JPS644760B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing optically active (S)-β-aminoglutaric acid monoalkyl ester. Optically active (S)-β-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 method for producing this substance is eagerly awaited. was. Ohno et al. used esterase derived from pig liver to optically inactive β-benzyloxycarbonylaminoglutarate dimethyl ester (), which is easily obtained by organic synthesis.
One methyl ester group is stereospecifically hydrolyzed to produce optically active (S)-β-benzyloxycarbonylaminoglutarate monomethyl ester (), and further catalytic reduction is performed to convert benzyloxy as an amino-protecting group. We have developed a method for producing optically active (S)-β-aminoglutarate monomethyl ester () by removing the carbonyl group. (However, Z is

[Formula]Me is _CH3 . Ohno et al. also suggested the use of not only animal enzymes but also enzymes derived from microorganisms, which would be advantageous on an industrial scale. Therefore, the present inventors conducted research on a method using microorganisms capable of mass production, and found that only one ester group of β-protected aminoglutarate dialkyl ester was stereospecifically hydrolyzed, and (S)-β -We have discovered that there are many microorganisms that have the ability to convert into protected aminoglutaric acid monoalkyl esters, and have completed the present invention. That is, the present invention is based on the following formula (In the formula, R is an alkyl group having 1 to 4 carbon atoms, and A is an amino-protecting group that can be removed by catalytic reduction or mild hydrolysis.) Candeida spp., Pichia spp., Trichosporon spp., Geotrichum spp., Aspergillus spp., Absidia spp., Acteinomucor spp., Helicosterium spp., Mucor spp., Morteierella spp., Pecilomyces spp. , Chigorhynchus spp.
Microorganisms belonging to the genus Fusarium, Acremotheum, Circinella, Cunninghamella, Rhizotpus, Penicillium, Proteus, Micrococcus, Hafnia, Brevibacterium,
or Lord Cotchus Lord Claws IFO3338
to act on the culture solution, bacterial cells or treated bacterial cells,
The following formula is characterized in that the amino protecting group (A) is then removed by a conventional method. The present invention relates to a method for producing an optically active (S)-β-aminoglutaric acid monoalkyl ester (wherein R has the same meaning as above). 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 removed by catalytic reduction, such as an aralkyl group such as a benzyl group or 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
From the viewpoint of ease of hydrolysis, methyl group is most preferred. Examples of microorganisms that have 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 include: Candida arborea IAM4147 (FERM BP−
1888), Candida Rugoza
rugosa) IFO0750, Pichia farinosa (Pichia farinosa) IFO0534, Trichosporon
Trichosporon beigelii
ATCC22310, Trichosporon brassicae IFO1584, Geotrichum vanryiae
CBS439.64, Aspergillus niger IAM3008, Absidia
Hyalospora (Absidia hyalospora)
HUT1025, Actinomucor repens HUT1049, Helicostylum nigricans
nigricans) HUT1106, Mucor alternans HUT1115, Mortierella isabellina
HUT1110, Paecilomyces Varioti HUT4018, Zygorhynchus moelleri
HUT1305, Fusarium merismoides IFO30040, Acremonium basilisporum
bacillisparum IFO9387, Circinella mucoroides HUT1079, Cunninghamella elegans
elegans) HUT1098, Mucor javanicus HUT1155, Rhizopus javanicus HUT1252,
Penicillium daigitatum
digitatum) IFO9370, Proteus mirabilis (Proteus mirabilis) IFO3849, Micrococcus luteus (Micrococcus luteus) IFO12708,
Hafnia alvei IRO3731,
Brevibacterium linens IFO12142, Rhodococcus
rhodochrous) IFO3338. IAM: Institute of Applied Microbiology, The University of Tokyo IFO: Institute of Fermentation, HUT: Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University CBS: Centralbureau voor Schimmelcultures
Baarn These microorganisms are usually cultured in liquid media, but can also be cultured on solid surfaces. The culture medium is appropriately blended with normally assimilated organic and inorganic carbon sources, nitrogen sources, vitamins, minerals, etc., and the culture temperature is 20 to 50°C and the pH is in the range of 3 to 11. 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 for hydrolysis. Preferably, after concentrating the bacterial cells by centrifugation,
A method in which the reaction substrate is added to a highly concentrated bacterial body fluid is preferable for recovering the product after the reaction. Furthermore, the cells can be used as freeze-dried cells for convenience in handling, and can also be used as crushed cells or cell extracts. The concentration of β-protected aminoglutaric acid dialkyl ester, which is a reaction substrate, in the reaction solution is from 0.01%.
It can be used at concentrations as high as 50%. 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 amylglutaric 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,
Extract with ethyl acetate. After dehydration with anhydrous sodium sulfate and concentration, an oily (S)-β-protected aminoglutaric acid monoalkyl ester is obtained. This is purified by column chromatography on silica gel prepared with benzene, and when the solvent is removed, (S)-β-protected aminoglutaric acid monoalkyl ester is obtained as white crystals. Then, deprotection is performed by 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 etc., the optical rotation (-) A (S)-β-aminoglutaric acid monoalkyl ester having the following formula can be easily obtained. The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples. Example 1 A nutrient liquid medium consisting of the following composition was prepared, and 2
After dispensing 400ml each into Sakaguchi flasks, the mixture was sterilized at 120°C for 15 minutes. [Medium composition] Glucose 4%, yeast extract
0.3%, meat extract 0.3%, peptone 0.3%,
(NH ₄ ) ₂ PO ₄ 0.2%, KH ₂ PO ₄ 0.1%, PH 7.0 Separately, 10 ml of the inoculum of the microorganism shown in Table 1, which had been precultured in a medium with the same composition, was added to the culture medium. and cultured with shaking at 30°C for 24 hours. Ten strains of each strain were cultured to obtain four culture fluids of each strain, from which bacterial bodies were collected by centrifugation. The bacterial cells were dissolved in M/15 phosphate buffer (PH).
7.0) β suspended in 1 and dissolved in 30 ml acetone
- 3 g of benzyloxycarbonylaminoglutarate dimethyl ester were added. This was placed in a 3-volume mini-jar fermenter and reacted at 30°C for 6 hours with stirring. After the reaction, the supernatant obtained by centrifugation was adjusted to pH 1 with sulfuric acid, and extracted with 2 portions of ethyl acetate. After dehydration with anhydrous sodium sulfate, the solvent was removed under reduced pressure. This was loaded onto a silica gel column 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 Rf value determined by NMR spectrum and silica gel thin layer chromatography (ethyl acetate: ethanol: water = 5:1:1) was consistent with the standard product prepared using pig liver esterase by the method of Ohno et al. Furthermore, when we measured the optical rotation angle, we found that [α] ²⁵ _D = +0.55 to 0.71° (C = 6,
CHCl ₃ ), all of which were stereoselectively hydrolyzed (S)-β-benzyloxycarbonylaminoglutarate monomethyl ester. 200 mg of each monomethyl ester obtained by each microbial reaction as described above was dissolved in 20 ml of methanol,
Add 40 mg of 10% palladium on carbon and incubate for 30 minutes under a hydrogen stream.
Stir for a minute. When the reaction solution was filtered and concentrated, 75 to 97 mg of white crystals of (S)-β-aminoglutaric acid monomethyl ester were obtained, and the NMR spectrum of this product was entirely consistent with that of the authentic product. In addition, these (S)-β-aminoglutarate monomethyl esters are [α] ²⁵ _D = −5.40 to 5.61° (C = 3.H ₂ O)
showed that.

【table】

[Table] The amount of aminoglutarate monomethyl ester is shown.
Example 2 Candeida Arborea IAM4147 (FERM
BP-1888), Pichia falinosa IFO0534, and Geotrichum vanliae CBS439.64 were cultured in the same manner as in Example 1 to prepare a bacterial cell suspension. Add 3 g of β-t-butoxycarbonylaminoglutarate dimethyl ester to each bacterial cell suspension 1,
30°C under stirring in a 3 volume mini-jar fermenter
The mixture was allowed to react for 6 hours. After the reaction, extraction and purification were carried out in the same manner as in Example 1, further deprotection was performed using trifluoroacetic acid, and isolation and purification was performed using ion exchange chromatography. -β-aminoglutarate monomethyl ester was obtained as white crystals. These NMR spectra all match those of the standard specimen, and the optical rotation is [α] ²⁵ _D = -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 Candeida Arborea IAM4147 (FERM
BP-1888), Pichia falinosa IFO0534, and Geotrichum vanliae CBS439.64 were cultured in the same manner as in Example 1, and two cell suspensions were prepared for each strain. Each bacterial cell suspension was divided into two equal parts, 3 g of β-benzyloxycarbonylglutarate diethyl ester was added to one, and 3 g of β-t-butoxycarbonylaminoglutarate diethyl ester was added to the other. Under stirring in a mini jar at 24 °C
Allowed time to react. After the reaction, extraction and purification and deprotection were performed in the same manner as in Examples 1 and 2, resulting in
An oily substance was obtained from each reaction solution (Table 3).
This NMR spectrum matched that of the standard β-aminoglutarate monoethyl ester. As a result of measuring these optical rotations, [α] ²⁵ _D = −3.80 to 3.85° (C
= 4, H ₂ O), and it was confirmed that it was (S)-β-aminoglutarate monoethyl ester.

【table】

Claims (1)

[Claims] Linear formula () (In the formula, R is an alkyl group having 1 to 4 carbon atoms, and A is an amino-protecting group that can be removed by catalytic reduction or mild hydrolysis.) Candeida spp., Pichia spp., Trichosporon spp., Geotrichum spp., Aspergillus spp., Absidia spp., Acteinomucor spp., Helicosterium spp., Mucor spp., Morteierella spp., Pecilomyces spp. , Chigorhynchus spp.
Microorganisms belonging to the genus Fusarium, Acremotheum, Circinella, Cunninghamella, Rhizotpus, Penicillium, Proteus, Micrococcus, Hafnia, Brevibacterium,
or Lord Cotchus Lord Claws IFO3338
to act on the culture solution, bacterial cells or treated bacterial cells,
The following formula () is characterized in that the amino protecting group (A) is then removed by a conventional method. (wherein R has the same meaning as above) A method for producing an optically active (S)-β-aminoglutaric acid monoalkyl ester. 2. The manufacturing method according to claim 1, wherein in formula (), A is a benzyloxycarbonyl group and R is a methyl group or an ethyl group. 3. The manufacturing method according to claim 1, wherein in formula (), A is a t-butoxycarbonyl group and R is a methyl group or an ethyl group.