JPH0634733B2 - Process for producing (R) -γ-azido-β-hydroxybutyric acid ester - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing (R) -γ-azido-β-hydroxybutyric acid ester by allowing a microorganism to act on γ-azidoacetoacetic acid ester. (R) -γ-azido-β-hydroxybutyric acid ester is useful as a raw material for synthesizing L-carnitine.

[Problems to be Solved by the Prior Art and Invention] γ-azidoacetoacetic acid ester is chemically reduced to give γ-
When producing azido-β-hydroxybutyric acid ester,
There is a drawback that side reactions easily occur and the yield of the target product is low. Moreover, when chemically reduced, a mixture of (R) and (S) isomers is produced, and the yield of the (R) isomer is 50% at the maximum. In order to solve these problems, it is considered to utilize the action of microorganisms, and JP-A-61-63639 discloses an example of using S. cerevisiae. The present inventors have made γ-azidoacetoacetic acid ester act with a microorganism of the genus Sporoboromyces,
The inventors have found that (R) -γ-azido-β-hydroxybutyric acid ester can be obtained more efficiently, and completed the present invention.

[Means for solving problems]

The present invention provides a γ-azidoacetoacetic acid ester as (R) -γ-
A culture solution of a microorganism of the genus Sporoboromyces having the ability to convert to azido-β-hydroxybutyric acid ester, a bacterial cell or a treated product of the bacterial cell is allowed to act on γ-azidoacetoacetic acid ester, and the product is collected. (R) -γ-azido-β-hydroxybutyric acid ester.

The starting material γ-azidoacetoacetic acid ester used in the present invention is represented by the general formula: N ₃ —CH ₂ CO.CH ₂ COOR (wherein R is any organic residue such as an alkyl group, a phenyl group, and an aryl group). A)).

The γ-azidoacetoacetic acid ester used in the present invention is, for example, 1 to 20 at a temperature of 20 to 70 ° C. in a water-alcohol solution of γ-haloacetoacetic acid ester and sodium azide.
It is carried out by reacting for a time.

Next, as the microorganism used in the present invention, those belonging to the genus Sporobolomyces and having the ability to reduce γ-azidoacetoacetate to (R) -γ-azido-β-hydroxybutyrate Any of them can be used. An example is Sporoboromyces Salmonicolo.
r) IFO1038 may be mentioned. This strain is stored in the Fermentation Research Institute (IFO) under the deposit number 1038 and is a strain that can be easily obtained as needed. As a general property, Sporoboromyces can mutate by natural or artificial means, but all those that reduce γ-azidoacetoacetic acid ester to (R) -γ-azido-β-hydroxybutyric acid ester It can be used for the production method of the invention.

The microorganism used in the present invention can be cultured according to a conventional method. The medium used for culturing is an ordinary medium containing a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of microorganisms. In addition, the addition of organic micronutrients such as vitamins and amino acids often produces desirable results.

Culturing is carried out under aerobic conditions for 1 to 10 days while controlling pH to 3 to 8 and temperature to 10 to 40 ° C. For the reaction, any of a culture solution of a microorganism and cultured cells separated and collected from the culture solution can be used. In addition, as the treated cells, dry cells obtained by freeze-drying, acetone-drying, etc., disrupted cells obtained by grinding or self-digesting the cells, ultrasonication, etc., as well as γ-azidoacetate An enzyme protein group having an enzymatic activity for converting an acetate ester into (R) -γ-azide-β-hydroxybutyrate ester, further, an immobilized product of these bacterial cells or a treated bacterial cell, and others can be used.

γ-azidoacetoacetic acid ester as (R) -γ-azido-β
The method for converting into hydroxybutyric acid ester is to contact the γ-azidoacetoacetic acid ester with a culture solution of the above-mentioned microorganism in the aqueous medium, cells, treated cells, or those obtained by immobilizing these by a known method. Good.

Water, a buffer solution, and a water-containing organic solvent can be used as the aqueous medium in the reaction.

In order to make the above-mentioned microorganism act on γ-azidoacetoacetic acid ester, it is usually carried out while controlling the pH within the range of 3 to 8 and the reaction temperature within the range of 10 to 60 ° C.

The γ-azidoacetoacetic acid ester is added to the reaction system as it is, or after being dissolved in a solvent or dispersed. The ester concentration in the reaction system is usually in the range of 0.001 to 50% by weight. The addition of such γ-azidoacetoacetic acid ester is possible at any stage of the reaction and can be performed collectively, continuously,
It can be implemented by any means of division.

During the reaction, sugars such as glucose, microbial nutrients,
The reaction can be carried out in the presence of a surfactant and the like.
The reaction time can be adjusted depending on the concentration conditions, but at most 4
After the reaction for about 8 hours, γ-azidoacetoacetic acid ester is converted to (R) -γ-azido-β-hydroxybutyric acid ester.

The (R) -γ-azido-β-hydroxybutyric acid ester thus obtained is collected from the culture solution or the reaction solution by a conventional method such as centrifugation or ultrafiltration of the cells or treated cells. Then, a usual method such as a method of removing with an organic solvent such as ether, carbon tetrachloride, benzene, ethyl acetate and the like can be employed.

〔Example〕

Next, the method of the present invention will be described in more detail with reference to Examples.

Example 1 5 ml of a medium (pH 6) containing 5% by weight of glucose and 5% by weight of corn steep liquor was placed in a test tube, and Sporobolomyces Salmoni
color) IFO1038 was inoculated and shake culture was performed at 28 ° C. for 24 hours to obtain a seed culture solution.

Next, 100 ml of the medium having the same composition as described above was placed in a 500 ml Sakaguchi flask, 5 ml of the seed culture was added, and shaking culture was performed at 28 ° C. for 64 hours.

Ethyl .gamma.-azidoacetoacetate (1.0 g) was added to this system over 4 hours, and the mixture was further shake-cultured for 4 hours to carry out a reaction.

The obtained reaction solution was sterilized by a centrifuge and then extracted with 300 ml of ethyl acetate (100 ml × 3 times). Anhydrous magnesium sulfate was added to this ethyl acetate layer for dehydration, followed by concentration under reduced pressure to obtain 0.95 g of an oily product. This product and IR (Shimadzu IR-435), NMR (JEOL GX-27)
0), and confirmed by HPLC (ODS column), it was confirmed to be ethyl γ-azido-β-hydroxybutyrate.

NMR δ (in CDCl ₃ ); δ (ppm) 1.29 ppm (3H, t) 3.52 (2H, s) 4.13 (2H, s) 4.20 (2H, q) IR 2120 cm ⁻¹ (N 3 N, stretching) HPLC analysis conditions Column: ODS (ULTRON C ₁₈ x 25 cm, Shinwa Kako) Flow rate: 2 ml / min Mobile phase: Methanol / water = 10/90 Temperature: 35 ° C Detection: 220 nm RT (min) 15.6 (γ-azidoacetoacetate) 17.6 (γ -Ethyl azido-β-hydroxybutyrate) Further, when converted into MTPA ester and the optical purity was measured by HPLC, it was ee (R) = 83%.

HPLC analysis conditions Column: URTRON SIL × 25 cm Flow rate: 1.5 ml / min Eluent: Water / tetrahydrofuran / methanol = 300
0/50 / 0.5 Temperature: 25 ° C. Detection: 217 nm (+) R.V. when using MTPA chloride. T
(Min) 30 ((S) -γ-azido-β-hydroxybutyrate ethyl) 33 ((R) -γ-azido-β-hydroxybutyrate ethyl) Also, the purity was 95% from C-13 NMR.

Example 2 Using the γ-azidoacetoacetic acid ester shown in the table as a substrate, the reaction was carried out in the same manner as in Example 1, and the corresponding γ-azido-
A β-hydroxybutyric acid ester was obtained. The results are shown in the table.

In addition, when the substrate is octyl ester, at the start of the reaction
0.5 ml of 10% Tween 80 (KAO-ATLAS) was added and reacted.

Example 3 1 g of ethyl acetate was added to 10 g of wet cells obtained in the same manner as in Example 1.
Add and mix 0.1 ml NaHCO ₃ and add 20 ml of 0.1M NaHCO ₃ , and keep the mixture near neutral with ammonia water (5%).
Mix at 8 ° C. for 8 hours and wash with 0.1 M NaHCO ₃ . After freezing and thawing of the obtained mixed solution twice, 0.1M NaHCO 3 was added.
Add 20 ml of ₃ and mix with ammonia water (5%) at 28 ° C until the viscosity decreases while keeping the neutrality, and remove the insoluble matter by centrifugation (28,000 G, 30 minutes) to obtain the crude enzyme solution. 10 ml was obtained.

To this crude enzyme solution, 10 ml of 0.1M phosphate buffer (pH 6.0), NA
After adding 500 mg of DPH (Sigma Co.), 50 mg of ethyl γ-azidoacetoacetate was added over 4 hours, and after reacting for 4 hours, the reaction solution was analyzed by the same method as in Example 1, The yield of ethyl γ-azido-β-hydroxybutyrate was 95%. Furthermore, when the optical purity was measured by the MTPA ester method, it was ee (R) = 95%.

〔The invention's effect〕

According to the present invention, from γ-azidoacetoacetic acid ester (R)
-Γ-azido-β-hydroxybutyric acid ester can be obtained in high yield, which is industrially advantageous.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 645)

Claims (1)

[Claims] 1. A method for converting γ-azidoacetoacetic acid ester into (R) -γ
-Azido-β-hydroxybutyric acid ester, characterized in that the culture solution of a microorganism of the genus Sporoboromyces, bacterial cells or a treated product of bacterial cells is allowed to act on γ-azidoacetoacetic acid ester to collect the product. (R) -γ-azido-β-hydroxybutyric acid ester.