JPH01211494A - Production of optically active alpha-hydroxyoxide - Google Patents

Abstract

PURPOSE:To produce in high efficiency the title alpha-hydroxyoxide useful as a raw material for circulatory remedies, by assymetric reduction of an alpha-oxoxide represented by a specific general formula with the culture product (or its treated product) from a strain belonging to the Lactobacillus. CONSTITUTION:A culture product or its treated product from a strain belonging to the Lactobacillus and an alpha-oxoxide of formula I (R1 is aralkyl or acyl; R2 is H, lower alkyl or aralkyl; X is alkylene) are brought into contact with each other to carry out assymetric reduction of the oxoxide to produce the objective optically active alpha-hydroxyoxide (or its salt) of formula II (assymetric carbon denoted by * represents R-coordination or S-coordination; R1, R2 and X are each the same as that in the formula I). A typical strain to be used in said assymetric reduction is e.g., Lactobacillus casei subsp. casei.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is directed to the use of optically active
- A method for producing a hydroxy acid compound.

More specifically, the present invention relates to Lactobacillus (Lactobacillus).
A culture of a strain belonging to the genus Bacillus or a processed product thereof, and a compound represented by the general formula [wherein R1 represents aralkyl or acyl, R7 represents hydrogen, lower alkyl or aralkyl, and X represents alkylene]. The compound represented by the general formula (1) is asymmetrically reduced by contacting with
- coordination, and other symbols have the same meanings as above], or a salt thereof.

Background Art As a method for producing an optically active α-hydroxy acid compound from an α-oxo acid compound by asymmetric reduction using microorganisms or enzymes, there is a method using baker's yeast (European Patent Publication EP-A-0187037, JP 1986-14
9658, etc.), a method using L-2-hydroxyisocaproic acid dehydrogenase derived from Lactobacillus conrusus (Schutte, 11゜et at,
: Appl, Microbiol, Bio
technol, +9°167 (1984)),
Leuconostoc
oenos), o Iconostoc 0 mesenteroides (Leuconostoc mesenteroi)
des).

Leuconostoc lactis
lactjs), Lactobacillus casei subspecies pseudoplantarum
uscasei 5ubsp, pseudopl
antarum), Lactobacillus casei subspecies rhamnosus
casei 5ubsp, rhamnosus) or Lactobacillus casei subspecies lactosus (Lactobacillus casei 5ubsp, rhamnosus)
5absp.

A method using D-2-hydroxyisocaproate dehydrogenase of bacterial origin belonging to P. alactosus (
Hummel W, et al: Appl.
Microbiol, Bio-technol
D- Or L-hydroxycaproate dehydrogenase is expressed by the formula (
It is not known at all whether the piperidine derivative represented by 1) or the derivative obtained by esterifying its carboxyl group has reactivity in asymmetric reduction with respect to an α-carbonyl group.

Problems to be Solved by the Invention The present invention provides a piperidine derivative represented by formula (II) that is useful as a raw material for the synthesis of angiotensin-converting enzyme inhibitors, which are therapeutic agents for cardiovascular diseases such as hypertension, heart disease, and stroke. optically active α-(R)-hydroxy acid or α
In order to produce -(S)-hydroxy acid, a piperidine derivative having a corresponding α-carbonyl group represented by formula (1) is asymmetrically reduced, and α represented by formula (II) is
- Provides a method for efficiently producing hydroxy acid compounds.

Means for Solving the Problems The present inventors have actively searched for microorganisms that can efficiently produce the corresponding optically active α-hydroxy acid by reducing the α-carbonyl group of piperidine derivatives, and have set out to obtain the same. As a result of this success and further research, we have completed the present invention.

The microbial strain used in the asymmetric reduction reaction of the present invention includes Lactobacillus bacteria, particularly Lactobacillus casei.
Subspecies casei (Lacto-bacillus)
s casei 5ubsp, casei) or Lactobacillus sp.
US sp, ) is preferably used, but the strain is not particularly limited, and even strains newly isolated from soil, food, animals and plants can be used to asymmetrically reduce the compound represented by formula (1). Any compound capable of producing the compound represented by formula (II) can be used in the method of the present invention. In addition, there are strains that have been artificially mutated by UV irradiation or treatment with mutagens, and other microorganisms that have been artificially extracted and incorporated with gene fragments necessary for expressing the reduction activity. Even bacterial cells can be used in the method of the present invention.

A specific example of a strain used for asymmetric reduction of the α-carbonyl group of the piperidine derivative represented by formula (1) is Lactobacillus casei 5.
ubsp.

casei I F 0 12004 strain or Lac
tobac i-11us sp, IP 0
There are 3,954 stocks. This strain is included in the [List of Cultures] published by the Fermentation Research Institute.
or Cultures), 7th edition, 1984J61
These are known strains listed on pages 62 and 62, respectively.
It is easily available from the Fermentation Research Institute. Among these, the former is preferably used to obtain an optically active α-(R)-hydroxy acid compound, and the latter is preferably used to obtain an α-(S)-hydroxy acid compound.

To cultivate these microbial strains, conventional static culture,
This can be carried out continuously or intermittently by shaking culture, aerated agitation culture, solid state culture, or the like. The medium used may have a normal composition that allows the microorganisms used to grow, and the carbon source may be selected from among carbohydrates, fats and oils, fatty acids, organic acids, alcohols, etc., and may be used singly or in combination. used as Nitrogen sources include organic nitrogen sources such as peptone, soybean flour, cottonseed flour, cornstarch liquor, yeast extract, meat extract, malt extract, and urea, as well as inorganic sources such as ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphorus. Nitrogen sources may be used alone or in combination as appropriate. In addition to a carbon source and a nitrogen source, essential growth factors and growth-promoting substances such as minerals, amino acids, and vitamins necessary for growth are preferably added to the medium.

p) during culture (and for the purpose of foam management, caustic alkaline solution, sodium carbonate solution, calcium salts can be added as appropriate, and addition of an antifoaming agent is also effective.Furthermore,
It is also effective to aerate nitrogen gas or carbon dioxide gas to make the environment anaerobic.

The culture temperature may be selected to be suitable for the growth of the microorganism used, and is usually 15°C to 55°C, preferably 25°C.
It is advantageous to culture at between 45°C and 45°C. The culture is continued for a sufficient period of time for the growth of the microorganism used, and is usually 5 to 48 hours.

After culturing in this manner, the strain may be used as a cultivated culture, or by some separation method such as centrifugation, sedimentation, flocculation, filtration using a porous membrane, polymer membrane, ceramic membrane, etc. The microbial cells isolated by the method or the processed product thereof are subjected to an asymmetric reduction reaction. The "processed product" used in the present invention refers to the culture obtained as described above subjected to physicochemical treatments such as filtration, centrifugation, ultrasonic treatment, French press treatment, osmotic pressure treatment, freeze-thaw treatment, alumina grinding treatment, etc. It refers to bacterial cells obtained by lytic enzyme treatment, surfactant treatment, organic solvent treatment, etc., or crushed bacterial cells containing an enzyme having the activity of asymmetrically reducing the α-carbonyl group of compound (I). Furthermore, the enzyme obtained by purification by the method described later, or immobilized bacterial cells or the enzyme can also be used. The enzyme can be used by subjecting the bacterial cells obtained above to, for example, freeze-thawing treatment, grinding treatment, ultrasonication treatment,
Solubilization can be achieved by subjecting to physicochemical treatments such as osmotic pressure treatment, cell wall membrane dissolution treatment, and surfactant treatment. In addition, the solubilized enzyme is further subjected to conventional enzyme purification methods such as protamine treatment, salting out, organic solvent treatment, isoelectric focusing precipitation, electrophoresis, ion exchange chromatography, gel filtration, affinity chromatography, and crystallization. Purification can be achieved by appropriately combining them.

The enzymes obtained are natural polymers such as agar and carrageenan,
It can be encircled and immobilized on a synthetic polymer such as polyacrylamide or urethane resin, or it can be bonded and immobilized on a carrier such as activated carbon, ceramic, dextran, agarose-based material, or porous glass.

Examples of the lower alkyl group represented by R3 in formula (1) include alkyl groups having about 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 5ec-butyl, and tert-butyl. Also,
Examples of the aralkyl group represented by R3 or R include phenyl lower ( 01-4) Alkyl groups. Examples of the acyl group represented by R1 include lower (C, ~
,) alkanoyl (e.g. acetyl, propionyl), benzoyl.

Phenyl lower (C3-4) alkoxycarbonyl (e.g.
benzyloxycarbonyl), lower (C+~4) alkoxycarbonyl (e.g. tert-butoxycarbonyl)
Examples include acyl groups such as Examples of the alkylene chain represented by , ethylmethylene, dimethyltetramethylene, and other divalent groups. The alkylene chain has an unsaturated bond within the chain (e.g.
double bond, triple bond). Among these compounds, 6-(1-acyl-4-piperidyl)
-2-oxohexanoic acid or its lower (C4-4) alcohol ester is preferred, and lower (CI,) alcohol ester of 6-(1-benzyloxycarbonyl-4-piperidyl)-2-oxohexanoic acid is particularly preferred. . The concentration of compound (1) in the reaction solution in the reaction carried out by bringing compound (1) into contact with a culture of a strain belonging to the genus Lactobacillus or a treated product thereof is preferably 0.
It is preferably 1% to 20%, more preferably 0.5% to 5%. Furthermore, it is preferable to add a carbon source that can be assimilated by the microorganisms (e.g., carbohydrates, fats and oils, fatty acids, organic acids, alcohols) together with the compound represented by formula (1) for the purpose of hydrogen donation. Minerals and vitamins may be added to promote this.

The reaction temperature may be within a range that allows the asymmetric reduction reaction to proceed, but it is usually preferably in the range of 20 to 60°C, more preferably in the range of 25 to 45°C. The pH range is preferably pH 2 to 1O, more preferably pH 5 to 7. During the reaction, it is desirable to stir the reaction solution appropriately in order to improve the contact between the microbial cells or their processed product and the compound represented by formula (1). Preferably, the atmosphere is maintained in an anaerobic state, for which purpose nitrogen gas or carbon dioxide gas may also be passed through.

The reaction is continued for a period of time until the α-oxo acid compound represented by formula (I) is asymmetrically reduced, but is usually 2 to 10 hours.
It is 0 hours.

After the reaction, the optically active α-hydroxy acid compound produced can be easily recovered and collected by combining conventional purification methods such as extraction and concentration.

When R8 is hydrogen, the compound represented by formula (n) may form a salt with an alkali metal (e.g., sodium, potassium), and the salt of the compound represented by formula (n) can be formed by the above reaction and Although it may be obtained by the purification method itself, it can also be obtained by adding a base to the compound represented by formula (II), if necessary.

Effect Compound (n) obtained by the present invention is subjected to a halogenation reaction or a sulfonylation reaction known per se to obtain a compound of the general formula [wherein W is halogen (e.g. chlorine) or a formula R-sot
A compound represented by -o- (wherein R represents lower (CI, 4) alkyl, trifluoromethyl, phenyl or p-)lyl, and other symbols have the same meanings as above) (European Patent Publication E)
P-A-0156455, JP-A-60-211668, etc.). Furthermore, using compound (I[l), angiotensin-converting enzyme inhibitor (European patent publication EP-
A-0156455, JP-A-60-231668, European Patent Publication EP-A-0187037, JP-A-62-149658, etc.) can be easily synthesized.

EXAMPLES The content of the present invention will be explained in more detail with reference to Examples below, but these are merely illustrative of the content of the present invention and do not limit the scope of the present invention.

Example 1 Lactobacillus casei 5 grown by puncture culture in commercially available CAM agar medium (Hinaga Pharmaceutical)
ubsp, caseiIFo 12004 strain with 2% glucose, 1% Ehrlich meat extract, 1% polypeptone, 0.5% yeast extract, 0.2% dipotassium phosphate,
Tween 80 0.1%, sodium acetate 0.5%, triammonium citric acid 0.2%, magnesium sulfate (7 hydrate) 0
20ml of sterile seed medium consisting of 0.02% manganese sulfate (approximately tetrahydrate) and 0.05%! The mixture was inoculated into 15 200M Erlenmeyer flasks containing the following, and cultured statically at 37°C for 24 hours. 3 d of this seed culture was transplanted into 83 200 d-volume pleated Erlenmeyer flasks containing 60 volumes of a sterile medium having the same composition, and cultured stationary at 37°C. Ethyl 6-(1-benzyloxycarbonyl-4-
piperidyl)-2-oxohexanoate (purity 68.
2%) 1%, glucose 3%, and calcium carbonate 2% were added to each flask, and the reaction was allowed to proceed until 96 hours with rotational shaking at 190 rpm. This reaction completed liquid is
The extract was sequentially extracted with 412.3Q and 2Q ethyl acetate, washed with water, dehydrated and concentrated to obtain 50.1 g of oily compound (A).

This was dissolved in ethyl acetate and washed with diluted sodium bicarbonate solution to obtain 23.5 g of composite material (B) from the ethyl acetate layer. Also, the diluted sodium bicarbonate aqueous layer is acidified with hydrochloric acid and extracted with ethyl acetate, and the oil obtained is ethanol! 00-, dissolved in toluene 100d and p
- 3.0 g of toluenesulfonic acid was added and refluxed for 8 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 120% of ethyl acetate, washed with water, diluted sodium bicarbonate solution and water, and the ethyl acetate was concentrated under reduced pressure to obtain 23.3 g of composite material (C).

When the content of ethyl 6-(1-benzyloxycarbonyl-4-piperidyl)-2-hydroxyhexanoate in compounds (B) and (C) was measured by high-performance liquid chromatography, they were 44.2% and 81%, respectively. The yield was 82.9%. Also these (-)-
When α-methoxy-α-trifluoromethylphenylacetic acid ester was derived and quantified by high performance liquid chromatography, the enantiomeric excess (%e, e,) was 94.4% and 49.6%, respectively, and (R ) The total yield is 6
It was 9.0%.

Example 2 Lactobacillus grown in the same manner as in Example I
casei 5ubsp, casei IF
12,004 strain seed cultures were transplanted into three 200-capacity pleated Erlenmeyer flasks containing 20 d of sterile medium with the same composition, and after 16 hours of static culture at 37°C, 6-(1-benzyloxycarbonyl-4-piperidyl)-2-oxohexanoic acid alkyl ester 1%, glucose 3%, and calcium carbonate 2% were added to shaker, model 221 Brunswick Scientific. While blowing nitrogen gas into the G-24 box at a rate of 1Q1 minute, the reaction was carried out by rotary shaking at 300 rpm for up to 48 hours, and the yield of the reduction product and the (R) integral enantiomer were determined in the same manner as in Example 1. Excess rate (e.

When the yield of (R) was investigated from (e,), the results shown in the table were obtained.

Table α-oxonis α-hydroxye (R) monolithic methyl ester 83.2 68
．． 1 Propyl ester 81.671.9 Butyl ester 82.3 71.5 Example 3 Lactobacillus grown in the same manner as in Example 1
Seed culture 2 of ssp, I P 0 3954 strain
Fnl was transplanted into a pleated Erlenmeyer flask containing 40 volumes of sterile medium with the same composition as the seed medium, except that cornstarch liquor was used instead of Ehrlich meat extract, and cultured statically at 37°C. Ethyl 6-(1-benzyloxycarbonyl-4-piperidyl)-2-oxohexanoate (purity 68.2%) 22 hours after the start of culture
2%, glucose 5%, calcium carbonate 3%,
The reaction was continued for 66 hours with rotational shaking at 190 rpm. The reaction solution was extracted twice with an equal amount of ethyl acetate, washed with water, dehydrated, and concentrated to obtain 770 mg of an oily compound.Ethyl 6-(l-benzyloxycarbonyl-4-piperidyl)- The content of 2-hydroxyhexanoate was measured to be 56.8%, and the yield was 80.2%. This one is (R)
Integrated 18.2%, (S) Integrated St.

The composition ratio was 8%.

Effect of the invention

Claims (1)

[Claims] A culture of a strain belonging to the genus Lactobacillus or a processed product thereof and a general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) [In the formula, R_1 represents aralkyl or acyl, R_
2 represents hydrogen, lower alkyl or aralkyl, and X represents alkylene],
There are general formulas, mathematical formulas, chemical formulas, tables, etc., which are characterized by asymmetric reduction of the compound represented by general formula (I) (II) [In the formula, the asymmetric carbon indicated by (*) is R -coordination or S
- coordination, and other symbols have the same meanings as above] A method for producing an optically active α-hydroxy acid compound or a salt thereof.