JPH0353890A - Production of 4-halo-3-hydroxybutyronitrile - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a synthetic intermediate for pharmaceuticals from an inexpensive raw material under mild condition in high yield by treating an epihalohydrin with a dehalogenation enzyme originated from microorganism in the presence of an alkali metal cyanide. CONSTITUTION:The objective compound is produced by treating an epihalohydrin with a dehalogenation enzyme originated from microorganism in the presence of an alkali metal cyanide preferably at 5-50 deg.C and pH4-10. The dehalogenation enzyme is e.g. an enzyme produced by Corynebacterium sp. N-2354 (FERM P-10673) which is a new microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing 4-halo-3-hydroxybutyronitrile. More specifically, the present invention relates to a method for biochemically producing 4-halo-3-hydroxybutyronitrile from epihalohydrin in the presence of alkali cyanide by the action of a dehalogenase derived from a microorganism.

4--Halo-3-hydroxybutyronitrile is a compound with two different functional groups, so it is a useful substance as a raw material for various pharmaceuticals and physiologically active substances.
It is known to be particularly useful as a raw material for L-carnitine (see JP-A-57-165352).
.

(Prior art and problems) Conventionally, methods for producing 4-halo-3-hydroxybutyronitrile include methods (1) to (3) shown below.
method is known.

(]) 1. A method of heating and reacting 3-dichloro-2-propanol with an alkali cyanide in an aqueous solution (see Japanese Patent Publication No. 36-21718).

(2) A method in which 3-chloro-1,2-propanediol is reacted with tosyl chloride to tosylate the alcohol at the 1st position, and then reacted with an alkali cyanide (JP-A-57-
(See Publication No. 165352).

(3) A method in which epichlorohydrin and hydrocyanic acid are reacted in the presence of a catalytic amount of potassium cyanide (F.
non) et al. Bailten des sochetes des.
Shigokes Helgies (llull. Soc. Cb
im. (Belges), Vol. 72, 166-
]. 77 (1963)).

However, method (1) has a low yield of about 40%;
Method (2) involves two steps and is a complicated reaction, and the overall yield is low at about 45%. Method (3) uses hydrocyanic acid, which is dangerous to operate and handle, and produces by-products. This method has problems such as the difficulty in controlling the reaction conditions to prevent the survival of the microorganisms, and cannot be said to be an advantageous method for industrial implementation. Furthermore, all of the methods (11 to (3)) are chemical preparation methods, and optically active substances cannot be obtained from zoicopol or racemic raw materials.

(Summary of the Invention) Therefore, the present inventors focused on the usefulness of 4-halo-3-hydroxybutyronitrile, particularly the fact that the optically active form is useful as an intermediate for various pharmaceutical crystal synthesis, and -Hello-3-
We have conducted extensive research on the manufacturing method for hydroxypetitic acid. As a result, we discovered a new manufacturing method that utilizes the enzymatic action of microorganisms. Zuna 3 That is, the present invention converts shrimp yakuchihydrin into 4-halo-3-hydroxybutyknitrile by the action of a microorganism-derived dehalogenase in the presence of alkali cyanide,
This is a method for producing 4-halo-3-hydroxybutyronitrile, which is characterized by collecting the 4-halo-3-hydroxybutyronitrile.

- Generally, the enzyme that converts halogen into hydroxyl is known as dehalogenase [Enzyme Handbook,
627 pages (Asakura Shoten), T. Yokota et al., Agricultural Natural and Hyological Chemistry (Yagri)
c. Biol. Chem. ) Vol. 50+45346
0 (1986)), the reaction of converting 1,3-dihalo-2-propanol into 3-halo-1,2-propanediol using a substrate was completely unknown, and was discovered for the first time by the present inventors. and filed a patent application earlier (Patent Application Hei 1-
100173). However, even more surprisingly, it was discovered that when this enzyme was applied to shrimp yakuchihydrin in the presence of alkali cyanide, epihalohydrin was converted into 4-halo-3-hydroxybutyronitrile through ring-opening cyanation. This led to the present invention. According to the method of the present invention, the reaction can be carried out extremely efficiently at room temperature and near neutral pl+, which is advantageous compared to chemical methods. For the first time, it became possible to produce nitriles from inexpensive epihalohydrins.

(Detailed Description of the Invention) The dehalogenase used in the present invention is an enzyme that can ultimately convert shrimp yakuchi hydrin into 4 halo-3-hydroxybutyronitrile in the presence of alkali cyanide. Specifically, for example, N-2354, a microorganism belonging to the genus Corynebacterium newly isolated and discovered by the present inventors.
Strains and microorganisms belonging to the genus Microbacterium, N-4
Examples include enzymes produced by the 701 strain and the like. These microorganisms were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken), respectively, as Microbiological Research Institute No. 10673 (Corynebacterium sp. N-2354) and Bacterium sp. N-470
1), and its mycological properties are as shown below.

N-2354 Morphology Peripheral cells of a colony Durham staining Spore movement Oxidase catalase OF Growth under anaerobic conditions Cell wall diamino acid glycolyl test Starch decomposition Gelatin Liquefaction Hydrogen sulfide production Peptone Sodium thiosulfate Production of methyl red levan Production of rods No elongation + Not observed Diaminobutyric acid (acetyl type) Growth in the presence of NaCl 3% 5% Acid production Inulin mannitol Mannose meletitose N-4701 Morphology Peripheral cells of colony Durham staining Spore transport Color of motile flagella hair colony Oxidase catalase OF Growth under anaerobic conditions Polymorphic bacillus elongation → - Not observed - Top pole to side hair yellow-orange + + O Concentration of meso-diaminopimelic acid in whole cell hydrolyzate Diagonal acids present in cell walls Lysine glycolyl test + (glycolyl type) starch degradation Gelatin liquefaction Nitrate reduced use of arginine Hydrogen desulphide production Urea decomposition skim Glycerol glucose + sucrose + trehalose + raffinose
+ 8 Mycological properties as described in Herger's Manual of
Systematic hatterology Vol. 2 (
1986) (Bergy's Manual of
Systematic Bacteriology
Vol. 2 (1986)),
Strain N-2354 is Corynebacterium spp. and N470
Each strain was identified as a bacterium belonging to the genus Microbacterium.

As the culture medium for culturing the above-mentioned microorganisms, any medium can be used as long as these microorganisms can grow. For example, carbon sources include sugars such as glucose, fructose, shakerose, and maltose, organic acids such as acetic acid and citric acid, alcohols such as ethanol and glycerol, and nitrogen sources include peptone, meat extract, yeast extract, and protein hydrolysates. In addition to general natural nitrogen sources such as , ξ-anoic acids, various inorganic and organic acid ammonium salts can be used, and inorganic salts, trace metal salts, vitamins, etc. can be used as appropriate. At this time, in order to induce high enzyme activity,
1.3-dichloro-2-propanol, 3-chloro-1
．． It is also useful to add 2-propanediol or the like to the medium.

The above microorganisms may be cultured by conventional methods, such as p114
~10, aerobically at a temperature range of 20~40°C 10~
Incubate for 96 hours.

Epihalohydrins used in the present invention include ebichlorohydrin and ebipromohydrin. Further, the alkali cyanide includes potassium cyanide, sodium cyanide, and the like.

By treating epi-octalohydrin with dehalogenase, 4-
- Halo-3-hydroxybutyronitrile can be obtained by adding a substrate and alkali cyanide (hereinafter referred to as "substrate") to a culture solution of microorganisms cultured as described above or a suspension of microbial cells obtained by centrifugation, etc. etc.),
A method of adding a substrate, etc. to a bacterial cell-treated product (for example, a bacterial cell crush product, a bacterial cell extract such as crude enzyme or purified enzyme, etc.) or a suspension of bacterial cells fixed by a conventional method or a bacterial cell-processed product, etc. , during the cultivation of microorganisms) J. There is a method in which a substance is added to the culture solution and the reaction is carried out simultaneously with culturing.

The concentration of the substrate in the reaction solution is not particularly limited, but
It is preferably 0.1 to 10 (W/V)%, and the amount of alkali cyanide used is usually 1 to 3 times the amount (mol) of the substrate. Substrates and the like can be added to the reaction solution all at once or in portions.

The reaction temperature is preferably 5 to 50°C, and the reaction ρII is preferably 4 to 100).

Although the reaction time varies depending on the concentration of the substrate, etc., the concentration of bacterial cells, and other reaction conditions, it is generally preferable to set the conditions so that the reaction is completed in 1 to 120 hours.

The 4-halo-3-hydroxybutylene nitrile thus produced and accumulated in the reaction solution can be collected and purified using known methods. For example, after removing bacterial cells from the reaction solution using a method such as centrifugation, extraction is performed with a solvent such as ethyl acetate, and the solvent is removed under reduced pressure.
--A syrup of halo-3-hydroxybutyronitrile can be obtained. Further, this syrup can be further purified by distilling it under reduced pressure.

Hereinafter, the present invention will be specifically explained with reference to Examples. 1 isn't it.

Example 1 Glucose 1%, peptone 0. 5%, meat extract 0.3
%, yeast extract 0. A medium containing 3% was adjusted to pH 7.0, dispensed into 500mffi Erlenmeyer flasks in 100d portions, sterilized at 120'C for 15 minutes, and then sterilized with a membrane filter. -Kuroro 1.
0.2-propanediol aqueous solution. 8 ml was added.

The above medium was inoculated with N-4701 strain and incubated at 30℃ for 48 hours.
A shaking culture was performed for hours. The cells were collected by centrifugation from this culture solution, and 20 m
The bacterial cells were suspended in M phosphate buffer (pH 7.0) and disrupted according to a conventional method. After dialysis, an enzyme solution was obtained which was partially purified by DEAE-Sephacel column chromatography. IM phosphate buffer (pl1 8.0
) Add 10 m of the above enzyme solution to 40-, and add 0.0 m of shrimp chlorohydrin to this. 5 g and potassium cyanide 0.
35 g was added and stirred at 20°C to carry out a reaction.

After 5 hours, the purified 412-3-hydroxybutyroni-1-lyl was quantified using gas chromatography, and the molar yield based on the charged shrimp chlorohydrin was 62.5%.

Example 2 The N-2354 strain was inoculated into a medium obtained in the same manner as in Example 1, and cultured with shaking at 30°C for 48 hours. Centrifuge 140 m of this culture solution to collect bacterial cells, and
M Tris 11cI buffer (pif 8.0) 140
After washing once with 35 ml of IMU phosphate buffer (pl.
The bacterial cells were suspended in 18.0). To this suspension was added 0.35 g of epicurostohydrin and 0.25 g of potassium cyanide.
g was added thereto, and the reaction was carried out by stirring at 20°C for 5 hours.

After the reaction, the amount of 4-chloro-3-hydroxybutyronitrile produced was determined by gas chromatography.
The molar yield based on the charged shrimp chlorohydrin is 55.
It was 6%.

Claims (2)

[Claims] (1) Epihalohydrin is converted into 4-halo-
A method for producing 4-halo-3-hydroxybutyronitrile, which comprises converting it into 3-hydroxybutyronitrile and collecting the same. (2) The microorganism is Corynebacterium (Coryneba).
cterium genus or Microbacterium (Mic
2. The method according to claim 1, wherein the plant belongs to the genus Robacterium.