JP4565335B2 - Method for producing optically active glycerol acetonide ester - Google Patents

Description

  The present invention relates to a method for producing an optically active glycerol acetonide isobutyric acid ester (also known as 2,2-dimethyl-1,3-dioxolane-4-methanol, hereinafter sometimes abbreviated as GAIB), and more particularly glycerol. A racemic form of acetonide isobutyrate is used as a substrate, a specific natural ester hydrolase is allowed to act in an aqueous solvent, and glycerol acetonide isobutyrate is stereoselectively selected as glycerol acetonide (hereinafter referred to as GA). The present invention relates to a process for producing an optically active glycerol acetonide isobutyric acid ester, which is hydrolyzed (sometimes abbreviated) and recovers the optically active glycerol acetonide isobutyric acid ester remaining from the reaction solution.

  Optically active glycerol acetonide and its ester form are useful compounds as pharmaceutical intermediates. As the production method, a method of synthesizing from 1,2-diol or mannitol is known as a chemical production method. However, it is difficult to say that it is practical because it requires a multi-step process and an optically active substance is required as a starting material.

  For optical resolution by biological production methods, enzymes such as esterification reaction using lipase or lipase AK derived from alkaline genus (Patent Document 2), esterification reaction using lipase MY, etc. (Patent Document 3) are used. Many esterification reactions in organic solvents are known, but it is not possible to obtain ester forms with high optical purity (> 98% ee). In addition, asymmetric hydrolysis of glycerol acetonide benzoate by non-patent coagulant esterase (Non-patent Document 1), asymmetric hydrolysis of glycerol acetonide acetyl ester (Patent Document 4), lipase AK derived from Pseudomonas genus, etc. Although there are known transesterification reactions of racemic glycerol acetonide ester (Non-patent Document 2), there is no knowledge about optical resolution of glycerol acetonide isobutyric acid ester (isobutyric acid ester) in any report .

JP 2004-147646 A JP-A-5-192186 JP-A-2-227097 European Patent Publication No. 1413627 Francisco Molinari et al., Enzyme and Microbial Technology, 1996, 19: p551-556. Eero. Vanttinen et al., Tetrahedron Asymmetry, 1997, Volume 8: p923-933.

  An object of the present invention is to provide an optically active glycerol acetonide isobutyric acid ester having a high optical purity by utilizing an enzyme derived from the natural world which can be easily prepared or obtained at low cost and has excellent stereoselectivity. Another object is to provide a method for industrially efficiently obtaining glycerol acetonide. When using a biological technique, the solvent used in the reaction is generally roughly divided into an organic solvent and water, but it is preferable to use water in consideration of price, safety, disposal and the like. However, since glycerol acetonide is water-soluble, the optically active glycerol acetonide extraction step after the reaction is difficult.

  Although the present inventors hardly dissolve glycerol acetonide isobutyrate in water, it is stereoselective by reacting with a specific ester hydrolase derived from nature shown in the present invention in a reaction system using water as a solvent. It was found that the optically active glycerol acetonide isobutyric ester remaining after hydrolysis was easily recovered, and the present invention was completed.

That is, the present invention
Item 1:
Glycerol acetonide isobutyrate ester racemate is treated with an ester-hydrolyzing enzyme derived from rabbit liver or an ester-hydrolyzing enzyme derived from microorganisms belonging to the genus Serratia, Alkaligenes, Barkholderia, or Enterobacter. Act in,
A method for producing an optically active glycerol acetonide isobutyric acid ester, wherein a racemic form of the compound is stereoselectively hydrolyzed and a remaining optically active glycerol acetonide isobutyric acid ester is recovered;
Item 2:
The ester hydrolase is an enzyme derived from rabbit liver or Serratia marcescens, and the remaining optically active glycerol acetonide isobutyrate is (R) -glycerol acetonide isobutyrate The manufacturing method of claim | item 1,
Item 3:
Ester hydrolase
The method for producing (R) -glycerol acetonide isobutyric acid ester according to Item 2, which is Esterase Rabbit Liver (derived from rabbit, manufactured by SIGMA) or LipaseSM (derived from Serratia Mars essence, manufactured by Tanabe Seiyaku Co., Ltd.),
Item 4:
The ester hydrolase is an enzyme derived from a microorganism belonging to the genus Alkagenes, Barkholderia, or Enterobacter, and the remaining optically active glycerol acetonide isobutyrate is (S) -glycerol acetonide isobutyrate The manufacturing method of claim | item 1 which is these,
Item 5:
Item 5. The (S) -glycerol acetonide according to Item 4, wherein the ester hydrolase is LipaseQLM (derived from the genus Alcaligenes, manufactured by Meishoku Sangyo Co., Ltd.) or LipaseSL (derived from Barkholderia cepacia, manufactured by Meishou Sangyo Co., Ltd.). A method for producing isobutyric acid ester,
Item 6:
The ester hydrolase is an enzyme derived from a microorganism belonging to the genus Enterobacter, and the following (a) amino acid sequence described in SEQ ID NO: 1,
(B) an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1, and has an activity of chlorohydrin and hydroxycarboxylic acid ester asymmetric hydrolase ,
Item 5. A method for producing (S) -glycerol acetonide isobutyric acid ester according to Item 4, which is an enzyme (EnHCH, manufactured by Daiso Corporation) consisting of any amino acid sequence of
Item 7:
Item 7. The production method according to any one of Items 1 to 6, wherein in the step of causing the ester hydrolase to act, the pH of the enzyme is controlled using aqueous ammonia.
About.

  According to the present invention, a glycerol acetonide isobutyric acid ester having high optical purity can be practically efficiently obtained by using an enzyme that can be easily prepared or obtained at low cost. Further, the reaction process is easy and the extraction process can be easily performed with a non-hydrophilic solvent such as hexane.

  The ester hydrolase derived from nature used in the present invention is an ester hydrolase derived from a rabbit liver, or an ester hydrolyzate derived from a microorganism belonging to the genus Serratia, Alkaligenes, Barkholderia, or Enterobacter. An enzyme, which is a glycerol acetonide isobutyric acid racemate, is reacted with the enzyme in an aqueous solvent to hydrolyze the racemate stereoselectively to produce optically active glycerol acetonide and optically active glycerol acetonate. Doisobutyric acid ester can be produced. The optically active glycerol acetonide isobutyric acid ester remaining after the hydrolysis reaction can be easily recovered by extraction with a non-hydrophilic solvent. In addition, R-form or S-form optically active glycerol acetonide isobutyrate can be selectively left by the enzyme to act.

Glycerol acetonide isobutyrate ester racemate is reacted with the enzyme in an aqueous solvent to leave (R) -glycerol acetonide isobutyrate ester, an ester hydrolase derived from rabbit liver, or An enzyme derived from Serratia marcescens,
Specifically, ester hydrolase such as Esterase Rabbit Liver (derived from rabbit, manufactured by SIGMA) or LipaseSM (derived from Serratia Mars essence, manufactured by Tanabe Seiyaku) is exemplified.

An ester hydrolase that causes the enzyme to act on a racemic glycerol acetonide isobutyrate ester in an aqueous solvent to leave (S) -glycerol acetonide isobutyrate ester may be an alkaligenes genus or a bulk formol. It is not limited as long as (S) -glycerol acetonide isobutyric acid ester can remain in an ester hydrolase derived from a microorganism belonging to the genus Delia cepacia, Enterobacter,
Specifically, commercially available enzymes such as LipaseQLM (derived from the genus Alkaligenes, manufactured by Meika Sangyo Co., Ltd.), LipaseSL (derived from Barkholderia cepacia, manufactured by Meishou Sangyo Co., Ltd.), or hydroxycarboxylic acid ester hydrolase EnHCH (Enterobacter) Examples include ester hydrolysis such as genus origin, manufactured by Daiso Corporation.

Among the ester hydrolases, the hydroxycarboxylic acid ester hydrolase EnHCH is an enzyme derived from the genus Enterobacter, and the amino acid sequence shown in the following (a) SEQ ID NO: 1,
(B) an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence has chlorohydrin and hydroxycarboxylate asymmetric hydrolase activity ,
It is an enzyme consisting of any one of the amino acid sequences.

The hydrolase EnHCH is a genetic recombinant (Escherichia coli DH5α (pKK-EnHCH1: this) in which a hydroxycarboxylate ester hydrolase gene derived from the microorganism DS-S-75 (FERM BP-5494) belonging to the genus Enterobacter is incorporated. The strain can be efficiently prepared by using FERM BP-08466 dated September 13, 2002, which is deposited at the National Institute of Advanced Industrial Science and Technology Patent Property Deposit Center).

Racemic glycerol acetonide isobutyric acid ester can be easily synthesized from a commercially available racemic glycerol acetonide and isobutyric acid chloride using a known esterification method. For example, it can be obtained by a method of esterification using a catalyst such as 4-dimethylaminopyridine. The resulting solution containing glycerol acetonide isobutyric acid ester is preferably purified with a silica gel column or the like.

  The naturally occurring ester hydrolase is particularly preferably optically resolved when acting on glycerol acetonide isobutyrate, and other ester forms of glycerol acetonide such as propionate (abbreviated as GAPr), An optically active ester cannot be obtained in a high yield even when it is allowed to act on racemates such as butanoic acid ester (abbreviated as GABu), benzoic acid ester (abbreviated as GABz), and pivalic acid ester (abbreviated as GAPv).

The usage form of the above-mentioned naturally occurring ester hydrolase is not particularly limited, and as long as it has the activity described above, any of the immobilized enzyme, the microbial cell containing the enzyme, the cell culture, or the treated cell product It may be used in the form of

  The amount of the enzyme used in the present invention and the amount of the racemate vary depending on the type of enzyme used, and may be appropriately used.

  In the present invention, glycerol acetonide isobutyric acid ester is reacted with the enzyme in an aqueous solvent. As the aqueous solvent, water or a mixed solvent of water and an organic solvent is used. As the organic solvent, an organic solvent that forms a two-layer system with water, such as butyl acetate, is used. Usually, the enzyme is added to an aqueous solution or buffer containing racemic glycerol acetonide isobutyrate. It is desirable to stir appropriately after the addition.

  In order to perform a stable enzyme reaction, it is preferable to maintain the pH in the reaction solution within a certain range. A suitable pH range is preferably 5 to 9, but it is preferable to bring the pH within the optimum range of the enzyme depending on the enzyme used. In order to control the reaction solution to an optimum pH, it is preferable to titrate with a suitable acid or alkali. Suitable acids to be used are, for example, hydrochloric acid, phosphoric acid and the like. Examples of suitable alkali are sodium hydroxide, aqueous ammonia, calcium carbonate and the like. It is particularly preferable to use ammonia water.

  Moreover, it is preferable to adjust the temperature of the reaction solution within a certain range for the purpose of efficiently exhibiting the activity of the enzyme used. Depending on the enzyme used, the temperature range to be prepared is preferably 20-60 ° C.

  The enzyme reaction time varies depending on the type of enzyme used, the amount of enzyme, the amount of racemic glycerol acetonide isobutyrate, the reaction temperature, and the pH. The optical purity of the remaining glycerol acetonide isobutyrate is 98% e.e. by analysis such as liquid chromatography. e. At that point, the reaction ends. It is necessary to terminate the reaction before the enzyme activity is lost.

  After the enzyme is allowed to act, the reaction solution contains the remaining optically active forms of glycerol acetonide isobutyric acid ester, GA, and isobutyric acid. In order to obtain an optically active glycerol acetonide isobutyric acid ester, the reaction solution can be fractionated using a separation method well known to those skilled in the art, such as a solvent extraction method and a column chromatography method. When using a solvent extraction method, a glycerol acetonide isobutyric acid ester syrup can be easily obtained by adding a non-hydrophilic solvent such as n-hexane or n-heptane to the reaction solution and removing the oil layer solvent under reduced pressure. Obtainable. For further purification, extraction, distillation, various chromatographic operations and the like may be performed.

  The optically active glycerol acetonide isobutyric acid ester thus obtained can be obtained as an optically active GA by hydrolysis using an acid, base or the like by a method known to those skilled in the art. S-form GA is obtained from R-form glycerol acetonide isobutyrate, and R-form GA is obtained from S-form glycerol acetonide isobutyrate.

  Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples, but the present invention is not limited to these examples.

The GA ester, GA quantification, and GA ester optical purity measurement in the reaction solutions in Examples and Comparative Examples were performed as follows.
1) Quantitative determination of GA ester and GA Gas chromatography conditions Column carrier: Silicone OV1701 UniportHP
2% 60/80 mesh (GL Science Co., Ltd.)
Column temperature: 120 ° C
Injection temperature: 240 ° C
Detection: FID: 240 ° C
2) Measurement of optical purity of GA ester Column for high performance liquid chromatography: Chiralcel OB-H 0.46φ × 25 cm (manufactured by Daicel Chemical Industries)
Moving bed: hexane: isopropyl alcohol (100: 1)
Flow rate: 0.5 mL / min Detection: UV 210 nm

(Reference example)
Racemic glycerol acetonide isobutyrate used in the optical resolution reaction was synthesized as follows. 200 ml of methylene chloride, 73.5 g of triethylamine, 1.48 g of 4-dimethylaminopyridine, 80.0 g (0.605 mol) of GA are added to the flask, and isobutanoic acid chloride is added at 0-10 ° C. with stirring. 64.5 g (0.605 mol) was added dropwise. After the reaction for 2 hours, 225 g of ion-exchanged water was added and stirred, and the upper layer was removed. Next, 240 g of 1N hydrochloric acid was added and stirred, the upper layer was removed, 225 g of 5% (w / w) sodium bicarbonate was added and stirred, and the upper layer was removed. Further, 225 g of ion-exchanged water was added and stirred, and the operation of removing the upper layer was performed until there was no unreacted GA in the lower layer, and then the lower layer was concentrated with an evaporator to obtain 122 g of concentrated syrup.

  A glass column tube having a diameter of 6 cm was filled with 500 g of silica gel (1001 manufactured by Daiso), and 200 ml of hexane: ethyl acetate (9: 1) was used as the moving bed. The concentrated syrup was applied to a 55 g column, and the eluted solution was concentrated with an evaporator. As a result, 53 g of purified syrup was obtained. As a result of chemical purity analysis by gas chromatography, the purity of glycerol acetonide isobutyrate was 93.1%.

Example 1
Screening for an enzyme capable of optical resolution of glycerol acetonide isobutyrate (GAIB) was performed as follows. To 5 ml of 20 mM sodium phosphate buffer (pH 7.0), 250 mg of calcium carbonate, 50 mg of racemic glycerol acetonide isobutyrate (final concentration 1% (w / v)) and 50 mg of each enzyme were added, Shake for 48 hours at ° C. After completion of the reaction, the produced GA was quantified by gas chromatography.

  Of the enzymes used above, EnHCH was prepared by the following method. In a 200 ml nutrient medium consisting of 2% (w / v) peptone, 1% (w / v) yeast extract, 0.5% (w / v) glycerin, 0.005% (w / v) ampicillin sodium, Recombinant Escherichia coli DH5α (pKK-EnHCH1) introduced with a gene encoding EnHCH enzyme is cultured aerobically at 37 ° C., and the resulting culture is collected by centrifugation, and the pellet is frozen for 24 hours. After drying, the powder ground in a mortar was used as the EnHCH enzyme sample. An EnHCH enzyme sample (0.8 g) was obtained from 200 ml of a culture solution having a microbial turbidity (OD) of 8.8.

  As a result of screening for 40 types of enzymes, 24 types of enzymes showed a conversion rate from glycerol acetonide isobutyrate to GA of 50% or more within 48 hours. Here, the conversion rate means that when the glycerol acetonide isobutyrate molar amount before the reaction is defined as 100 with respect to the produced GA molar amount as a result of degradation of glycerol acetonide isobutyric acid ester to GA by the enzymatic reaction. The relative value of. When one of the racemates is divided as in this reaction, the ideal conversion rate is 50%, and if the conversion rate of glycerol acetonide isobutyrate is not more than 50%, highly optically pure glycerol acetonide isobutyrate Cannot be obtained. However, if the conversion rate is too high, the yield of the remaining optically active glycerol acetonide isobutyric acid ester becomes poor. For reactions with a conversion rate of 50% or more, extraction was performed with an equal amount of hexane to the reaction solution, and concentration was performed to obtain glycerol acetonide isobutyrate ester syrup remaining after the reaction. About this, the optical purity was measured by the high performance liquid chromatography. As a result of obtaining the E value, when three kinds of enzymes (LipaseQLM, LipaseSL, EnHCH) showing high values in the enzyme group that leaves the S-isomer glycerol acetonide isobutyrate are used, the optical purity is 99% e.e. e. The above S-form glycerol acetonide isobutyrate was obtained at a conversion rate of 70% or less. In addition, 98% e.e. of two enzymes, Esterase Rabbit River and Lipase SM. e. The above R-form glycerol acetonide isobutyric acid ester was able to remain. The results are shown in Table 1 below.

(Comparative Examples 1-4)
Esterase Rabbit Liver, LipaseSM, LipaseQLM, LipaseSL, and DAISOLipase (EnHCH), which showed high stereoselectivity for glycerol acetonide isobutyric acid ester, were used for other esters of GA, namely GAPr (propionic acid ester, comparison) The reactivity with respect to Example 1), GABu (butanoic acid ester, comparative example 2), GABz (benzoic acid ester, comparative example 3), GAPv (pivalic acid ester, comparative example 4) was examined. The racemates of each ester were synthesized in the same manner as in the Reference Example except that isobutanoic acid chloride and propionic acid, butanoic acid, benzoic acid, and pivalic acid chloride were reacted in equimolar amounts. The same procedure as in Example 1 was carried out except for the racemate used and the final concentration charged. The results are shown in Tables 2 to 4 below.

Comparative Example 1

Comparative Example 2

Comparative Example 3

Comparative Example 4

  As a result of the reaction, for the reaction of GAPr and GABu, the Esterase Rabbit River does not have a conversion rate of 50% or more even after 48 hours, and for other enzymes, the conversion rate becomes 70% or more within 2 hours. The optical purity and E value of the ester were much lower than those of glycerol acetonide isobutyric acid ester, indicating that the stereoselectivity was low. Regarding the reaction of GABz, it was relatively high stereoselectivity with enzymes other than Esterase Rabbit River, but 98% e. e. In addition, it took 120 hours when 1% (w / v) was charged. Regarding GAPv reaction, only Lipase SL had a conversion rate of 51.9% in 48 hours, but the optical purity was low.

Example 2
In a 100 ml nutrient medium consisting of 2% (w / v) peptone, 1% (w / v) yeast extract, 0.5% (w / v) glycerin, 0.005% (w / v) ampicillin sodium, Recombinant Escherichia coli DH5α (pKK-EnHCH1) was cultured under aerobic shaking at 37 ° C. To 400 ml of 20 mM sodium phosphate buffer (pH 6.5), 75 g of racemic GAIB and 100 ml of the above culture solution were added and reacted at 30 ° C. Since the pH decreased with the progress of the reaction, the pH was controlled to 6.5 with 25% (w / w) NaOH. During and after the reaction, the amount of GA produced was quantified by gas chromatography. Further, sterilization was performed by centrifugation, and n-hexane extraction was performed on the supernatant. Concentration was performed using an evaporator to obtain (S) -GAIB syrup. The optical purity of the extracted (S) -GAIB was determined.

Example 3
The pH was controlled in the same manner as in Example except that 14% (w / w) aqueous ammonia was used. As a result, when aqueous ammonia was used, the reaction rate was higher than when NaOH was used, and the increase in optical purity of (S) -GAIB was accelerated.
The results of Example 2 and Example 3 are shown in Table 6 below.

  The isobutyric acid ester of optically active glycerol acetonide obtained by the present invention is a useful compound as a pharmaceutical intermediate. According to the present invention, high-purity glycerol acetonide isobutyrate can be efficiently produced by using an enzyme that can be easily prepared or inexpensively obtained.

Claims (6)

Glycerol acetonide isobutyrate ester racemate is treated with an ester hydrolase derived from rabbit liver or Serratia marcescens in an aqueous solvent to hydrolyze the racemate of the compound stereoselectively. A method for producing (R) -glycerol acetonide isobutyrate, which is decomposed and recovers the remaining optically active glycerol acetonide isobutyrate. Ester hydrolase
Esterase by Rabbit Liver (rabbit, SIGMA Co.), or LipaseSM (from Serratia Mars essence, Tanabe Seiyaku Co., Ltd.) which is, according to claim 1 (R) - method of manufacturing glycerol acetoacetic Nai de isobutyrate. A racemic glycerol acetonide isobutyrate ester is allowed to react with an ester hydrolase derived from microorganisms belonging to the genus Alkagenes, Barkholderia, or Enterobacter in an aqueous solvent, and the racemate of the compound is stereoselectively selected. And (S) -glycerol acetonide isobutyric acid ester production method, wherein the remaining optically active glycerol acetonide isobutyric acid ester is recovered . The (S) -glycerol acetonate according to claim 3 , wherein the ester hydrolase is LipaseQLM (derived from the genus Alkaligenes, manufactured by Meika Sangyo Co., Ltd.) or LipaseSL (derived from Barkholderia cepacia, manufactured by Meishou Sangyo Co., Ltd.). Method for producing doisobutyric acid ester. The ester hydrolase is an enzyme derived from a microorganism belonging to the genus Enterobacter, and (a) the amino acid sequence set forth in SEQ ID NO: 1,
(B) an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence has chlorohydrin and hydroxycarboxylate asymmetric hydrolase activity ,
The method for producing (S) -glycerol acetonide isobutyric acid ester according to claim 3 , which is an enzyme (EnHCH, manufactured by Daiso Co., Ltd.) comprising any one of the amino acid sequences. The production method according to any one of claims 1 to 5 , wherein in the step of causing the ester hydrolase to act, ammonia water is used to control the pH of the enzyme to the optimum pH.