JPH0678B2 - Process for producing optically active 3-phenylglycidate compounds - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for producing an optically active trans-3-phenylglycidate compound.

[Prior art]

3-Phenylglycidic acid ester compounds include (2R, 3
There are a total of four stereoisomers, one pair of enantiomers in the (S) form and the trans form of the (2S, 3R) form and one pair of the enantiomers in the (2R, 3R) form and the (2S, 3S) form of the cis form. Among them, the optically active trans-3-phenylglycidic acid ester compound is an important compound as a synthetic intermediate for diltiazem hydrochloride and various other pharmaceutical compounds useful as a coronary vasodilator, but conventionally, a method for producing this compound was used. For example, a method of hydrolyzing trans-3- (4-methoxyphenyl) glycidic acid methyl ester to a corresponding carboxylic acid, optically resolving this carboxylic acid with optically active amines, and then esterifying it (Japanese Patent Application Laid-Open No. 2000-242242).
60-13775 and 60-13776) are known.

[Technical issues to be solved]

However, the above method has a large number of steps, and the desired optically active trans-3- (4-methoxyphenyl)
The problem is that glycidic acid methyl ester can only be obtained as an oily substance of low purity.

[Structure and Effect of Invention]

The present inventors have conducted extensive studies to solve such problems, and as a result, from racemic trans-3-phenylglycidic acid ester compounds, the desired optically active trans-
The inventors have found a method for obtaining 3-phenylglycidic acid ester compounds at once and as crystals, and have completed the present invention.

That is, according to the present invention, the optically active trans-3-phenylglycidic acid ester compound has the general formula (However, ring A is a phenyl group which may have a substituent,
R represents a lower alkyl group. ) In the racemic trans-3-phenylglycidic acid ester represented by the formula, a culture solution, a microbial cell or a treated product of a microorganism belonging to the genus Corynebacterium or Serratia having the ability to asymmetrically hydrolyze the ester bond. Alternatively, it can be produced by reacting an enzyme derived from the microorganism to hydrolyze one of the optically active substances, and then separating and collecting the antipode from the reaction solution.

In the method of the present invention, even when the glycidic acid ester represented by the general formula [I] has a substituent selected from a lower alkyl group, a lower alkoxy group and a halogen atom on the ring A, It can be carried out similarly. Examples of such a substituent include a methyl group, a methoxy group or a chloro atom at the 4-position. The substituent R is a lower alkyl group, for example, a methyl group, an ethyl group, an isopropyl group or a t-butyl group.

In the present invention, the racemic trans-
The 3-phenylglycidic acid ester compound [I] includes not only those containing the (2S, 3R) -isomer and (2R, 3S) -isomer in equal amounts, but also these optically active substances in appropriate proportions. Any of them can be used.

The enzyme source for asymmetrically hydrolyzing the ester bond of the glycidic acid ester compound [I] is known as a culture solution of a microorganism belonging to the genus Corynebacterium or the genus Serratia, a microbial cell or a treated microbial cell, as well as the microbial cell. Purified or partially purified lipase or esterase obtained by the method can be used.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium alkanolyticum (Corynebacterium
bacterium alkanolyticum) ATCC 21511, Corynebacterium hydrocarbocluster
hydrocarboclastum) ATCC 15592, Corynebacterium primorioxy
dans) ATCC 31015 etc. can be mentioned. On the other hand, specific examples of the microorganism belonging to the genus Serratia include Serratia liquefaciens ATCC 2759.
2. Serratia marcescens
ATCC 13880, ATCC 14764, ATCC 19180, ATCC 210
74, the same ATCC 27117, the same ATCC 21212 and the like. These may be wild strains or mutant strains, or may be derived from these microorganisms by bioengineering techniques such as gene recombination and cell fusion.

In addition, the culture solution and bacterial cells of the above-mentioned microorganism are, for example, a medium in which the microorganism is usually used in this field, for example, a medium containing a conventional carbon source, nitrogen source and inorganic salts, at room temperature or under heating (preferably about It can be obtained by culturing at 20 to 40 ° C.) under aerobic conditions at a pH of about 5 to 8, and if necessary, separating and collecting cells from the culture solution by a conventional method.

When culturing, racemic trans-3-
Phenylglycidic acid ester compound [I] was added to about 0.00
The enzyme activity can be increased by adding 1% or more, especially about 0.1 to 1%.

Examples of the treated product of the microbial cells include freeze-dried microbial cells of the above-mentioned microorganism, acetone-dried microbial cells, autolyzed microbial cells, microbial cell extracts, microbial cell grinds, and sonicated products of the microbial cells. can give. Furthermore, the microbial cells or treated cells of the present invention should be immobilized by known methods such as polyacrylamide method, sulfur-containing polysaccharide gel method (carrageenan gel method, etc.), alginic acid gel method, agar gel method, etc. You can also

The asymmetric hydrolysis reaction according to the present invention can be carried out by using a racemic trans-3-phenylglycidic acid ester compound [I] in an appropriate solvent, an enzyme, a culture solution of a microorganism, a bacterial cell or a microorganism collected from the culture solution. It can be carried out by contacting the body-treated product.

The concentration of the substrate is generally 0.05 to 20%, preferably 0.5 to 5%, and the reaction is carried out at room temperature or under heating, preferably 10 to 50%.
Suitably progresses at ℃, particularly preferably 25-40 ℃. In the reaction, the pH of the reaction solution is 5-10, especially 6-
It is preferable to adjust it to be 9. In this case, since most of the substrates are poorly soluble in water, the reaction is preferably carried out in water or a two-phase solvent system of an aqueous solvent and an organic solvent. Examples of the organic solvent include benzene, toluene, xylene, carbon tetrachloride, chloroform, dichloromethane, trichloroethylene, chlorobenzene, ethyl acetate, butyl acetate, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, t-butyl alcohol, diethyl. Examples thereof include ether, diisopropyl ether, methyl ethyl ketone, and methyl isobutyl ketone, with ethyl acetate, carbon tetrachloride, and toluene being particularly preferable.
When using a microbial cell or a culture solution thereof as an enzyme source, if the above reaction is carried out in the presence of a surfactant,
The reaction time can be shortened and the yield of the optically active trans-3-phenylglycidic acid ester compound [I] can be increased. As such a surfactant, cetylpyridinium bromide, cetyltrimethylammonium bromide, polyethylene glycol, polyoxyethylene octylphenyl ether, sodium lauryl sulfate or the like can be used, and the addition amount thereof is about 0.0001 with respect to the reaction solution. It is preferably about 0.1%.

Optically active trans-3-phenylglycidic acid ester compound [I] from the hydrolysis reaction liquid thus obtained
Can be easily isolated by a conventional method. For example, when the hydrolysis reaction is carried out in a water-organic solvent two-phase system, one of the optically active forms of the 3-phenylglycidic acid ester compound [I] is hydrolyzed and transferred to the water layer to carry out the reaction. Since the other optically active substance which does not receive remains in the organic solvent, the optically active trans-3-phenylglycidic acid ester compound can be collected as crystals by separating the organic solvent layer and concentrating at source pressure. it can.

According to the above method of the present invention, an optically active trans-3-phenylglycidate compound [I] can be obtained in a short step, and the enzyme of the present invention has good stereoselectivity for a substrate, and the substrate is stable. Since the optimum pH of the enzyme and the optimum pH of the enzyme are almost the same, a highly purified target product can be obtained in a high yield, which is an industrially advantageous production method.

Experimental Example 1 <Purpose> The stereoselectivity of a substrate of the enzyme of the present invention and a control enzyme was examined.

<Enzyme used> Enzyme of the present invention: Each microorganism is used as a medium (composition; dextrin 1
%, Ammonium sulfate 0.2%, mist S 2%, KH ₂ PO ₄ 0.1%, Mg
SO ₄ / 7H ₂ O 0.05%, CaCl ₂ / 2H ₂ O 0.01%, FeSO ₄ / 7H ₂
O 0.001%, Tween80 0.5%, Calarin 102 0.1%, pH
After culturing in 7.0) at 30 ° C. for 18 hours, the culture solution was centrifuged, and the supernatant was used as it was (hereinafter, the same).

Control enzyme: a commercially available enzyme was used (hereinafter the same).

<Experimental method> 3.75 ml of 0.4 M Tris-HCl buffer containing 2 mM calcium chloride
And toluene solution containing 1.5 g of racemic trans-3- (4-methoxyphenyl) glycidic acid methyl ester 7.5 m
Mix l and pre-incubate at 30 ° C for 10 minutes. To this, 3.75 ml of the enzyme solution was added to adjust the pH of the reaction solution to the optimum pH of the enzyme, and then the hydrolysis reaction was carried out by stirring at 30 ° C. and 1400 rpm for 3 hours.

The amount of enzyme used was such that the hydrolysis rate of the substrate was 40 to 50% in the reaction for 3 hours.

After the reaction, the recovered trans-3- (4-methoxyphenyl) glycidic acid methyl ester was analyzed by high performance liquid chromatography using Chiralcel OJΦ4.6 × 250 mm manufactured by Daicel Chemical Industries, Ltd. And the optical purity of (2R, 3S) -3- (4-methoxyphenyl) glycidic acid methyl ester was determined, and from these, the E value [Journal of American Chemical Society, Vol. 104, pp. 7294-7299 ( 1982)] And the stereoselectivity of each enzyme with respect to the substrate was judged using this as an index.

<Results> The results are shown in Table 1 below.

<Discussion> From Table 1 above, it can be seen that when the enzyme derived from the microorganisms of the genus Serratia and Corynebacterium of the present invention is used, an extremely high E value is exhibited as compared with the case where each of the control enzymes is used. Recognize.

This means that the enzymes derived from microorganisms of the genus Serratia and Corynebacterium have extremely excellent stereoselectivity in the hydrolysis reaction of trans-3- (4-methoxyphenyl) glycidic acid methyl ester. It is shown.

Experimental Example 2 <Purpose> The stability of the enzyme of the present invention, the control enzyme, and the substrate in each pH region (24 hours after the enzyme and 6 hours after the substrate) were examined.

<Enzyme used> Enzyme of the present invention: enzyme derived from Serratia marcescens ATCC 27117 Control enzyme: lipase OF-360 [from Kyandeida Silindracia, manufactured by each sugar industry] <Experimental method> (a) pH stability of enzyme 600 10 ml of the enzyme solution of the unit was added to 10 ml of 0.2M aqueous buffer solution having the pH shown in Table 2 and left standing at 30 ° C. for 24 hours to obtain an enzyme solution. On the other hand, it contains 2 mM calcium chloride.
3.75 ml of 4M aqueous buffer was added to racemic trans-3- (4-
(Methoxyphenyl) glycidic acid methyl ester (1.5 g) was mixed with 7.5 ml of a toluene solution and preincubated at 30 ° C. for 10 minutes, and an aqueous solution containing 2 ml of the above enzyme solution was added thereto.
75 ml was added, and the mixture was stirred at 30 ° C. and 1400 rpm for 7.5 minutes.
(2S, 3R) -3- (4-methoxyphenyl) in the reaction solution
Glycidic acid methyl ester was measured by high performance liquid chromatography, and the residual enzyme activity was calculated from this.

In the above, one unit is (2S, 3R) -3- (4
-Methoxyphenyl) glycidic acid methyl ester 1μ
It represents the amount of enzyme required to decompose M in 1 minute (hereinafter the same).

(b) Substrate pH stability 7.5 ml of 0.2 M aqueous buffer was added to 0.75 g of racemic trans-3.
Mixed with 7.5 ml of a toluene solution containing methyl (-methoxyphenyl) -glycidate. The mixture was stirred at 30 ° C. and 1400 rpm for 6 hours. Then, by high performance liquid chromatography, the remaining trans-
The ratio (%) of 3- (4-methoxyphenyl) glycidic acid methyl ester was measured.

As an aqueous buffer solution, hydrochloric acid-glycol (glycoco
l) Buffer (pH 2), Mc Ilvain
e) Buffer solution (pH 3 to 7), Tris-HCl buffer solution (pH 8 to
9), and glycine-sodium hydroxide buffer (pH 10
~ 11) was used.

<Results> The results are as shown in Table 2 below.

<Discussion> From the above results, the enzyme of the present invention has the highest residual rate of enzyme activity at pH (8), which has the highest residual rate of the substrate, and is effective in carrying out the asymmetric hydrolysis reaction while avoiding the degradation of the substrate. Conveniently, the control enzyme has a pH that significantly degrades the substrate.
In (6), the enzyme activity is considerably reduced, which is not desirable for efficiently carrying out the asymmetric hydrolysis reaction.

Experimental Example 3 <Purpose> The optimum pHs of the enzyme of the present invention and the control enzyme were examined.

<Enzyme used> Enzyme of the present invention: enzyme derived from Serratia marcescens ATCC 27117 Control enzyme: lipase OF-360 [from Candida Silindracia, manufactured by Meito Sangyo] <Experimental method> 0.4 M aqueous buffer solution containing 2 mM calcium chloride 3.75 ml was mixed with 7.5 ml of a toluene solution containing 1.5 g of racemic trans-3- (4-methoxyphenyl) glycidic acid methyl ester and preincubated at 30 ° C. for 10 minutes. to this
Add 3.75 ml of an aqueous solution containing 50-70 units of enzyme,
After stirring at 1400 rpm for 7.5 minutes at ℃, trans-3- (4-methoxyphenyl) glycidic acid methyl ester was measured by high performance liquid chromatography, and the enzyme activity was determined based on this.

In addition, each buffer used in Experimental Example 2 was used as the aqueous buffer.

<Results> The results are shown in Table 3 below.

(Note): For each enzyme activity in the table, the highest value measured is 100
And expressed as a percentage.

<Discussion> From Table 3 above, the enzyme of the present invention has the most stable substrate at pH 9
It can be seen that the optimum pH for activity expression exists in the vicinity, and asymmetric hydrolysis can be efficiently carried out.

Experimental Example 4 [Half-life of enzyme activity] <Purpose> The half-life of enzyme activity was examined for the enzyme of the present invention and the control enzyme.

<Enzyme used> Enzyme of the present invention: Serratia marcescens ATCC 27117-derived enzyme Control enzyme: Lipase OF-360 [from Kyandeida Silindracia, manufactured by Meito Sangyo] <Experimental method> 0.2M aqueous solution containing 1700 unit enzyme 50 ml of buffer solution and 50 ml of toluene solution were mixed. The half-life of the enzyme activity was calculated by stirring the mixture at 30 ° C. at 1400 rpm and measuring the enzyme activity every 3 days in the same manner as in Experimental Example 3.

<Results> The results are as shown in Table 4 below.

<Discussion> It can be seen from Table 4 above that the enzyme of the present invention has a half-life that is about 3 times longer than that of the control enzyme and that the stability is extremely good.

Example 1 Glucose 0.5%, peptone 1%, meat extract 1%, yeast extract
50 ml of a medium consisting of 1.25% and sodium chloride 0.5% (pH 7.
0) was placed in a 500 ml shake flask and sterilized at 120 ° C for 10 minutes. Serratia marcescens ATCC 271
One platinum loop was inoculated with 17 and cultured with shaking at 30 ° C. for 20 hours. The cells collected from the above culture solution 5.4 by centrifugation were suspended in physiological saline and then collected by centrifugation. The cells were suspended in 10 mM phosphate buffer solution 1.8 (pH 7.5) containing 0.01% cetyltrimethylammonium bromide, and racemic 3- (4-
(2S, 3R) -3-was added to carbon tetrachloride (1.8 g) containing 7.2 g of methoxyphenyl) glycidic acid methyl ester, followed by asymmetric hydrolysis reaction at 30 ° C. for 3 days.
(4-Methoxyphenyl) glycidic acid methyl ester was completely decomposed. The carbon tetrachloride layer was separated and then concentrated under reduced pressure to obtain 3.0 g of (2R, 3S) -3- (4-methoxyphenyl) glycidic acid methyl ester as crude crystals. After adding 10 ml of isopropyl alcohol to 3.0 g of the crude crystals, the mixture was heated and dissolved at 80 ° C. for 20 minutes while stirring.
The mixture was gradually cooled from 80 ° C to 20 ° C over 3 hours, then ice-cooled for 1 hour, and the precipitated crystals were collected by filtration (2R, 3
2.9 g of prismatic crystals of S) -3- (4-methoxyphenyl) glycidic acid methyl ester were obtained.

MP: 87-88 ° C ▲ [α] ²⁰ _D ▼: −207.08 ° (C = 1, methanol) Purity: 100% Elemental analysis value: Theoretical value C: 63.45, H: 5.81, O: 30.74 Measurement value C: 63.44 , H: 5.80, O: 30.76 Infrared absorption spectrum: Fig. 1 Nuclear magnetic resonance spectrum: Fig. 2 Mass spectrum: Fig. 3 Example 2 Bacteria shown in Table 5 below were added to the medium shown in Example 1. The cells were inoculated and cultured at 30 ° C for 20 hours. The bacterial cells collected from 45 ml of the above culture solution by centrifugation were suspended in physiological saline and further collected by centrifugation. 15 ml of 10 mM phosphate buffer containing 0.01% cetyltrimethylammonium bromide (pH 7.5)
The mixture was suspended in 15 ml of carbon tetrachloride containing 60 mg of racemic 3- (4-methoxyphenyl) glycidic acid methyl ester, and subjected to asymmetric hydrolysis reaction at 30 ° C. for 3 days. After the reaction, the carbon tetrachloride layer was separated to obtain (2R, 3S) -3-
A (4-methoxyphenyl) glycidic acid methyl ester-containing reaction solution was obtained. The content of the (2R, 3S) isomer in this reaction solution is as shown in Table 5 below, and the antipode (2S, 3R) isomer was hardly detected in the reaction solution.

The amount of the optically active substance was determined by high performance liquid chromatography using Chiralcel OIΦ 4.6 × 250 mm manufactured by Daicel Chemical Industries, Ltd.

[Brief description of drawings]

Figure 1 is the infrared absorption spectrum of (2R, 3S) -3- (4-methoxyphenyl) glycidic acid methyl ester, Figure 2 is the nuclear magnetic resonance spectrum of the same compound, and Figure 3 is the mass spectrometry of the same compound. It is a spectrum.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12P 41/00 C12R 1: 425)

Claims (4)

[Claims] 1. A general formula [I] (However, ring A is a phenyl group which may have a substituent,
R represents a lower alkyl group. ) To racemic trans-3-phenylglycidic acid ester represented by the following, a culture solution of a microorganism belonging to the genus Corynebacterium or the genus Serratia having the ability to asymmetrically hydrolyze an ester bond, a microbial cell or a treated product of the microbial cell, or Of an optically active trans-3-phenylglycidic acid ester compound, characterized in that an enantiomer is separated and collected from the reaction solution after hydrolyzing one of the optically active substances by causing an enzyme derived from the microorganism to act. Manufacturing method. 2. The configuration of the antipodes to be separated and collected is (2R,
The method according to claim 1, which is 3S). 3. The production method according to claim 1, wherein the enzyme is esterase or ribase. 4. The method according to claim 1, wherein the microorganism is a microorganism belonging to the genus Serratia.