KR20030082730A - Method of optical resolution by enzyme using solvent-free two-phase system - Google Patents

Abstract

PURPOSE: A method of optical resolution by an enzyme using a solvent-free two-phase system is provided, thereby cheaply carrying out the optical resolution without causing environment pollution. CONSTITUTION: A method of optical resolution by an enzyme comprises the steps of: forming a solvent-free two-phase system consisting a racemic ester layer as a reaction substrate and a water layer; adding an enzyme to the system to optic selectively hydrolyze the racemic ester; and separating the hydrolysate from the system during the hydrolysis reaction, wherein the racemic ester is ketoprofen ester, ibuprofen ester, or naproxen ester; the enzyme is lipase, esterase, or protease having the optical selectivity to racemic ester; the separation is carried out by evaporation of the hydrolysate or solubilization of hydrolysate in other system; and the hydrolysis temperature is 30 to 100 deg. C.

Description

Method of optical resolution by enzyme using solvent-free two-phase system

The present invention relates to an optical splitting method for separating racemic mixtures, and more particularly, it is possible to separate racemic esters into individual optically pure enantiomers by hydrolysis using enzymes. To optical splitting method.

Most nonsteroidal anti-inflammatory drugs, such as ketoprofen, ibuprofen, sperprofen, and naproxen, have pharmacological activity only in ( S ) -enantiomer and need to be separated purely.

In addition, since the use of ( R ) -enantiomers is increasing, optical splitting of such racemic drugs appears to be essential (Journal of Molecular Catalysis B: Enzymatic 10, 2000, p 523-529).

The study of the optical splitting of these materials has been directed to the separation of pure enantiomers by hydrolysis of the esterifications made through esterification reactions in solvents (Biotechnology Letters 21, 1999, p143-). 146).

However, in this method, the reaction volume increases due to the organic solvent used to effectively dissolve the ester, and the concentration of the reaction substrate is lowered, so that the low concentration enzyme reaction proceeds. Problems such as causing environmental pollution during the treatment of organic solvents are expected. In particular, this problem will appear to be a bigger problem when the reaction scale is increased for mass production.

As another optical splitting method, there is a method of optical splitting by forming an ideal system with reactants that do not mix with enzymes. In this case, enzymes are used by using various membrane reactors such as chamber tube membranes and polymer membranes. (Journal of Membrane Science 125, 1997, p177-187).

Application examples of the solvent-free system are the production of glycerides or wax esters using fatty acids and glycerol, and the application to optical splitting is still under way.

An object of the present invention for solving the above problems is to separate the optically active material into each enantiomer in a simpler manner by using a solvent-free ideal system composed only of a reaction substrate and an enzyme without a solvent.

Figure 1a is a schematic diagram showing a reaction-free solvent-free ideal optical splitting system,

Figure 1b is a schematic diagram showing a solvent-free ideal optical splitting system after the reaction proceeds,

2 is a diagram showing the hydrolysis reaction conversion rate of ketoprofen decyl ester over time.

In order to achieve the above object, the present invention provides a solvent-free two-phase system consisting of a racemic ester layer and a water layer as a reaction substrate in a method for optically dividing a racemic ester using an enzyme. Forming; Adding an enzyme to the system to optically hydrolyze the racemic ester; And simultaneously causing the hydrolysis product to be separated in the system in which the reaction takes place at the same time as the hydrolysis reaction.

In this case, the racemic ester is not particularly limited as long as the ester has optical activity, but according to a preferred embodiment of the present invention, the racemic ester may be a nonsteroidal anti-inflammatory analgesic agent, and more particularly, ketoprofen ester, ibuprofen ester, and soup. Lofen ester, naproxen ester and the like. The enzyme having optical selectivity is not particularly limited as long as it is an enzyme that exhibits optical selectivity with respect to the reactant, and may be a lipase, an esterase, a protease, or the like.

More preferably, the enzyme is an enzyme in immobilized form with hydrophobicity. Through this configuration, the enzyme stays in the ester layer, thereby increasing the efficiency of the enzyme.

According to a preferred embodiment of the present invention, the separation step may be achieved by vaporizing or dissolving the hydrolysis product into another system.

At this time, the temperature range of a preferable hydrolysis reaction is 30-100 degreeC, and it is preferable that a water layer contains a base further. Through this configuration, the product carboxylic acid and alcohol can be better separated from the ester layer. At this time, the base is not particularly limited, but is preferably NaHCO ₃ .

Hereinafter, the configuration and operation of the present invention will be described in detail.

In order to obtain an optically pure material, the optical division is performed through a solvent-free ideal system consisting of ester, water, and enzyme, which are reaction substrates without solvent.

By the solvent-free ideal system of the present invention, enzymatic optical cleavage of esters can be performed with high selectivity. Although there is no restriction | limiting in the ester compound which can be optically divided, Ketoprofen ester, ibuprofen ester, a sperpropene ester, a naproxen ester, etc. are preferable. These compounds are nonsteroidal anti-inflammatory analgesics, which can perform optical splitting by optically hydrolyzing these compounds by the solvent-free ideal system.

The process of the invention preferably removes the product from the system in which the reaction takes place in order to promote the hydrolysis reaction of the compounds and to obtain higher reaction conversion. Since most enzymatic reactions are reversible reactions, removal of the product can increase conversion and reaction rates because the chemical equilibrium favors the desired reaction.

In the case of an ester compound, when a hydrolysis reaction occurs, carboxylic acid and alcohol are produced. Carboxylic acids in these products can be removed by transfer to another system using a base, and alcohols can be removed by vaporizing or dissolving. By removing the reaction product in this way, an efficient hydrolysis reaction can be induced.

The optical splitting method of the present invention will be described in more detail by way of example.

In order to explain the method of the present invention, ketopropene ester is used as the ester to be optically divided. Ketoprofen ester can be easily obtained by the method as described in well-known literature.

In addition, for the explanation of the method of the present invention, by using the immobilizing enzyme Novozym 435 ( Candida antarctica type B lipase) as a hydrolase, the enzyme is naturally maintained in the ester layer without any special device as shown in FIGS. 1A and 1B. To make it possible. Novozym 435 as used herein is a lipolytic enzyme immobilized in a hydrophobic acrylic resin, available from Novo Industries (Denmark).

First, the ketopropene ester layer and the water layer are formed, and when Novozym 435 is added thereto, hydrolysis reaction occurs in the ester layer, and as the reaction proceeds as shown in FIGS. 1A and 1B, the selectivity to Novozym 435 is increased. The ( R ) -ketoprofen ester represented is dissolved into the water layer as it becomes ( R ) -ketoprofen through a hydrolysis reaction.

As described above, the system in which the reaction takes place in order to obtain a high reaction conversion, that is, the carboxylic acid which is the reaction product in the ester layer in FIG. 1A, is separated into a water layer using a base to induce an efficient hydrolysis reaction. That is, NaHCO ₃ which is weakly basic in water is added to dissolve the reaction product ketopropene (2- (3-benzolphenyl) propionic acid).

Therefore, as the reaction proceeds, as shown in FIG. 1B, ( R ) -ketoprofen is present in the water layer, and ( S ) -ketoprofen decyl ester is present in the ester layer. The (S) - When using enzymes in the selectivity to ketoprofen in contrast (S) in the case of the above-ketoprofen is present in water, the upper level, the (R) - only the ketoprofen ester is left .

In addition, depending on the characteristics of the alcohol constituting the ester, the alcohol, another product produced by the hydrolysis reaction may further exhibit this reaction promoting effect by partially vaporizing at the reaction temperature or recording in water.

In this way, racemic ketoprofen esters can be more easily separated into ketoprofen in optically pure ( R ) and ( S ) forms.

With respect to the optical splitting by this method, ibuprofen esters, sperpropene esters, naproxen esters, etc. belonging to the arsenic salt analgesics showed similar reactivity.

This enzymatic reaction process can be represented by the following scheme 1.

(In the formula, Ar (Ketoprofen), (Ibuprofen), (Spropene), or (Naproxen), R ₁ represents substituted or unsubstituted alkyl or aryl)

By attempting the optical splitting reaction of the solventless system of the present invention, various advantages can be obtained unlike the conventional method.

First, high productivity can be expected by enabling high concentration reaction, and second, since no solvent including organic solvent is used, the reaction volume can be minimized and the apparatus and design costs involved in the reaction can be reduced. Third, since the system is composed of only the reaction substrate, it is easy to separate and recover the product. Fourth, an environmentally friendly reaction system can be configured because no organic solvent is harmful to the environment.

Therefore, the new solvent-free ideal system developed to obtain optically pure material is easy to configure and economical, and also easy to separate products. Therefore, it can be easily applied to the enzymatic reaction of various materials including nonsteroidal anti-inflammatory analgesics. .

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1

Optical splitting of ketoprofen decyl ester

A solvent-free biphasic system was constructed with only 2 ml (4.9 mmol) of racemic ketoprofen decyl ester and 25 ml of solution containing the weak base 1N NaHCO ₃ .

The reaction was started by adding 0.3 g of the hydrophobic immobilizing enzyme Novozym 435. The reaction was carried out in a thermostat reactor maintained at 70 ^° C., 150 rpm.

Over time, samples were taken from the water and ester layers, respectively, and dissolved in water and acetonitrile to investigate the reactivity of the ketoprofen decyl ester with hydrolysis.

The results are shown in FIG. As shown in FIG. 2, the reaction proceeded relatively rapidly within 24 hours after the reaction.

Example 2

The hydrolysis reaction was carried out in the same manner as in Example 1 except that 1N NaHCO ₃ was not added.

Example 3

The hydrolysis reaction was carried out in the same manner as in Example 1 except that 1N Na ₂ CO ₃ was used.

Example 4

The hydrolysis reaction was carried out in the same manner as in Example 1 except that 1N NaOH was used.

The conversion rates of the hydrolysis reactions performed in Examples 1 to 4 above were as shown in Table 1 below.

division Example 2 Example 1 Example 3 Example 4 Water layer pH 6.8 8.7 11.2 13. Reaction conversion rate, C 0.08 0.23 0.15 0.09

Compared with the result of Example 2 using pure distilled water, it was found that the reaction conversion was improved by about 3 times in Example 1 when NaHCO ₃ , which was about base, was added. When Na ₂ CO ₃ was added, the degree of reaction conversion could be increased to a certain degree.

Example 5-7

Hydrolysis was carried out in the same manner as in Example 1 except that the reaction temperature was set to 30 ° C (Example 5), 50 ° C (Example 6), and 90 ° C (Example 7).

The reaction conversion rate and optical selectivity of the hydrolysis reaction in Example 5-7 were as shown in Table 2 below.

division 30 ° C. (Example 5) 50 ° C. (Example 6) 70 ° C. (Example 1) 90 ° C. (Example 7) Reaction Conversion Rate, C 0.07 0.18 0.23 0.16 Optical selectivity, E 6.0 5.0 5.3 4.8

Example 8-10

Ketoprofen butyl ester (Example 8), ketoprofen hexyl ester (Example 9), and ketoprofen octyl ester (Example 10) were also used in the same manner as in Example 1 using a solvent-free ideal system. It was decomposed and optical division was performed.

The reaction conversion rate and optical selectivity of the hydrolysis reaction in Example 8-10 were as shown in Table 3 below.

division Ketoprofenbutyl ester (Example 8) Ketoprofenhexyl ester (Example 9) Ketoprofenoctyl ester (Example 10) Ketopropenedecyl ester (Example 1) Reaction conversion rate, C 0.50 0.32 0.23 0.23 Optical selectivity, E 7.2 6.4 5.1 5.3

As shown in Table 3, the shorter the length of the alcohol constituting the ketoprofen ester showed a tendency to show a higher reaction conversion rate. Ketoprofen octyl and decyl esters, consisting of 8 and 10 carbons, show the same reaction conversion rates. The reason why the reaction conversion rate is high in a short alcohol substance such as ketoprofen butyl ester is that the reaction substrate is able to bind well with the active site of the enzyme, and the boiling point of the reaction product alcohol is relatively low so that it partially evaporates at the reaction temperature. It can also be dissolved in water, so that the reaction with the reaction product ketoprofen can be removed from the ester layer where the reaction takes place, leading to a reversible reaction (esterification, hydrolysis reaction) towards the hydrolysis reaction. It was seen because there was.

Example 11-13

Ibuprofen butyl ester (Example 11), sperpropen butyl ester (Example 12), and naproxen butyl ester (Example 13) belonging to the arsenic salt analgesic series, using the solvent-free ideal system in the same manner as in Example 1 Hydrolysis was performed to perform optical cleavage.

The reaction conversion rate and optical selectivity of the hydrolysis reaction in Examples 11-13 were as Table 4 below.

division Ibuprofenbutyl ester (Example 11) Spropenebutyl Ester (Example 12) Naproxenbutyl Ester (Example 13) Reaction Conversion Rate, C 0.47 0.23 0.37 Optical selectivity, E 6.8 5.6 6.2

Ibuprofen butyl esters exhibited similar reaction conversions to ketoprofen butyl esters, while sperprofen butyl esters and natroxene butyl esters showed relatively low reactivity. This was seen as a property of the reactants.

The results showed that the reaction conversion rate and the optical selectivity are very superior to the optical splitting method using the conventional organic solvent. As an example using the conventional organic solvent, ibuprofen ethyl ester was enzymatic optical splitting in the presence of methanol. (Monatschefte fur Chemie 131, 2000, 633-638) According to this report, the enzyme similar to the enzyme (Novzym 435) used in the embodiment of the present invention, ie lipase produced by the same Candida genus microorganism When ibuprofen ethyl ester was subjected to enzymatic optical split using (CAL-B) in the presence of methanol as an organic solvent, the reaction conversion rate was 0.33 and the optical selectivity was 2 or less. It can be seen that it is excellent.

As described above, the solvent-free ideal system is a relatively simple optical splitting system consisting only of a reaction substrate and an enzyme, and is expected to be widely applied to the production of various optical medicines including nonsteroidal anti-inflammatory drugs using enzymes. In addition, it is considered to be of great industrial value because it can be widely applied to hydrolysis reaction of various ester materials using various enzymes.

Claims (7)

In the method of optically separating the racemic ester using an enzyme, Forming a solvent-free two-phase system consisting of a racemic ester layer and a water layer as a reaction substrate; Adding an enzyme to the system to optically hydrolyze the racemic ester; And And simultaneously causing the hydrolysis product to be separated in the system in which the reaction takes place, simultaneously with the hydrolysis reaction. The method of claim 1, wherein the racemic ester is ketoprofen ester, ibuprofen ester, sperpropene ester, or naproxen ester. The method of claim 1, wherein the enzyme is a lipase, esterase, or protease that exhibits optical selectivity to the racemic ester. The optical splitting method of racemic ester according to claim 1, wherein the enzyme is an immobilized form of enzyme having hydrophobicity. The optical separation method of racemic ester according to claim 1, wherein the separation step is performed by vaporizing or dissolving the hydrolysis product into another system. The optical splitting method of racemic ester according to claim 5, wherein the temperature range of the hydrolysis reaction is 30 to 100 ° C. 6. The method of optical separation of racemic esters according to claim 5, wherein the water layer further comprises a base.