JPS6384496A - Production of 4-oxopimelic acid monoester - Google Patents

Abstract

PURPOSE:To produce a large amount of the titled substance in high yield and purity, by reacting a microorganism of the genus Achromobacter, etc., with a 4-oxipimelic acid diester expressed by a specific general formula. CONSTITUTION:A microorganism, e.g. Achromobacter lyticus IFO12725, Pseudomonas diminute IFO13181, Chromobacterium chocolatum IFO3578, etc., of the genus Achromobacter, Chromobacterium, Flavobacterium, Gluconobacter, Pseudomonas, Arthrobacter, Bacillus or Corynebacterium is cultivated. A 4- oxopimelic acid diester expressed by formula I (R represents 1-4C lower alkyl group), together with an aqueous solution of a buffer, is added and reacted with the resultant microbial cells. After removing the microbial cells from the culture, the unreacted diester is extracted and the obtained substance is extracted with an organic solvent and dried to afford the 4-oxopimelic acid monoester.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing 4-oxopimelic acid monoester using microorganisms.

[Problems to be solved by conventional techniques and inventions In recent years, the synthesis of physiologically active substances has been actively researched.
Optically active substances are important intermediate raw materials in their synthesis. However, normally obtained compounds have 6 bodies with levorotatory properties and 9 bodies with dextrorotatory properties in a ratio of 1=1.
Since it is a racemic substance containing a ratio of Therefore, many compounds that can be easily synthesized asymmetrically, that is, 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 (It) to (2).

The (R)-γ-butyrolactone-γ-propionic acid ester of the formula (1) 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 (II) include the following.

(1) Intermediate raw material for jasmolactone (main component of jasmine oil, a natural fragrance) (Fragrance Journal Nα77 (198B) pp. 124-129) (2) Raw material for elderaturide, an insect pheromone (Agricultural Bio Logical Chemistry - (Agri
c, Biol, Chea+,) 4 Niji, 2775~
277B (198B)) (3) Raw material for steganodon, a drug for leukemia (
Tetrahedron Letters
etters) 21.2709 (1980)) The above compound (2) can be obtained by intramolecular Friedel-Krach reaction of the ester of the present invention (Kemissi J-'Berichte (1980)).
CheIl, Ber, ) 100.297a ~ 297
7 (1967)), which is a raw material for the antitumor substance Coriolin (The e-Journal of Organic Chemistry (J, Org, Chcv,) 47).
, 2820 (1982)). Further, the compound N can be obtained by reducing the compound surface with a microorganism having a reducing ability or an asymmetric reducing reagent, and then further lactonizing it. The compound N is derived from Coreylactone, which is used as a raw material for prostaglandin.
on) can be induced into one volume of precursor material (journal/
of the American Chemical Society (J.
AIQer, Ches.

Soc, ) 95.8832 (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 (Journal φ of the American Chemical Society (J, Amer, Chem, Soc,
) 107, 1385-1389 (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 focused on the above-mentioned problems and produced 4-oxopimelic acid monoester with high yield and high purity under mild conditions and with simple operations. We conducted extensive research to develop the method. As a result, based on biochemical methods, 4-
It was discovered that the corresponding monoester can be converted to oxopimelic acid diester by the action of microorganisms, leading to the completion of the present invention.

That is, the present invention provides general formula (■): RO2CCll2CH2ecH2CII2C02R(1
) (in the formula, R represents a lower alkyl group of C1 to C4), a 4-oxopimelic acid diester of the Achromobacter genus,
Chromobacterium (Chroa+obacter1u
m) Genus 1 Flavobacterium
lum), Gluconobacter (Gluconobacter)
ter) genus, Pseudomonas
) genus, Arthrobacter (Artl+robacter)
), Bacillus genus, and Corynebacterium genus Corynebacterium (Corynebacterium+). Regarding the method.

4-oxopimelic acid diester (1), which is the starting material of the present invention, can be purified, 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, after dissolving the furfuryl acrylic acid obtained in the first reaction in a C1 to C4 alcohol, in the presence of a strong acid,
4-oxopimelic acid diester can be obtained by heating under reflux. By appropriately selecting and using the alcohol, the corresponding ester compound can be changed (Journal of Chemical Society U.

Chem, Soc,) 78.3425 (195B))
.

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 converted.

[Operations and Examples] The obtained 4-oxopimelic acid diester can be converted into a monoester by bringing the diester into contact with bigliba disterase or the cells of a microorganism by an appropriate method. Specific examples of microorganisms used include microorganisms with strong esterase activity, such as Achromobacter Φlyticus (Achromobacter 1).
ytlcus) IP0127251272B, Pseudomonas dlml
Nuta) IPO1318113182, Chromobacterium chocolat
um) IFO3578, Flavobacterium lutescens
s) IFO3084, Gluconobacter dixyocet
onicus) IFO3271, Pseudomonas dinitrificans (Pseudollonas din
ltr1f'1cans) IPO13302, Arthrobacter nicotianae
icotianae) IAM 12342, Bacillus
Bacillus pumillus IFo
3813, Corynebacterium 0 fuasciens (C
orynebacterium! 'asklens)J
Examples include AM 1072.

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,
The bacterial cells are grown by shaking or stirring culture for a certain period of time in a medium containing meat extract, glucose, casein decomposition product, etc. Then, 4-oxopimelic acid diester is added and further culture is performed. Furthermore, after the bacterial cells have been grown, the bacterial cells are separated by an operation such as centrifugation, and the starting material is 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. Good too.

Culture temperature is usually 20-50°C, preferably 25-40°C
The culture time is usually 1 to 120 hours, preferably 2 hours.
~72 hours is appropriate. 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, P
It can be used by being immobilized on synthetic polymers such as VA-based resins, super absorbent resins, and photocurable resins by an appropriate method. After removing the bacterial cells from the obtained culture by, for example, centrifugation, the supernatant is adjusted to pH 7 to 1o, and unreacted diester is extracted with a residual solvent in a conventional manner. After that, the aqueous solution is
The mixture is adjusted to H2-4, extracted again with an organic solvent, dried, and the solvent is distilled off to obtain 4-oxopimelic acid monoester.

Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these.

Example 1 A loopful of Pseudomonas diminuta IF013181 cells cultured on a peptone meat extract agar slant medium at 30°C for 48 hours was inoculated into a 2X18c+n test tube into which 10ml of a medium having the composition shown in Table 1 was dispensed. A seed culture solution was prepared by performing shaking culture at ℃ for 24 hours.

Table 1 5. Dispense 100 ml of the same liquid medium as the above composition.
1 ml of the above inoculum solution was inoculated into a 00 ml Sakaro flask 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 350 ml of 0.2M phosphate buffer (pH 8.5).
4-oxopimelic acid diethyl ester 17
．． 5 g was added and reacted at 30"C for 72 hours. After the reaction was completed, the pH was adjusted to 9.0 with KOH, and unreacted 4-oxopimelic acid diethyl ester was extracted and removed, and the aqueous layer was diluted to pH 2 with 11 CI. , 0 and extracted with ethyl ether.The ether layer was dried over sodium sulfate and then distilled off under reduced pressure to give 4-oxopimelic acid monoethyl ester 12.
．． 5. (yield 81%).

The material is nearly pure from NMR and TLC;
The melting point was 67-68°C.

'HNMR(80MIlz, CDCla)δF
1.28 (t, 3H), 2.74 (m, 8H
), -4,15(Q, 2H), 10.95(s
.

l11) IR (neat) (cm-'): 3100, 1
740, 171O1142゜Example 2 Pseudomonas diminuta IF01 cultured in Example 1
3181 cells were added to 0.2M phosphate buffer (p117
, 5) After suspending in 350 ml, 10.0 g of 4-oxopimelic acid dimethyl ester was added, and the mixture was reacted at 30°C for 72 hours. After the reaction was completed, the pl was adjusted to 9.0 with KOH, and unreacted 4-oxopimelic acid dimethyl ester was extracted and removed, and the aqueous layer was adjusted to pH 2.0 with HCI and extracted with ethyl ether. After drying the ether layer with sodium sulfate, it was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (MeOtl/chloroform-1710).
Colorless oily 4-oxopimelic acid monomethyl ester 5
．． 6 g was obtained (yield 60%). The material is NMR and T
Almost pure from LC.

')I NMR (60MHz 5CDCI! 3)
δ; 2.132 (m, 8H), 3.62 (s
, 311), 6.14(s, IH) IR(nea
t) (cm'): 3180.1730.1710
, ■410410 Pseudomonas ψ Jiminuta IP01 cultured in Example 1
A loopful of bacterial cells of 3181 was inoculated into a 2 x 18 cm test tube into which 3% plain heart infusion loml was dispensed, and cultured with shaking at 30°C for 24 hours to prepare an inoculum solution.

5% Plain Heart Infusion 100ml
The above inoculum solution 1 was dispensed into a 500 ml Sakalo flask.
ml was inoculated and cultured at 30°C for 24 hours. Total 700
Bacterial cells obtained by shaking culture of ml were collected by centrifugation and collected in 0.2M phosphate buffer (pH 8,0)3.
After suspending in 50 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, pn was adjusted to 9.0 with KOH, unreacted 4-oxopimelic acid dipropyl ester was extracted and removed, and the aqueous layer was adjusted to pH 2.0 with HCI) and extracted with ethyl ether. The ether layer was dried over sodium sulfate and then evaporated under reduced pressure to obtain 13.2 g of 4-oxopimelic acid monopropyl ester as a pale yellow oil (yield 7).
8%).

The material was nearly pure from NMR and TLC.

II NMR (80MIIz, CDCJ3)
δ: 0.8B (t, 3H), 1.88 (m
, 211), 2.74 (m, 88), 4.05 (t
.

2H), 10.18(s, IH)IR(neat
) (cm-1) :3200.1735.1720
．． 1420 Example 4 Pseudomonas diminuta IF01 cultured in Example 3
3181 cells in 0.2M phosphate buffer (pH 8,
0) After suspending in 350 ml, 4-oxopimelic acid dibutyl ester 25. The same operation as in Example 1 was carried out except that Og was added, and 15.5 g of 4-oxopimelic acid monobutyl ester, which was a pale yellow oil, was obtained (yield 77%).
). The material was nearly pure from NMR and TLC.

'HNMR (60MHz 5CDCJ3) δ:
0.95 (t, 311), 1.85 (11,411
), 2.87(+, 811), 4
．． 07(t.

211), 7.54 (s 5III) IR (ne
at)(c+n-1):3220.1740.172
0.1410 Example 5 A loopful of Flavobacterium lutecens IFO3085 cells cultured on a peptone meat extract agar slant medium at 28°C for 24 hours was placed in a 2 x 18 cm square. Inoculate test tubes and incubate at 30℃ for 24 hours.
An inoculum solution was prepared by performing shaking culture for a period of time.

5. Dispense 100 ml of the same liquid medium as the above composition.
1 ml of the above inoculum solution was inoculated into a 00 ml Sakaro flask and cultured at 30°C for 36 hours. A total of 1g of bacterial cells obtained by shaking culture was collected by centrifugation, and 0.2M
After suspending in 500 ml of phosphate buffer (1) 118.0), 5.0 g of 4-oxopimelic acid diethyl ester
was added and reacted at 30°C for 72 hours. After the reaction was completed, 1.2 g of 4-oxopimelic acid monoethyl ester was obtained after processing in the same manner as in Example 1 (yield 27%).
. The material was nearly pure by NMR and TLC, with a melting point of 67-B8°C.

'HNMR (80MHz, CDCJ3) δ:
1.28 (t, 3H), 2.74 (+, 8H)
,' 4.15(q, 2H), 10.95(s
.

lII) IR(neat) (, cm-1): 3100.174
0.1710.1420 Example 6 After suspending 375 g of Chromobacterium cocolatum IF0 cells cultured in the same manner as in Example 5 in 500 ml of 0.2 M phosphate buffer (pH 7.5),
-The same operation as in Example 5 was carried out except that 2.0 g of oxopimelic acid diethyl ester was added, and 0.8 g of 4-oxopimelic acid monoethyl ester was obtained (yield 34%).
). The material was nearly pure by NMR and TLC, with a melting point of 67-68<0>C.

'HNMR (60MHz %CDC13) δ: 1.
2g (t, 311), 2.74 (a, 8H),
4.15 (q, 2H), 10.95 (s.

IH) IR (neat) (cm'): 3100.1740.
1710.1420 Example 7 0.05M phosphoric acid buffer y (pH 8,0) 100
Dispense ml into a 300 ml Erlenmeyer flask and incubate at 25°C.
After incubation in a thermostatic water bath, 1800 units of Big River Esterase (manufactured by Sigma) and 1 g of 4-oxopimelic acid diethyl ester were added and stirred. A total of 4.0 g of 4-oxopimelic acid diethyl ester was added and reacted for 6 hours while keeping the pH at 7.0 to 8.0 with dilute 011 aqueous solution. After the reaction was completed, the p) was adjusted to 110.0 with a dilute KOH aqueous solution and treated in the same manner as in Example 1 to obtain 3.4 g of 4-oxopimelic acid monoethyl ester (yield: 9B%). The material is nearly pure from NMR and TLC, with a melting point of 67-68°C.
Met.

'HNMR (BOMHz, CDCJ3) δ
: 1.28 (t, 3H), 2.74 (Ill,
8H), 4.15(q, 2H), 10.95(s
.

IH) IR (neat) (cm'): 3100.17
40.1710.1420 [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 (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R represents a lower alkyl group of C_1 to C_4) Bacter (Achromobacter) genus,
Chromobacterium
) genus, Flavobacterium
um) genus, Gluconobacter (Gluconobacter)
er) genus, Pseudomonas
4, characterized in that it is brought into contact with a culture of a microorganism or its bacterial cells belonging to a genus selected from the group consisting of the genus Arthrobacter, Bacillus, and Corynebacterium. - A method for producing oxopimelic acid monoester.