JPS62228292A - Biochemical separation of (+)-3-alkyl substituted-5-hydroxymethyloxazolidin-2-one - Google Patents

Abstract

PURPOSE:To make it possible to readily separate the (+)-isomer from a mixture thereof with (-)-isomer of a 3-alkyl substituted-hydroxymethyloxazolidin-2-one with a high efficiency, by utilizing esterase produced by a microorganism. CONSTITUTION:An organic carbolic acid ester of a mixture of the (+)-isomer with the (-)-isomer of a 3-alkyl substituted-5-hydroxymethyloxazolidin-2-one expressed by the formula (R is alkyl) is brought into contact with esterase produced by a microorganism in a hydrous organic solvent. Thereby, since only the ester bond of the (+)-isomer is selectively hydrolyzed and the (-)-isomer remains unreacted, the (+)-isomer can be selectively separated.

Description

[Detailed description of the invention] 3. Detailed explanation of H■ (Industrial application field) The present invention utilizes esterase produced by microorganisms to
±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one to (+)-3-alkyl-substituted-5
The present invention relates to a biochemical separation method for (+)-3-alkyl-substituted-5 to hydroxymethyloxazolidin-2-one that selectively separates -hydroxymethyloxazolidin-2-one.

(Prior Art) 3-Alkyl-substituted-5-hydroxymethyloxazolidin-2-one has an asymmetric carbon atom at the 5th position due to its chemical structure. Therefore, 3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one has a (+) form and a (-) form.
Optical isomers exist.

Among the (+) forms, (+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one is.

It is an important synthetic intermediate of l-propranolol.

L-propranolol has an antiarrhythmic effect because it has a antagonistic inhibitory effect on adrenergic receptors.

Strong antihypertensive effect. Therefore, β-propranolol is
Shows remarkable effects as an antiarrhythmia drug and antihypertensive drug. Furthermore, (+)-3-(t-butyl)-5-hydroxymethyloxazolidin-2-one is a synthetic intermediate for optically active carteolol, which has remarkable antihypertensive and cardiac depressant effects. For this reason, attempts have been made to separate only the (+) form from (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one.

As a method for separating the (+) isomer from (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one, there is a method of deriving it from D-mannitol (chem
, & Pharm Bulletin, 29+ (
12) 3593, (1981)). However, such a method requires a large number of steps and requires a large amount of toxic lead tetranosate.

JP-A-59-31692 and JP-A-59-3169
Publication No. 3 discloses that (±)-3-alkyl-substituted-5-hydroxymethyloxazolidine derivatives are asymmetrically hydrolyzed by the action of esterases produced by microorganisms to produce (+)-3-alkyl-substituted-5-hydroxymethyloxazolidine derivatives. -Although methods for obtaining hydroxymethyloxazolidin-2-one have been disclosed, all methods involve dispersing a poorly water-soluble substrate in water and performing asymmetric hydrolysis, which makes the operation complicated and The recovery rate of (+) bodies is low.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to
-Hydroxymethyloxazolidin-2-one to (+
)-3-Alkyl-substituted-5-hydroxymethyloxazolidin-2-one can be obtained easily and in high yield (+)
An object of the present invention is to provide a biochemical separation method for -3-alkyl-substituted-5-hydroxymethyloxazolidin-2-ones.

(Means for Solving the Problems) The present invention is directed to the production of an organic carboxylic acid ester of (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one by a microorganism in a water-containing organic solvent. By contacting with esterase, only the ester bond of the (+) form is hydrolyzed, and the (-) form remains unreacted.

(±)-3-alkyl substituted-5-hydroxymethyloxazolidin-2-one to (+)-3-alkyl substituted-
5-Hydroxymethyloxazolidin-2-one can be selectively separated. It was completed based on the inventor's knowledge.

The biochemical separation method of (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one of the present invention comprises an esterase, an immobilized product of esterase, a microorganism having esterase-producing ability, an immobilized product of the microorganism, and At least one selected from the group consisting of immobilized products of processed microorganisms.

(±)-3-Alkyl-substituted-5-hydroxymethyloxazolidin-2-one is contacted in a water-containing organic solvent, thereby achieving the above object.

For example, esterases produced from microorganisms belonging to the genus Brevibacterium, Rhodotorula, Enterobacter, Corynebacterium, Rhizopus, etc. are used.

Examples of microorganisms having esterase-producing ability include microorganisms belonging to the genus Brevibacterium, genus Rhodotorula, genus Enterobacter, genus Corynebacterium, and genus Rhizopus. However, without limitation, (±)-3-alkyl-substituted-5-hydroxymethyloxazolidine-2-
Any microorganism can be used as long as it produces an esterase for asymmetrically hydrolyzing organic carboxylic acid esters.

Examples of microorganisms used in the present invention are shown below.

All of these microorganisms are publicly known.9 For example, the Fermentation Research Institute (I
FO).

(1) Brevibacterium genus Brevibacterium,
IFO-12128 (Brevibacteri
um protophorimae) (2) Rhodotorula sp. Rhodotorula chixensis bur minuta
IFO-0932 (Rhodotorula
texensis var m1nuta) (3)
Enterobacter genus Enterobacter 7E Kenneth
IFO-13534 (Enterobacterium
(4) Corynebacterium svetonicum
IFO-13763 (Corynebacter
(5) Rhizopus cinesis
IFO-4168 (Rhizopus chi
(±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one has 2 e.g.
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one, (±)-3-(t-butyl)
-5-hydroxymethyloxazolidin-2-one. Such compounds have the following structural formulas.

Here, R represents an alkyl group.

(±)-3-Alkyl-substituted-5-hydroxymethyloxazolidin-2-one organic carboxylic acid esters are (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one and carbon number 1, respectively. It is obtained by reacting a chloride of an aliphatic monocarboxylic acid having ˜7 and a monoester monochloride of an aliphatic dicarboxylic acid having 3 to 7 carbon atoms. Examples of aliphatic monocarboxylic acids having 1 to 7 carbon atoms include acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and heptanoic acid.

Contains acrylic acid and crotonic acid. Examples of aliphatic dicarboxylic acids having 3 to 7 carbon atoms include malonic acid, succinic acid, glutaric acid, pimelic acid, and maleic acid.

Examples include fumaric acid. These carboxylic acids.

It is easily converted into acid chloride by a known method of reaction with thionyl chloride. Acid chlorides of carboxylic acids may be substituted with alkyl groups and/or halogens.

Examples of solvents that can sufficiently dissolve the ester and whose water content does not impair the esterase activity of microorganisms include n-heptane, isoheptane, n-hexane, isohexane, n-pentane, isopentane, and n-heptane. - Aliphatic chain hydrocarbons such as octane and isooctane, aliphatic cyclic hydrocarbons such as cyclobencune, cyclohexane and cycloheptane, aromatic hydrocarbons such as benzene, toluene and xylene, methanol and ethanol.

Alcohols such as n-propanol, isopropanol, n-butanol and isobutanol, ketones such as acetone and methyl isopucovir ketone, and ethers such as tetrahydrofuran, dimethyl ether, diethyl ether and dioxane.

Esters such as methyl acetate, ethyl acetate, and butyl acetate, halogenated hydrocarbons such as carbon tetrachloride, chloroform, methylene chloride, and methane chloride, dimethylformamide,
Examples include dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, and acetonitrile. These organic solvents may be used alone or in combination. The amount of water contained in the organic solvent for hydrolyzing the ester may be extremely small. Even if an organic solvent has low solubility in water, a saturated amount of water is sufficient. Alternatively, the reaction may be carried out in a two-layer system of an organic solvent and water.

In this case, the pH of the aqueous system is 6-8. Preferably it is adjusted to 7-8. If the pH is outside this range, the progress of the enzymatic reaction will be hindered.

The esterase or microorganism in the present invention is:

For example, a culture of bacterial cells cultivated in a liquid medium, bacterial cells isolated from a culture medium, purified esterase or crude esterase separated from bacterial cells or culture liquid based on various enzyme separation methods, and an esterase-containing extract.

It is used in the form of processed products such as concentrated liquid. It is also possible to use immobilized products in which esterase, microorganisms, and processed products of microorganisms are immobilized.

9 for carriers for immobilization of esterases and microorganisms.
Examples include natural products such as alginic acid, carrageenan, collagen, cellulose, acetylcellulose, agar, cellophane, and collodion, and synthetic polymers such as polyacrylamide, polystyrene, polyethylene glycol, polypropylene glycol, polyurethane, and polybutadiene.

The amount of esterase and microorganism immobilized on the carrier depends on the type of carrier and microbial cells, the purity of esterase, and the like. However, 1 to 5 g of carrier is usually used per 1 g of microbial cells (wet basis). Also, for esterase, 910
(2) It is appropriate to use 0.1 to 2 g of carrier relative to the amount of protein.

There is no particular limitation on the concentration of the organic carboxylic acid ester of (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one in a water-containing organic solvent.
．． High concentrations such as 1-70% can also be used.

The above ester is one example.

Esterase protein amount: 0.1 per 1 mg protein
-2g, preferably 0.5-1g. In microorganisms that have esterase-producing ability.

1 for 1 g of microorganisms (lyophilized standard), e.g. 0.1 to
2 g of ester are used.

In the present invention, the reaction temperature in the hydrolysis reaction of the organic carboxylic acid ester of (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one is 20 to 4
The temperature is adjusted to 5°C, preferably 25 to 35°C. 2
If the temperature falls below 0°C, the hydrolysis reaction will not proceed sufficiently.

When the temperature exceeds 45°C, the activity of esterase decreases and the progress of the 9 reaction is hindered.

A suitable reaction time is 5 to 72 hours. but.

One reaction time can be shortened by increasing the amount of microorganisms or the amount of esterase. The reactor used for the reaction may be either an 8-batch type or a continuous flow type. When using a continuous flow reactor.

The space velocity of the substrate liquid is preferably in the range of 0.1 to 5 hr-'.

From the reaction liquid selectively hydrolyzed by the method of the present invention, (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one and (-)-3-alkyl-substituted-5-hydroxymethyloxazolidine The separation of -2-one from the organic carboxylic acid ester is carried out, for example, in the following manner: After the reaction solution is washed with an aqueous sodium bicarbonate solution, the aqueous layer is removed and the organic layer is dehydrated with nitric acid. The organic solvent is distilled off, and 1 g of the obtained oil is subjected to column chromatography using 30 g of silica gel for column chromatography (Wako Gel C-200) and ethyl acetate as a developing solvent. Hydrolyzed by column chromatography (+)
The body and the unhydrolyzed (-) body are separated.

Among the (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-ones thus obtained, particularly (+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one is Halogenated by reaction with thionyl. By coupling this (+)-3-isopropyl-5-methyloxazolidin-2-one chloride with sodium naphthoxide and further opening the ether ring, j2-propranolol is obtained. During the reaction process, the optical rotation changes from dextrorotation to dextrorotation.

(Example) The present invention will be described below with reference to examples.

Next 50"L acrylamide 723■ and N-N'-methylenebisacrylamide 38Akebono were dissolved in potassium phosphate buffer (
pH 8, 0, 50mM) was dissolved in 2.3ml. Brevibacterium protophorima
eriumprotophorimae IFO-12
128) 720 mg of wet microbial cells were suspended in 2 ml of potassium phosphate buffer (pHB, 0.50 mM) and mixed with the acrylamide solution.

Add 0.1 mj2 of N-N-N'・N''-tetramethylenediamine aqueous solution (0.1 g/ml) and ammonium persulfate aqueous solution (50 mg/m (1) to the mixed solution.
After adding 0.1 ml of the mixture, the mixture was left to stand at 0° C. for 5 minutes and then at room temperature for 1 hour under a nitrogen gas atmosphere to obtain an immobilized product of microbial cells. This immobilized product was cut into cubes of 21 x 2 mm x 2 m.

Acetone/water mixed solvent (377 volume ratio) to 100 mff.

20 g of acetate of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one as substrate
was dissolved. Add the cut pieces of the above immobilized product to this solution, and shake at 30°C for 48 hours (1805 trokesaki
n) L was reacted.

After the reaction is completed, the immobilized microorganisms are removed.

After adding an equal amount of ether to the reaction solution, the ether layer was washed with a saturated aqueous solution of sodium hydrogen carbonate.

The aqueous layer was repeatedly extracted with ether, the ether layers were collected, and then dried over sulfur salt. Distill the ether.

Column chromatography was performed using 30 g of silica gel per 1 g of the obtained oil. As a result, (+)-3-isopropyl-5-hydroxymethyloxazolidine-2
8.5 g of -on was obtained [α].

=+46.8', c; 2.0% CI, OH)
.

In place of acetic acid ester of large carbonaceous (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one, (sat)
The same operation as in Example 1 was carried out except that the acetate ester of -3-(t-)ethyl)-5-hydroxymethyloxazolidin-2-one was used. (+)-
8.0 g of 3-(t-butyl)-5-hydroxymethyloxazolidin-2-one was obtained ([α].=+4
6. P, c; 2.0% CH, OH).

Fruit 1B Note 1 As a microorganism, Rhodotorula texensis var minuta (Rhodotorula texensis)
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as in Example 1, except that var m1nutaIFO-0932) was used. As a result, the optical rotation [α] P= +46
．． (+) of 7° (C; 2.0% CI (:l011)
7.5 g of -3-isopropyl-5-hydroxymethyloxazolidin-2-one was obtained.

Example 4 As a microorganism, Enterobacter aerogenes (En
terobacter aerogenes TFO-
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as in Example 1, except that 13534) was used. As a result, the optical rotation [α] P=+44.3'(c;'1.0%
(+)-3-isopropyl- of CII ff O11)
5-Hydroxymethyloxazolidin-2-one 6.5
g was obtained.

Corynebacterium su, Pedonicum (
Corynebacterum spedonicus
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as in Example 1, except that m IFO-13763) was used.

As a result, 2 optical rotations [α) P=+46.1” (c;
(+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one in 2.0% CH, 011)8.
3g was obtained.

Rhizopus chinensis (Rhizopus
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as in Example 1, except that S. schinensis IFO-4768) was used. As a result, the optical rotation [α) g'= +
43.5'(c; 2,0% CH30H) (+
6.0 g of )-3-isopropyl-5-hydroxymethyloxazolidin-2-one were obtained.

In the same manner as in Example 1, except that propionate ester of the above compound was used in place of acetate ester of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one (± )-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated. the result.

Optical rotation (α) o'=+42.1@<c; 2.0
(+)-3-isopropyl-5- of %C1l, 011)
6.0 g of hydroxymethyloxazolidin-2-one was obtained.

Example 8 (±)-Produced in the same manner as in Example 1, except that acetic acid ester of the above compound was used instead of acetic acid ester of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one. Separation of 3-isopropyl-5-hydroxymethyloxazolidin-2-one was carried out. As a result, the optical rotation [α) g'= +44.3'(c
; 2.0% C1130 old (+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one 5
．． 0 g was obtained.

Example 9 (±) Same as Example 1 except that valerate of the above compound was used instead of acetate of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one (±) -3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated. As a result, the optical rotation (α) ft5= +43.5° (C;
5.
5g was obtained.

Example 1 except that the malonic acid ester of the above compound was used in place of the acetic acid ester of Shinhan Bakan (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one.
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as above. As a result, optical rotation (α) '8' = +38.5' (
c; 5.5 g of (+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one with 2.0% CH3θ11) was obtained.

Example 1 except that the succinate ester of the above compound was used in place of the acetate ester of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one.
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as above. As a result, the optical rotation [α] P = + 46.8° (c
(+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one in 2.0% CH30H)8.
2g was obtained.

Tsugan Masuhi Instead of acetone/water mixed solvent (377 volume ratio).

Separation of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was performed in the same manner as in Example 1, except that a methanol/water mixed solvent (174 volume ratio) was used. As a result, optical rotation [α) 1t5= +
(+)- of 46.7°(c; 2.0%CH30H)
5.7 g of 3-isopropyl-5-hydroxymethyloxazolidin-2-one were obtained.

Disaster U Instead of acetone/water mixed solvent (377 volume ratio).

Dimethyl sulfoxide (DMSO>/water mixed solvent (1
(/4 volume ratio) was used in the same manner as in Example 1 to separate (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one.

As a result, optical rotation [α) fj' = ``46.5''
6.5 g of (+)-3-isopropyl-5-hydroxymethyloxazolidin-2-one in (C; 2.0% CJhOH) was obtained.

Tsubame ■dan Instead of acetone/water mixed solvent (377 volume ratio).

Separation of (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was carried out in the same manner as in Example 1, except that an acetonitrile/water mixed solvent (377 volume ratio) was used. the result.

Optical rotation [α)! lS= +46.7° (c; 2.0
(+)-3-isopropyl- of %CI+ 3011)
5-Hydroxymethyloxazolidin-2-one 7.5
g was obtained.

charcoal charcoal Instead of acetone/water mixed solvent (377 volume ratio).

Dimethylformamide (DMF)/water mixed solvent (17
(±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one was separated in the same manner as in Example 1, except that 4 volume ratio) was used.

As a result, the optical rotation [α) P= +42.3° (c;
(+)-3-isopropyl- of 2.0%C1hOII)
5-Hydroxymethyloxazolidin-2-one 6.0
g was obtained.

(Effects of the Invention) According to the present invention, from (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one to (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one, -one is easily separated in high yield. Among the (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-ones obtained, (+
)-3-isopropyl-5-hydroxymethyloxazolidin-2-one is l-propranolol (which is
It is useful as a synthetic intermediate for antiarrhythmia drugs and antihypertensive drugs. Also.

(+)-3-(t-Butyl)-5-hydroxymethyloxazolidin-2-one can be effectively used as an intermediate for the synthesis of optically active forms of carteolol, which have remarkable antihypertensive and cardiac depressant effects.

that's all

Claims (1)

[Scope of Claims] 1. At least one selected from the group consisting of esterase, an immobilized product of esterase, a microorganism having esterase-producing ability, an immobilized product of the microorganism, and an immobilized product of a processed product of the microorganism, (±) Biochemical separation of (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-ones comprising contacting -3-alkyl-substituted-5-hydroxymethyloxazolidin-2-ones in an aqueous organic solvent. Law. 2. The esterase is an esterase produced from a microorganism belonging to at least one of the genera Brevibacterium, Rhodotorula, Enterobacter, Corynebacterium, and Rhizopus. Biochemical separation method of (+)-3-alkyl substituted-5-hydroxymethyloxazolidin-2-ones as described. 3. Claim 1, wherein the microorganism is an esterase produced from a microorganism belonging to at least one of the genus Brevibacterium, Rhodotorula, Enterobacter, Corynebacterium, and Rhizopus.
A biochemical separation method for (+)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one as described in Section 1. 4. The (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one is (±)-3-isopropyl-5-hydroxymethyloxazolidin-2-one and (±)-3-(t- (+)-3-alkyl-substituted-5-hydroxymethyloxazolidine-2 according to claim 1, which is at least one of (butyl)-5-hydroxymethyloxazolidin-2-one.
- Biochemical separation method of on. 5. The organic carboxylic acid ester of (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one has a carbon number of (±)-3-alkyl-substituted-5-hydroxymethyloxazolidin-2-one and The compound according to claim 1, which is an ester obtained by reacting a chloride of an aliphatic monocarboxylic acid having 1 to 7 carbon atoms and a monoester monochloride of an aliphatic dicarboxylic acid having 3 to 7 carbon atoms. +) Biochemical separation method of -3-alkyl-substituted-5-hydroxymethyloxazolidin-2-ones. 6. (+)- according to claim 5, wherein the monocarboxylic acid is at least one of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, heptanoic acid, acrylic acid, and crotonic acid. Biochemical separation method of 3-alkyl substituted-5-hydroxymethyloxazolidin-2-ones. 7. The (+)-3-alkyl-substituted compound according to claim 5, wherein the dicarboxylic acid is at least one of malonic acid, succinic acid, glutaric acid, pimelic acid, maleic acid, and fumaric acid. Biochemical separation method of 5-hydroxymethyloxazolidin-2-one. 8. The organic solvent is n-heptane, isoheptane, n-heptane,
-hexane, isohexane, n-pentane, isopentane, n-octane, isooctane, cyclopentane, cyclohexane, cycloheptane, benzene, toluene,
Xylene, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetone, methyl isopropyl ketone, tetrahydrofuran, dimethyl ether, diethyl ether, dioxane, methyl acetate, ethyl acetate, butyl acetate, carbon tetrachloride, chloroform, chloride The (+)-3-alkyl substituted - according to claim 1, which is at least one member selected from the group consisting of methylene, methane chloride, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, and acetonitrile. 5
- Biochemical separation method of hydroxymethyloxazolidin-2-one.