JP2018154559A - Method for isolating r-isomer of 3 hydroxy butyric acid ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for isolating R-isomer of 3-hydroxy butyric acid ester in order to enhance the optical purity of the R-isomer of 3-hydroxy butyric acid ester.SOLUTION: There is provided a method for isolating R-isomer of 3-hydroxy butyric acid ester including: an acylation step A of chemically acylating the hydroxy group at the 3-position of the 3-hydroxy butyric acid ester containing R-isomer and S-isomer; a deacylation step B of deacylating an acylated product of the R-isomer of 3-hydroxy butyric acid ester with lipase to produce the R-isomer of 3-hydroxy butyric acid ester; and a distillation step C of isolating the R-isomer of 3-hydroxy butyric acid ester from an acylated product of the S-isomer of 3-hydroxy butyric acid ester by distillation.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for isolating a 3-hydroxybutyric acid ester of R form in order to improve the optical purity of the R-form of 3-hydroxybutyric acid ester.

  Since 3-hydroxybutyric acid and its salt (hereinafter referred to as 3HB, also referred to as 3HB, especially its monomer) is a substance that is originally present in the human body, it has high biocompatibility and is a sugar. Expected to be a groundbreaking energy source instead of quality. In addition, it has been found that 3HB not only serves as a mere energy source but also acts as a signal transmitter that affects the expression of various genes and the activity of proteins. 3HB is known to improve cognitive function and long-lasting memory by inhibiting histone deacetylase, for example, by regulating gene expression, and its effectiveness in preventing Alzheimer's has been confirmed. For example, 3HB produced by the intake of medium-chain fatty acids contained in coconut oil and metabolism in the body has the effect of improving the symptoms of Alzheimer's disease and diabetic patients who cannot use carbohydrates in the brain and body. It has been known. In addition, 3HB is converted to energy more rapidly than carbohydrates in the body and has the effect of suppressing the absorption of fat and sugar into cells, so it can be applied to energy substances for athletes, diet and health foods. Can be expected.

  3HB is obtained, for example, by performing a fermentation process in which 3HB-producing halomonas bacteria are added, separating and removing halomonas bacteria from the obtained fermentation broth, and purifying. The fermentation process can be performed as a process in which 3HB-producing halomonas bacteria are added as they are to a raw material liquid containing a saccharide nutrient source such as fruit juice, and aerobic fermentation and anaerobic fermentation are sequentially performed (Patent Document 1). . As a result, the sugar is converted to 3HB and produced in the fermentation broth.

  3HB is known to be usable as a raw material for biodegradable resins and 3HB esters (for example, Ethyl 3-Hydroxybutyrate (EHB)), and is also useful in industrial applications. It is growing. 3HB ester is a compound that can be used as a highly safe solvent and detergent, and is also being applied as a pharmaceutical, agricultural chemical, food additive, and the like.

Japanese Unexamined Patent Publication No. 2013-081403

3HB has R and S optical isomers, and 3HB obtained by a fermentation process using microorganisms is mainly in R form. However, there was a limit in improving the optical purity of the R-form by the fermentation process.
Moreover, when 3HB was purified, 3HB was sometimes distilled after being converted to EHB (3HB ester) as an ethyl form. At this time, R and S optical isomers also exist in EHB, and it was required to improve the optical purity of R from the state of EHB.

  Accordingly, an object of the present invention is to provide a method for isolating the R-form 3-hydroxybutyrate ester in order to improve the optical purity of the R-form of 3-hydroxybutyrate ester.

  In order to achieve the above object, the first characteristic configuration of the method for isolating the R-hydroxy 3-hydroxybutyrate according to the present invention is to chemistry the hydroxyl group at the 3-position of the 3-hydroxybutyric ester in which the R-form and S-form are mixed. An acylation step of acylating, a deacylation step of deacylating an acylated product of R-hydroxy 3-hydroxybutyrate by lipase to produce R-hydroxy 3-hydroxybutyrate, and R-hydroxy 3-hydroxybutyrate And a distillation step of separating the ester from the acylated product of S-hydroxy 3-hydroxybutyrate by distillation.

  In this configuration, first, in the 3-hydroxybutyric acid ester in which the R-form and the S-form are mixed, the acylation step in which the hydroxyl group at the 3-position in both the R-form and the S-form 3-hydroxybutyrate is chemically substituted with an acyl group I do. As a result, the acylated product of the R-form 3 hydroxybutyrate ester and the acylated product of the S-form 3 hydroxybutyrate ester are mixed. In this state, a deacylation step is performed in which the acylated product of R-hydroxy 3-hydroxybutyrate is deacylated with lipase to produce R-hydroxy 3-hydroxybutyrate. In the deacylation step, the lipase selectively reacts with the acylated product of R-hydroxy 3-hydroxybutyrate, thereby deacylating the acylated product of R-hydroxy-3-hydroxybutyrate to give R-hydroxy 3-hydroxybutyrate. Can be produced. By the deacylation step, the R-form 3-hydroxybutyric acid ester produced in the deacylation step and the S-form 3-hydroxybutyrate ester produced in the acylation step are mixed. In this state, R-form 3-hydroxybutyrate ester can be isolated by performing a distillation step for separating R-form 3-hydroxybutyrate ester and S-form 3-hydroxybutyrate ester acylated product.

  Therefore, the present invention can provide a method for isolating the R-hydroxy 3-hydroxybutyrate capable of improving the optical purity of the R-hydroxy 3-hydroxybutyrate to an extremely high level (close to 100%).

  The second characteristic configuration of the method for isolating R-hydroxy 3-hydroxybutyrate according to the present invention is that the ester of 3-hydroxybutyrate is an alkyl ester having 1 to 4 carbon atoms.

  In this configuration, the optical purity of the R form of 3HB ester that can be used as a highly safe solvent and cleaning agent, that is, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, propyl 3-hydroxybutyrate, and butyl 3-hydroxybutyrate is extremely high. (Approx. 100%).

  The third characteristic configuration of the method for isolating the R-hydroxy 3-hydroxybutyrate according to the present invention is that the alkyl ester is an ethyl ester.

  In this configuration, the optical purity of the R-form of ethyl 3-hydroxybutyrate can be improved to an extremely high level (close to 100%).

  The fourth characteristic configuration of the method for isolating R-hydroxy 3-hydroxybutyrate according to the present invention is that the deacylation step is performed in the presence of alcohol.

  According to this configuration, it is possible to prevent hydrolysis of the ethyl ester of the acylated product of S-form ethyl 3-hydroxybutyrate in the deacylation step, for example.

  The fifth feature of the method for isolating the R-hydroxy 3-hydroxybutyrate according to the present invention is that acylation of the R-hydroxy 3-hydroxybutyrate by lipase is performed among the 3-hydroxybutyric acid ester in which the R-form and S-form are mixed. An acylation step, an R-form 3-hydroxybutyrate ester acylate and an S-form 3-hydroxybutyrate ester are separated by distillation, and the R-form 3-hydroxybutyrate acylate is chemically separated. And a deacylation step for deacylation.

  In this configuration, first, in the 3-hydroxybutyric acid ester in which the R-form and the S-form are mixed, the R-form 3-hydroxybutyrate is subjected to the reaction, and the hydroxyl group at the 3-position of the R-form 3-hydroxybutyrate is acyl group. An acylation step for substituting is performed. In the acylation step, the lipase selectively reacts with the 3-hydroxybutyric acid ester in the R form, whereby the hydroxyl group at the 3-position of the 3-hydroxybutyric acid ester in the R form can be substituted with an acyl group. As a result, an acylated product of R-hydroxy 3-hydroxybutyrate and S-hydroxy 3-hydroxybutyrate are mixed. In this state, the acylated product of the R-form 3 hydroxybutyrate ester and the S-form 3 hydroxybutyrate ester can be isolated by distillation to isolate the R-form 3-hydroxybutyrate ester acylated product. it can. An R-form 3-hydroxybutyrate ester can be prepared by performing a deacylation step in which the acylated product of the R-form 3-hydroxybutyrate separated by the distillation step is chemically deacylated.

  Therefore, the present invention can provide a method for isolating the R-hydroxy 3-hydroxybutyrate capable of improving the optical purity of the R-hydroxy 3-hydroxybutyrate to an extremely high level (close to 100%).

1 is a schematic view showing a method for isolating R-form ethyl 3-hydroxybutyrate of the present invention. FIG. It is the schematic which shows the method of isolating the R 3 form ethyl 3-hydroxybutyrate of another embodiment. It is the graph which showed the analysis result by the gas chromatograph of R-EHB which is a standard product. It is the graph which showed the analysis result by the gas chromatograph of S-EHB which is a standard product. It is the graph which showed the result of the analysis by the gas chromatograph of the reaction material after acetylation (R-EHB). It is the graph which showed the result of the analysis by the gas chromatograph of the reaction material after acetylation (S-EHB). It is the graph which showed the result of the analysis by the gas chromatograph of the reaction material after a lipase process (R-EHB). It is the graph which showed the result of the analysis by the gas chromatograph of the reaction material after a lipase process (S-EHB).

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Preferred embodiments are described below, but these embodiments are described in order to more specifically illustrate the present invention, and various modifications can be made without departing from the spirit of the present invention. The present invention is not limited to the following description.

  The present invention is a method for isolating a 3-hydroxybutyric acid ester of R form from a 3-hydroxybutyric acid ester in which R form and S form are mixed. The ester of 3-hydroxybutyric acid ester can be an alkyl ester having 1 to 4 carbon atoms. That is, in the case of an alkyl ester having 1 carbon, methyl 3-hydroxybutyrate is obtained. In the case of an alkyl ester having 2 carbons, ethyl 3-hydroxybutyrate is obtained. In the case of an alkyl ester having 3 carbons, propyl 3-hydroxybutyrate is obtained. In the case of this alkyl ester, butyl 3-hydroxybutyrate is obtained.

  In the present embodiment, a case where ethyl 3-hydroxybutyrate (EHB) is used as the 3-hydroxybutyric acid ester will be described.

  FIG. 1 shows an outline of the present invention. In the figure, R-EHB represents R-form of ethyl 3-hydroxybutyrate, and S-EHB represents S-form of ethyl 3-hydroxybutyrate.

  The method includes an acylation step A in which the hydroxyl group at the 3-position of ethyl 3-hydroxybutyrate in which R-form and S-form are mixed, and acylation of R-form ethyl 3-hydroxybutyrate by lipase is deacylated. And a deacylation step B for producing R-form ethyl 3-hydroxybutyrate, and a distillation step C for separating R-form ethyl 3-hydroxybutyrate from an acylated product of S-form ethyl 3-hydroxybutyrate by distillation.

(Acylation step A)
In the acylation step A, acylation is performed by chemically substituting the hydroxyl group (—OH) at the 3-position in the R-form and S-form of ethyl 3-hydroxybutyrate with an acyl group (RCO—). As used herein, “chemically acylating” refers to acylating with a reagent such as an acylating agent without using an enzyme. In the present embodiment, the case where acetic anhydride as an acylating agent and triethylamine as a base are used as reagents for acylation will be described, but the present invention is not limited thereto.

For example, acetic anhydride is added to R-EHB or S-EHB in an amount of about 1-3 equivalents, preferably 2 equivalents, and triethylamine is added in an amount of about 0.05-0.5 equivalents. What is necessary is just to add so that it may become 0.1-0.2 equivalent preferably.
In the acylation step A, the reaction is preferably carried out at 40 to 60 ° C., preferably 55 ° C., and the reaction time is preferably about 5 to 18 hours.

  By performing the acylation step, an R-form ethyl 3-hydroxybutyrate acylate and an S-form ethyl 3-hydroxybutyrate acylate are mixed.

(Deacylation step B)
In the deacylation step B, R-form ethyl 3-hydroxybutyrate is deacylated with lipase to produce R-form ethyl 3-hydroxybutyrate. The deacylation step is preferably performed in the presence of an alcohol. Alcohols that can be used in this step include, but are not limited to, methanol, ethanol, propanol and butanol. In this embodiment, a case where ethanol is used will be described.

  Moreover, the lipase which can be used at this process can use the lipase of microorganisms, an animal, and a plant origin. For example, lipases of microbial origin may utilize lipases that can be isolated from eukaryotic organisms such as fungi or yeast or prokaryotic organisms such as bacteria. Specifically, bacterial lipases derived from the genus Bacillus or Pseudomonas, lipases derived from fungi such as Aspergillus, or lipases derived from yeasts such as Candida or Yerrowia. Particularly preferred.

  Some lipases are commercially available, such as lipase PS Amano SD, lipase AS Amano, lipase AYS Amano, lipase AK Amano (manufactured by Amano Enzyme), or Novozyme 435 (manufactured by Novozyme). However, it is not limited to these. The lipase may be in a liquid form or a solid form.

  In this step, the above lipase selectively reacts with an acylated product of R-form ethyl 3-hydroxybutyrate, thereby deacylating the acylated product of R-form ethyl 3-hydroxybutyrate to obtain R-form ethyl 3-hydroxybutyrate. Can be produced.

  In the deacylation step B, the reaction is preferably carried out at 20 to 40 ° C., preferably 30 ° C., and the reaction time is preferably about 1 to 18 hours.

  In addition, by using ethanol in this step, hydrolysis of the ethyl ester of the acylated product of S-form ethyl 3-hydroxybutyrate can be prevented. In addition, this step is generally performed under atmospheric pressure and appropriately under an inert gas such as nitrogen or argon, but is not limited to these conditions.

  By performing the deacylation step B, the R-form 3-hydroxybutyrate prepared in the deacylation step B and the S-form ethyl 3-hydroxybutyrate prepared in the acylation step A coexist. It becomes a state.

(Distillation process C)
In the distillation step C, the R-form ethyl 3-hydroxybutyrate prepared in the deacylation step B and the S-form ethyl 3-hydroxybutyrate prepared in the acylation step A are separated by distillation.

  By this step, R-form ethyl 3-hydroxybutyrate can be isolated by separating reaction product lipase, acetic anhydride, triethylamine, ethanol, and ethyl acetate in addition to acylated product of S-form ethyl 3-hydroxybutyrate. .

  The distillation in this step is not particularly limited as long as it is a distillation under normal conditions that can separate at least R-form ethyl 3-hydroxybutyrate and S-form ethyl 3-hydroxybutyrate. Specifically, since the boiling point of the R-form ethyl 3-hydroxybutyrate is different from the boiling point of the acylated product and reaction residue of the S-form ethyl 3-hydroxybutyrate, these can be easily separated by known distillation. Can do.

  As described above, the optical purity of the R form of ethyl 3-hydroxybutyrate (EHB) can be improved to an extremely high level (close to 100%) by performing acylation step A, deacylation step B, and distillation step C. A method for isolating R-form ethyl 3-hydroxybutyrate can be provided.

[Another embodiment]
The method for isolating R-form ethyl 3-hydroxybutyrate from R-form and S-form ethyl 3-hydroxybutyrate is not limited to the method described above, and can also be performed by the following method.

  That is, another method of the above-described method is an acylation step a in which R-form ethyl 3-hydroxybutyrate is acylated with lipase among 3-hydroxybutyrate in which R-form and S-form are mixed, and R-form 3 Distillation step b for separating ethyl acylbutyrate and S-form ethyl 3-hydroxybutyrate by distillation, and deacylation step c for chemically deacylating the separated R-form ethyl 3-hydroxybutyrate acylate (FIG. 2). Moreover, in this separate implementation method, after the deacylation step c, the distillation step d may be performed again to purify the R-form ethyl 3-hydroxybutyrate.

  In this embodiment as well, the R-form 3-hydroxybutyric acid ester can be an alkyl ester having 1 to 4 carbon atoms.

(Acylation step a)
In this separate implementation method, first, acylation step a is performed in which ethyl 3-hydroxybutyrate in which R-form and S-form are mixed is acylated with R-form ethyl 3-hydroxybutyrate by lipase.

  In the acylation step A described above, the hydroxyl group at the 3-position in the R-form and S-form ethyl 3-hydroxybutyrate is chemically substituted with an acyl group, whereas the acylation step a is performed in the R-form 3-hydroxybutyrate. The difference is that ethyl is the target of the reaction and the hydroxyl group at the 3-position of ethyl 3-hydroxybutyrate in the R form is substituted with an acyl group.

  As the lipase used in this step, the same lipase as the lipase used in the deacylation step B described above can be used. The lipase selectively reacts with R-form ethyl 3-hydroxybutyrate, whereby the hydroxyl group at the 3-position of R-form ethyl 3-hydroxybutyrate can be substituted with an acyl group. In this step, acetic acid is used together with lipase, but is not limited thereto.

  By performing the acylation step a, an acylated product of R-form ethyl 3-hydroxybutyrate and S-form ethyl 3-hydroxybutyrate are mixed.

(Distillation step b)
In the distillation step b, the R-form ethyl 3-hydroxybutyrate and the S-form ethyl 3-hydroxybutyrate prepared in the acylation step a are separated by distillation. According to this step, in addition to S-form ethyl 3-hydroxybutyrate, the reaction residue lipase and acetic acid can be separated to isolate the R-form ethyl 3-hydroxybutyrate acylate.

  The distillation in this step is not particularly limited as long as it is a distillation under normal conditions capable of separating at least an acylated product of R-form ethyl 3-hydroxybutyrate and S-form ethyl 3-hydroxybutyrate. Specifically, since the boiling point of the acylated product of R-form ethyl 3-hydroxybutyrate is different from the boiling point of S-form ethyl 3-hydroxybutyrate and reaction residue, these can be easily separated by known distillation. Can do.

(Deacylation step c)
In the deacylation step c, the acylated product of R-form 3-hydroxybutyrate separated in the distillation step b is chemically deacylated. In the present specification, “chemically deacylate” means deacylation with a reagent such as a deacylating agent without using an enzyme. In this step, by adding alcohol, the R-form ethyl 3-hydroxybutyrate can be chemically deacylated to produce R-form ethyl 3-hydroxybutyrate. As the alcohol, methanol, ethanol, propanol, butanol and the like can be used, but the alcohol is not limited thereto.

  As described above, the optical purity of the R-form of ethyl 3-hydroxybutyrate (EHB) can be improved to an extremely high level (close to 100%) by performing acylation step a, distillation step b, and deacylation step c. A method for isolating R-form ethyl 3-hydroxybutyrate can be provided.

(Distillation step d)
By performing the distillation step d after the deacylation step c, the R-form ethyl 3-hydroxybutyrate is purified. In this distillation step d, the reaction residue ethanol, acetic acid, and ethyl acetate can be separated to purify R-form ethyl 3-hydroxybutyrate.

  The distillation in this step is not particularly limited as long as the R-form ethyl 3-hydroxybutyrate and the reaction residue can be separated under normal conditions. Specifically, since the boiling point of the R-form ethyl 3-hydroxybutyrate and the boiling point of the reaction residue are different, they can be easily separated by known distillation.

  An acylation step A for chemically acylating the hydroxyl group at the 3-position of ethyl 3-hydroxybutyrate in which R-form and S-form are mixed, and acylation of R-form ethyl 3-hydroxybutyrate by lipase are deacylated below. A deacylation step B for producing R-form ethyl 3-hydroxybutyrate, and a distillation step C for separating R-form ethyl 3-hydroxybutyrate from an acylated form of S-form ethyl 3-hydroxybutyrate by distillation. Examples of methods for isolating R-form ethyl 3-hydroxybutyrate will be described.

  R-EHB (manufactured by Sigma-Aldrich), S-EHB (manufactured by Sigma-Aldrich) as a standard product, acetic anhydride (manufactured by Wako Pure Chemical Industries) of acetylating agent (acylating agent), and triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) as a base Were prepared in a vial with the reaction composition shown in Table 1 below, and reacted at 55 ° C. overnight (acylation step A). About 2 equivalents of acetic anhydride was added to and reacted with R-EHB or S-EHB, and the solution before and after the reaction was analyzed with a gas chromatograph, and further structural analysis was performed by NMR. NMR was performed by a known method.

Analysis conditions by gas chromatograph were as follows.
Packed column: Reoplex 400 2% Chromosorb G 60-80 Aw G-1130
Injection temperature: 180 ° C
Column temperature: 160 ° C
Detector FID temperature: 200 ° C
Carrier gas: N2
Injection volume: 1 μL

  As a result of analysis by gas chromatograph, the retention time (Rt) of the standard R-EHB was 2.863 (FIG. 3), and the retention time of S-EHB was 2.865 (FIG. 4). It was.

  As a result of gas chromatographic analysis of the reaction product after acetylation, in R-EHB, the peak at Rt 2.863 disappeared and a peak was confirmed at Rt 3.475 (FIG. 5). In S-EHB, the peak at Rt 2.865 disappeared and a peak was confirmed at Rt 3.476 (FIG. 6). Structural analysis of these reactants by NMR revealed that the hydroxyl group at the 3-position was acetylated (results not shown).

  150 mg of lipase PS Amano SD (manufactured by Amano Enzyme Co., Ltd.) is added to each of the acetylated reactants of R-EHB and S-EHB in the solid state, and the ethyl group does not deethylate. Ethanol (1.4 mL) was added to each and reacted at 30 ° C. overnight (deacylation step B).

  Analysis conditions by gas chromatography were the same as described above. As a pretreatment for analysis, in order to remove the solid lipase that did not dissolve, centrifugation (15000 rpm × 5 minutes) was performed, 1 mL of the supernatant was collected, 1 mL of chloroform was added, and filtration (0. 45 μm). The solution was analyzed by gas chromatography.

As a result of analysis by gas chromatograph, in the solution after the lipase reaction of acetylated-R-EHB, Rt. A peak of 3.475 (corresponding to Rt.3.663 in FIG. 7) remained, but Rt. 2.999 peaks were detected. As a result of NMR, this peak was found to be R-EHB (conversion rate: 39.5%) in which acetylated-R-EHB was deacetylated (results not shown).
On the other hand, in the solution after the lipase reaction of acetylated-S-EHB, Rt. A peak of 3.476 (corresponding to Rt.3.668 in FIG. 8) remained, and no new peak was detected.
As a result, it was recognized that R-EHB in the R form was deacetylated by lipase.

  In ethyl 3-hydroxybutyrate in which R-form and S-form are mixed, it is considered that the reaction product after performing the acylation step A and deacylation step B shows the following composition.

  That is, by subjecting the composition shown in Table 2 to simple distillation (EHB was isolated under conditions of a bath temperature of 100 ° C. and a pressure of 10 mmHg or less, and the tower top temperature was 60 to 65 ° C.) (distillation step C). In Table 2, reaction residues such as EHB (R-form ethyl 3-hydroxybutyrate) and Ethyl-3-Acetoxybutyrate (S-form ethyl 3-hydroxybutyrate) and acetic anhydride can be separated. The body's ethyl 3-hydroxybutyrate could be isolated.

  INDUSTRIAL APPLICABILITY The present invention can be used in a method for isolating an R-form 3-hydroxybutyrate in order to improve the optical purity of the R-form of 3-hydroxybutyrate.

A Acylation step B Deacylation step C Distillation step

Claims (5)

An acylation step of chemically acylating the hydroxyl group at the 3-position of a 3-hydroxybutyric acid ester in which R-form and S-form are mixed;
A deacylation step of deacylating an acylated product of R-hydroxy 3-hydroxybutyrate by lipase to produce R-hydroxy 3-hydroxybutyrate;
And a distillation step of separating the R-form 3-hydroxybutyrate ester from the acylated product of the S-form 3-hydroxybutyrate by distillation.   The method for isolating an R-form 3-hydroxybutyrate ester according to claim 1, wherein the ester of the 3-hydroxybutyrate ester is an alkyl ester having 1 to 4 carbon atoms.   The method for isolating an R-form 3-hydroxybutyrate according to claim 2, wherein the alkyl ester is an ethyl ester.   The method for isolating the 3-hydroxybutyric acid ester of R form according to claim 3, wherein the deacylation step is carried out in the presence of an alcohol. Of the 3-hydroxybutyric acid ester in which R-form and S-form are mixed, an acylation step of acylating R-form 3-hydroxybutyric acid ester with lipase;
A distillation step of separating the acylated product of R-form 3-hydroxybutyrate and S-form 3-hydroxybutyrate by distillation;
A method for isolating the R-hydroxy 3-hydroxybutyrate ester, which comprises chemically deacylating the acylated product of the R-hydroxy 3-hydroxybutyrate ester that has been separated.

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