JPH044872B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing 4-oxopimelic acid monoester using microorganisms. [Prior Art and Problems to be Solved by the Invention] In recent years, the synthesis of physiologically active substances has been actively studied, and optically active substances are important intermediate raw materials when synthesizing them. However, the compound usually obtained is a racemate containing the d-form with levorotatory properties and the l-form with dextrorotatory properties in a 1:1 ratio, so optical resolution is required to obtain optically active substances. Therefore, it is difficult to synthesize optically active substances. Therefore, many compounds that can be easily synthesized asymmetrically, ie, substances that can be used as raw materials for optically active substances, are still unknown. 4-oxopimelic acid monoester, which is the object compound of the present invention, can be used as a raw material for various organic compounds, and is particularly useful as a precursor for optically active substances or raw materials thereof. For example, it is useful as a precursor for the following compounds () to (). (R)-γ-butyrolactone- of the above formula ()
γ-propionate ester can be easily obtained by treating the monoester of the present invention with a microorganism having a reducing ability. Examples of uses of the compound of formula () include the following. (1) Intermediate raw material for diasmolactone (main component of diasmine oil, a natural fragrance) (Fragrance Journal No. 77 (1986) 124-129) (2) Raw material for eldanolide, an insect pheromone (Agricultural Bio Logical Chemistry (Agric.Biol.Chem.) 49 , 2775-2776
(1986)) (3) Raw material for steganodon, a drug for leukemia (Tetrahedron Letters)
Letters) 21 , 2709 (1980)) The above compound () is selected by intramolecular Friedel-Crafts reaction of the ester of the present invention (Chem.Ber. 100 , 2973~
2977 (1967)), which is the raw material for the antitumor substance Coriolin (The Journal of
Organic Chemistry (J.Org.Chem.)
47, 2820 (1982)). Further, the compound () can be obtained by reducing the compound () with a microorganism having a reducing ability or an asymmetric reducing reagent and then further lactonizing it. The compound () can be derived from the aforementioned body substances () and () of Coreylactone, which is used as a raw material for prostaglandin (Journal of
The American Chemical Society (J.
Amer.Chem.Soc.) 95 , 6832 (1973)). As described above, 4-oxopimelic acid monoester is important as a precursor of various useful substances. Conventional methods for obtaining monoesters from aliphatic dicarboxylic acid diesters include chemical partial hydrolysis, or mixing diester and dicarboxylic acid in the same ratio and heating in the presence of a catalyst such as a Lewis acid. There is. However, in order to obtain the desired monoester from the compound obtained by these methods, distillation and other complicated separation operations are required, and these methods are still impractical. Another method has been reported in which a dicarboxylic acid is adsorbed on alumina and then a monoester is synthesized with diazomethane (J.Amer.Chem.Soc. ) 107 , 1365–1369 (1985)).
However, according to this method, the target product can only be synthesized on a benchtop experimental scale. As described above, a method for producing 4-oxopimelic acid monoester in high yield, high purity, and in large quantities has not been developed. [Means for Solving the Problems] The present inventors have focused on the above-mentioned problems and have succeeded in producing 4-oxopimelic acid monoester in high yield and purity under mild conditions and through simple operations. We conducted extensive research to develop a manufacturing method. the result,
Based on biochemical techniques, the inventors discovered that the corresponding monoester can be obtained by allowing microorganisms to act on 4-oxopimelic acid diester, leading to the completion of the present invention. That is, the present invention provides general formula (): (In the formula, R represents a _C1 to _C4 lower alkyl group excluding a t-butyl group.) A culture of a microorganism or its cells belonging to a genus selected from the group consisting of the genus Flavobacterium, Gluconobacter, Pseudomonas, and Bacillus, or Pitogriver The present invention relates to a method for producing 4-oxopimelic acid monoester, which comprises bringing it into contact with an esterase. 4-oxopimelic acid diester (), which is the starting material of the present invention, can be obtained, for example, by the following steps. (In the formula, R is the same as above.) In the first reaction, furfural and malonic acid are dehydrated and condensed in the presence of a basic catalyst such as ammonia, primary or secondary amine to produce furfuryl acrylic acid. sell. The amine used in this case is preferably pyridine. In the latter reaction, the furfuryl acrylic acid obtained in the first reaction is dissolved in a C ₁ to _{C 4} alcohol excluding t-butyl alcohol, and then heated under reflux in the presence of a strong acid to form 4-
Oxopimelic acid diester can be obtained. By appropriately selecting and using alcohols here, corresponding ester compounds can be obtained. Journal of the American Chemical Society (J.Am.Chem.Soc.) 78 , 3425
(1956)). Note that the method for synthesizing 4-oxopimelic acid diester is not limited to the above method, and any method may be used as long as the diester can be obtained. [Operations and Examples] The obtained 4-oxopimelic acid diester can be converted into a monoester by bringing the diester into contact with Pitugu liver esterase or microbial cells or cultures by an appropriate method. . Specific examples of microorganisms used include microorganisms with strong esterase activity, such as Achromobacter lyticus.
IFO12725 12726, Pseudomonas diminuta IFO 13181 13182,
Chromobacterium chocolatum IFO 3758,
Flavobacterium lutescens IFO 3084, Gluconobacter lutescens
dixyocetonicus) IFO 3271, Pseudomonas
Pseudomonas (Pseudomonas)
dinitrificans) IFO 13302, Bacillus pumilus (Bacillus pumilus) IFO 3813, etc. In order to produce the 4-oxopimelic acid monoester of the present invention, the above-mentioned microorganism is first inoculated into a suitable medium and cultured using a conventional culture method for bacteria. For example, microbial cells are grown by performing shaking or stirring culture for a certain period of time in a medium containing meat extract, glucose, casein decomposition product, and the like. Then, 4-oxopimelic acid diester is added and further culture is performed. Furthermore, after growing the bacterial cells and separating them by operations such as centrifugation,
The starting material may be newly added to the bacterial cells together with an aqueous buffer solution having a pH of 5 to 9, preferably 6 to 8, and reacted. The culture temperature is usually 20-50℃, preferably 25-40℃, and the culture time is usually 1-120℃.
A suitable time is preferably 2 to 72 hours. The method for bringing the starting material into contact with the bacterial cells may be any method as long as it does not cause significant damage to the bacterial cells. immobilization carrier, or urethane-based,
It can be used by being immobilized on synthetic polymers such as PVA, super absorbent resin, and photocurable resin by an appropriate method. After removing the bacterial cells from the resulting culture by, for example, centrifugation, the supernatant is adjusted to pH 7-10, and unreacted diesters are extracted with an organic solvent in a conventional manner. After that, adjust the aqueous solution to PH2-4,
Extract with organic solvent again, dry, and evaporate the solvent to obtain 4
-Oxopimelic acid monoester is obtained. Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these. Example 1 A bacterial ear of Pseudomonas diminuta IFO 13181 cultured on a peptone meat extract agar slant medium at 30°C for 48 hours was placed in a 2 x 18 cm test tube into which 10 ml of a medium having the composition shown in Table 1 was dispensed. The seeds were inoculated and cultured with shaking at 30°C for 24 hours to prepare an inoculum solution. [Table] 500ml of the same liquid medium as the above composition was dispensed.
Inoculate 1 ml of the above inoculum solution into a ml Sakaguchi flask and heat at 30°C.
The cells were cultured for 24 hours. A total of 700ml of bacterial cells obtained by shaking culture were collected by centrifugation.
After suspending in 350ml of 0.2M phosphate buffer (PH6.5),
17.5 g of 4-oxopimelic acid diethyl ester was added and reacted at 30°C for 72 hours. After the reaction is complete, check the pH
After adjusting the pH to 9.0 with KOH and extracting and removing unreacted 4-oxopimelic acid diethyl ester, the aqueous layer was adjusted to pH 2.0 with HCl and extracted with ethyl ether. The ether layer was dried over sodium sulfate and then evaporated under reduced pressure to obtain 12.5 g of 4-oxopimelic acid monoethyl ester (yield: 81%). The material is nearly pure from NMR and IR, with a melting point of 67-68°C
It was hot. ^1H NMR (60MHz, _CDCl3 ) δ: 1.28 (t,
3H), 2.74 (m, 8H), 4.15 (q, 2H), 10.95 (s,
1H) IR (neat) (cm ^-1 ): 3100, 1740, 1710, 1420 Example 2 Pseudomonas diminuta cultured in Example 1
After suspending the IFO 13181 cells in 350 ml of 0.2 M phosphate buffer (PH7.5), 10.0 g of 4-oxopimelic acid dimethyl ester was added and reacted at 30°C for 72 hours. After the reaction was completed, the pH was adjusted to 9.0 with KOH.
After extracting and removing unreacted 4-oxopimelic acid dimethyl ester, the aqueous layer was adjusted to pH 2.0 with HCl and extracted with ethyl ether. The ether layer was dried over sodium sulfate, then evaporated under reduced pressure, and the residue was purified by silica gel chromatography (MeOH/chloroform = 1/10) to obtain 5.6 g of 4-oxopimelic acid monomethyl ester as a colorless oil (yield:
60%). The material was nearly pure from NMR and IR. ^1H NMR (60MHz, _CDCl3 ) δ: 2.62 (m,
8H), 3.62 (s, 3H), 6.14 (s, 1H) IR (neat) (cm ^-1 ): 3180, 1730, 1710, 1410 Example 3 Pseudomonas diminuta cultured in Example 1
A loopful of IFO 13181 bacterial cells was inoculated into a 2 x 18 cm test tube containing 10 ml of 3% Brain Heart Infusion, and cultured with shaking at 30°C for 24 hours to prepare an inoculum solution. . 5% Brain Heart Infusion 100
Dispense 1 ml of the above seed culture solution into a 500 ml Sakaguchi flask.
ml was inoculated and cultured at 30°C for 24 hours. A total of 700 ml of bacterial cells obtained by shaking culture were collected by centrifugation and added to 0.2M phosphate buffer (PH8.0) at 350 mL.
ml, 20.0 g of 4-oxopimelic acid dipropyl ester was added, and the mixture was reacted at 30°C for 72 hours.
After the reaction was completed, the pH was adjusted to 9.0 with KOH, and the unreacted 4
- After extracting and removing oxopimelic acid dipropyl ester, the aqueous layer was adjusted to pH 2.0 with HCl and extracted with ethyl ether. After drying the ether layer with sodium sulfate, it was distilled off under reduced pressure to obtain 4-oxopimelic acid monopropyl ester, which was a pale yellow oil.
g (yield 78%). The substance is NMR and IR
It was almost pure. ^1H NMR (60MHz, _CDCl3 ) δ: 0.86 (t,
3H), 1.68 (m, 2H), 2.74 (m, 8H), 4.05 (t,
2H), 10.18 (s, 1H) IR (neat) (cm ^-1 ): 3200, 1735, 1720, 1420 Example 4 Pseudomonas diminuta cultured in Example 3
Example 1 except that 25.0 g of 4-oxopimelic acid dibutyl ester was added after suspending IFO 13181 cells in 350 ml of 0.2 M phosphate buffer (PH8.0).
Perform the same operation as above to obtain 4, which is a pale yellow oily substance.
-15.5 g of oxopimelic acid monobutyl ester was obtained (yield 77%). The material was nearly pure from NMR and IR. ^1H NMR (60MHz, _CDCl3 ) δ: 0.95 (t,
3H), 1.65 (m, 4H), 2.67 (m, 8H), 4.07 (t,
2H), 7.54 (s, 1H) IR (neat) (cm ^-1 ): 3220, 1740, 1720, 1410 Example 5 Flavobacterium lutecens IFO cultured on peptone meat extract agar slant medium at 28°C for 24 hours
3084 bacterial cells and a loopful of culture medium 10 with the composition shown in Table 1
ml into 2 x 18 cm test tubes and incubate at 30℃.
An inoculum solution was prepared by culturing with shaking for 24 hours. 500ml of the same liquid medium as the above composition was dispensed.
Inoculate 1 ml of the above inoculum solution into a ml Sakaguchi flask and heat at 30°C.
The cells were cultured for 36 hours. 0.2M of bacterial cells obtained from a total of 1 shaking culture were collected by centrifugation.
After suspending in 500ml of phosphate buffer (PH8.0), 4-
Add 5.0 g of oxopimelic acid diethyl ester,
The reaction was carried out at 30°C for 72 hours. After the reaction, Example 1
After treatment in the same manner as above, 1.2 g of 4-oxopimelic acid monoethyl ester was obtained (yield: 27
%). The material was nearly pure by NMR and IR, with a melting point of 67-68°C. ^1H NMR (60MHz, _CDCl3 ) δ: 1.28 (t,
3H), 2.74 (m, 8H), 4.15 (q, 2H), 10.95 (s,
1H) IR (neat) (cm ^-1 ): 3100, 1740, 1710, 1420 Example 6 Chromobacterium tiyocolatum IFO 3758 cells cultured in the same manner as in Example 5 were
After suspending in 500ml of 0.2M phosphate buffer (PH7.5),
The same operation as in Example 5 was carried out except that 2.0 g of 4-oxopimelic acid diethyl ester was added, and 4-oxopimelic acid diethyl ester was added.
-0.6 g of oxopimelic acid monoethyl ester was obtained (yield 34%). The material was nearly pure by NMR and IR, with a melting point of 67-68°C. ^1H NMR (60MHz, _CDCl3 ) δ: 1.28 (t,
3H), 2.74 (m, 8H), 4.15 (q, 2H), 10.95 (s,
1H) IR (neat) (cm ^-1 ): 3100, 1740, 1710, 1420 Example 7 100ml of 0.05M phosphate buffer (PH8.0) at 300
ml Erlenmeyer flask, and after incubation in a thermostatic water bath at 25° C., 1600 units of Pitugu River esterase (manufactured by Sigma) and 1 g of 4-oxopimelic acid diethyl ester were added and stirred. Rare
While keeping the pH between 7.0 and 8.0 with KOH aqueous solution, total
4.0 g of 4-oxopimelic acid diethyl ester was added and reacted for 6 hours. After the reaction is complete, dilute KOH
After adjusting the pH to 10.0 with an aqueous solution and treating in the same manner as in Example 1, 3.4 g of 4-oxopimelic acid monoethyl ester was obtained (yield 96%). The material was nearly pure by NMR and IR, with a melting point of 67-68°C. ^1H NMR (60MHz, _CDCl3 ) δ: 1.28 (t,
3H), 2.74 (m, 8H), 4.15 (q, 2H), 10.95 (s,
1H) IR (neat) (cm ^-1 ): 3100, 1740, 1710, 1420 Example 8 Transfer 500 ml of the culture obtained in Example 1 to Erlenmeyer flask No. 1, and add 3.4 g of 4-oxopimelic acid diethyl ester. The reaction was carried out at 30°C for 72 hours while keeping the pH at 8.0 with 0.5N KOH. After the reaction was completed, unreacted 4-oxopimelic acid diester was extracted and removed, and the aqueous layer was adjusted to pH 2.0 with HCl and extracted with ethyl ether. After drying the ether layer with sodium sulfate, it was distilled off under reduced pressure to obtain 3.0 g of 4-oxopimelic acid monoethyl ester with a melting point of 67-68°C.
(yield 81%). The material was nearly pure from NMR and IR. The NMR and IR values showed the same values as in Example 1. [Effects of the Invention] According to the method of the present invention, 4-oxopimelic acid monoester can be easily synthesized under mild reaction conditions with high yield and high purity.

Claims (1)

[Claims] 1 General formula (): (In the formula, R represents a _C1 to _C4 lower alkyl group excluding a t-butyl group.) Genus Flavobacterium, Genus Gluconobacter, Genus Pseudomonas and Bacillus
1. A method for producing 4-oxopimelic acid monoester, which comprises bringing a microorganism belonging to a genus selected from the group consisting of the following genus into contact with a culture or its cells, or pitugerver esterase.