JPS61242585A - Production of gamma-halogenated-beta-hydroxybutyric acid - Google Patents

Abstract

PURPOSE:To produce the titled compound useful as a synthetic intermediate for L-carnitine, etc., easily in high yield, by treating gamma-halogenated crotonic acid in aqueous medium with a microbial strain capable of converting the gamma- halogenated crotonic acid to optically active gamma-halogenated-beta-hydroxybutyric acid. CONSTITUTION:The objective compound can be produced by treating a gamma- halogenated crotonic acid in an aqueous medium with a microbial strain capable of converting gamma-halogenated crotonic acid to optically active gamma-halogenated-beta- hydroxybutyric acid (e.g. Alcaligenes faecalis), and centrifuging the reaction liquid to remove the microbial cells. L-carnitine is obtained from the titled compound by adding an aqueous solution of trimethylamine to the compound, refluxing at 80 deg.C for 5hr, and distilling out excess trimethylamine under reduced pressure.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing optically active γ-halogenated-β-hydroxybutyric acid. More specifically, optically active γ-halogenated β-β is produced by treating microorganisms capable of converting γ-halogenated crotonic acid into optically active γ-halogenated β-hydroxybutyric acid in an aqueous medium. - Concerning the production method of hydroxybutyric acid.

γ-halogenated β-hydroxybutyric acid is a useful intermediate raw material in the synthesis of various optically active natural products or physiologically active substances such as pharmaceuticals. For example, it can be easily obtained by reacting L-carnitine, γ-halogenated-β-hydroxybutyric acid, which is well known as a stomachic digestive agent, with excess trimethylamine.

(Prior art) As a method for synthesizing L-carnitine, Japanese Patent Application Laid-Open No. 57-397
No. 91 and JP-A-59-192095 are known.

JP-A No. 57-39791 discloses that in addition to the substrate γ-butirobetaine, sodium 2-oxoglutamate,
A method for synthesizing L-carnitine characterized in that a solution of a reducing agent and a ferrous ion source dissolved in a hydroxyl-donating solvent is brought into contact with a phase consisting of a substance released from spores of N. rosbora crap. Published in Japanese Unexamined Patent Publication No. 59-
No. 192095 discloses that L-carnitine is converted into L-carnitine by the action of microorganisms that have the ability to convert crotonbetaine into L-carnitine.
A method for synthesizing monocarnitine is shown.

(Problems to be Solved by the Invention) However, JP-A-57-39791 has problems such as a complicated reaction system and a low yield.

Furthermore, JP-A-59-192095 also has problems such as low yield. That is, the reality is that there is still no satisfactory method for synthesizing L-carnitine.

(Means for solving the problem) As a result of various studies under the circumstances described above, the inventors of the present invention found that L.
-We have discovered an excellent method for producing an intermediate that is easily converted to carnitine, and have completed the present invention. That is, when converting γ-halogenated crotonic acid to optically active γ-halogenated β-hydroxybutyric acid, it has the ability to convert γ-halogenated crotonic acid to optically active γ-halogenated β-hydroxybutyric acid. Optically active γ-halogenated-β characterized by allowing microorganisms to act in an aqueous medium
- A method for producing hydroxybutyric acid is provided.

The present invention will be explained in more detail below.

A microorganism having the ability to convert γ-halogenated crotonic acid to optically active γ-halogenated β-hydroxybutyric acid when converting γ-halogenated crotonic acid to optically active γ-halogenated β-hydroxybutyric acid. A method for causing γ-halogenated crotonic acid to act in an aqueous medium is to bring the γ-halogenated crotonic acid into contact with the cells, culture solution, or treated product of the microorganism.

Examples of microorganisms having the ability to convert γ-710-genated crotonic acid to optically active γ-halogenated-β-hydroxybutyric acid used in the present invention include Alcaligenes genus, Brera (
Bullera) genus, Acinetonokuta (Ac1n)
Etobacter ) genus, Cryptococcus (cry
ptococcus) genus, Candi
da), Debaryomyces (, Debaryomyces
ces), Kloeckera J, Hanseniaspora
Genus, Agrobacterium
m) genus, Pachy-solen
Genus, Arthrobacter, Pichia, Nocardia
-3rdia) genus, Protaminono (Prot.
amin-obacter) genus, Lipomyces (Li
Pomyces) genus, Rhodotorula
Genus ula J, Rhodosporidium (phodospo
ridium) genus, Saccha omyces (Saccha
romyces), xanthomonas (xanthomonas)
monas ) genus, Vibrio genus, Achromobacter (Achromobacter J genus,
Genus Erwinia, Bacillus
illus), Brevibacterium (Brevib
acterium ) genus, Corynebacterium (Oor
ynebacterium), Schizosaccharomyces x, (schizosaccharom-yces)
), xloeckera , Torulopsis , Pseudomonas , Micrococcus , Chiono (
Th1obacillus) genus, Trichospoon (T
richosporon), Proteus (Prot.
eus) genus, Streptomyces (Streptomyces)
yces) genus, Serratia (5erratia) genus,
Salmonella genus, Hansenula genus, Sporidiobolus genus, Escheri-chia genus, Enterobacter genus, Endomyces genus myces) genus, Kluyveromyces (
Kluyveromyces), Helicostylum, Tremella (Tr)
em-ella), Geotrich
um ) genus, Debaryomyces
es ) genus, Microbacterium (Microbacterium
erium) genus, Kuribia genus,
Examples include the genus Aeromonas, the genus Azoobacter, and the genus Staphylococcus.

In order to obtain cells of these microorganisms, it is sufficient to culture them using a normal medium and adding γ-...rogenated crotonic acid from the beginning of the culture or during the culture. The medium used for culturing this microorganism can be any of natural medium, semi-synthetic medium, synthetic medium, etc. That is, γ
- Halogenated organic acids, carbon sources such as alcohols, peptone, meat extract, yeast extract, dried yeast, soybean meal, urea,
Nitrogen sources such as thiourea, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds, and inorganic salts such as phosphates, nitrates, carbonates, and chlorides of magnesium, manganese, potassium, calcium, iron, etc., are used. Also, amino acids, vitamins, nucleic acids and related compounds may be added.

Cultivation is carried out in accordance with the usual aerobic culture method, and for example, the pH of the culture solution is preferably 4 to 8, and the culture temperature is preferably 20 to 50°C. Moreover, the culture can be suitably carried out for a period of 12 hours to 10 days.

The method of using the bacterial cells obtained as described above to act on γ-halogenated crotonic acid is to culture them using the method shown above and then apply γ-halogenated crotonic acid from the beginning of the culture, or How to add and make it work in the middle of
Alternatively, one inch of the culture fluid after completion of culture, or bacterial cells isolated from the culture fluid by a method commonly used in the field, or washed bacterial cells, or a processed product of bacterial cells, such as freeze-dried bacterial cells,
In addition to acetone-dried bacterial cells, bacterial cells that have been brought into contact with toluene, surfactants, etc., bacterial cells that have been treated with lysozyme, bacterial cells that have been treated with ultrasound, mechanically ground bacterial cells, and other bacterial cells that have been treated with these bacterial cells, The obtained γ-halogenated crotonic acid is converted into an optically active γ
- Dissolve γ-halogenated crotonic acid with the enzyme protein portion having the enzymatic activity to convert it into halogenated β-hydroxybutyric acid, as well as these bacterial cells, immobilized enzymes, insolubilized products of bacterial cell processing, etc. Or suspend the aqueous medium at 10°C.
What is necessary is just to stir or stand still while adjusting the temperature to ~80 degreeC and keeping pH at 4-8. After 5 to 200 hours in this manner, optically active γ-halogenated-β was added to the aqueous medium.
- A method for producing and accumulating hydroxybutyric acid is used. The aqueous medium used includes water, buffer solutions, organic solvents such as ethanol, etc., such as phosphates, carbonates,
acetate, tartrate, lactate, borate, maleate,
Buffers such as citrate, succinate, biphthalate, Tris, etc., or organic solvents miscible with water, alcohols such as methanol, ethanol, propatool, propylene glycol, esters such as methyl acetate, Typical examples include ketones such as acetone, amides such as acetamide, and the above can be used.

Furthermore, if necessary, nutrients, antioxidants, surfactants, coenzymes, hydroxylamine, metal ions, etc. necessary for the growth of microorganisms can be added to the aqueous medium.

The thus obtained optically active γ-halogenated
β-Hydroxybutyric acid is easily converted into various optically active natural products or physiologically active substances such as pharmaceuticals by well-known methods. For example, L-carnitine can be easily synthesized by reaction with excess trimethylamine, and L-γ-amine-β-hydroxybutyric acid, which is used as a pharmaceutical, can be easily synthesized by reaction with excess ammonia.

(Effects of the Invention) As shown above, the present invention is an efficient method for synthesizing intermediate raw materials useful in synthesizing various optically active natural products or physiologically active substances such as pharmaceuticals. It is extremely useful in industry.

The present invention will be explained in more detail below using Examples.

Example 1 Glycerin 10g/11 Asparagine 27/l, K2H
P0.1y/1XFeSO, 7H200, 1f
f/l.

MnCt2*4H,,00,1y/LXZn+sO,・
7H3O[], 1 ff7t, yeast extract 27/l, peptone 2f! /L and γ-chlorocrotonic acid 31iI/
l (pn7.o) into a 500ml shaker flask at 100Wl/! After sterilizing at 120℃ for 15 minutes,
The microorganisms shown in Table 1 which had been precultured in a broth medium at 60°C for 12 hours were inoculated, and cultured with shaking at 60°C for 24 hours.

Bacterial cells were collected from this culture solution by centrifugation, washed once with the same physiological saline as the culture solution, and collected. 0.5 containing this bacterial body and 502/l of γ-chlorocrotonic acid
Add to 100 liters of M phosphate buffer (pH 7.0) and add 6
The reaction was carried out at 0°C for 10 hours.

After removing the bacterial cells from the reaction solution by centrifugation, the reaction product was identified by gas chromatography mass spectrometry and NMR1/R1 elemental analysis, and was found to be β-hydroxy-γ-chlorobutyric acid. 6 in this liquid
10 ml of 0% trimethylamine was added, and the mixture was stirred under reflux at 80° C. for 5 hours, and then excess trimethylamine was distilled off under reduced pressure. The reaction product thus obtained was analyzed by the method of David, Jie, Bisian et al. (Met-
hod of enzymatic analysis
, Volume 4, 2nd edition>, 1974, 1758,
However, as shown in Table 1, L-carnitine was detected.

Claims (1)

[Claims] When converting γ-halogenated crotonic acid into optically active γ-halogenated β-hydroxybutyric acid, γ-halogenated crotonic acid is converted into optically active γ-halogenated β-hydroxybutyric acid.
An optically active γ-
Method for producing halogenated-β-hydroxybutyric acid.