JP2019030249A - Production method optical activity 2, 2-disubstituted-3-hydroxypropionic acid or salt thereof - Google Patents

Abstract

To provide a method for efficiently producing optical activity 2, 2-disubstituted-3-hydroxypropionic acid which is useful as a synthetic raw material or synthetic intermediate for a medicine or the like, or salt thereof.SOLUTION: The production method of optical activity 2,2-disubstituted-3-hydroxypropionic acid or salt thereof comprises a step for acting an oxidase source to the 2,2-disubstituted-1,3-propanediol.SELECTED DRAWING: None

Description

  The present invention relates to a method for efficiently producing optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof useful as a synthetic raw material for pharmaceuticals and the like and a synthetic intermediate.

  2,2-disubstituted-3-hydroxypropionic acid is an important compound as a synthetic raw material or synthetic intermediate for pharmaceutical products. In particular, the synthesis of optically active compounds is generally time consuming and expensive, so that an optically active form of 2,2-disubstituted-3-hydroxypropionic acid in which the carbon at the 2-position is an asymmetric carbon is efficiently produced. The technology to manufacture is highly useful.

  Non-Patent Document 1 describes a method of converting 2,2-disubstituted-1,3-propanediol into optically active 2,2-disubstituted-3-hydroxypropionic acid using a copper chloride complex. . However, this method requires many steps and is wasteful because the racemic body is optically resolved, and a high cost is required for the treatment of the waste liquid containing the metal catalyst.

  On the other hand, it is advantageous to use an enzyme source such as a microorganism or an enzyme for the stereoselective oxidation reaction (Non-patent Document 2). For example, Non-Patent Document 3 describes a method of oxidizing 2-methyl-1,3-propanediol with an Acetobacter bacterium, and Non-Patent Document 4 describes 2-butyl-1,3-propanediol. A method of oxidizing with Acetobacter bacteria or Pseudomonas bacteria is described, and Non-Patent Document 5 discloses 2-ethyl-1,3-propanediol and 2-isopropyl-1,3-propanediol as Acetobacter bacteria. And a method of oxidizing with Gluconobacter bacteria.

Lee Ji-Young et al., Journal of American Chemical Society, 133, 1772 (2011) By Andrea Schmid, edited by Karlheinz Drauz et al., “Oxidation of alcohols”, Wiley-VCH, p. 1108-1170 (2002). Elisabetta Brenna et al., ChemCatChem, 8, 3796 (2016) Kohichi Mitsukuura et al., Applied Microbiology and Biotechnology, 76, 61 (2007) Hiromichi Ohta et al., Chemistry Letters, 8, 1379 (1979)

As described above, a method for obtaining optically active 2-substituted-3-hydroxypropionic acid by oxidizing 2-substituted-1,3-propanediol with a microorganism has been conventionally developed. However, it is known that synthesis of a compound having an asymmetric quaternary carbon such as optically active 2,2-disubstituted-3-hydroxypropionic acid is generally particularly difficult.
Under the circumstances described above, the present invention provides a method for efficiently producing optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof useful as a synthetic raw material or synthetic intermediate for pharmaceuticals and the like. Objective.

The inventors of the present invention have made extensive studies to solve the above problems. As a result, it was found that if 2,2-disubstituted-1,3-propanediol, which is inexpensive, is oxidized with an oxidase, 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof can be efficiently produced. The present invention has been completed.
Hereinafter, the present invention will be described.

[1] A method for producing optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof,
The optically active 2,2-disubstituted-3-hydroxypropionic acid is represented by the following general formula (I):

[Wherein, R ¹ and R ² independently represent an _optionally substituted C _1-7 alkyl group. However, R ¹ and R ² are different from each other. ]
A method comprising a step of allowing an oxidase source to act on 2,2-disubstituted-1,3-propanediol represented by the following general formula (II).

［式中、Ｒ¹とＲ²は前記と同義を示す。］

[Wherein, R ¹ and R ² are as defined above. ]

  [2] The method according to [1] above, wherein the enzyme source is one or more microorganisms selected from the group consisting of the genus Rhodococcus and the genus Agromyces.

  [3] The method according to [2] above, further comprising culturing the microorganism in the presence of 2,2-diethyl-3-hydroxypropanol, and using the obtained cultured microorganism as the oxidase source.

  [4] The method according to [3] above, wherein the enantiomeric excess of the 2,2-disubstituted-3-hydroxypropionic acid represented by the general formula (I) is 50% ee or more.

  [5] The method according to any one of [1] to [4], wherein the oxidase source is Rhodococcus sp.

  [6] The method according to any one of [1] to [4], wherein the oxidase source is Agromyces sp.

[7] The method according to any one of [1] to [6], wherein R ¹ is ethyl.

[8] The method according to any one of [1] to [7], wherein R ² is methyl.

[9] Optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof obtained by the method according to any one of [1] to [8].
[10] A pharmaceutical product using the optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof according to [9] above as a raw material.

  According to the method of the present invention, an inexpensive prochiral 2,2-disubstituted-1,3-propanediol can be used as a raw material compound, and optically active 2,2-disubstituted-3- using an oxidase. Hydroxypropionic acid or a salt thereof can be easily produced. Therefore, the present invention is very useful industrially as a method for producing optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof useful as a synthetic raw material or synthetic intermediate for pharmaceuticals.

  The process for producing an optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof according to the present invention comprises 2,2-disubstituted-1,3-propanediol represented by the general formula (II): An optically active 2,2-disubstituted-3-hydroxypropionic acid represented by the general formula (I) or a salt thereof is produced by acting an oxidase source. Hereinafter, the present invention will be described in detail based on embodiments.

In the present disclosure, the “C _1-7 alkyl group” refers to a linear, branched or cyclic monovalent aliphatic hydrocarbon group having 1 to 7 carbon atoms. For example, linear C _1-7 alkyl groups that are methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl; isopropyl, isobutyl, s-butyl, t-butyl, isopentyl And branched C _1-7 alkyl groups such as isohexyl and isoheptyl; and cyclic C _1-7 alkyl groups such as cyclopropyl, cyclohexyl, cyclopentyl and cyclohexyl. A C _1-6 alkyl group is preferable, a C _1-4 alkyl group is more preferable, and a C _1-2 alkyl group is still more preferable. R ¹ and R ² are independently preferably methyl, ethyl, n-propyl or n-butyl, more preferably methyl, ethyl or n-butyl, still more preferably methyl or ethyl, and particularly preferably ethyl. preferable. R ² is particularly preferably methyl.

The C _1-7 alkyl group may have a substituent. Examples of the substituent include a halogeno group, an imino group (—NH—), an ether group (—O—), a sulfide group (—S—), an amino group (—NH ₂ ), a hydroxyl group, a thiol group, a nitro group, and a cyano group. And one or more substituents selected from the group consisting of carboxy groups. Examples of the “halogeno group” include a fluoro group, a chloro group, a bromo group, and an iodo group. The number of substituents is not particularly limited as long as substitution is possible, but may be, for example, 1 or more and 5 or less, preferably 1 or more and 3 or less, more preferably 1 or 2, and even more preferably 1. When the C _1-7 alkyl group has two or more substituents, the substituents may be the same as or different from each other.

The 2,2-disubstituted-1,3-propanediol (hereinafter abbreviated as “propanediol (II)”) represented by the general formula (II), which is a raw material compound, is an easily available compound, and is commercially available. If there is something, it may be purchased and used, or it can be easily synthesized by those skilled in the art. For example, if a base such as sodium ethoxide or sodium hydride is used, protons can be easily extracted from the 2nd position of the malonic acid diester, and the 2nd position is alkylated by the action of a halogenated C _1-7 alkane. Can be By reducing the 2,2-disubstituted malonic acid diester obtained by repeating such a reaction using a reducing agent such as lithium borohydride (LiBH ₄ ) or lithium aluminum hydride (LiAlH ₄ ), a desired propanediol is obtained. (II) can be obtained.

  As the oxidase source used in the present invention, propanediol (II) is an optically active 2,2-disubstituted-3-hydroxypropionic acid (hereinafter referred to as “hydroxypropionic acid (I)” represented by the general formula (I). For example, as long as it has the above-mentioned ability, it may be an oxidase itself or a microbial cell having the ability to produce an oxidase. In addition, a culture solution of the microorganism, a processed product of the microorganism, or a transformant introduced with DNA encoding the oxidase may be used. Alternatively, two or more types may be used in combination, and the oxidase source may be immobilized by a known method so that it can be used repeatedly.

  The microbial cell-treated product is not particularly limited as long as it has the above-mentioned ability. For example, a dried microbial cell or a surfactant obtained by dehydration with acetone or diphosphorus pentoxide or drying using a desiccator or a fan. Examples include treated products, lysed enzyme treated products, immobilized cells or cell-free extract preparations obtained by disrupting cells. The immobilization can be carried out by methods well known to those skilled in the art, such as a crosslinking method, a physical adsorption method, and a comprehensive method.

  The transformant containing the DNA encoding the oxidase can be used for the production of hydroxypropionic acid (I) as a processed product, not to mention the transformant cell itself. The treated product of the transformant here means, for example, cells treated with a surfactant or an organic solvent, dried cells, disrupted cells, crude cell extracts, etc., and immobilizing them by known means. Means something.

  A microorganism having the ability to convert propanediol (II) to hydroxypropionic acid (I) can be found, for example, by the method described below. Per liter glucose 40 g, yeast extract 3 g, diammonium hydrogen phosphate 6.5 g, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.8 g, zinc sulfate heptahydrate 60 mg, iron sulfate heptahydrate After sterilizing 5 mL of liquid medium (pH 7) containing 90 mg, copper sulfate pentahydrate 5 mg, manganese sulfate tetrahydrate 10 mg, and sodium chloride 100 mg, aseptically inoculating the microorganism to be tested, 30 Incubate at 2-3C with shaking for 2-3 days. Thereafter, the cells are collected by centrifugation, suspended in 0.5 to 5 mL of phosphate buffer, added to a test tube containing 0.5 to 25 mg of propanediol (II) in advance, and at 30 ° C. for 2 to 3 days. Shake. Under the present circumstances, what dried the microbial cell obtained by centrifugation in the desiccator or acetone can also be used. Furthermore, an organic solvent may be allowed to coexist when reacting these microorganisms or processed products thereof with propanediol (II). After the conversion reaction, extraction is performed with an appropriate organic solvent, and the extract is analyzed by high performance liquid chromatography or the like. When hydroxypropionic acid (I) is detected, the test microorganism is determined as a microorganism having the above-mentioned ability.

  Any microorganism can be used as the microorganism that can be used in the present invention as long as it has the ability to convert propanediol (II) to hydroxypropionic acid (I), and preferably, the genus Rhodococcus, and / or Or the microorganisms which belong to the genus Agromyces (Agromyces) are mentioned. More preferably, Rhodococcus sp. Rhodococcus erythropolis, Rhodococcus rhodochrous, Agromyces sp. More preferably, Rhodococcus sp. NCIMB11215 strain, Rhodococcus sp. NCIMB11216 strain, Rhodococcus erythropolis NCIMB11148 strain, Rhodococcus rhodochrous J1 strain, Rhodococcus sp. 2N strain, Agromyces sp. E2 strain is mentioned.

  These microorganisms can generally be obtained from stocks that are readily available or purchased, but can also be isolated from nature. It is also possible to obtain a strain having properties more advantageous for this reaction by causing mutation in these microorganisms. Examples of the source of microorganisms include NCIMB Ltd (Aberdeen, UK), Biotechnology Center, Biotechnology Center, National Institute for Product Evaluation and Technology (Kisarazu City, Chiba Prefecture), and the like.

  The above microorganism may be cultured in the presence of an inducer, and the obtained cultured microorganism or a processed product thereof may be used as an oxidase source. Such cultured microorganisms are preferable because the oxidase may be induced to increase the ability.

  For culture of microorganisms, any medium can be used without limitation as long as it contains a nutrient source that can be assimilated by these microorganisms. For example, a medium in which a carbon source, a nitrogen source, a nutrient source, and the like are appropriately blended can be used. Examples of the carbon source include sugars such as glucose, sucrose and maltose; organic acids such as lactic acid, acetic acid, citric acid and propionic acid; alcohols such as ethanol and glycerin; hydrocarbons such as paraffin; soybean oil and rapeseed oil And oils and the like; or mixtures thereof. Examples of the nitrogen source include ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor and the like. Examples of nutrient sources include inorganic salts and vitamins.

The oxidase that converts propanediol (II) to hydroxypropionic acid (I) can be induced by adding an inducer during culture. Any inducer can be used as long as it exhibits the above-mentioned action. For example, as a preferred inducer, in addition to propanediol (II), propane having R ¹ = R ² in the general formula (II) can be used. Diol (II ′) can be mentioned. More specifically, 2,2-diethyl-3-hydroxypropanol, 2-ethyl-2-methyl-3-hydroxypropanol, 2,2-dimethyl-3-hydroxypropanol, 2-methyl-3-hydroxypropanol, Examples include 2-butyl-3-hydroxypropanol, 3-hydroxypropanol, 2-amino-2-methyl-3-hydroxypropanol, and glycerol. More preferable are 2,2-diethyl-3-hydroxypropanol and 2-ethyl-2-methyl-3-hydroxypropanol, and more preferable is 2,2-diethyl-3-hydroxypropanol. According to the experimental findings by the present inventors, 2,2-diethyl-3-hydroxypropanol was particularly excellent as an inducer of oxidase and was induced by achiral 2,2-diethyl-3-hydroxypropanol. Even with an oxidase, propanediol (II) can be enantioselectively oxidized.

  When an inducer is added to the culture solution, the concentration of the inducer may be adjusted as appropriate, and may be, for example, 0.01% by mass or more and 10% by mass or less. As the said density | concentration, 0.05 mass% or more is preferable, 0.1 mass% or more is more preferable, 5 mass% or less is preferable, 3 mass% or less is more preferable, 1 mass% or less is more preferable, 0.5 mass% or less is especially preferable.

  The culture of microorganisms can be usually carried out under general conditions. For example, it is preferably aerobically cultured for 10 to 96 hours in a pH range of 4.0 to 9.5 and a temperature range of 20 ° C to 45 ° C.

  When oxidase itself is used as the oxidase source, it may be purified by a conventional method from a culture solution of a microorganism that produces oxidase. Of course, an inducer may be added to the culture solution as described above. The oxidase may be produced by a genetic engineering technique. Further, when oxidase is accumulated in the microbial cells, treated microbial cells such as microbial cells and dried cells can be used as the oxidase source. When the components in the culture solution have an adverse effect on the reaction, it is possible to use bacterial cells or processed bacterial cells obtained by separating the culture solution by centrifugation or filtration. When microorganisms release oxidase from the cells, a culture solution of the microorganism, a concentrate or a dried product of the culture solution can be used as the oxidase source. Or you may use the culture solution itself containing microorganisms as an oxidase source.

  In the present invention, oxidase is allowed to act on propanediol (II) to oxidize one hydroxymethyl group of propanediol (II) to obtain hydroxypropionic acid (I).

  Any solvent can be used as the solvent for the oxidation reaction as long as the oxidase works. Generally, water or an aqueous buffer is used. However, if propanediol (II) is difficult to dissolve only in water or an aqueous buffer, a small amount of a water-miscible organic solvent such as methanol, ethanol, or isopropanol is added as long as the activity of the oxidase is maintained. May be.

  The concentration of propanediol (II) in the reaction solution for the oxidation reaction may be appropriately adjusted. For example, it can be 0.01 w / v% or more and 50 w / v% or less, preferably 0.1 w / v. It is v% or more and 30 w / v% or less. Propanediol (II) may be added all at once at the beginning of the reaction, or may be added in portions as the reaction proceeds. As for the oxidase source, an appropriate amount to be used varies depending on the form of the oxidase source, and therefore it may be appropriately determined by preliminary experiments or the like.

  Specific reaction conditions may be determined and adjusted as appropriate. For example, the reaction temperature can usually be 10 ° C. or higher and 60 ° C. or lower, preferably 20 ° C. or higher and 40 ° C. or lower. The pH of the reaction solution may be appropriately determined within a range in which the oxidase source to be used can exhibit oxidative activity, and may be, for example, 2.5 or more and 9 or less, preferably 5 or more and 9 or less. The reaction is usually carried out with shaking or stirring with aeration. The reaction time is appropriately determined depending on the substrate concentration, the amount of oxidase source, and other reaction conditions. Usually, it is preferable to set each condition so that the reaction is completed in 2 hours or more and 168 hours or less.

  In the oxidation reaction from propanediol (II) to hydroxypropionic acid (I) according to the present invention, generally, an aldehyde is once generated from an alcohol, and further oxidized to generate a carboxylic acid. According to the present invention, hydroxypropionic acid (I) can be obtained from propanediol (II), but there is a possibility that aldehyde as a reaction intermediate can be obtained by appropriately adjusting the reaction conditions.

  After completion of the reaction, the target compound hydroxypropionic acid (I) or a salt thereof may be purified from the reaction solution by a conventional method. For example, the solids such as bacterial cells can be separated directly from the reaction solution, extracted with a solvent such as ethyl acetate, toluene, t-butyl methyl ether, and hexane, dehydrated, and purified by distillation or silica gel column chromatography. For example, highly pure hydroxypropionic acid (I) or a salt thereof can be easily obtained.

  Whether the resulting compound is hydroxypropionic acid (I) or a salt thereof depends on the type of cation present in the reaction solution and the purification conditions. Examples of the salt of hydroxypropionic acid (I) include alkali metal salts such as lithium salts, sodium salts and potassium salts; magnesium salts; alkaline earth metal salts such as calcium salts, strontium salts and barium salts. it can.

  Since oxidase is used in the present invention, there is a possibility that hydroxypropionic acid (I) having high optical purity can be obtained from propanediol (II) which is a prochiral body. Which enantiomer of hydroxypropionic acid (I) is obtained and its optical purity can be adjusted by the oxidase source used. In the present disclosure, “optical activity” means not only the case where only one enantiomer is selectively obtained and the enantiomeric excess is 100% ee, but one enantiomer is excessive relative to the other. It means that there should be. The optical purity of the resulting hydroxypropionic acid (I) is preferably 30% ee or more, 50% ee or more, or 70% ee or more in terms of enantiomeric excess, 80% ee or more, 90% ee or more, or 95% ee or more. More preferably, it is 98% ee or more, 99% ee or more, or 99.5% ee or more.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

Example 1: Reaction by Rhodococcus bacteria (1) Oxidation reaction of 2,2-diethyl-3-hydroxypropanol In 1.0 L of distilled water, 5.0 g of polypeptone, 5.0 g of meat extract, sodium chloride 0 g, yeast extract 3.0 g, 2,2-diethyl-3-hydroxypropanol 3.0 g were mixed to adjust the medium (pH 7.0). Rhodococcus sp. Strain 2N was cultured at 28 ° C. and 120 rpm for 2.5 days using a shake flask containing 40 mL of the above medium. The centrifuged cells were washed with a 0.15 M sodium chloride aqueous solution and then suspended in 4 mL to obtain a cell suspension.
Prepare 4 mL of the reaction solution containing 60 mM 2,2-diethyl-3-hydroxypropanol, 100 mM potassium phosphate buffer (pH 8.0), and 25 v / v% cell suspension at a final concentration, and prepare a 50 mL sample bottle. The reaction was carried out at 30 ° C. and 120 rpm for 72 hours. When the reaction solution was analyzed by HPLC under the following conditions, it was confirmed that 2,2-diethyl-3-hydroxypropionic acid was produced at a conversion rate of 93.3%.
Column: YMC-Triart C18
Solvent: 50 mM NaH ₂ PO ₄ / H ₃ PO ₄ (pH 2.8): acetonitrile = 4: 1
Flow rate: 1.0 mL / min
Detection wavelength: 210nm

(2) Oxidation reaction of 2-ethyl-2-methyl-3-hydroxypropanol Above (except that 2-ethyl-2-methyl-3-hydroxypropanol was used instead of 2,2-diethyl-3-hydroxypropanol) The reaction was carried out in the same manner as 1). As a result, it was confirmed that 64.7% ee (R) -2-ethyl-2-methyl-3-hydroxypropionic acid was produced at a conversion rate of 47.0%.

(3) Oxidation reaction of 2-ethyl-2-methyl-3-hydroxypropanol Instead of Rhodococcus sp 2N strain, Rhodococcus sp. NCIMB 11215 strain, Rhodococcus sp. NCIMB 11216 strain, or Rhodococcus sp. When the erythropolis (Rhodococcus erythropolis) NCIMB 11148 strain was tested in the same manner as in (2) above, the same stereoselective oxidation activity as in the 2N strain was observed against the diol.

Example 2: Reaction with bacteria belonging to the genus Agromyces In 1.0 L of distilled water, 10.0 g of glucose, 10.0 g of peptone, 6.0 g of sodium chloride, 2.0 g of casein peptone, 2.0 g of yeast extract, 2, A medium (pH 7.5) was prepared by mixing 2.0 g of 2-diethyl-3-hydroxypropanol. Agromyces sp. E2 strain was cultured at 28 ° C. and 120 rpm for 2 days using a shake flask containing 40 mL of the above medium. The centrifuged cells were washed with 0.15 M sodium chloride aqueous solution and then suspended in 4 mL to obtain a cell suspension.
Prepare 4 mL of a reaction solution containing 10 mM 2-ethyl-2-methyl-3-hydroxypropanol, 100 mM potassium phosphate buffer (pH 7.0), and 25 v / v% cell suspension at a final concentration. The sample bottle was reacted at 35 ° C. and 120 rpm for 72 hours. The reaction solution was analyzed under the same conditions as in Example 1 (1). As a result, 32.5% ee (S) -2-ethyl-2-methyl-3-hydroxypropionic acid with a conversion rate of 62.5% was obtained. Was confirmed to be generated.

Example 3: Improving reactivity with inducers of Rhodococcus bacteria and Agromyces bacteria In Example 1 (1) and Example 2, 2,2-diethyl-3-hydroxypropanol was added to the medium. A cell suspension was prepared in the same manner except that it was not added, and the reaction was carried out using the resulting cell suspension, but 2,2-diethyl-3-hydroxypropanol and 2-ethyl- 2-Methyl-3-hydroxypropanol was not oxidized. Therefore, it was considered that the enzymes that oxidized 2,2-diethyl-3-hydroxypropanol and 2-ethyl-2-methyl-3-hydroxypropanol in Example 1 and Example 2 were both inductive enzymes. .

Example 4: Improvement of reactivity by inducer of Rhodococcus genus bacteria In Example 1 (1) above, 3.0 g of 2,2-diethyl was added to a medium for culturing Rhodococcus sp. 2N strain. Cell suspensions were prepared in the same manner except that various diols were added in place of -3-hydroxypropanol to a final concentration of 0.3 w / v% and the culture was continued. Next, 4 mL of a reaction solution containing 2,2-diethyl-3-hydroxypropanol having a final concentration of 0.13 w / v, 100 mM potassium phosphate buffer (pH 8.0), and 25 v / v% cell suspension is prepared. And it was made to react at 30 degreeC and 120 rpm for 24 hours using a 50 mL sample bottle. After the reaction, the produced 2,2-diethyl-3-hydroxypropionic acid was quantified to examine the oxidation activity. Table 1 shows the relative activity when the activity when 2,2-diethyl-3-hydroxypropanol is used as the oxidase inducer is 100% and another diol is used as the inducer.

  As shown in Table 1, 2,2-diethyl-3-hydroxypropanol acted as the most effective oxidase inducer.

Example 5: Improvement of reactivity by inducer of bacteria belonging to the genus Agromyces (Agromyces) In Example 2 above, 2.0 g of 2,2-diethyl-3- (2) -diethyl-3- (2) was added to a medium for culturing Agromyces sp. Strain E2. Cell suspensions were prepared in the same manner except that various diols were added in place of hydroxypropanol to a final concentration of 0.2 w / v% and the culture was performed. Next, 4 mL of a reaction solution containing 2,3-diethyl-3-hydroxypropanol having a final concentration of 0.13 w / v, 100 mM potassium phosphate buffer (pH 7.0), and 25 v / v% cell suspension is prepared. And it was made to react at 35 degreeC and 120 rpm for 65 hours using a 50 mL sample bottle. After the reaction, the produced 2,2-diethyl-3-hydroxypropionic acid was quantified to examine the oxidation activity. Table 2 shows the relative activity when the activity when 2,2-diethyl-3-hydroxypropanol is used as the oxidase inducer is 100% and another diol is used as the inducer.

  As shown in Table 2, 2,2-diethyl-3-hydroxypropanol also acted as the most effective inducer in the case of Rhodococcus bacteria.

Example 6: Substrate specificity of Rhodococcus genus bacteria In Example 1 (1) above, the cell reaction was carried out using various diols having a concentration of 10 mM instead of 2,2-diethyl-3-hydroxypropanol. It was. Table 3 shows the relative activity when the activity when 2,2-diethyl-3-hydroxypropanol was used was 100%.

  As shown in Table 3, it was proved that even an oxidase induced by 2,2-diethyl-3-hydroxypropanol exhibits oxidative activity against other 1,3-diol compounds.

Example 7: Substrate specificity of bacteria belonging to the genus Agromyces In the above Example 1 (1), the cell reaction using various diols having a concentration of 10 mM instead of 2-ethyl-2-methyl-3-hydroxypropanol Went. Table 4 shows the relative activity with 100% activity when 2-ethyl-2-methyl-3-hydroxypropanol was used.

  As shown in Table 4, it was proved that even an oxidase induced by 2,2-diethyl-3-hydroxypropanol exhibits oxidative activity against other 1,3-diol compounds.

Example 8: Enzyme activity of Rhodococcus genus Bacteria obtained by culturing Rhodococcus sp. Strain 2N in the same manner as in Example 1 (1) above was obtained at a final concentration of 1 mM dithiothreitol. In 50 mM potassium phosphate buffer (pH 7.0) and crushed using an ultrasonic crusher. The disrupted solution was centrifuged at 12000 rpm and 4 ° C. for 30 minutes, and the supernatant was obtained as a cell-free extract. 8 mL of a reaction solution containing 10 mM 2-ethyl-2-methyl-3-hydroxypropanol, 100 mM potassium phosphate buffer (pH 8.0), cell-free extract 25 v / v% (v / v), 1 mM NAD ⁺ The enzyme reaction was performed at 30 ° C. and 120 rpm for 24 hours using a glass test tube. The reaction solution after the reaction was analyzed by HPLC in the same manner as in Example 1, and production of 2-ethyl-2-methyl-3-hydroxypropionic acid was confirmed.

Example 9: Substrate specificity of bacteria belonging to the genus Agromyces (Agromyces) The cells obtained by culturing the Agromyces sp. E2 strain in the same manner as in Example 2 above contain 1 mM dithiothreitol at a final concentration. The suspension was suspended in 50 mM potassium phosphate buffer and crushed using an ultrasonic crusher. The disrupted solution was centrifuged at 12000 rpm and 4 ° C. for 30 minutes, and the supernatant was obtained as a cell-free extract. In a reaction solution containing 10 mM 2-ethyl-2-methyl-3-hydroxypropanol, 100 mM potassium phosphate buffer (pH 7.0), cell-free extract 25 v / v%, 1 mM NAD ⁺ , The reaction was performed at 30 ° C. and 120 rpm for 24 hours. The reaction solution was analyzed by HPLC in the same manner as in Example 1 to confirm the formation of 2-ethyl-2-methyl-3-hydroxypropionic acid.

Example 10: Isolation of reaction product and confirmation of structure Using cells obtained by culturing in the same manner as in Example 1 (1) and Example 2, 100 mM 2,2-diethyl-3-hydroxypropanol After microbial cell reaction by shaking at 30 ° C. for 96 hours with 50 mL of the reaction solution containing 2N, 2N hydrochloric acid was added to the reaction solution supernatant to adjust the pH to 3.0. After extracting the product with diethyl ether, the organic layer was concentrated, and a 1M aqueous sodium hydroxide solution was added to the concentrate to dissolve it to obtain a neutral aqueous solution. The aqueous solution was dried under reduced pressure and washed with diethyl ether to obtain a white solid. The resulting white solid was analyzed by NMR and identified as the sodium salt of 2,2-diethyl-3-hydroxypropionic acid.
¹ H NMR (600 MHz, D ₂ O): δ (ppm) 0.59 (3H × 2, t, J = 7.2 Hz), 1.29 (3H × 2, q, J = 7.7 Hz), 3 .47 (2H, s)
¹³ C NMR (150 MHz, D ₂ O): δ (ppm) 8.23, 26.24, 52.56, 61.44, 184.34

Example 11: Isolation of reaction product and confirmation of structure 20 mM 2-ethyl-2-methyl-3-methylation was carried out using the cells obtained by culturing in the same manner as in Example 1 (1) and Example 2. After the bacterial cell reaction was carried out by shaking with 30 mL of a reaction solution containing hydroxypropanol at 30 ° C. for 20 hours, 6N hydrochloric acid was added to the supernatant of the reaction solution to adjust the pH to 3.0. After extracting the product with diethyl ether, the organic layer was concentrated. Silica gel column chromatography was performed, and fractions in which carboxylic acid was detected by TLC analysis were collected and concentrated. As a result of structural analysis using NMR, the reaction product was identified as 2-ethyl-2-methyl-3-hydroxypropionic acid.
¹ H NMR (600 MHz, CDCl ₃ ): δ (ppm) 0.92 (3H, t, J = 14.1 Hz), 1.25 (3H, s), 1.68 (2H, dq, J = 12. 0 Hz), 3.55 (1 H, d, J = 18.0 Hz), 3.73 (1 H, d, J = 18.0 Hz)
¹³ C NMR (150 MHz, CDCl ₃ ): δ (ppm) 8.53, 18.90, 28.35, 47.76, 67.65, 181.17

Example 12: Detection of Aldehyde Intermediate The reaction solution after cell reaction using Rhodococcus sp. 2N strain and Agromyces sp. E2 strain was extracted with ethyl acetate and 2 under acidic conditions. , 4-Dinitrophenylhydrazine was added and reacted. The reaction solution was subjected to preparative TLC, and the isolated compound was identified as hydrazone by NMR analysis. Therefore, it was confirmed that an aldehyde was formed as a reaction intermediate.

Claims (8)

A method for producing an optically active 2,2-disubstituted-3-hydroxypropionic acid or a salt thereof,
The optically active 2,2-disubstituted-3-hydroxypropionic acid is represented by the following general formula (I):

[Wherein, R ¹ and R ² independently represent an _optionally substituted C _1-7 alkyl group. However, R ¹ and R ² are different from each other. ]
A method comprising a step of allowing an oxidase source to act on 2,2-disubstituted-1,3-propanediol represented by the following general formula (II).

[Wherein, R ¹ and R ² are as defined above. ]   The method according to claim 1, wherein the enzyme source is one or more microorganisms selected from the group consisting of the genus Rhodococcus and the genus Agromyces.   The method according to claim 2, further comprising the step of culturing the microorganism in the presence of 2,2-diethyl-3-hydroxypropanol, and using the obtained cultured microorganism as the oxidase source.   The method according to claim 3, wherein the enantiomeric excess of the 2,2-disubstituted-3-hydroxypropionic acid represented by the general formula (I) is 50% ee or more.   The method according to any one of claims 1 to 4, wherein the oxidase source is Rhodococcus sp.   The method according to claim 1, wherein the oxidase source is Agromyces sp. The method according to claim ^1, wherein R ¹ is ethyl. The method according to claim 1, wherein R ² is methyl.