JP5474280B2 - Process for producing optically active trans-form nitrogen-containing cyclic β-hydroxyester - Google Patents

Description

The present invention relates to a process for producing an optically active trans-form nitrogen-containing cyclic β-hydroxyester. The optically active trans-form nitrogen-containing cyclic β-hydroxy ester is a useful compound as a synthetic raw material and intermediate for pharmaceuticals, agricultural chemicals and the like.

  As a method for producing a substance similar to the compound of the present invention, the following methods are conventionally known.

For example, as a method for producing 1-substituted-4-hydroxypyrrolidine-3-carboxylic acid ester,
(1) A method of reducing 1-benzyl-4-pyrrolidinone-3-carboxylic acid ethyl ester to 1-benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester with lithium borohydride (Non-patent Document 1),
(2) Racemic trans-1-benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester is optically resolved with Novozyme 435 lipase, and optically active trans-1-benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl A method of obtaining an ester (Patent Document 1),
There is. However, in the method (1), the product is a mixture of a cis form and a trans form, and the obtained trans form is a racemate. In the method (2), it is necessary to synthesize racemic trans-1-substituted-4-hydroxypyrrolidine-3-carboxylic acid ester, which is a raw material, by a complicated operation, and in an optical resolution reaction using lipase. Thus, the yield is at most 50%.

As a method for producing 1-substituted-4-hydroxypiperidine-3-carboxylic acid ester,
(3) A method of stereoselectively reducing 1-substituted-4-piperidinone-3-carboxylic acid ester to optically active cis-1-substituted-4-hydroxypiperidine-3-carboxylic acid ester by the action of baker's yeast ( Non-Patent Documents 2, 3, 4),
(4) Racemic trans-N-substituted-4-benzoyloxypiperidine-3-carboxylic acid methyl ester is optically resolved by the action of porcine liver lipase to produce optically active trans-N-substituted-4-hydroxypiperidine- A method for obtaining 3-carboxylic acid methyl ester (Non-Patent Document 5),
(5) Racemic trans-1-Boc-4-hydroxypiperidine-3-carboxylic acid ethyl ester is optically resolved by the action of lipase to produce optically active trans-1-Boc-4-hydroxypiperidine-3-carboxylic acid A method of obtaining an ethyl ester (Non-Patent Document 6),
It has been known.

  However, 95% or more of the nitrogen-containing cyclic β-hydroxyester obtained by the method (3) is a cis form. In the methods (4) and (5), it is necessary to synthesize the racemic trans-1-substituted-4-piperidinone-3-carboxylic acid ester as a raw material by a complicated operation, and an optical system using a lipase. Since it is a resolution reaction, the yield is at most 50%.

Thus, the optically active trans-1-substituted-4-hydroxypyrrolidine-3-carboxylic acid ester and trans-1-substituted-4-hydroxypiperidine-3-carboxylic acid ester by reduction reaction using an asymmetric catalyst. No manufacturing method has been reported.
International Publication No. 2005/033076 Pamphlet J. et al. Org. Chem, 30, 740 (1965) Helvetica. Chimica. Acta, 70, 1605 (1987) Tetrahedron: Asymmetry, 4,625 (1993) J. et al. Chem. Soc. Perkin. Trans1,3673 (1998) Tetrahedron: Asymmetry, 11, 4397 (2000) Tetrahedron: Asymmetry, 15, 3281 (2004)

  An object of the present invention is to provide a method by which an optically active trans-form nitrogen-containing cyclic β-hydroxy ester useful as an intermediate of a pharmaceutical can be easily produced from inexpensive and readily available raw materials.

  As a result of repeated studies to develop an efficient process for producing an optically active trans-form nitrogen-containing cyclic β-hydroxyester, the present inventors have determined that only the carbonyl group of the optically active trans-form nitrogen-containing cyclic β-ketoester is present. The present inventors have completed the present invention by discovering a method of stereoselectively reducing and converting into optically active trans-form nitrogen-containing cyclic β-hydroxyesters.

  The present invention has one or more of the following features.

  That is, the present invention relates to the general formula (1);

(Wherein R ¹ represents a linear or branched alkyl group; R ² represents an amino-protecting group; m represents 1 or 2) or a nitrogen-containing cyclic β-ketoester represented by The salt is subjected to an asymmetric reduction reaction to give a general formula (2);

(Wherein R ¹ _, R ² and m are the same as above) or general formula (3);

(Wherein R ¹ _, R ² and m are the same as above), which is converted into any of the optically active trans-form nitrogen-containing cyclic β-hydroxy esters represented by the above formula (2) And an optically active trans-form nitrogen-containing cyclic β-hydroxyester represented by (3).

  Preferably, the above-mentioned production method is characterized in that an enzyme source having the ability to stereoselectively reduce a carbonyl group is allowed to act as the catalyst.

More preferably, the compound is 1-substituted-4-hydroxypyrrolidine-3-carboxylic acid ester or 1-substituted-4-hydroxypiperidine-3-carboxylic acid ester, and the enzyme source is a genus Candida . , Kluyveromyces (Kluyveromyces) genus, Lipomyces (Lipomyces) genus, Rodotorura (Rhodotorula) genus Saccharomyces (Saccharomyces) genus Trichosporon (Trichosporon) genus, shake Bun di Monas (Brevundimonas) genus Cellulomonas (Cellulomonas) genus, Deboshia ( Devosia spp.), Micrococcus (Micrococcus) genus Serratia (Serratia) genus, sphingolipid Baku It is a genus (Sphingobacterium) manufacturing process is of microbial origin selected from the group consisting of the genus.

In another preferred embodiment, oxidized nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD ⁺ ) and / or oxidized nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP ⁺ ) are reduced to their respective reduced forms. In the above-mentioned production method, an enzyme that reduces to a coexistent substrate and a substrate for the reduction coexist.

  The present invention provides an efficient and industrial method for producing optically active trans-form nitrogen-containing cyclic β-hydroxy esters useful as synthetic raw materials and intermediates for pharmaceuticals and agricultural chemicals.

  Hereinafter, the present invention will be described in detail.

  Nitrogen-containing cyclic β-ketoesters serving as substrates for the asymmetric reduction reaction of the present invention are represented by the general formula (1);

(Wherein R ¹ represents a linear or branched alkyl group; R ² represents an amino-protecting group; m represents 1 or 2) or a nitrogen-containing cyclic β-ketoester represented by Its salt. The cyclic moiety of the general formula (1) is a 5-membered ring with m = 1 or a 6-membered ring with m = 2.

R ^{1 in the} general formula (1) is preferably a linear or branched alkyl group having 1 to 10 carbon atoms.

More preferably, R ^{1 in the} general formula (1) is a methyl group or an ethyl group.

In addition, R ^{2 in the} general formula (1) is preferably a general amino protecting group. For example, there are protecting groups described in “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS” (John Wiley & Sons, Inc.). That is, t-butoxycarbonyl group, methoxycarbonyl group, ethoxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, benzyloxycarbonyl group, paramethoxybenzyloxycarbonyl group, paranitrobenzyloxycarbonyl group, formyl group, acetyl group , Propanoyl group, t-butyroyl group, methoxyacetyl group, fluoroacetyl group, difluoroacetyl group, trifluoroacetyl group, chloroacetyl group, pivaloyl group, benzoyl group, benzyl group, α-methylbenzyl group, trityl group, benzhydryl group , A protecting group selected from the group consisting of a paranitrobenzyl group and a paramethoxybenzyl group.

More preferably, R ^{2 in the} general formula (1) is a benzyl group.

  Examples of the compound represented by the formula (1) include 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester and 1-benzyl-4-piperidinone-3-carboxylic acid ethyl ester.

  In the present invention, using a catalyst, the carbonyl group of the compound represented by the general formula (1) is asymmetrically reduced, and the general formula (2):

(Wherein R ¹ _, R ² and m are the same as above) or general formula (3);

(Wherein R ¹ _, R ² and m are the same as described above), and are converted into an optically active trans-form nitrogen-containing cyclic β-hydroxyester.

  The catalyst is preferably an enzyme that stereoselectively reduces a carbonyl group.

  Further, the general formulas (2) and (3) are specifically trans-1-benzyl-4-hydroxypyrrolidine-3-carboxylic acid methyl ester or trans-1-benzyl-4-hydroxypiperidine-3-carboxylic acid. Corresponds to acid ethyl ester.

  In addition, "optical activity" here means that the optical purity of one optical isomer is high among various possible optical isomers.

  In the present invention, the optically active trans-form nitrogen-containing cyclic β-hydroxy ester represented by the general formula (2) or (3) has an optical purity of 50% e.e. e. Or more, more preferably 90% e.e. e. Or more, more preferably 99% e.e. e. That's it.

  Further, the trans isomer ratio of the nitrogen-containing cyclic β-hydroxy ester produced by asymmetric reduction is preferably 20% or more, more preferably 50% or more, and further preferably 90% or more.

  The trans isomer ratio can be determined by analyzing nitrogen-containing cyclic β-hydroxy ester by high performance liquid chromatography or the like, and “trans product peak area ÷ (trans isomer peak area + cis isomer peak area) × 100”. .

Moreover, optical purity was calculated | required from the following formula.

In the trans isomer, the optical isomer represented by the general formula (2) is the main product:
(Optical purity of optical isomer represented by general formula (2) (% ee) = {(peak area of general formula (2)) − (peak area of general formula (3))} ÷ {(general Peak area of formula (2)) + (peak area of general formula (3))} × 100

Among trans isomers, when the optical isomer represented by the general formula (3) is the main product:
(Optical purity of optical isomer represented by general formula (3) (% ee) = {(peak area of general formula (3)) − (peak area of general formula (2))} ÷ {(general Peak area of formula (2)) + (peak area of general formula (3))} × 100

The enzyme source used in the present invention may be derived from a microorganism having the ability to asymmetrically reduce the nitrogen-containing cyclic β-ketoesters to optically active trans-form nitrogen-containing cyclic β-hydroxyesters.

  As used herein, the term “derived from a microorganism” may be a cell of the microorganism itself, a culture solution of the microorganism, or a treated product of the microorganism, or an enzyme obtained from the microorganism. Also included are transformants into which a DNA encoding an enzyme having the above reducing activity has been introduced.

  The microbial cell processed product is not particularly limited. For example, a dried microbial cell obtained by dehydration using acetone or diphosphorus pentoxide or drying using a desiccator or a fan, a surfactant-treated product, or a lysed enzyme-treated product. Examples thereof include immobilized cells or cell-free extract preparations obtained by disrupting cells. Furthermore, an enzyme that catalyzes the asymmetric reduction reaction may be purified from the culture and used.

  These may be used alone or in combination of two or more. In addition, these enzyme sources may be immobilized and used by a known method.

  Microorganisms having the ability to asymmetrically reduce nitrogen-containing cyclic β-ketoesters to optically active trans-form nitrogen-containing cyclic β-hydroxyesters can be found by the method described below.

  For example, it is performed as follows. 40 g glucose, 3 g yeast extract, 6.5 g diammonium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 0.8 g magnesium sulfate heptahydrate, 60 mg zinc sulfate heptahydrate, 90 mg iron sulfate heptahydrate, sulfuric acid Put 5 ml of liquid medium (pH 7) consisting of 5 mg of copper pentahydrate, 10 mg of manganese sulfate tetrahydrate, and 100 mg of sodium chloride (each per liter) into a test tube and sterilize it, then aseptically inoculate the microorganism. Incubate with shaking at 30 ° C. for 2 to 3 days.

Thereafter, the bacterial cells were collected by centrifugation, suspended in 1-5 ml of phosphate buffer containing 2-10% glucose, and 2.5-25 mg of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester in advance. Shake at 30 ° C for 2-3 days in addition to the test tube. Under the present circumstances, what dried the microbial cell obtained by centrifugation in the desiccator or acetone can also be used. Furthermore, NAD ⁺ and / or NADP ⁺ and glucose dehydrogenase and glucose, or formate dehydrogenase and formic acid are added when reacting these microorganisms or processed products thereof with nitrogen-containing cyclic β-ketoesters. Also good. Further, an organic solvent may coexist in the reaction system.

  After the conversion reaction, extraction is performed with a suitable organic solvent, and the resulting 1-benzyl-4-hydroxypyrrolidine-3-carboxylic acid methyl ester is analyzed by high performance liquid chromatography or the like.

Any microorganism can be used as the microorganism that can be used in the present invention as long as it has an ability to asymmetrically reduce nitrogen-containing cyclic β-ketoesters to trans-form nitrogen-containing cyclic β-hydroxyesters. da (Candida) genus Kluyveromyces (Kluyveromyces) genus, Lipomyces (Lipomyces) genus, Rodotorura (Rhodotorula) genus Saccharomyces (Saccharomyces) genus Trichosporon (Trichosporon) genus, shake Bun di Monas (Brevundimonas) genus Cellulomonas (Cellulomonas ) genus, Deboshia (Devosia spp.), Micrococcus (Micrococcus) genus Serratia (Serratia) genus, sphingolipid Examples include microorganisms belonging to the genus Sphingobacterium .

In particular, when the absolute configuration is to be converted to a trans-form nitrogen-containing cyclic β-hydroxy ester having the general formula (2), the genus Candida, the genus Lipomyces, the genus Rhodotorula , Trichosporon (Trichosporon) genus, shake Bun di Monas (Brevundimonas) genus Cellulomonas (Cellulomonas) genus, Deboshia (Devosia spp.), Serratia (Serratia) genus, sphingomyelin genus (Sphingobacterium) microorganism belonging to the genus is preferable. More preferably, Candida-Magnolie (Candida magnoliae), Candida parapsilosis (Candida parapsilosis), Lipomyces-Sutakei (Lipomyces starkeyi), Rodotorura-Guruchinisu (Rhodotorula glutinis), Trichosporon asteroid scan (Trichosporon asteroides), Burebundi Monas-Diminuta (Brevundimonas diminuta), Cellulomonas sp (Cellulomonas sp.), Deboshia-Ribofurabina (Devosia riboflavina), Serratia marcescens (Serratia marcescens), sphingomyelin bacterin Such as Umm Supirichiboramu (Sphingobacterium spiritivorum), and the like.

In particular, when the absolute configuration is to be converted to a trans-form nitrogen-containing cyclic β-hydroxyester having the general formula (3), the genus Candida, the genus Kluyveromyces , the saccharomyces ( Microorganisms belonging to the genus Saccharomyces, the genus Trichosporon, the genus Brevundimonas, the genus Devosia and the genus Micrococcus are preferred.

More preferably, the candy da Magnolie (Candida magnoliae), Candida Maris (Candida maris), Candida parapsilosis (Candida parapsilosis), Kluyveromyces Porisuporasu (Kluyveromyces polysporus), Saccharomyces Bayanasu (Saccharomyces bayanus), Saccharomyces cerevisiae (Saccharomyces cerevislae), Trichosporon Kyutaneumu (Trichosporon cutaneum), Bacillus megaterium (Bacillus megaterium), shake Bun di Monas-Diminuta (Brevundimonas diminut ), Deboshia-Ribofurabina (Devosia riboflavina), and the like, such as Micrococcus luteus (Micrococcusluteus).

  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.

  For culturing these microorganisms, any medium can be used as long as it contains a nutrient source that can be assimilated by these microorganisms. For example, 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, fats and oils such as soybean oil and rapeseed oil, Or a carbon source such as a mixture thereof; a nitrogen source such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor; and other nutrient sources such as other inorganic salts and vitamins; -A normal mixed medium can be used. What is necessary is just to select these culture media suitably according to the kind of microorganisms to be used.

  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. In the case of reacting microorganisms with nitrogen-containing cyclic β-ketoesters, the culture solution containing the microorganisms can usually be used as it is for the reaction, but a concentrate of the culture solution can also be used. . In addition, when a component in the culture solution adversely affects the reaction, a bacterial cell or a processed bacterial cell obtained by treating the culture fluid by centrifugation or the like can also be used.

  The microbial cell processed product is not particularly limited. For example, a dried microbial cell obtained by dehydration using acetone or diphosphorus pentoxide or drying using a desiccator or a fan, a surfactant-treated product, or a lysed enzyme-treated product. Examples thereof include immobilized cells or cell-free extract preparations obtained by disrupting cells. Furthermore, an enzyme that catalyzes the asymmetric reduction reaction may be purified from the culture and used.

  In the reduction reaction, the nitrogen-containing cyclic β-ketoester as a substrate may be added all at the beginning of the reaction or may be added in portions as the reaction proceeds. The temperature during the reaction is usually 10 to 60 ° C., preferably 20 to 40 ° C., and the pH during the reaction is in the range of 2.5 to 9, preferably 5 to 9. The amount of the enzyme source in the reaction solution may be appropriately determined according to the ability to reduce these substrates. The substrate concentration in the reaction solution is preferably 0.01 to 50% (W / V), more preferably 0.1 to 30% (W / V). 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 enzyme source, and other reaction conditions. Usually, it is preferable to set each condition so that the reaction is completed in 2 to 168 hours.

  In order to promote the reduction reaction, it is preferable to add an energy source such as glucose, ethanol or isopropanol to the reaction solution at a ratio of 0.5 to 30% because excellent results can be obtained. Complements such as reduced nicotinamide / adenine dinucleotide (hereinafter abbreviated as NADH), reduced nicotinamide / adenine dinucleotide phosphate (hereinafter abbreviated as NADPH), etc., which are generally required for reduction by biological methods. The reaction can also be promoted by adding an enzyme. In this case, specifically, these are added directly to the reaction solution.

Further, in order to promote the reduction reaction, it is preferable to perform the reaction in the presence of an enzyme that reduces NAD ⁺ and / or NADP ⁺ to the respective reduced form and the substrate for the reduction, because excellent results are obtained. . For example, glucose dehydrogenase as the enzyme that reduces to the reduced form, glucose coexists as the substrate for reduction, or formate dehydrogenase as the enzyme that reduces to the reduced form, and formic acid as the substrate for reduction, respectively Coexist.

  Even when a transformant containing DNA encoding the enzyme is used instead of the enzyme that catalyzes the reduction reaction of the present invention, trans-form nitrogen-containing cyclic β-hydroxyesters can be produced in the same manner. Further, even when a transformant containing both the DNA encoding the enzyme and the DNA encoding a polypeptide having a coenzyme regeneration ability is used, the optically active trans-form nitrogen-containing cyclic β-hydroxy ester is similarly obtained. Can be produced. In particular, when a transformant containing both the DNA encoding the enzyme and the DNA encoding a polypeptide capable of coenzyme regeneration is used, an enzyme for regenerating the coenzyme is separately prepared and added. Therefore, the trans isomer-containing cyclic β-hydroxyester can be produced more efficiently.

  A transformant containing DNA encoding the polypeptide of the present invention, or a transformant containing both DNA encoding the polypeptide of the present invention and DNA encoding a polypeptide having a coenzyme regeneration ability, Needless to say, the cultured cells can be used for the production of trans-form nitrogen-containing cyclic β-hydroxyesters as treated products. 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 transformant containing both the DNA encoding the enzyme and the DNA encoding a polypeptide having coenzyme regeneration ability encodes the DNA encoding the enzyme and a polypeptide having coenzyme regeneration ability In addition to incorporating both of the DNA into the same vector and introducing it into the host cell, these two types of DNA are incorporated into two different vectors of different incompatibility groups, respectively. It can also be obtained by introducing it into the same host cell.

Examples of vectors incorporating both the DNA encoding the enzyme and the DNA encoding a polypeptide having coenzyme regeneration ability include the oxidoreductase gene derived from Candida magnolia described in WO01 / 040450 Examples include pNTCRG in which a Bacillus megaterium-derived glucose dehydrogenase gene is introduced into the expression vector pNTCR. An example of a transformant containing both the DNA encoding the enzyme and the DNA encoding a polypeptide having a coenzyme regeneration ability is E. coli. obtained by transforming E. coli HB101; E. coli HB101 (pNTCRG). This transformed microorganism is deposited under the accession number FERM BP-6898 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at 1st, 1st, 1st, 1st East, Tsukuba, Ibaraki, Japan.

  Culture of transformants containing DNA encoding the enzyme, and culture of transformants containing both DNA encoding the enzyme and DNA encoding a polypeptide having a coenzyme regeneration ability allow them to grow As long as it is normal, it can be carried out using a liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like.

  Furthermore, it is also effective to add a surfactant such as Triton (manufactured by Nacalai Tesque), Span (manufactured by Kanto Chemical Co., Ltd.), Tween (manufactured by Nacalai Tesque) to the reaction solution. In addition, an organic solvent insoluble in water such as ethyl acetate, butyl acetate, isopropyl ether, toluene, hexane, or the like is added to the reaction solution in order to avoid inhibition of the reaction by the substrate and / or the alcohol that is the product of the reduction reaction. May be. Furthermore, for the purpose of increasing the solubility of the substrate, an organic solvent soluble in water such as methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide and the like can be added.

  The collection of trans-form nitrogen-containing cyclic β-hydroxyesters produced by the reduction reaction is not particularly limited, but ethyl acetate, toluene, t-butyl methyl ether, hexane, etc. directly from the reaction solution or after separating the cells, etc. Extraction with the above solvent, followed by dehydration and purification by distillation, silica gel column chromatography or the like makes it possible to easily obtain highly pure trans-form nitrogen-containing cyclic β-hydroxyesters.

EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited at all by these Examples. In the following description, “%” means “% by weight” unless otherwise specified.

Reference Example 1 Preparation of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt 1-Benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt is disclosed in JP-A-54-16466. The compound was synthesized with reference to the method disclosed in 1).

First, 15.0 g of benzylamine was dissolved in 13.8 g of methanol, 12.7 g of methyl acrylate was added dropwise little by little, and the mixture was reacted at 35 ° C. with stirring for 5 hours. After concentrating the product at 90 ° C., 24.7 g of toluene and 17.7 g of potassium carbonate were added and heated to 90 ° C. After heating, 20.8 g of methyl monochloroacetate was added dropwise and reacted for 17 hours. After the reaction, the mixture was cooled to room temperature and quenched by adding 49.5 g of water. The organic layer was separated and concentrated with an evaporator. To 40.5 g of this concentrated solution, 46.3 g of toluene and 36.5 g of 28% sodium methoxide were added and reacted at 25 ° C. The produced crystals were separated by filtration and then vacuum-dried to obtain 23.6 g of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt.

Reference Example 2 Preparation of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester 10 g of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt was dissolved in 50 ml of water, and 6N HCl was dissolved. And adjusted to pH 7. To this solution, 100 ml of t-butyl methyl ether was added for extraction, and the organic layer was evaporated under reduced pressure to obtain oily 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester.

Reference Example 3 Preparation of 1-benzyl-4-pyrrolidinone-3-carboxylic acid ethyl ester Org. Chem. 30, 740 (1964).

  First, 20.0 g of benzylamine was dissolved in 18.4 g of ethanol, 19.6 g of ethyl acrylate was added dropwise little by little, and the mixture was reacted at 35 ° C. with stirring for 5 hours. After concentrating the product at 90 ° C., 38.7 g of toluene and 18.7 g of potassium carbonate were added and heated to 90 ° C. After heating, 38.7 g of methyl monochloroacetate was added dropwise and reacted for 17 hours. After the reaction, the mixture was cooled to room temperature and quenched by adding 77.4 g of water. The organic layer was separated and concentrated with an evaporator. 100 g of toluene and 8 g of t-butoxy potassium were added to 20 g of this concentrated solution, and reacted at 5 ° C. with stirring. 50 g of water was added to the reaction solution, and the aqueous layer was separated. Next, the product was extracted from the aqueous layer with 50 g of diethyl ether saturated with hydrochloric acid. The organic layer was concentrated with an evaporator to obtain 11 g of 1-benzyl-4-pyrrolidinone-3-carboxylic acid ethyl ester.

Reference Example 4 Purification of Reductase Derived from Breven Dimonas diminuta The present inventors converted ethyl N-Boc-2-amino-3-cyclohexyl-3- oxopropionate to N-Boc-2-amino-3. -Reductase RBD derived from Brevundimonas diminuta which reduces to ethyl cyclohexyl-3-hydroxypropionate converts 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester to (3S, 4R) -4- It has been found to convert to hydroxypyrrolidine-3-carboxylic acid methyl ester. A method for purifying the reductase RBD used in Example 6 will be described below.

First, from the blur Boon di Sphingomonas-Diminuta (Brevundimonas diminuta) NBRC12697 strain was isolated a polypeptide having reductase activity were purified to single. Unless otherwise noted, purification operations were performed at 4 ° C.

  The reductase activity was calculated as follows. First, an appropriate amount of a crude enzyme solution and a 100 mM phosphate buffer (pH 6.5) are added to a test tube to make a total volume of 0.5 ml. Further, 1 mg of NADH and 0.25 mg of ethyl N-Boc-2-amino-3-cyclohexyl-3-oxopropionate as a substrate were added and reacted at 30 ° C. for 2 hours with shaking. After the reaction, 1 ml of ethyl acetate was added for extraction.

  The produced N-Boc-2-amino-3-cyclohexyl-3-hydroxypropionate was quantified by capillary gas chromatography (column: InertCAP5 (ID 0.25 mm × 30 m) manufactured by GL Science Co., Ltd.), column temperature: 200 ° C. , Carrier gas: helium (70 kPa), detection: FID).

  Enzyme activity was calculated from the amount of ethyl N-Boc-2-amino-3-cyclohexyl-3-hydroxypropionate produced. The activity for producing 1 μmol of N-Boc-2-amino-3-cyclohexyl-3-hydroxypropionate per minute under the reaction conditions was defined as 1 unit.

(Microbial culture)
From 5L jar fermenter (manufactured by Maruhishi Bioengineer), meat extract 10g, peptone 10g, yeast extract 5g, sodium chloride 3g, Adecanol LG-109 (manufactured by NOF Corporation) 0.1g (all per liter) 3 L of a liquid medium (pH 7) to be prepared was prepared and steam sterilized at 120 ° C. for 20 minutes. This medium was inoculated with 15 ml of a culture solution of Brevundimonas diminuta NBRC12697 previously cultured in the same medium, with a stirring speed of 450 rpm, an aeration rate of 0.9 NL / min, and 30 ° C. Culture was performed for 16 hours.

(Preparation of cell-free extract)
Bacteria were collected from the culture broth by centrifugation, and washed with 0.8% aqueous sodium chloride solution. The cells are suspended in a 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol, disrupted using a SONIFIER 250 ultrasonic crusher (manufactured by BRANSON), and then centrifuged to separate the bacteria. A cell-free extract was obtained by removing body residues.

(DEAE-TOYOPEARL column chromatography)
The above cell-free extract is applied to a DEAE-TOYOPEARL 650M (Tosoh Corp.) column (400 ml) pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol. Minutes were adsorbed. After washing the column with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 0.3 M).

(Phenyl-TOYOPEARL column chromatography)
In the active fraction obtained by DEAE-TOYOPEARL column chromatography, ammonium sulfate was dissolved to a final concentration of 1.0 M and glycerol was dissolved to a final concentration of 10%, 1.0 M ammonium sulfate and 5 mM β-mercaptoethanol and The active fraction was adsorbed by applying to a Phenyl-TOYOPEARL 650M (manufactured by Tosoh Corporation) column (50 ml) pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 10% glycerin. After washing the column with the same buffer, the active fraction was eluted with a linear ammonium sulfate gradient (from 1.0 M to 0 M). The active fractions were collected and dialyzed overnight against 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin.

(5′-AMP Sepharose column chromatography)
The active fraction obtained by Phenyl-TOYOPEARL column chromatography was pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin 5′-AMP Sepharose 64B (Amersham) The product was applied to a column (14 ml) manufactured by Bioscience Co., Ltd. to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 2 M) to obtain a purified preparation of a single polypeptide electrophoretically.

(Example 1) Production of (3S, 4R) or (3R, 4S) -4-hydroxypyrrolidine-3-carboxylic acid methyl ester Glucose 40 g, yeast extract 3 g, diammonium hydrogen phosphate 6.5 g, dihydrogen phosphate 1 g of potassium, 0.8 g of magnesium sulfate heptahydrate, 60 mg of zinc sulfate heptahydrate, 90 mg of iron sulfate heptahydrate, 5 mg of copper sulfate pentahydrate, 10 mg of manganese sulfate tetrahydrate, 100 mg of sodium chloride (any 5 ml of a liquid medium (pH 7) having a composition of 1 liter) was dispensed into a large test tube and steam sterilized at 120 ° C. for 20 minutes.

  These liquid media were aseptically inoculated with one platinum loop of the microorganisms shown in Table 1 and cultured with shaking at 30 ° C. for 72 hours. After culture, each culture solution was centrifuged to collect the cells, and the cells were suspended in 0.6 ml (pH 6.5) of 100 mM phosphate buffer containing 1% glucose. This bacterial cell suspension was added to a test tube containing 6 mg of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester in advance, and reacted at 30 ° C. for 24 hours. After the reaction, 0.6 ml of t-butyl methyl ether was added to each reaction solution and mixed well.

The organic layer diluted with 2-propanol was analyzed under the following analysis conditions to determine the conversion rate to 4-hydroxypyrrolidine-3-carboxylic acid methyl ester, trans isomer ratio, and optical purity. In addition, the ratio of the trans body when the sum of the peak areas of the cis body and the trans body in the following analysis condition 1 was defined as 100% was defined as the trans body ratio.

[High-performance liquid chromatography analysis condition 1 (for conversion rate, trans body ratio calculation)]
Column: Develosil ODS HG-3 (150 mm × 4.6 mm) manufactured by Nomura Chemical Co., Ltd.
Eluent: 10 mM potassium phosphate buffer / methanol = 6/4
Flow rate: 0.6 ml / min
Detection: 210nm
Retention time: cis body 18min, trans body 24min

[High-performance liquid chromatography analysis condition 2 (for optical purity calculation)]
Column: Daicel Chemical Industries, Ltd. Chiralpak OB-H (250 mm × 4.6 mm)
Eluent: n-hexane / 2-propanol = 95/5
Flow rate: 1 ml / min
Detection: 210nm
Retention time: cis body 24min and 34min, (3S, 4R) body 26min, (3R, 4S) body 40min

The results are summarized in Table 1.

(Example 2) Production of (3S, 4R) or (3R, 4S) -4-hydroxypyrrolidine-3-carboxylic acid methyl ester 10 g of meat extract, 10 g of peptone, 5 g of yeast extract, 3 g of sodium chloride (all per 1 L) 7 ml of a liquid medium (pH 7) having the following composition was dispensed into a large test tube and steam sterilized at 120 ° C. for 20 minutes. These liquid media were aseptically inoculated with one platinum loop of the microorganisms shown in Table 2 and cultured with shaking at 30 ° C. for 72 hours.

  After culture, each culture solution was centrifuged to collect the cells, and the cells were suspended in 0.6 ml (pH 6.5) of 100 mM phosphate buffer containing 1% glucose. This bacterial cell suspension was added to a test tube containing 6 mg of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester in advance, and reacted at 30 ° C. for 24 hours.

  After completion of the reaction, analysis was performed in the same manner as in Example 1, and the conversion rate and the optical purity of trans-4-hydroxypyrrolidine-3-carboxylic acid methyl ester were analyzed.

  The results are summarized in Table 2.

(Example 3) Production of (3S, 4R) or (3R, 4S) -4-hydroxypyrrolidine-3-carboxylic acid methyl ester A liquid consisting of 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl (each per liter). 50 ml of the medium (pH = 7) was dispensed into a 500 ml Sakaguchi flask and steam sterilized at 120 ° C. for 20 minutes. Each liquid E. coli shown in Table 3 was aseptically inoculated into these liquid culture medium and cultured with shaking at 37 ° C. for 72 hours.

  After culture, 0.9 ml of each culture solution, 10 mg of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt, 1 mg of NAD (or NADP), 10 mg of glucose, 1M phosphate buffer (pH 6.5) 1 ml was added to the test tube and reacted at 30 ° C. for 24 hours.

  After completion of the reaction, analysis was conducted in the same manner as in Example 1 to analyze the conversion rate and the optical purity of the product.

  The results are summarized in Table 3. The recombinant bacteria indicated by the deposit number is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan.

(Example 4) Production of (3S, 4S) or (3R, 4R) -4-hydroxypiperidine-3-carboxylic acid ethyl ester A liquid comprising a composition of 16 g of tryptone, 10 g of yeast extract and 5 g of NaCl (all per 1 L). 50 ml of the medium (pH = 7) was dispensed into a 500 ml Sakaguchi flask and steam sterilized at 120 ° C. for 20 minutes. These liquid media were aseptically inoculated with one platinum loop of various recombinant E. coli shown in Table 3 and cultured with shaking at 30 ° C. for 72 hours.

  After culturing, 0.9 ml of each culture solution is added to a test tube containing 10 mg of 1-benzyl-4-piperidinone-3-carboxylic acid ethyl ester in advance, 1 mg of NAD (or NADP), 10 mg of glucose, 1M phosphate buffer solution (PH 6.5) 0.1 ml was added and reacted at 30 ° C. for 24 hours.

After the reaction, 0.6 ml of t-butyl methyl ether was added to each reaction solution and mixed well. The organic layer diluted with 2-propanol was analyzed under the following analysis conditions to determine the conversion rate, the trans isomer ratio of trans-4-hydroxypiperidine-3-carboxylic acid ethyl ester, and the optical purity.

[High-performance liquid chromatography analysis condition 3 (conversion rate)]
Column: Develosil ODS HG-3 (150 mm × 4.6 mm) manufactured by Nomura Chemical Co., Ltd.
Eluent: 10 mM potassium phosphate buffer / acetonitrile = 6/4
Flow rate: 0.7 min / ml
Detection: 210nm
Retention time: 1-benzyl-4-hydroxypiperidine-3-carboxylic acid ethyl ester 8 min

[High-performance liquid chromatography analysis condition 4 (for trans body ratio, optical purity calculation)]
Column: Daicel Chemical Industries, Ltd. Chiralcel OJ (250 mm × 4.6 mm × 2)
Eluent: n-hexane / 2-propanol = 9/1
Flow rate: 0.5 ml / min
Detection: 210nm
Retention time: cis body 39min, cis body 40min, (3S, 4S) body 30min, (3R, 4R) body 36min

The results are summarized in Table 4. The recombinant bacteria indicated by the deposit number is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan.

(Example 5) Production of (3S, 4R) -4-hydroxypyrrolidine-3-carboxylic acid ethyl ester Liquid medium (pH = 7) having a composition of 16 g of tryptone, 10 g of yeast extract and 5 g of NaCl (all per 1 L). 50 ml was dispensed into a 500 ml Sakaguchi flask and steam sterilized at 120 ° C. for 20 minutes. These liquid media were inoculated with Escherichia coli HB101 (pNTCRG) described in the pamphlet of International Publication No. WO01 / 040450 and cultured with shaking at 37 ° C. for 72 hours.

After culturing, 0.9 ml of each culture solution, 10 mg of 1-benzyl-4-pyrrolidinone-3-carboxylic acid ethyl ester, NADP ⁺ 1 mg, glucose 10 mg, 0.1 M phosphate buffer (pH 6.5) 0.1 ml were added to a test tube. In addition, the reaction was performed at 30 ° C. for 24 hours.

After completion of the reaction, analysis was conducted in the same manner as in Example 1 to determine the amount of product (3S, 4R) -4-hydroxypyrrolidine-3-carboxylic acid ethyl ester and its optical purity. Ratio 79%, optical purity 99% e.e. e. That was all.

(Example 6) Production of (3S, 4R) -4-hydroxypyrrolidine-3-carboxylic acid methyl ester In 50 ml of 100 mM phosphate buffer (pH 6.5), 3.5 g of glucose, Brevundimonas diminuta ( Brevundimonas) diminuta ) Add oxidoreductase RBD 1000 U prepared from NBRC12697 strain by the method shown in Reference Example 4, glucose dehydrogenase “GLUCDH“ Amano2 ”(manufactured by Amano Enzyme) 50 mg, NAD ⁺ 5 mg, and stir at 30 ° C. While 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt was added 4 times by 1 g every 2 hours. Meanwhile, the reaction was maintained at pH 6.5 with 5N NaOH and 5N H ₂ SO ₄ .

After the reaction for 24 hours, 200 ml of t-butyl methyl ether was added to the reaction solution for extraction, and the organic layer was distilled off under reduced pressure to give oily (3S, 4R) -4-hydroxypyrrolidine-3-carboxylic acid. 2.3 g of methyl ester was obtained. Chemical purity is 71.5%, trans isomer ratio is 86.1%, optical purity is 99.9% e.e. e. That was all.

(Example 7) Production of (3S, 4R) -4-hydroxypyrrolidine-3-carboxylic acid methyl ester Liquid medium (pH = 7) having a composition of tryptone 16 g, yeast extract 10 g, NaCl 5 g (all per 1 L). 50 ml was dispensed into a 500 ml Sakaguchi flask and steam sterilized at 120 ° C. for 20 minutes. This liquid medium was inoculated with Escherichia coli HB101 (pNTCRG) described in WO 01/040450, and cultured with shaking at 37 ° C. for 18 hours.

Add 3 mg of NADP ⁺ and 3.5 g of glucose to 50 ml of the obtained culture solution, and add 1 g of 1-benzyl-4-pyrrolidinone-3-carboxylic acid methyl ester sodium salt 4 times every 2 hours while stirring at 30 ° C. did. During the reaction, the pH of the reaction solution was maintained at 6.5 with 5N NaOH and 5N H ₂ SO ₄ .

  After completion of the reaction, the reaction solution was extracted by adding 200 ml of t-butyl methyl ether, and the organic layer was distilled off under reduced pressure and purified by silica gel column chromatography to obtain oily (3S, 4R) -4-hydroxypyrrolidine. 0.94 g of -3-carboxylic acid methyl ester was obtained. The trans isomer ratio is 99.3%, and the optical purity is 99.9% e.e. e. That was all.

Claims (8)

General formula (1);

(Wherein R ¹ represents a linear or branched alkyl group; R ² represents an amino-protecting group), and the carbonyl group of the nitrogen-containing cyclic β-ketoester or salt thereof is stereoselected. General formula (2) by acting an enzyme source having the ability to reduce oxidatively;

(Wherein R ¹ _and R ² are as defined above), wherein the enzyme source is Escherichia coli HB101 (pNTCRG); FERM BP-6898, Escherichia coli HB101 (pNTRGG1); FERM BP-7858, Lipomyces-Sutakei (Lipomyces starkeyi) IFO 0678, Sphingobacterium-Supirichiboramu (Sphingobacterium spiritivorum) IFO 14948, or Trichosporon Kyutaneumu (Trichosporon cutaneum) of IFO 1198 It is one of the fungus body, the culture solution, and the processed product thereof. The process for producing an optically active trans-form nitrogen-containing cyclic β- hydroxyester. General formula (1);

(Wherein R ¹ represents a linear or branched alkyl group; R ² represents an amino-protecting group), and the carbonyl group of the nitrogen-containing cyclic β-ketoester or salt thereof is stereoselected. A general formula (3) by acting an enzyme source having the ability to reduce oxidatively;

(Wherein R ¹ _and R ² are the same as above), wherein the enzyme source is Escherichia coli HB101 (pTSBG1); FERM BP-7119, Brevundimonas diminuta IFO 3140, Kluyveromyces polysporus IFO 0996, Saccharomyces bay Saccharomyces Trichosporon asteroids oides) A method for producing an optically active trans-form nitrogen-containing cyclic β-hydroxyester, characterized in that it is any of the cells of IFO 0173, a culture solution, and a processed product thereof. General formula (4);

(Wherein R ¹ represents a linear or branched alkyl group; R ² represents an amino-protecting group), and the carbonyl group of the nitrogen-containing cyclic β-ketoester or salt thereof is stereoselected. A general formula (5) by acting an enzyme source having the ability to reduce oxidatively;

(Wherein R ¹ _and R ² are the same as above), wherein the enzyme source is Escherichia coli HB101 (pTSCS); FERM BP-10024, Escherichia coli HB101 (pNTDRG1); FERM BP-8458, Escherichia coli HB101 (pNTSGGG1); FERM P-18449 cells, culture fluid, and any of those processed products A process for producing an optically active trans-form nitrogen-containing cyclic β-hydroxyester. General formula (4);

(Wherein R ¹ represents a linear or branched alkyl group; R ² represents an amino-protecting group), and the carbonyl group of the nitrogen-containing cyclic β-ketoester or salt thereof is stereoselected. General formula (6) by acting an enzyme source having the ability to reduce oxidatively;

(Wherein R ¹ _and R ² are the same as above), wherein the enzyme source is Escherichia coli HB101 (pNTS1G); FERM BP-5835, Escherichia coli HB101 (pNTFPG); FERM BP-7117, Escherichia coli HB101 (pNTRGG1); FERM BP-7858, Escherichia coli HB101 (pTSBG1 and FER, 19) A process for producing an optically active trans-form nitrogen-containing cyclic β-hydroxyester, characterized in that The production method according to any one of claims 1 to 4, wherein R ¹ is a linear or branched alkyl group having 1 to 10 carbon atoms. The production method according to claim 5, wherein R ¹ is either a methyl group or an ethyl group. An enzyme that reduces oxidized nicotinamide / adenine dinucleotide and / or oxidized nicotinamide / adenine dinucleotide phosphate to each reduced form and a substrate for the reduction coexist, The manufacturing method in any one of Claims 1-6. 8. The enzyme that reduces oxidized nicotinamide / adenine dinucleotide and / or oxidized nicotinamide / adenine dinucleotide phosphate to the respective reduced form is glucose dehydrogenase or formate dehydrogenase. Manufacturing method.