JPH0242477B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing optically active β-(S)-aminoglutaric acid monoalkyl ester. Optically active β-(S)-aminoglutarate monoalkyl ester is a substance useful as a raw material for the synthesis of carbapenems such as thienamycin that require optical activity, and the development of an easy and inexpensive manufacturing method for this substance is eagerly awaited. It had been. Ohno et al. used porcine liver-derived esterase to asymmetrically hydrolyze optically inactive β-benzyloxycarbonylaminoglutarate dimethyl ester (), which is easily obtained by organic synthesis. β-
(S)-benzyloxycarbonylaminoglutarate monomethyl ester () is prepared, and the benzyloxycarbonyl group as an amino protecting group is removed by catalytic reduction, resulting in optically active β-(S)-
We have developed a method to make aminoglutarate monomethyl ester (). however,

[Formula]Me= _CH3 . Furthermore, Ohno et al. suggest the use of not only animal enzymes but also enzymes derived from microorganisms, which may be advantageous when carried out on an industrial scale. Therefore, the present inventors conducted research on a method for performing asymmetric hydrolysis using microorganisms that can be mass-produced.
It has been shown that there are many microorganisms that have the ability to stereospecifically hydrolyze only one ester group of β-protected aminoglutarate dialkyl ester and exchange it into β-(S)-protected aminoglutarate monoalkyl ester. Heading Completing the Invention. In other words, the present invention is expressed by the following formula (In the formula, R is an alkyl group having 1 to 4 carbon atoms,
A is an amino protecting group that can be removed by catalytic reduction or mild hydrolysis. ), which has the ability to selectively hydrolyze only one ester group (R) in the β-protected dialkyl aminoglutarate ester of genus Tropisis, genus Debaryomyces, genus Endomyces, genus Satucharomycopsis, and Cryptococcus. Culture solution, cells or bacteria of microorganisms belonging to the genera Pachyzolens, Sporobolomyces, Syringospora, Corynebacterium, Pseudomonas, Arthrobacter, Bacillus, Staphylococcus, Streptococcus The following formula, characterized in that the amino protecting group (A) is removed by a conventional method, (wherein R has the same meaning as above) The present invention relates to a method for producing an optically active β-(S)-aminoglutaric acid monoalkyl ester. The amino protecting group (A) in the β-protected aminoglutarate dialkyl ester used as the starting compound of the present invention can be removed by catalytic reduction, and can be a benzyl group, an aralkyl group such as a benzhydryl group, or a benzyloxycarbonyl group. Aralkyloxycarbonyl groups such as the following are preferred, but t-butoxycarbonyl groups and the like which can be eliminated by mild hydrolysis may also be used. Further, R may be any alkyl group having 1 to 4 carbon atoms, but a methyl group is most preferable in view of ease of hydrolysis. As a microorganism that has the ability to selectively hydrolyze only one ester group of the β-protected aminoglutarate dialkyl ester used in the present invention and convert it into β-(S)-protected aminoglutarate monoalkyl ester, For example, Torulopsis candida IFO00380, Torulopsis inconspica
inconspicua) IFO0621, Torulopsis pinus IFO0741, Debaryomyces marama
IFO0668, Debaryomyces hansenii IFO 0794, Endomycestetrasperma CBS 765, 70, Saccharo-
mycopsis lipolmtica) IFO 0746, Cryptococcus albidus
IFO0378, Pachysolen tannophilus IFO 1007, Sporobolomyces
salmonicolor) IAM 12249, Syrigonspora,
Syringospora claussenii
IFO0759, Corynebacterium sepedonicum IFO13763,
Corynebacterium xerosis IFO 12684, Corynebacterium spenicum
spenicum) IFO3306, Pseudomonas riboflavina (Pseudomonas riboflavina) IFO13584,
Pseudomonas solanacearum IFO3898, Pseudomonas aeruginosa
aeruginosa) IFO13130, Arthrobacter paraffineus
ATCC21218, Bacillus megaterium ATCC10778, Staphylococcus aureus
aureus) IFO3060, and Streptococcus faeca-lis (Streptococcus faeca-lis) IFO12964. IAM: Institute of Applied Microbiology, University of Tokyo IFO: Fermentation Research Institute CBS: Centraalbureau voor Schim−
melcultures Baarn These microorganisms are usually cultured in liquid media, but can also be cultured on solid surfaces. The culture medium contains organic and inorganic carbon sources, nitrogen sources, vitamins, minerals, etc. that can be normally assimilated, and the culture temperature is 20-50℃.
A pH range of 3 to 11 is used. Furthermore, the growth of microorganisms can be promoted by aeration and stirring. In the hydrolysis reaction of β-protected aminoglutarate dialkyl ester, which is a reaction substrate, the reaction substrate is added to the medium at the start of culture and the hydrolysis reaction is carried out in parallel with the culture, or as described above. There is a method in which the obtained culture solution, bacterial cells, or treated bacterial cells are brought into contact with a reaction substrate to perform hydrolysis. Desirably, a method of concentrating the bacterial cells by centrifugation or the like to obtain a highly concentrated bacterial body liquid and adding a reaction substrate to this liquid is preferable for recovering the product after the reaction. Furthermore, the cells can be used as freeze-dried cells for convenience of handling, and can also be used as crushed cells or cell extracts. The concentration of β-protected aminoglutarate dialkyl ester, which is a reaction substrate, in the reaction solution is 0.01 to 50.
It can be used up to concentrations as high as %. The solubility of β-protected aminoglutarate dialkyl ester in water is generally low, but this does not interfere with this reaction as long as sufficient contact with the bacterial cells or treated bacterial cells is maintained by stirring. .
It is even better if a hydrophilic solvent such as acetone or a surfactant is added to an extent that does not interfere with the reaction. The pH used for the hydrolysis reaction is in the range of 3 to 11, preferably in the range of 6 to 8.
When reacting at high concentrations, the product β-protected aminoglutaric acid monoalkyl ester gradually accumulates in the reaction solution and the pH decreases, so maintain the optimal pH with an appropriate neutralizing agent. is preferable. The hydrolysis reaction is usually carried out at a temperature in the range of 15 to 50°C, but a temperature suitable for the strain used is adopted. A common method may be used to isolate the β-protected aminoglutaric acid monoalkyl ester produced by the hydrolysis reaction from the reaction solution. For example, after removing insoluble substances such as bacterial cells from the reaction solution by centrifugation, the pH of the reaction solution is adjusted to 1 and extracted with ethyl acetate. Next, after dehydration with anhydrous sodium sulfate, the mixture is concentrated to obtain an oily β-(S)-protected aminoglutaric acid monoalkyl ester.
This is purified by column chromatography on silica gel prepared with benzene, and when the solvent is removed, β-(S)-protected aminoglutaric acid monoalkyl ester is obtained as white crystals. Then, deprotection is performed using a known catalytic reduction method such as dissolving in methanol and reducing with hydrogen under a palladium-carbon catalyst, or depending on the protecting group, hydrolysis with trifluoroacetic acid or the like. β-(S)-aminoglutaric acid monoalkyl ester having the following formula can be easily obtained. EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited only to these Examples. Example 1 A nutrient liquid medium consisting of the following composition was prepared, and 2
After dispensing 400 ml each into Sakaguchi flasks, the mixture was sterilized at 120°C for 20 minutes. (Medium composition) Glucose 4%, yeast extract
0.3%, meat extract 0.3%, peptone 0.3%, ( _NH4 )
PO ₄ 0.2%, KH ₂ PO ₄ 0.1%, PH7.0 Apart from this,
10 ml of the inoculum of the microorganism shown in Table 1 pre-cultured in a medium with the same composition was inoculated into the culture medium and incubated at 30°C.
A shaking culture was performed for 24 hours. For each strain
Cultivate 10 cells at a time to obtain 4 cultures of each bacteria, suspend the cells in 1 part M/15 phosphate buffer (PH7.0) by centrifugation, and add β-benzyloxycarbonyl dissolved in 30 ml acetone. 3 g of aminoglutarate dimethyl ester was added. Pour this into a 3-volume mini-jar fermenter and heat at 30℃ for 6 hours while stirring.
Allowed time to react. After the reaction, the supernatant obtained by centrifugation was adjusted to pH1 with sulfuric acid.
The mixture was extracted with 2 portions of ethyl acetate. Thereafter, it was dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. This was loaded onto silica gel prepared by suspending it in benzene, and eluted with a benzene:acetone (10:1) mixture. The β-benzyloxycarbonylaminoglutarate monomethyl ester fractions were collected and the solvent was removed under reduced pressure to obtain white crystals of β-benzyloxycarbonylaminoglutarate monomethyl ester. The MNR spectrum and the Rf value determined by silica gel thin layer chromatography (ethyl acetate: ethanol: water = 5:1:1) were consistent with the standard product prepared using pig liver esterase by the method of Ohno et al. In addition, when we measured the optical rotation angle, we found that [α] _D ²⁵ = +0.55 to 0.71° (C =
6, CHCl ₃ ), all of which were stereoselectively hydrolyzed β-(S)-benzyloxycarbonylaminoglutarate monomethyl ester. β-(S) obtained by each microbial reaction as described above
-Dissolve 200 mg each of benzyloxycarbonylaminoglutarate monomethyl ester in 20 ml of methanol, add 40 mg of 10% palladium on carbon, and stir for 30 minutes under a hydrogen stream. After filtering and concentrating the reaction solution, 75 to 97 mg of white crystals of β-(S)-aminoglutaric acid monomethyl ester were obtained.
All NMR spectra were consistent with the standard product. In addition, these β-(S)-aminoglutarate monomethyl esters have [α] _D ²⁵ = −5.40 to −5.61° (C =
3, H ₂ O).

[Table] -The amount of aminoglutarate monomethyl ester is shown.
Example 2 Torulopisis pinus IFO0741, Debaryomyces marama IFO0668, and S. riboflavina IFO13584 were cultured in the same manner as in Example 1 to prepare a bacterial cell suspension. β-t for each bacterial cell suspension 1
-Butoxycarbonylaminoglutarate dimethyl ester was added in 3 g portions, stirred in a 3-volume mini jar fermenter, and reacted at 30°C for 6 hours.
After the reaction, extraction and purification was performed in the same manner as in Example 1, and further deprotection was performed using trifluoroacetic acid, followed by isolation and purification using ion exchange chromatography.
Each β-(S)-aminoglutaric acid monomethyl ester was obtained as white crystals in the amounts shown in Table 2. All of these NMR spectra agree with the standard specimen, and the optical rotation is [α] _D ²⁵ = −5.50 to −5.61° (C
= 3, H ₂ O), and the product was confirmed to be β-(S)-aminoglutarate monomethyl ester.

[Table] Example 3 Torulopsis binus IFO0741, Debaryomyces marama IFO0668, and Pseudomonas ribflavina IFO13584 were cultured in the same manner as in Example 1, and two cell suspensions were prepared for each strain. Divide each bacterial cell suspension into two equal parts, and one half contains 3 g of β-benzyloxycarbonylglutarate diethyl ester,
To the other side, 3 g of β-t-butoxycarbonylaminoglutarate diethyl ester was added, and the pH was adjusted to 7.0.
The reaction was carried out at 30° C. in a mini-jar fermenter for 24 hours with stirring. After the reaction, extraction and purification and deprotection were performed in the same manner as in Examples 1 and 2, and as a result, oily substances were obtained from each reaction solution. This NMR spectrum matched that of the standard β-aminoglutarate monoethyl ester. As a result of measuring these light exposure degrees, [α] _D ²⁵ = −3.80 to −3.85° (C = 4, H ² O)
It was confirmed that it was β-(S)-aminoglutaric acid monoethyl ester.

Claims (1)

[Claims] Linear formula () (In the formula, R is an alkyl group having 1 to 4 carbon atoms,
A is an amino-protecting group that can be removed by catalytic reduction or mild hydrolysis). Genus, Debaryomyces, Endomyces, Satucharomycopsis, Cryptococcus, Pachyzolens, Sporobolomyces, Syringospora, Corynebacterium, Pseudomonas, Arthrobacter, Bacillus, Staphylococcus The following formula (), characterized in that a culture solution, bacterial cells, or treated bacterial cells of a microorganism belonging to the genus Kass or Streptococcus is applied, and then the amino protecting group (A) is removed by a conventional method. (wherein R has the same meaning as above) A method for producing an optically active β-(S)-aminoglutaric acid monoalkyl ester. 2. The manufacturing method according to claim 1, wherein A in the formula () is a benzyloxycarbonyl group, and R in the formula () is a methyl group or an ethyl group. 3. The manufacturing method according to claim 1, wherein A in the formula () is a t-butoxycarbonyl group, and R in the formula () is a methyl group or an ethyl group.