JPH02238895A - Production of optically active alpha-halogenocarboxylic acid - Google Patents

Abstract

PURPOSE:To industrially advantageously obtain the title compound to be used as a raw material for medicines by acting on a (R,S)alpha-halogenocarboxylic acid microorganisms classified as e.g. Candida sp. and having prior metabolic ability for the (S)-modification to take the (R)-modification accumulated. CONSTITUTION:The objective (R)-halogenocarboxylic acid can be obtained by acting on a (R,S) alpha-halogenocarboxylic acid of the formula (n is 1-3; X is halogen) microorganisms classified as e.g. Candida, Cryptococcus, Debaryomyces, Endomyces, Geotrichum, Hansenula, Kluyveromyces, Rhodosporidium, Pichia, Rhodotorula, Saccharomyces, Saccharomycopsis, Torulopsis, Trichosporon, Actinomucor, Anixiella sp. and having prior metabolic ability for the (S)- modification to take the (R)-modification accumulated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing an optically active α-halogenocarboxylic acid. Optically active α-halogenocarboxylic acids (hereinafter referred to as α-haloacids) are useful substances as raw materials for the synthesis of optically active physiologically active compounds such as pharmaceuticals and agricultural chemicals. [Prior art and problems] As a method for producing optically active α-haloacids, there is a method of deriving optically active amino acids or optically active α-hydroxyacids as raw materials [for example, “Journal of American Chemical Society, vol. 76, p. 6054 (1954) and JP-A-57-42945, etc.], a method of optically resolving racemic α-halo acids using optically active 1-(1-naphthyl)ethylamine, etc. 61-227549) is known. However, in the former method, the production of the optically active material as a raw material is not necessarily economical;
It has problems such as racemization being observed in some parts during halogenation. In the latter method, the racemic α-haloacid as a raw material can be obtained industrially at a relatively low cost, but the separation operation is complicated and it cannot be said to be an economical production method. Therefore, the present inventors have developed optical activity (R) by utilizing microorganisms having stereospecific α-halo acid dehalogenase.
-Planned the production of haloacids. Conventionally, it has been known that there are many microorganisms that have the ability to dehalogenate organic halogen compounds, and the present inventors catalyzed the reaction to dehalogenate α-halo acids and give α-hydroxy acids. Many microorganisms have been reported to possess α-haloate dehalogenase. However, the α of these microorganisms
- Regarding the stereospecificity of octaloyl dehalogenase, Pse
Only udomonas has been investigated. Its content is I'seudosonas deh
European Journal of Biochemistry, Volume 21, Page 99 (1)
971) ), Pseudomonas putid
a (Agricultural Biological Chemistry J, 461', p. 837 (1982)
), Pseudomonas sρ, [The Journal of Biological Chemistry, 24
3t! , p. 428 (1968)) has shown that α-halo acid dehalogenase acts only on (S)-based α-halo acids. However, Pseudo+mon
as sp. strain 113 (Japanese Patent Publication No. 1983-
125691), which act on both the (R) and (S) forms and have no stereospecificity, and α-octatate dehalogenase is not necessarily (S) specific. [Means for Solving the Problems] In view of the above circumstances, the present inventors have discovered that stereospecific,
When we searched for α-halo acid dehalogenase-producing microorganisms that are easy to use industrially, we found that various microorganisms preferentially metabolize (S) mono-α-halo acids and accumulate (R) mono-α-halo acids. We have discovered that this can be done and completed the present invention. That is, the present invention is based on the general formula (!)C. Hz--+ CH COOHX (in the formula, n is an integer of 1 to 3, and X represents a halogen atom)
The (R,S) α-halo acid represented by Candida spp.
Waliptochosoccus, Debaryomyces, Endomyces, Geotrichum, Hansenula, Thalyveromyces, Rhodosboridium, Pichia, Rhodotorula, Saso. Calomyces, Satucharomycopsis, Torulopsis, Trichosboro, Actinomucor, Anixiera, Aspergillus, Arthroderma, Basoxella, Chronostathys, Echinopodosvora, Henneromyces, Gerasinospora,
A microorganism that belongs to at least one genus selected from the genus Gliocephalotritium, Gongronella, Pecilomyces, and Penicillium and has the ability to preferentially metabolize α-halo acids in the (S) body is activated and accumulated. (R
) The method for producing (R)-octactic acid is characterized by collecting α-haloacid. Among the microorganisms that can be used in the present invention, (S)
Any microorganism can be used as long as it preferentially metabolizes mono-α-octactic acid, such as Candida humicola IFO.
0760, Crybutococcus albidus (Cry
ptococcus albidus) IFO 03
78, Debaryomyces hansenii (Loebaryo
IF0 0018
, Endomyces opertensis (Endo*yces)
Geotrlchus Ioubi (Ovetensis) IP0 1201%
eri) CBS 252+6L Hansenu-la nonfermentans
enLans) IFO 1473, Kluyveromyces fr
IFO 0288, Pichia membranaefaciens (Pic-hia mes+br
anaefaciens) IFO 0864, Rhodosporidium tonorelloides (Rhodospori
dius+ toreroids) IFO 04
13. Rhodo-tor
ula glutinis) IPO 1099, Saccharomyces luxii (Saccharoa+yc
es rouxii) IFO 0493, Saccharom-yc
opsis lipolytica) IP0 154
B. Torulopsis sorbophila
S sorbophila) IFO 1583, Trichosboron fermentans (Trichospor
IFO 1199
, Act4nomu elegans
cor elegans) IFO 4022, Anixiella indic
a) IFO 30246, Aspergillus cellulosae
IP0 4040% Arthroderma ancinatum (
Arthroderma ancii+atam) I
FO 7865, Backus
ella circina) IPO 923l1 Chronostathys cylindrosvora (Clono-stac
hys cylindrospora) IFO 70
66, Echinobodosvora jamaicensis (Ech
inopodospora jam-aicensis
) IFO 30406, Fennellomyces Iinderi
IFO 6409, Geracinosvora cerealis (Ge
lasinospora cerealis) IFO
6759, Gliocephalotrichum c.
ylindrosp-orum) IFO 9326,
Gongronella batleri (Gongr-onella)
butleri) IP0 8080% Paecilomyces farimosus (Paecilomyces farimos)
mosus) IFO 8108, Penicillium cla-vif
orma) IP0 5739 and other microorganisms. In the present invention, in order to react the substrate (R,S)-α-octaloic acid with the microorganism, the substrate is added to the culture medium of the microorganism from the beginning, and the method is carried out simultaneously with the aerobic cultivation of the microorganism. Or a culture solution obtained by culturing microorganisms in advance aerobically,
Furthermore, a method is used in which bacterial cells are collected by centrifugation, etc., and then suspended in an appropriate reaction solution or reacted with immobilized bacterial cells in a water-insoluble carrier such as carrageenan gel or polyacrylic acid amide gel. be able to. The medium used for culturing the microorganisms may be any medium as long as it contains a nutrient source that allows the microorganisms used to grow. For example, carbon sources include carbohydrates such as glucose and sucrose, organic acids and their salts such as acetic acid, lactic acid, and succinic acid, and nitrogen sources include organic nitrogen sources such as yeast extract, casamino acids, cornstarch liquor, and peptone. , ammonium sulfate, ammonium phosphate, etc.
If necessary, vitamins or trace amounts of organic or inorganic metal salts can be added. The reaction between microorganisms and (R,S)-α-haloacids is caused by pll5
The temperature is preferably in the range of 20 to 45°C, more preferably 25 to 36°C, and the temperature is preferably in the range of 25 to 36°C. Good. The culture time is preferably 24 to 72 hours, and the concentration of the substrate added to the reaction solution is in the range of 0.1 to 30% by weight, and is selected depending on the capacity of the Kokutai used. The α-haloacid in the reaction solution can be easily quantified by gas chromatography [column PAL-M toχTP^ (0.5 m), temperature 160°C]. In addition, the optical purity of the remaining α-haloacid was analyzed by deriving it into an anilide,
High performance liquid chromatography [Daicel OD, hexane-isopropanol (20:1), flow rate 0.8 m/day
n, detection 280 mm). After the reaction, the optically active α-haloacid is collected from pl1 of the reaction solution.
After making it acidic to 2 or less and salt-saturated with ammonium sulfate, etc., extract with an organic solvent such as ethyl acetate or methylene chloride, collect the solvent layer, and remove the solvent. Further, by purifying this by distillation, a pure optically active α-haloacid can be obtained. [Examples] Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples. Example 1 Glucose 40g, (NI+4)lPO4 13g
SKll! PO4 7g, MgSOa 41lx
0 0.8g, ZnSOa 4tlx0 60m
g, FeSOa ・71IxO 90 mg
, CuSO4-5}1x0 5 mg, Mn
SOi ・4+1!0 10 mg, NaCI 0
．． A culture medium consisting of 1 g of yeast extract (3 g per 1 liter) was set to pl+7.2, and 30 g of this was put into a 500 volume flask and sterilized, then inoculated with the microorganisms shown in Table 1 and incubated at 30°C. The culture was incubated with shaking for 20 to 48 hours until the growth of the bacteria reached its maximum. Next, bacteria were collected by centrifugation, (R,
S) 0.5% by weight of monoα-chloropropionic acid.
Suspended in 1 M phosphate buffer (pH 7.0) 30-
The reaction was carried out at 30°C with shaking. Analyzing α-chloropropionic acid using gas chromatography over time,
The reaction was stopped when the remaining amount was less than 50%. The reaction solution was adjusted to pH 2 with sulfuric acid, saturated with ammonium sulfate, extracted with twice the amount of ethyl acetate, the solvent layer was dehydrated with anhydrous sodium sulfate, the solvent was removed under reduced pressure, and optically active (R) α-chloroprobionic acid was extracted. I got it. The optical purity analyzed by liquid chromatography is shown in Table 1. Example 2 3j! of the same medium 16 as in Example 1! Pichia membranaefaciens IF0 0864, Candida humicola IFO 0760, Thalyveromyces fragilis IFO 0288, Safcharomyces luxi-I after sterilization.
FO 0493, Actinomucor elegans IF
0 4022, Penicillium tarabiforma IFO 5
739 strains and 500 rpm+
, IVVM, and cultured at 30°C for 16 to 24 hours.

To this was added 0.5% (R,S)-α-chlorobutyric acid (
5g) was added and the reaction was carried out under the same conditions for 60 hours while maintaining the pH at 7. Next, pl+ of the reaction solution was added to ρ112 with sulfuric acid.
The mixture was then saturated with ammonium sulfate and extracted twice with equal volumes of ethyl acetate. After dehydrating the ethyl acetate layer with anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain an oily substance. This was distilled under reduced pressure (70℃/288}1g), colorless and transparent (R).
Mono-α-chlorobutyric acid was obtained. The yield and optical purity were as shown in Table 2. In the above, Pichia menplanaefaciens I
The specific optical rotation of (R)-α-chlorobutyric acid obtained by F0 0864 was [α);'+15.53@C=2 methanol. [Effects of the Invention] As described above, according to the present invention, high optical purity (R)-α-octactic acid can be industrially advantageously produced by a simple method.

Patent applicant Kanebuchi Chemical Industry Co., Ltd.

Claims (1)

[Claims] 1. General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, n is an integer from 1 to 3, and X represents a halogen atom)
(R, S) α-halogenocarboxylic acid represented by Candida sp., Walliptococcus sp., Debaryomyces sp., Endomyces sp., Geotrichum sp., Hansenula sp.
Kluyveromyces, Rhodosporidium, Pichia, Rhodotorula, Saccharomyces, Saccharomycopsis, Torulopsis, Trichosporon, Actinomucor, Anixiera, Aspergillus, Arthroderma, Baccella, Chronostathis , Echinopodospora, Henneromyces, Gerasinospora, Gliocephalotrichium, Gongronella, Pecilomyces, and Penicillium, and preferentially uses (S) α-halogenocarboxylic acids. A method for producing (R)-halogenocarboxylic acid, which comprises collecting accumulated (R)-α-halogenocarboxylic acid by applying a microorganism capable of metabolic decomposition. 2. The manufacturing method according to claim 1, wherein in the general formula (I), the halogen atom is CI or Br. 3. The compound represented by the general formula (I) is α-chloropropionic acid, α-bromopropionic acid, α-chlorobutyric acid,
The manufacturing method according to claim 1 or 2, wherein the at least one selected from α-bromobutyric acid. 4. The microorganisms are Candida humicola, Cryptococcus albidus, Debaryomyces hansenii,
Endomyces obetensis, Geotrichum robieri, Hansenula nonfermentans, Kluyveromyces fragilis, Pichia membranae faciens, Rhodosporidium toruloides, Rhodotorula glitinis, Saccharomyces ruxii, Saccharomycopsis lipolytica, Torulopsis sorbophila, Trichosporon fermentans, Actinomucor elegans, Anixiera indica,
Aspergillus cellulosa, Arthroderma ancinatum, Baccella circina, Chronostathis cylindrospora, Echinopodospora chamaiscensis,
The method according to claim 1, 2 or 3, wherein the method is selected from Henneromyces Linderi, Gerasinospora cerealis, Gliocephalotritium cylindrosporium, Gongronella batleri, Pecilomyces farimosus, and Penicillium claviforma.