JPH04126089A - Production of 4-oxopimelic acid monoester - Google Patents

Abstract

PURPOSE:To obtain the subject monoester in high purity in high yield by bringing a specific mixture into contact with lipase, etc., derived from genus Achromobacter and reacting. CONSTITUTION:Microorganism selected from a group of genus Achromobacter, genus Chromobacterium, genus Flavobacterium, genus Gluconobacter, genus Pseudomonas, genus Arthrobacter, genus Bacillus and genus Corynebacterium is cultured in a medium containing meat extract, etc., and strain is separated from the resultant cultured substance, then a buffer solution of pH5-9 is added to the strain to obtain a strain suspension (A). Next, a mixture (B) of a 4- oxopimelic acid diester expressed by formula I (R is 1-4C lower alkyl) and a 4,4-ethylenedioxypimelic acid diester derivative expressed by formula II is added to the suspension A and reacted at 20-50 deg.C for 1-120hr to obtain a reaction product (C). Then, strain is removed from the component C, thus the resultant supernatant is adjusted to pH7-10 and the unreacted 4,4-ethylenedioxypimelic acid diester is removed by extraction, then remaining suspension is adjusted to pH2-4, thus extracted and purified to recover a 4-oxopimelic acid monoester expressed by formula III.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for selectively producing 4-oxopimelic acid monoester using microorganisms, their cells, or lipases extracted from them.

(Prior art) In recent years, the synthesis of physiologically active substances has been actively researched.
When synthesizing them, substances with photoactive activity are important intermediate raw materials. However, it is difficult to synthesize optically active substances, and only racemates are generally obtained through normal operations. Therefore, compounds that facilitate asymmetric synthesis, ie, substances that can serve as raw materials for optically active substances, are important.

By the way, 4-oxopimelic acid monoester, which is the target substance of the present invention, is an optically active substance fsl -f-
1-γ-butyrolactone-γ-3-propatul, (formerly
(+)-γ-Butyrolactone-γ-3-propion, etc., can be easily synthesized and used as a raw material for vine production.Furthermore, the optically active substance can be used as a raw material for various organic compounds, especially for physiologically active substances, fragrances, etc. It is a precursor and a vine. Further, other compounds separated by the method of the present invention, namely 4,4-ethylenedioxypimelic acid diester, can also be used as raw materials for various organic compounds in the same manner as described above.

Specific examples of these uses include, for example, the following.

(1) Intermediate raw material for jasmolactone (main component of jasmine oil, a natural fragrance) (Fragrance Journal, Vol. 77, pp. 124-129 (19861)).

(2) Raw material of elderturide, an insect pheromone ¥4 (
Agricultural Biological, Chemistry
-(Agric, Biol, Chel+, )
Volume 49, P2775 ~2776+198
61).

(3) Raw material for steganodon, a drug for leukemia (
Tetrahedron Letters
Letters) 2 I@, p 27o9+1qg
(See o+).

(4) Raw material for the pheromone of the Japanese bean (the temple opened in 1977)
(See Publication No. 157055).

Conventionally, methods for obtaining monoesters from aliphatic dicarboxylic acids (diesters) include chemical partial hydrolysis, or mixing diesters and dicarboxylic acids in the same ratio and using a hornworm medium such as a Lewis acid. There is a method of heating in the presence of. To obtain the desired monoester from the compounds obtained by these methods,
One problem is that it requires distillation and other complicated separation operations, and it still lacks consistency. Another method reported is to adsorb cafrebonic acid on alumina and then synthesize a monoester with siabumethane (Journal of the American Chemical
Society (J, /mer, chem, soc,
) Volume 107, P1365-1369 f19851)
. However, this method has the problem that the target product can only be synthesized on a desktop experimental scale.

Recently, JP-A-63-84496, JP-A-64-1
No. 0993 and Japanese Patent Application Laid-Open No. 1-132393 disclose inventions relating to a method for producing 4-oxopimelic acid monoester by a biochemical method that focuses on the above-mentioned problem. However, in these methods, it is still desired to improve the yield of the target product and simplify the reaction operation.

(Problems to be Solved by the Invention) The present invention uses a mixture of 4-oxopimelic acid diester and 4,4-ethylenedioxypimelic acid diester as a starting material, which is an intermediate raw material for synthesizing the useful compound. The object of the present invention is to provide a manufacturing method for easily obtaining 4-oxopimelic acid monoester in high yield and purity by treating the mixture under mild conditions.

(Means for Solving the Problems) However, the present inventors have focused on the problems of the prior art, and as a result of intensive studies to obtain 4-oxopimelic acid monoester in high yield and high purity, they have identified We have found that this objective can be achieved by employing a specific biochemical approach using starting materials. The present invention was completed based on this knowledge.

That is, the present invention (in both formulas, R represents lower alkyl having 1 to 4 carbon atoms)
A mixture of 4-oxopimelic acid diester derivatives represented by Achromobacter
er), Chromobacterium (Chromobacterium
cteriun), genus Flavono\cteriun (Fl
avabacterium), Gluconobacter (G
luconobacter), Pseudomona (Ps.
eudomonas), Arthlobacter (Arth
robacter) l:):, Bacillus (Bacillus)
lus, Corynebacterium h.
T, erjum; selectively hydrolyzes only the 4-oxopimelic acid diester of the general formula (1) by contacting the cells of a culture of microorganisms selected from the group consisting of the genus or lipase extracted therefrom; , - Martyr's Day 2 ROOCCH2CH2CCH, CH2C0OH (for) (
In the formula, R is the same as above. ) The present invention relates to a method for producing 4-oxopimelic acid monoester, which is characterized in that the onoester is separated and purified.

The configuration of the present invention will be explained below.

The starting materials in the present invention are 4-oxopimelic acid diester (I) of general formula (I) and 4-oxopimelic acid diester (I) of general formula (II).
- A mixture of ethylenedioxypimelic acid diesters, and not only the mixture itself but also each component is a useful compound. For example, 4,4-ethylenedioxypimelic acid diester is converted to 5-hydroxybicyclo[4,1,0]]butane-2 as described in JP-A-1-283248.
-one itself is suitable as a raw material for the production of natural physiologically active compounds (see note), which is important as a raw material for the synthesis of natural physiologically active compounds. Therefore, in producing 4-oxopimelic acid monoester, as in the present invention,
It is advantageous to use the mixture consisting of the general formula (1) and the general formula tn+ as a starting material as is for the following reasons. That is, it is possible to efficiently produce the target product, 4-oxopimelic acid monoester, and at the same time, it is possible to effectively utilize the residual component, 4,4-ethyl-dioxybimelic acid diester. Therefore, JP-A-63-84496, JP-A-64-10
In the method 1 described in JP-A No. 993, JP-A No. 11239, when the above-mentioned mixture is employed as the starting material, the compound of formula (I) and the compound of formula (II) are simultaneously The disadvantage is that selective hydrolysis as in the present invention is not allowed. In contrast, in the present invention, only the compound of general formula (1) is selectively hydrolyzed, so there is no need for the operation of isolating compound (1) in advance, which is required in the prior art. Furthermore, it is easy to separate and purify the produced target compound ([1) and the residual component Compound (Il), and as a result, it is superior in terms of the yield of the target product and the reaction operation.

The mixture, which is the starting material of the present invention, is produced, for example, as follows.

First, furfural and malonic acid are subjected to a dehydration condensation reaction in the presence of a basic catalyst such as ammonia, primary, secondary amine, etc. to obtain furyl acrylic acid. Pyridine is preferred as the amine. Then, the obtained furyl acrylic acid is dissolved in an alcohol having 1 to 4 carbon atoms, and then heated under reflux in the presence of a strong acid such as hydrochloric acid to obtain the 4-oxopimelic acid diester of the general formula (I). can. By appropriately selecting and using alcohols, corresponding ester compounds can be obtained (Journal of the American Chemical Society, Vol. 78, P3425f+956).

Next, this diester is heated and refluxed in benzene in the presence of an acid catalyst such as p-h luenesulfonic acid with the addition of ethylene glycol. A portion of the 4,4-ethylenedioxypimelic acid diester is produced, resulting in a mixture of compounds of general formulas II) and (II). Incidentally, the method for synthesizing the mixture is not limited to the above-mentioned method, and any method that yields the desired product can also be employed.

A selective hydrolysis reaction proceeds by contacting the resulting mixture of the compounds of general formula (I) and CI+) with a culture of microorganisms, their cells, or lipase extracted from them in an appropriate manner, Only the diester of general formula (r) in the mixture is of general formula (III), which is the object of the present invention.
It changes to 4-oxopimelic acid monoester.

The above microorganisms include microorganisms with strong esterase activity, Achromobacter archipelago
er 1yticus IFO1272512726)
Pseudomonas d.
imunuta TFO1318113+82),
Chromobacterium chocolatum (Chromobacterium
cterium chocolatum IFO3578
), Flavobacterium lutescens (Flavob
acterium 1utescens IFO308
4), Gluconobacter jixocetonicus (Gluc
onobacter dixyocetoniCus
rFo 327] ') Pseudomonas dinitrificans (rific
ans IFO13302), Arthrobacter nicotianae
nae JAM 12342), Bacillus pumilus (Bacillus pumilus IFO381)
3) Corynebacte J. um Fasciens (Cor.
ynebacterium+nfasciens IAM
1072) etc.

Among these, Pseudomonas diminuta is particularly preferred.

To produce 4-oxopimelic acid monoester by the selective hydrolysis described above, the microorganisms described above are first inoculated into a suitable medium and cultured according to the usual culture method for bacteria. For example, bacteria may be grown by shaking or stirring culture for a certain period of time in a medium containing meat extract, glucose, casein decomposition products, etc., and the culture may be used as is for the reaction, or after further growth of the bacteria. After separating the bacterial cells by operations such as centrifugation, the bacterial cells are newly injected with p115~
9. The starting material may be added and reacted with an aqueous buffer solution, preferably p86-8. The culture temperature is usually 20 to 50°C, preferably 25 to 40°C, and the culture time is usually 1 to 120 hours, preferably 2 to 72 hours. The method of bringing the starting material into contact with the bacterial cells is as follows:
Any method may be used as long as it does not cause significant damage to the bacterial cells, such as known immobilization carriers such as carrageenan, collagen, alginic acid, agar, or urethane-based carriers.
It can be used by being immobilized on a synthetic polymer such as a PVA system, a super absorbent resin, or a photocurable resin by an appropriate method. After removing the bacterial cells from the obtained culture by, for example, centrifugation,
The supernatant was adjusted to pH 7 to 1O, and unreacted 4,4-ethylenedioxypimelic acid diester (11) was extracted with an organic solvent in a conventional manner, and the aqueous solution was adjusted to pH 12 to 4, and again ! When extracted with a solvent, dried, and distilled off, 4-
Oxopimelic acid monoester is obtained.

The same objective can also be achieved by using a pase extracted from the microorganisms or a commercially available lipase instead of the microorganisms. Commercial products of these enzymes include, for example, Lipase P (manufactured by Ohno Pharmaceutical Co., Ltd., trade name).
, Lipase SP (manufactured by Ohno Pharmaceutical Co., Ltd., trade name), Lipase Toyo (manufactured by Toyo Jozo Co., Ltd., trade name), Pancreatine Novase (manufactured by Ohno Pharmaceutical Co., Ltd., trade name), Lipase MY (manufactured by Ohno Pharmaceutical Co., Ltd., trade name)
Examples include 8 Sugar Sangyo Co., Ltd. (product name).

(Examples) Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited only to these Examples.

Example 1 A loopful of Pseudomonas diminuta rFo 1318 cultured on a peptone meat extract agar slant medium at 30°C for 48 hours was inoculated into a 2 x 18 cm test tube into which 10+wl of a medium having the composition shown in Table 1 was dispensed. A seed culture solution was prepared by culturing with shaking at 30° C. for 24 hours.

Table 1 1 ml of the above seed culture solution was inoculated into a 500 μl Sakaro flask into which 100 ml of the same liquid medium as the above composition had been dispensed.
Shaking culture was carried out at 30°C for 24 hours. A total of 700 ml of bacterial cells obtained by shaking culture were collected by centrifugation, and the cells were collected into 350 ml of 0.2M phosphate bag-fpH6.5.
After suspending in 1, 4-oxopimelic acid diester and 4,4
- Mixture of ethylenedioxypimelic acid diesters 17
．． 5 g was added and reacted at 30°C for 72 hours. After the reaction was completed, +11)1 was adjusted to 9.0 with KOH, and unreacted 4.
72 g of 4-ethylenedioxypimelic acid diester was extracted. 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 95 g of 4-oxopimelic acid monoethyl ester. The material was analyzed using NMR and TLC and was found to be pure as evidenced by the data below. In addition, 4-oxopimelic acid diethyl ester was not present in either the target product or the residual components, indicating that it had been selectively hydrolyzed almost completely to the above monoester.

4-oxopimelic acid monoethyl ester,
67-68℃ HNMRf60MHz, CDCl3)δ'], 28f
t, 3H), 2.74 (m, 8 old, 4.15tq,
2 old, +0.95 fs, IHIIRfneat)
3100,1740.1710 1420cm4.4
-Ethylenedioxypimelic acid diethyl ester HNMRf60MHz, CDCl l δ=1.
, 231 t, G t+ ), 1, 95ft, 4
H), 2.32 (t, 4H), 3.90 (
s, 4H) 4.08 fq, 4) 1) IRfneat,) 3100, 1740.17] [
1,1420 CII + Example 2 Pseudomonas diminuta IFO1 cultured in Example 1
318], 5 g of lipase P on a cloth plate was added in place of the bacterial cells of 3
50+nl of 0.2M phosphate buffer fp)17.5)
After digestion with 4-oxopimelic acid diethyl ester and 4
．． 17.5 g of a mixture of 4-ethylenedioxypimelic acid diethyl ester was added and reacted at 30"C for 72 hours. After the reaction, the pH was adjusted to 90 with OH, and unreacted 4.4-ethylene Dioxypimelic acid diester was extracted to obtain 7.5 g. The aqueous layer was adjusted to p) 12.0 with HCI and extracted with ethyl ether. The ether layer was dried over sodium sulfate, and then evaporated under reduced pressure. 9.8 g of 4-oxopimelic acid monoethyl ester was obtained.The material was analyzed using NMR and TLC and was found to be pure as evidenced by the following data.Also, 4-oxopimelic acid diethyl ester was not mixed in either the target product or the residual component, indicating that it had been selectively hydrolyzed almost completely to the above monoester.

4-oxopimelic acid monoethyl ester melting point 67~
68℃ HNMRi60MHz, CDCl δ・1.28
(t, 3H), 2.74℃m, 8H1,4,15(
q, 2) 11,](], 95 (s, I)IIT
R(neatl 3]00,1740,1710.14
20cm-4,4-ethylenedioxypimelic acid diethyl ester HNMRf60MHz, CDC131δ・1.23
ft, 6H), 1.95ft, '4H),
2.32 (t, 4H), 3.90 (s, 4t(
)4.08(q, 4H) IRfneat)3]00,1740.1710.14
2[]cm Example 3 Pseudomonas diminuta IFO1 cultured in Example 1
318] of 3% Plain Heart Infusion I was inoculated into a 2XI8c+n test tube, and cultured with shaking at 30°C for 24 hours to prepare an inoculum solution.

1rnl of the above was inoculated into a 500+nJ Sakaro flask into which 00ml of 5% Plain Heart Infusion was dispensed 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 the cells were collected using a 0.2 M phosphoric acid buff of 350 ml of inoculum fi 7-fp8.
8.0), a mixture of 4-oxopimelic acid dipropyl ester and 4.4-ethylenedioxypimelic acid dipropyl ester was suspended in 30. 72 at 30°C.
Allowed time to react. After the reaction was completed, p1 was adjusted to 90 with KOH, and unreacted 4,4-ethylenedioxypimelic acid dipropyl ester was extracted to obtain 122 g.
101 to adjust the pH to 2.0, and extracted with ethyl ether. After drying the ether layer with sodium sulfate, it was distilled off under reduced pressure.
152 g of 4-year-old xobimelic acid monobrobyl ester, a pale yellow oil, was obtained. The material is NMR and TL
As a result of analysis using C, it was found to be pure as evident from the data below. Furthermore, since 4-oxopimelic acid diethyl ester was not mixed in either the target product or the residual component, it was confirmed that it was almost completely selectively hydrolyzed to the above monoester.

4-oxopimelic acid monopropyl ester HNMRf
601JHz, CDC1,) δ=0.86ft, 3
H), 1.68℃m, 2 old, 2.74 (m,
8 old, 4.05 (t, 2 old, 10.18s, IH IRfneatl 3200, 1735.1720.1
420cm4.4-Ethylenedioxypimelic acid dipropyl ester HNMRf60MHz, CDCl-16・0.85(t
, 6H), 1.22 (m, 4H), 1.95
(t, 4H) , 2.32 (t, 4H) 3.91
(s, 4H), 4.08 (q, 4H) TRine
aLl 3200.17201420cm Example 4 Pseudomonas diminuta IFO1 cultured in Example 3
4.4-
Ethylenedioxypimelic acid dipropyl ester11.
5 g of 4-oxopimelic acid monopropyl ester were obtained. The material was analyzed using NMR and TLC and was found to be pure as evidenced by the data below. Also.

Since 4-oxopimelic acid diethyl ester was not mixed in either the target product or the residual component, it was confirmed that it was almost completely selectively hydrolyzed to the above monoester.

4-oxopimelic acid monopropyl ester HNMRi
60MHz, CDC131δ=0.86 (t, 3H)
, 1.68℃m, 2 old, 2.744 m, 8 old, 4
．． 05 (t, 2 old, +0. l1lfs, IH IRfneatl 3200.] 7735.1720.
1420cm44-Ethylenedioxypimelic acid dipropyl ester HNMRf60MHz, CDC15l δ ・0.8
5 (t, 6H), 1.22 (m, 4tl),
1.95 ft, 4H), 2.32 (t, 4H
), 3.91 (s, 4H), 4.08 (q,
4H) IR(neat) 3200. ]720.14
20 cll Example 5 Cells of Chromobacterium cocolatum IFO3758 cultured in the same manner as in Example 3 were suspended in 0.2 M phosphate buffer (p87.5) of 500 m, and then 4-
Mixture of oxopimelic acid dipropyl ester and 4,4-ethylenedioxypimelic acid dipropyl ester 30
．． The same operation as in Example 3 was carried out except that 0 g was used to obtain 0.5 g of 4,4-ethylenedioxypimelic acid dipropyl ester and 4.5 g of 4-oxopimelic acid monopropyl ester i. The material was analyzed using NMR and chromatography and was found to be pure as evidenced by the data below. Furthermore, since 4-oxopimelic acid diethyl ester was not mixed in either the target product or the residual component, it was confirmed that it was almost completely selectively hydrolyzed to the above monoester.

4-Oxopimelic acid monopropyl ester fusion, 6
7-68℃ HNMR (60MH2, CDCl3) δ ・0.8
6 (t, 3H), 1.681m, 2 old, 2.7
4 in+, 8) 11.4.05 (t, 2 old, 10
18(s IH) IRfneatl 3200, 1735.1720.
1420cm4.4-Ethylenedioxypimelic acid dipropyl ester HNMRi60MHz, CDC1xl δ・0.85
(t, 6H), 1.22(m, 4H), ], 9
5 (t, 4H), 2.32 (t, 4H) 3.9
1 (s, 4H), 4.08 (q, 4H)IRf
nati 3200, 1720.1420c+e (Effects of the Invention) According to the present invention, 4-oxopimelic acid diester and 4.
4-oxopimelic acid monoester can be obtained in high yield and purity by selectively hydrolyzing only 4-oxopimelic acid diester from a mixture of 4-ethylenedioxypimelic acid diesters under mild reaction conditions, and A great effect is achieved in that 4,4 ethylene dioxidipimelic acid diester, which is a residual component, can also be effectively utilized.

Claims (1)

[Claims] General formula: ▲ Numerical formula, chemical formula, table, etc. ▼ (I) 4-oxopimelic acid diester represented by (In both formulas, R represents a lower alkyl group having 1 to 4 carbon atoms) The mixture of derivatives was added to Achromobacter (Achromobacter).
er) genus, Chromobacterium (Chromobacterium
erium), Flavobacterium (Flavobacterium)
cterium), Gluconobacter (Glucon)
(obacter) genus, Pseudomonas (Pseudom
onas), Arthrobacter (Arthrobacter)
A culture of a microorganism selected from the group consisting of the genus Ter, Bacillus, and Corynebacterium, its bacterial cells, or lipase extracted therefrom is contacted with
Only the 4-oxopimelic acid diester of I) is selectively hydrolyzed to produce 4- 1. A method for producing 4-oxopimelic acid monoester, which comprises preparing 4-oxopimelic acid monoester and then separating and purifying the monoester.