JP4476963B2 - Optically active 3-N-substituted aminoisobutyric acids and salts thereof and process for producing them - Google Patents

Description

  The present invention relates to optically active 3-N-substituted aminoisobutyric acids and salts thereof that are extremely useful as raw materials or intermediates for pharmaceuticals, agricultural chemicals, liquid crystals and the like, and methods for producing them.

  It has been reported that racemic 3-N-substituted aminoisobutyric acids can be easily synthesized by adding an amine to a methacrylic acid ester (J. Org. Chem., 1958, 18, 898, Zh. Obshch Khim. 1957, 27, 3302, Liebigs Ann. Chem., 1958, 608, 22, Ann. Chim. (Paris), (12) 9, 1954, 674, JP 4-66580 and JP 54 No. -30178)), however, there is no description of these optically active substances.

  On the other hand, in Chem. Pharm. Bull., 1977, 25 (6), 1319, optically active 1-phenylethylamine is optically selectively added to methacrylonitrile, and α-methyl-β-alanine is added through two steps. A method of synthesis is disclosed. Furthermore, Tetrahedron Asymmetry, 1992, 3 (6), 723 also discloses a method of synthesizing optically active α-methyl-β-alanine through a six-step reaction using optically active asparagine as a raw material. However, there is no disclosure of a method for producing optically active 3-N substituted aminoisobutyric acids.

  In J. Am. Chem. Soc., 1989, 111, 6354, an acylase that is effective for optical resolution of 2-acylamino acids cannot be applied to optical resolution of 3-acylaminoisobutyric acid. Has been reported.

As a method for producing optically active 3-aminoisobutyric acid, a method using optically active 3-hydroxyisobutyric acid as a starting material described in JP-A-59-67252 is known, but it is difficult to say that it is an industrially advantageous method. . Also, optically active 3-N substituted aminoisobutyric acids are not obtained by this method.
Accordingly, development of optically active 3-N substituted aminoisobutyric acids and effective synthetic methods thereof is desired.

An object of the present invention is to provide optically active 3-N-substituted aminoisobutyric acids.
Moreover, the subject of this invention is providing the manufacturing method of optically active 3-N substituted amino isobutyric acid.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have completed the present invention by discovering an enzyme and a microorganism having an activity to optically hydrolyze a racemic ester of 3-N-substituted aminoisobutyric acid.

  The present invention relates to a general formula (1):

（式中、Ｒ¹は水素原子またはアルキル基を示し、Ｒ²およびＲ³はそれぞれ独立に水素原子、アシル基または置換もしくは非置換のアルキル基を示し、Ｒ²とＲ³とは直接または他の原子を介して互いに結合して両者に結合している窒素原子とともに３〜７員環を形成していてもよい。＊が付された炭素原子は不斉炭素原子である。）
で表される光学活性３−Ｎ置換アミノイソ酪酸類およびその塩である。

(In the formula, R ¹ represents a hydrogen atom or an alkyl group, R ² and R ³ each independently represents a hydrogen atom, an acyl group, or a substituted or unsubstituted alkyl group, and R ² and R ³ may be directly or otherwise. And a nitrogen atom bonded to both of them to form a 3- to 7-membered ring. The carbon atom marked with * is an asymmetric carbon atom.)
Are optically active 3-N-substituted aminoisobutyric acids and salts thereof.

  The present invention also provides a general formula (2):

（式中、Ｒ⁴はアルキル基を示す。Ｒ²およびＲ³は前記のとおりである。また、Ｒ²とＲ³とは直接または他の原子を介して互いに結合して両者に結合している窒素原子とともに３〜７員環を形成していてもよい。）
で表されるラセミ体３−Ｎ置換アミノイソ酪酸エステルを、エステル不斉加水分解酵素の存在下で不斉加水分解することを特徴とする、上記一般式(1)で表される光学活性３−Ｎ置換アミノイソ酪酸類およびその塩の製造方法である。

(In the formula, R ⁴ represents an alkyl group. R ² and R ³ are as defined above. Also, R ² and R ³ are bonded to each other directly or via other atoms. And may form a 3- to 7-membered ring together with the nitrogen atom.)
An optically active 3-amino acid represented by the above general formula (1), wherein the racemic 3-N-substituted aminoisobutyric acid ester represented by the above formula is asymmetrically hydrolyzed in the presence of an ester asymmetric hydrolase. This is a method for producing N-substituted aminoisobutyric acids and salts thereof.

  Furthermore, the present invention relates to a general formula (2):

（式中、Ｒ²、Ｒ³およびＲ⁴は前記のとおりである。また、Ｒ²とＲ³とは、直接または他の原子を介して互いに結合して両者に結合している窒素原子とともに３〜７員環を形成していてもよい。）
で表されるラセミ体３−Ｎ置換アミノイソ酪酸エステルを、アスペルギルス属(Aspergillus）属、リゾプス(Rhizopus)属、キャンディダ(Candida)属、バシラス(Bacillus)属、シュウドモナス(Pseudomonas)属またはエシェリキア(Esherichia)属に属し、エステル不斉加水分解能を有する微生物の培養物、菌体または菌体処理物の存在下で不斉加水分解することを特徴とする、上記一般式(1)で表される光学活性３−Ｎ置換アミノイソ酪酸類およびその塩の製造方法である。

(Wherein R ² , R ³ and R ⁴ are as defined above, and R ² and R ³ are bonded to each other directly or via other atoms and bonded to both) (A 3-7 membered ring may be formed.)
A racemic 3-N-substituted aminoisobutyric acid ester represented by the following genus: Aspergillus genus, Rhizopus genus, Candida genus, Bacillus genus, Pseudomonas genus or Esherichia An optical compound represented by the above general formula (1), characterized in that it is asymmetrically hydrolyzed in the presence of a culture, a microbial cell or a treated cell of a microorganism belonging to the genus and having ester asymmetric hydrolysis ability This is a method for producing active 3-N-substituted aminoisobutyric acids and salts thereof.

In particular, the present invention includes:
1. General formula (4):

（式中、Ｒ¹は水素原子またはアルキル基を示し、＊が付された炭素原子は不斉炭素原子である。）
で表される光学活性３−Ｎ置換アミノイソ酪酸類およびその塩。

(In the formula, R ¹ represents a hydrogen atom or an alkyl group, and the carbon atom marked with * is an asymmetric carbon atom.)
An optically active 3-N-substituted aminoisobutyric acid represented by the formula:

  2. General formula (5):

（式中、Ｒ¹は水素原子またはアルキル基を示し、＊が付された炭素原子は不斉炭素原子である。）
で表される光学活性３−Ｎ置換アミノイソ酪酸類およびその塩。

(In the formula, R ¹ represents a hydrogen atom or an alkyl group, and the carbon atom marked with * is an asymmetric carbon atom.)
An optically active 3-N-substituted aminoisobutyric acid represented by the formula:

  3. General formula (2):

（式中、Ｒ⁴はアルキル基を示し、Ｒ²およびＲ³は、それぞれ独立に水素原子、アシル基または置換もしくは非置換のアルキル基を示し、Ｒ²とＲ³とは直接または他の原子を介して互いに結合して両者に結合している窒素原子とともに３〜７員環を形成していてもよい。）
で表されるラセミ体３−Ｎ置換アミノイソ酪酸エステルを、アスペルギルス(Aspergillus）属、リゾプス(Rhizopus)属、キャンディダ(Candida)属、バシラス(Bacillus)属、シュウドモナス(Pseudomonas)属またはエシェリキア(Esherichia)属に属し、エステル不斉加水分解酵素を有する微生物の培養物、菌体または菌体処理物の存在下で不斉加水分解することを特徴とする、
一般式(1)：

(Wherein R ⁴ represents an alkyl group, R ² and R ³ each independently represents a hydrogen atom, an acyl group, or a substituted or unsubstituted alkyl group, and R ² and R ³ may be directly or other atoms. 3 to 7-membered rings may be formed together with the nitrogen atoms bonded to each other via
A racemic 3-N-substituted aminoisobutyric acid ester represented by the genus Aspergillus, Rhizopus, Candida, Bacillus, Pseudomonas or Esherichia Characterized in that it asymmetrically hydrolyzes in the presence of a culture, a microbial cell or a treated cell of a microorganism belonging to the genus and having an ester asymmetric hydrolase,
General formula (1):

（式中、Ｒ¹は水素原子またはアルキル基を示し、Ｒ²およびＲ³はそれぞれ独立に水素原子、アシル基または置換もしくは非置換のアルキル基を示し、Ｒ²とＲ³とは直接または他の原子を介して互いに結合して両者に結合している窒素原子とともに３〜７員環を形成していてもよい。＊が付された炭素原子は不斉炭素原子である。）
で表される光学活性３−Ｎ置換アミノイソ酪酸類およびその塩の製造方法。

(In the formula, R ¹ represents a hydrogen atom or an alkyl group, R ² and R ³ each independently represents a hydrogen atom, an acyl group, or a substituted or unsubstituted alkyl group, and R ² and R ³ may be directly or otherwise. And a nitrogen atom bonded to both of them to form a 3- to 7-membered ring. The carbon atom marked with * is an asymmetric carbon atom.)
A process for producing optically active 3-N-substituted aminoisobutyric acids and salts thereof represented by the formula:

4). The method for producing optically active 3-N-substituted aminoisobutyric acids and salts thereof according to the above 3, wherein in the general formula (1), either R ² or R ³ is a hydrogen atom and the other is an acyl group .

5). In the above general formula (1), R ² and R ³ are bonded to each other to form an alkylene group having 2 to 6 carbon atoms, and a nitrogen atom bonded to both forms a ring. A process for producing optically active 3-N-substituted aminoisobutyric acids and salts thereof as described in 1. above.

  6). Production of optically active 3-N-substituted aminoisobutyric acids and salts thereof according to any one of 3 to 5 above, wherein the microorganism is a transformant transformed with a gene encoding an ester asymmetric hydrolase Method.

  According to the present invention, optically active 3-N-substituted aminoisobutyric acids and salts thereof which are extremely useful as intermediates for optically active pharmaceuticals, optically active agricultural chemicals and the like can be provided. In addition, according to the production method of the present invention, such an optically active substance can be efficiently produced, and the produced optically active substance can be easily separated and purified. It is advantageous.

Hereinafter, the present invention will be described in detail.
In the general formula (1), (2), (4) or (5), the alkyl group represented by R ¹ and R ⁴ is usually an alkyl group having 1 to 4 carbon atoms and is linear. Any branched structure may be used. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl and the like.

In the general formula (1) or (2), the alkyl group represented by R ² and R ³ is usually an alkyl group having 1 to 10 carbon atoms, and has a linear or branched structure. Good. Specific examples include methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, decyl and the like. The alkyl group may be substituted, and examples of the substituent include a halogen group and a hydroxyl group.

In the above general formula (1) or (2), the acyl group represented by R ² and R ³ is usually an acyl group having 1 to 10 carbon atoms, and has a linear or branched structure. Good. Specific examples include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, decanoyl and the like.

In the above general formula (1) or (2), R ² and R ³ are bonded to each other directly or via other atoms to form a ^3- to 7-membered ring together with the nitrogen atom bonded to both. May be. Here, examples of other atoms include an oxygen atom, a nitrogen atom, and a sulfur atom. Examples of the ring formed by combining R ² and R ³ together with the nitrogen atom include aziridyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholidyl, piperazinyl, pyrazolidinyl and the like. Examples of 3-N-substituted aminoisobutyric acids forming such a ring include, for example, the following general formula (3):

（式中、ｎは２〜６の整数であり、Ｒ¹は前記のとおりである。）
で表される、上記一般式(1)中のＲ²とＲ³とが互いに結合して炭素原子数２〜６のアルキレン基を形成し、両者に結合している窒素原子とともに環を形成している化合物が挙げられる。

(In the formula, n is an integer of 2 to 6, and R ¹ is as described above.)
In the above general formula (1), R ² and R ³ are bonded to each other to form an alkylene group having 2 to 6 carbon atoms, and form a ring together with the nitrogen atom bonded to both. The compound which is mentioned.

  Examples of the salt of the optically active 3-N substituted aminoisobutyric acid of the present invention include sodium salt, potassium salt, lithium salt, magnesium salt, calcium salt, ammonium salt and the like as the carboxylate, and hydrochloride and odor as the amine salt. Illustrative are mineral acid salts such as hydrohalide, sulfate, nitrate, and organic acid salts such as acetate, propionate, butyrate, fumarate, malonate, oxalate, benzoate, etc. .

The optically active compound of the present invention can be produced by the following method.
As a raw material, a racemic 3-N-substituted aminoisobutyric acid ester represented by the general formula (2) is used. This racemic 3-N-substituted aminoisobutyric acid ester can be easily synthesized by a conventionally known method. That is, for example, it can be synthesized by reacting a methacrylate such as methyl methacrylate (MMA) with an amine such as piperidine, pyrrolidine or dimethylamine.

A racemic 3-acylaminoisobutyric acid ester in which R ² and / or R ³ is an acyl group can be produced by N-acylating and then esterifying the racemic 3-aminoisobutyric acid.

  Racemic 3-aminoisobutyric acid can be produced by a method of adding ammonia to methacrylic acid or a method of adding ammonia to methacrylonitrile followed by hydrolysis using acid, alkali, enzyme or the like. However, in these methods, it is difficult to selectively produce racemic 3-aminoisobutyric acid or 3-aminoisobutyronitrile. This is because 3-aminoisobutyric acid or 3-aminoisobutyronitrile produced in the ammonia addition reaction is more likely to react with methacrylic acid or methacrylonitrile, respectively, and a secondary amine and / or a tertiary amine is produced. It is. Separation of this secondary amine and / or tertiary amine from 3-aminoisobutyric acid or 3-aminoisobutyronitrile is difficult, and thus the highly purified racemic 3-aminoisobutyric acid or 3-amino It is difficult to obtain isobutyronitrile.

  Therefore, in order to selectively obtain racemic 3-aminoisobutyric acid, 2-chloropropionic acid or a derivative thereof is reacted with an alkali cyanide, and the obtained 2-methylcyanoacetic acid or a derivative thereof is converted into nickel, palladium-charcoal or the like. It is preferable to produce racemic 3-aminoisobutyric acid by a catalytic reduction method in the presence of a reduction catalyst.

  N-acylation of racemic 3-aminoisobutyric acid is usually carried out by dissolving racemic 3-aminoisobutyric acid in an aqueous alkali solution and then reacting with acyl chloride or acid anhydride.

  The esterification of racemic 3-acylaminoisobutyric acid obtained by N-acylation is usually performed by dissolving racemic 3-acylaminoisobutyric acid in alcohol and then adding a dehydrating agent such as thionyl chloride or dicyclohexylcarbodiimide. Is done. Moreover, it is preferable to use alcohol which has been dehydrated in advance.

  The enzyme used in the present invention is an ester asymmetric hydrolase having the ability to produce an optically active 3-N substituted aminoisobutyric acid and its enantiomer by asymmetric hydrolysis of racemic 3-N substituted aminoisobutyric acid ester Then, the kind of enzyme and its production source are not limited. For example, various lipases, proteases, esterases and the like can be used, and among them, alkaline protease, bromelain, trypsin, and neurase are preferable.

  As the ester asymmetric hydrolase, those isolated from microorganisms having an ester asymmetric hydrolysis ability can be used. As microorganisms having an ester asymmetric hydrolysis ability, they belong to the genus Aspergillus, Rhizopus, Candida, Bacillus, Pseudomonas, Esherichia, etc. Examples include microorganisms. Examples of the microorganism also include a transformant transformed with a gene encoding an ester asymmetric hydrolase. Examples of microorganisms belonging to the genus Aspergillus and having ester asymmetric hydrolysis ability include Aspergillus oryzae, Aspergillus saitoi, Aspergillus sojae, Aspergillus niger and the like. Is mentioned. Examples of microorganisms belonging to the genus Rhizopus and having an ester asymmetric hydrolysis ability include Rhizopus niveus. Examples of microorganisms belonging to the genus Candida and having an ester asymmetric hydrolysis ability include Candida antarctica. Examples of microorganisms belonging to the genus Bacillus and having an ester asymmetric hydrolysis ability include Bacillus subtilis, Bacillus licheniformis, Bacillus polymyxa, and the like. Examples of microorganisms belonging to the genus Pseudomonas and having an ester asymmetric hydrolysis ability include Pseudomonas putida FERM BP-3846. Examples of microorganisms belonging to the genus Escherichia and having an ester asymmetric hydrolysis ability include Escherichia coli FERM BP-3835 and the like. This Escherichia coli FERM BP-3835 is a microorganism transformed with a gene encoding an esterase derived from Pseudomonas putida FERM BP-3846.

  Moreover, a commercial item can be used as ester hydrolase. Specifically, lipase Amano A6 (trade name, manufactured by Amano Pharmaceutical, Aspergillus genus enzyme), lipase Amano AP6 (trade name, manufactured by Amano Pharmaceutical, Aspergillus genus enzyme), Amanonewase F (trade name, manufactured by Amano Pharmaceutical) , Rhizopus genus enzyme), SIGMA neurase (type XVIII) (trade name, Rhizopus genus enzyme), name sugar lipase OF (trade name, name sugar industry, Candida genus enzyme), SIGMA protease (type I) (Trade name, bovine pancreas-derived enzyme), SIGMA protease (type II) (trade name, Aspergillus genus enzyme), SIGMA protease (type XIII) (trade name, Aspergillus genus enzyme), SIGMA protease (type XV) (product) Name, enzyme derived from the genus Bacillus), SIGMA protease (type XVI) (trade name, Bacillus Derived enzyme), SIGMA protease (type XIX) (trade name, enzyme derived from Aspergillus), SIGMA protease (type XXIII) (trade name, enzyme derived from Aspergillus), SIGMA protease (type XXXI) (trade name, enzyme derived from the genus Bacillus) ), SIGMA trypsin (type II) (trade name, swine pancreatic enzyme), Fluka lipase (trade name, enzyme derived from Candida), SIGMA bromelain (trade name), Meiji Seika Alka Protease (trade name), NOVO Alcalase 2.5L (Brand name, Bacillus genus-derived enzyme), NOBO dirazyme 16.0L (Brand name, Bacillus genus-derived enzyme), NOVO esperase 8.0L (Brand name, Bacillus genus-derived enzyme), NOVO sabinase 16.0L (Brand name, Bacillus genus-derived enzyme) ), NOVO sabinase 6.0T (trade name, enzyme derived from the genus Bacillus), KH Seikagaku bi options hydrolase Conc (trade name, Bacillus derived enzyme), Nagase Seikagaku Denachimu AP (trade name, Aspergillus derived enzyme) and the like.

  In the production method of the present invention, when the above-mentioned ester asymmetric hydrolase is subjected to the reaction, the mode of use is not particularly limited as long as the enzyme exhibits activity, not only the purified enzyme but also the enzyme. Additives for stabilization, for example, polyhydric alcohols such as glycerol, ethylene glycol and propanediol; sugars such as lactose, sorbitol, cellulose and dextrin; may be used in a mixture with inorganic salts . In addition, a culture obtained by culturing a microorganism having an ester hydrolyzing ability as described above in a medium is used as it is, or a cell obtained from the culture by a collection operation such as centrifugation or a processed product thereof is also obtained. Can be used. Examples of treated cells include cells treated with acetone, toluene, etc., freeze-dried cells, disrupted cells, cell-free extracts obtained by disrupting cells, and crude enzyme solutions obtained by extracting enzymes from these. . Further, for example, the enzyme can be separated and purified by recovering the active fraction from the cell-free extract by gel filtration, ion exchange chromatography or the like. Furthermore, the enzyme or the microorganism can be recovered and reused after the completion of the reaction by immobilizing it on an appropriate carrier. Immobilization is performed by comprehensively immobilizing enzymes or microorganisms on a cross-linked acrylamide gel or the like, or by physically and chemically immobilizing them on a solid support such as an ion exchange resin or caustic soil.

  The microorganism can be cultured in a liquid medium or a solid medium. As a culture medium, what mix | blended suitably components, such as a carbon source, a nitrogen source, a vitamin, a mineral which a microorganism can normally utilize, is used. In order to improve the ester hydrolysis ability of microorganisms, it is also possible to add a small amount of an inducer such as a low molecular weight compound having an ester bond or an amide bond to the medium. Culturing is performed at a temperature and pH at which the microorganism can grow, but it is preferably performed under the optimal culture conditions for the strain to be used. In order to promote the growth of microorganisms, aeration and agitation may be performed.

  In order to optically hydrolyze the racemic 3-N-substituted aminoisobutyric acid ester, the racemic 3-N-substituted aminoisobutyric acid ester is dissolved or suspended in a solvent, and the ester asymmetric hydrolase or catalyst of the above microorganism is used as a catalyst. The culture, cells or treated cells are added, and the reaction is continued until about half of the 3-N-substituted aminoisobutyric acid ester is hydrolyzed while controlling the reaction temperature and, if necessary, the pH of the reaction solution. In some cases, the reaction may be stopped or reacted excessively at an early stage.

  As the reaction solvent, an aqueous medium such as ion-exchanged water or a buffer solution is usually used, but the reaction can also be performed in a system containing an organic solvent. The presence of an organic solvent in the reaction system often promotes selectivity, conversion rate, yield, and the like. When using a system containing an organic solvent as a reaction solvent, a solvent in which the organic solvent is dissolved in water may be used, or a solvent in which the organic solvent and water form a two-phase system may be used. Good. Examples of the organic solvent include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butyl alcohol, t-amyl alcohol, and other alcohols; diethyl ether, isopropyl ether, dioxane, and other ethers; acetone, methyl ethyl ketone, Ketones such as methyl isobutyl ketone; aromatic and aliphatic hydrocarbon solvents such as hexane, octane, benzene, and toluene can be appropriately used.

  The concentration of the substrate in the reaction solvent is not particularly limited and is usually 0.1 to 70% by weight. However, considering the solubility, reactivity, etc. of the racemic 3-N-substituted aminoisobutyric acid ester serving as the substrate, the concentration is 5 to 40%. % Is preferred.

The amount of enzyme in the reaction solvent may be appropriately adjusted according to the catalytic ability of the enzyme for the reaction, and is not particularly limited, but is preferably used in the range of 0.01 to 20% by weight, more preferably 0.01 to 5% by weight. % Range.
Moreover, 5-70 degreeC is preferable and the temperature of asymmetric hydrolysis reaction has more preferable 10-60 degreeC.

  Furthermore, the pH of the reaction solution varies depending on the optimum pH of the enzyme, microorganism, etc. used, but in general, it is preferably in the range of pH 6 to 9.5. By performing the reaction at a pH in this range, it is possible to suppress a decrease in optical purity due to a chemical hydrolysis reaction. Further, as the reaction proceeds, the pH of the reaction solution decreases due to the produced carboxylic acid. In this case, an appropriate neutralizing agent such as sodium hydroxide or potassium hydroxide is added to maintain the optimum pH. This often promotes the progress of the reaction.

  The reaction conditions such as reaction solvent, substrate concentration, reaction temperature, enzyme concentration, reaction solution pH and the like described above take into consideration the reaction yield, optical yield, strict stereoselectivity, etc. due to the difference in conditions. Thus, the conditions under which the target optically active substance can be collected most are selected. Usually, it is preferable to select reaction conditions such that the reaction is completed in 1 hour to 1 week, preferably 1 to 72 hours.

  Such an asymmetric hydrolysis reaction produces optically active 3-N-substituted aminoisobutyric acid. In addition, the unreacted residual substrate becomes an enantiomer ester of the produced optically active 3-N-substituted aminoisobutyric acid.

  Isolation of the produced optically active 3-N-substituted aminoisobutyric acid and its enantiomer ester from the reaction solution can be performed by a general isolation method such as distillation, extraction, column separation and the like. For example, when a microorganism is used as the catalyst, the microbial cells are removed by an operation such as centrifugation and filtration, and then extracted with a solvent such as hexane or ethyl acetate, thereby causing unreacted optically active 3-N. Substituted aminoisobutyric acid esters can be obtained.

  If the reaction solvent contains a large amount of organic solvent, the reaction solvent is once removed by distillation, and then combined with an appropriate organic solvent to form an optically active 3-N substituted aminoisobutyric acid and its enantiomer in the same manner. Esters can be extracted and separated.

  The obtained optically active 3-N-substituted aminoisobutyric acid can be converted to a 3-N-substituted aminoisobutyric acid ester while maintaining optical activity by esterification by a usual method. Further, the optically active 3-N-substituted aminoisobutyric acid ester can be converted to 3-N-substituted aminoisobutyric acid while maintaining the optical activity by hydrolysis by a usual method. Therefore, optically active 3-N substituted aminoisobutyric acids having an arbitrary configuration can be obtained.

  In these optically active 3-N substituted aminoisobutyric acids, amines and carboxylic acids may be present. In this case, amine salts and carboxylates can be formed by a conventional method.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
[Reference Example 1]
(Synthesis of racemic methyl 3-acetylaminoisobutyrate)
10 g of 3-aminoisobutyric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 120 ml of 1N aqueous sodium hydroxide solution, and 12 g of acetic anhydride was slowly added dropwise thereto and reacted at room temperature with stirring for 18 hours. After completion of the reaction, 2N hydrochloric acid was added to adjust the pH to 2.0. Extraction with the same amount of ethyl acetate as the obtained reaction mixture was performed three times. The organic phases were combined and dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. In this way, 9.2 g of racemic 3-acetylaminoisobutyric acid was obtained.

  66 g of anhydrous methanol was added to the obtained racemic 3-acetylaminoisobutyric acid, and 9.0 g of thionyl chloride was slowly added dropwise. After stirring at room temperature for 18 hours, the mixture was concentrated under reduced pressure. In this way, 6.9 g of racemic methyl 3-acetylaminoisobutyrate was obtained.

[Example 1]
(Synthesis of methyl d (+) 3-acetylaminoisobutyrate and l (-) 3-acetylaminoisobutyric acid)
Escherichia coli FERM BP-3835 was inoculated into 50 ml of LB medium (1% polypeptone, 0.5% yeast extract and 0.5% NaCl) containing 50 μg / ml ampicillin, and cultured with shaking at 37 ° C. for 24 hours. After completion of the culture, the culture solution was centrifuged to collect microbial cells. The whole amount of the obtained microbial cells was washed with ion exchange water. After washing, the cells are suspended in 50 ml of 50 mM phosphate buffer (pH 7.0), and 5 g of racemic methyl 3-acetylaminoisobutyrate is added to the cell suspension and reacted at 30 ° C. for 20 hours. It was. During the reaction, the pH of the reaction solution was adjusted to 7.0 by appropriately adding a 1N sodium hydroxide aqueous solution. After completion of the reaction, the cells were removed by centrifugation, and unreacted methyl 3-acetylaminoisobutyrate was extracted with ethyl acetate. The organic phase was dehydrated with anhydrous sodium sulfate and the solvent was evaporated off. In this way, 2.0 g of optically active methyl 3-acetylaminoisobutyrate was obtained.

  When the optical purity of the obtained optically active methyl 3-acetylaminoisobutyrate was determined by gas chromatography (column; CP-Chirasil Dex CB, manufactured by Chrome Pack), the d (+) isomer was 100% ee. .

  2N hydrochloric acid was added to the aqueous phase of the extraction residue in the above ethyl acetate extraction to adjust the pH to 2.0, and 3-acetylaminoisobutyric acid as a reaction product was extracted with ethyl acetate. The organic phase was dehydrated with anhydrous sodium sulfate and the solvent was evaporated off. In this way, 1.2 g of optically active 3-acetylaminoisobutyric acid was obtained.

The obtained optically active 3-acetylaminoisobutyric acid as a reaction product was treated with a methanol / hydrochloric acid mixed solution to form a methyl ester, and the optical purity was measured in the same manner as described above. The l (-) isomer 90% It was ee.
Hereinafter, physical property values of the obtained methyl d (+) 3-acetylaminoisobutyrate are shown.

( ¹ H-NMR spectrum (FIG. 1))
Solvent: CDCl ₃ , Internal standard: TMS, 270 MHz
δ _H 1.19 to 1.21 (3H, d, -CH ₃ )
δ _H 1.95 (3H, s, -COCH ₃ )
δ _H 2.62 to 2.78 (1H, m, -CH-)
δ _H 3.20-3.60 (2H, m, -CH _2- )
δ _H 3.70 (3H, s, -COOCH ₃ )
δ _H 6.00 (1H, b, -NH-)

( ¹³ C-NMR spectrum (FIG. 2))
Solvent: CDCl ₃ , Internal standard: TMS, 270 MHz
δ _C 14.9 (-CH ₃ )
δ _C 23.3 (-COCH ₃ )
δ _C 39.5 (-CH-)
δ _C 41.6 (-CH _2- )
δ _C 51.9 (-COOCH ₃ )
δ _C 170.2 (-CO-)
δ _C 176.1 (-COO-)

(Optical purity)
100% ee
(Specific rotation)
[Α] _D ²⁵ = + 53.12 (c = 2.9, MeOH)

[Reference Example 2]
(Synthesis of racemic 3-pyrrolidylisobutyric acid methyl ester)
While heating at 70 ° C. to 35.2 g (0.35 mol) of methyl methacrylate (MMA), 17 g (0.24 mol) of pyrrolidine was gradually added dropwise, and the reaction was continued until pyrrolidine disappeared. After completion of the reaction, the reaction mixture was distilled to obtain 31 g of racemic 3-pyrrolidylisobutyric acid methyl ester (1 to 3 mmHg, 55 to 58 ° C.).

[Reference Example 3]
(Synthesis of racemic 3-piperidylisobutyric acid methyl ester)
Piperidine 20 g (0.24 mol) was gradually added dropwise to methyl methacrylate (MMA) 35.2 g (0.35 mol) while heating at 70 ° C., and the reaction was continued until the piperidine disappeared. After completion of the reaction, the reaction mixture was distilled to obtain 30 g of racemic 3-piperidylisobutyric acid methyl ester (7-5 mmHg, 72-73 ° C.).

[Example 2]
(Synthesis of optically active 3-pyrrolidylisobutyric acid and its methyl ester)
In 75 g of t-butyl alcohol, 10 g of racemic methyl 3-pyrrolidylisobutyrate is dissolved, 7.5 g of water and 7.5 g of NOVO alcalase 2.5 L (enzyme derived from Bacillus licheniformis) are added at 30 ° C. The reaction was allowed for days. The obtained reaction mixture was filtered, and the filtrate was concentrated with an evaporator. To the obtained concentrated liquid, 50 ml of n-hexane and 50 ml of water were added for liquid separation, and the organic phase was recovered. After drying with sodium sulfate, the mixture was concentrated. In this way, 2.1 g of d (+) 3-pyrrolidylisobutyric acid methyl ester was obtained.

  The aqueous phase was neutralized and concentrated, and then dissolved in 100 ml of methanol. After insoluble matter was filtered off, 4 ml of sulfuric acid was added to carry out an esterification reaction. After completion of the reaction, the reaction mixture was concentrated with an evaporator and suspended in 60 ml of hexane. The extract was washed with a saturated aqueous sodium carbonate solution, dried over magnesium sulfate, and concentrated. In this way, 5.8 g of l (−) 3-pyrrolidylisobutyric acid methyl ester was obtained.

To 2.0 g of each of d (+) and l (−) 3-pyrrolidylisobutyric acid methyl ester, 10 ml of a 2.0N aqueous sodium hydroxide solution was added and stirred at room temperature. Then, it neutralized with sulfuric acid and concentrated to dryness. The soluble component was dissolved in about 10 ml of methanol, filtered, and concentrated to obtain 1.3 g of d (+) 3-pyrrolidylisobutyric acid and 1.3 g of l (−) 3-pyrrolidylisobutyric acid, respectively.
The specific rotation of each optically active 3-pyrrolidylisobutyric acid is shown in Table 1.

Example 3
(Synthesis of optically active 3-pyrrolidylisobutyrate)
10 g of racemic 3-pyrrolidylisobutyrate synthesized according to Reference Example 2 was dissolved in 90 ml of 100 mM phosphate buffer (pH 7), and 2.5 g of NOVO alcalase 2.5 L was added and reacted at 30 ° C. for 6 hours. The reaction mixture obtained was adjusted to pH 10 while cooling, and then 100 ml of hexane was added to recover unreacted butyl 3-pyrrolidylisobutyrate. The hexane phase was concentrated with an evaporator. In this way, 2.1 g of butyl d (+) 3-pyrrolidylisobutyrate was obtained. The specific rotation [α] _D ²⁴ was +20.1 (neat).

Example 4
(Synthesis of optically active 3-piperidylisobutyric acid and its methyl ester)
10 g of racemic methyl 3-piperidylisobutyrate was dissolved in 75 g of t-butyl alcohol, 7.5 g of water and 7.5 g of NOVO alcalase were added, and the mixture was reacted at 30 ° C. for 2 days. The obtained reaction mixture was filtered, and the filtrate was concentrated with an evaporator. To the obtained concentrated liquid, 50 ml of n-hexane and 50 ml of water were added for liquid separation, and the organic phase was recovered. After drying with sodium sulfate, the mixture was concentrated. In this way, 3.8 g of d (+) 3-piperidylisobutyric acid methyl ester was obtained.

  The aqueous phase was neutralized and concentrated, and then dissolved in 100 ml of methanol. After insoluble matter was filtered off, 4 ml of sulfuric acid was added to carry out an esterification reaction. After completion of the reaction, the reaction mixture was concentrated with an evaporator and suspended in 60 ml of hexane. The extract was washed with a saturated aqueous sodium carbonate solution, dried over magnesium sulfate, and concentrated. In this way, 3.4 g of l (−) 3-piperidylisobutyric acid methyl ester was obtained.

To each 2.0 g of d (+) and l (-) 3-piperidylisobutyric acid methyl ester, 10 ml of a 2.0N aqueous sodium hydroxide solution was added and stirred at room temperature. Then, it neutralized with sulfuric acid and concentrated to dryness. The soluble component was dissolved in about 10 ml of methanol, filtered, and concentrated to obtain 1.3 g of d (+) 3-piperidylisobutyric acid and 1.4 g of l (−) 3-piperidylisobutyric acid, respectively.
The specific rotation of each optically active 3-piperidylisobutyric acid is shown in Table 2.

Example 5
Racemic methyl 3-pyrrolidylisobutyrate is dissolved in 100 mM phosphate buffer (pH 7) so as to be 1% by weight, and the enzymes shown in Table 3 are appropriately added in the range of 0.1 to 1% by weight. Reacted for hours. The optical purity and concentration of each d (+) 3-pyrrolidylisobutyrate methyl were determined by gas chromatography (column: CP-Chirasil DEX CB 0.25 mm × 25 M, manufactured by Chrome Pack). The results are shown in Table 3.

Example 6
Racemic methyl 3-piperidylisobutyrate is dissolved in 1% by weight in 100 mM phosphate buffer (pH 7), and the enzymes shown in Tables 4 and 5 are appropriately added in the range of 0.1 to 1% by weight. The reaction was carried out at 30 ° C. for 1 day. The optical purity and concentration of each d (+) 3-pyrrolidylisobutyrate methyl were determined by gas chromatography (column: CP-Chirasil DEX CB 0.25 mm × 25 M, manufactured by Chrome Pack). The results are shown in Tables 4 and 5.

1 is a diagram showing a ¹ H-NMR spectrum of methyl d (+) 3-acetylaminoisobutyrate obtained in Example 1. FIG. 2 is a diagram showing a ¹³ C-NMR spectrum of methyl d (+) 3-acetylaminoisobutyrate obtained in Example 1. FIG.

Claims (2)

General formula (4):

(In the formula, R ¹ represents a hydrogen atom , and the carbon atom marked with * is an asymmetric carbon atom.)
An optically active 3-N-substituted aminoisobutyric acid represented by the formula: General formula (5):

(In the formula, R ¹ represents a hydrogen atom or an alkyl group, and the carbon atom marked with * is an asymmetric carbon atom.)
An optically active 3-N-substituted aminoisobutyric acid represented by the formula:

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