers were separated.

Subsequently, the ether layer was dried over magnesium sulfate, the ether was distilled off, and epichlorohydrin was distilled off to obtain 60.3 g. The purity of this epichlorohydrin is
The result measured by gas chromatography was 99.4% or more. Moreover, the specific optical rotation was as follows.

[α] W = +34.3° (C = 3.4.methanol) That is, the obtained epichlorohydrin is S-(
+)-epichlorohydrin, and its optical purity is 99
% or more.

Example 2 Same as Example 1, yeast extract 1.0%, polypeptone 1
．． ()%, glycerin 2.0%, pH 7.0 medium 2
1 was placed in a 51-volume jar fermentor and sterilized by heat in the usual manner. DS- was inoculated with the -838 strain and cultured under the same conditions as in Example 1 for 24 hours.

Next, add the synthetic medium 80.1 shown in Example 1 to a u-sized jar fermenter! and 160 g of calcium carbonate.

Racemic β-DC day 320rIJ1. Polypeptone 40
After heat sterilization, the above-mentioned culture was inoculated in the usual manner and reacted while culturing at a temperature of 30°C, 9 aeration, M40.l!/min, and a rotation speed of 300 rpm.

48 hours after the start of the reaction, the reaction solution is separated into supernatant, bacterial cells, and precipitate using a centrifuge, and the remaining β is removed from the supernatant.
-DC port was fractionated in the same manner as in Example 1, and R-(+)-β
-8148 g of DC was obtained.

The specific optical rotation of the obtained R-(+)-β-DC port is [α]
W=+1o, 4° (C=1.0, dichloromethane), and as a result of analysis in the same manner as in Example 1, the optical purity was 99% or more. Further, the conversion from R-(+)-β-DC to S-(+)-epichlorohydrin was carried out in the same manner as in Example 1, and the specific optical rotation [α] 'fl = +
34.3° (C=3.4, methanol) Optical purity 9
More than 9% S-(+)-epichlorohydrin was obtained.

(Effects of the Invention) According to the present invention, optically active R-(+)-2 can be produced more easily and with higher purity than 2,3-dichloro-1-propanol by using bacteria belonging to the genus Alcaligenes isolated from soil.
, 3-dichloro-1-propanol to optically active S
-(10)-Epichlorohydrin can be obtained.

[Brief explanation of drawings]

Figure 1 shows R, the raw material of the present invention obtained in Example 1.
-(+)-2,3-dichloro-1-propanol and a commercially available infrared absorption spectrum of the same substance. □ indicates commercially available β-DCH, and --1 indicates R-(10)-β-DC.

Claims (2)

[Claims] (1) Bacteria belonging to the genus Alcaligenes having the ability to assimilate S-(-)-2,3-dichloro-1-propanol, or cultured cells thereof, are grown in a culture medium to produce racemic 2,3-dichloro-1-propanol.
R-(+)-2 obtained by reacting with 1-propanol
, 3-dichloro-1-propanol with an alkali agent to obtain S-(+)-epichlorohydrin. (2) Claim 1, which uses a bacterium belonging to the genus Alcaligenes having the ability to assimilate S-(-)-2,3-dichloro-1-propanol, or a cultured cell thereof, after being immobilized.
Manufacturing method described in section.