WO2015141758A1

WO2015141758A1 - Asymmetric hydrolysis of 3-substituted glutarimide

Info

Publication number: WO2015141758A1
Application number: PCT/JP2015/058155
Authority: WO
Inventors: 増俊野尻; 雅俊大貫; 伸行堀之内; 慎日比; 順小川; 八十原　良彦
Original assignee: 株式会社カネカ
Priority date: 2014-03-20
Filing date: 2015-03-19
Publication date: 2015-09-24

Abstract

The present invention addresses the problem of providing a method for producing an optically active 3-substituted glutaric acid monoamide, which is useful as a pharmaceutical intermediate, said method being suitable for industrial uses. The present invention provides, as a solution for the above-described problem, a method for producing an optically active 3-substituted glutaric acid monoamide by subjecting an easily available 3-substituted glutarimide to an asymmetric hydrolysis with use of an enzyme source.

Description

Asymmetric hydrolysis of 3-substituted glutarimide

The present invention relates to a method for producing an optically active 3-substituted glutaric acid monoamide useful as a pharmaceutical intermediate.

For example, the following method is known as a method for producing an optically active 3-substituted glutaric monoamide.
1) A method for obtaining optically active 3-substituted glutaric acid monoamide by optical resolution of racemic 3-substituted glutaric acid monoamide by salt formation crystallization or the like (Patent Document 1) (Non-Patent Document 1).
2) An optically active 3-substituted glutaric acid monoamide obtained by obtaining an optically active 3-substituted glutaric acid monoester by asymmetric hydrolysis reaction of a 3-substituted glutaric acid diester using esterase or lipase and then amidating it. (Non-patent Document 2).

However, since the method 1) is an optical resolution method, a yield of 50% or more cannot be expected. On the other hand, the method 2) requires high pressure and low temperature conditions in the amidation of the subsequent step of the asymmetric hydrolysis reaction. .

As described above, the currently known methods for producing optically active 3-substituted glutaric monoamides are not advantageous methods for industrial production in terms of yield and reaction conditions.

International Publication No. 97/22578

In view of the above, an object of the present invention is to provide a method suitable for industrial use for producing an optically active 3-substituted glutaric acid monoamide useful as a pharmaceutical intermediate.

As a result of intensive studies based on the above problems, the present inventors obtained a microorganism that asymmetrically hydrolyzes 3-substituted glutarimide. By using the microorganism, optically active 3-substituted glutaric acid monoamide can be produced from 3-substituted glutarimide under mild reaction conditions, and the present invention has been completed.

That is, the present invention provides the following formula (1);

(In the formula, R may have an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, or an optionally substituted group. A preferable alkynyl group having 2 to 8 carbon atoms, an aryl group having 4 to 20 carbon atoms which may have a substituent, or an aralkyl group having 5 to 20 carbon atoms which may have a substituent. An enzyme source having asymmetric hydrolysis activity is allowed to act on the 3-substituted glutaric imide represented by the following formula (2);

(Wherein, * represents an asymmetric carbon atom, R is the same as above), and relates to a process for producing an optically active 3-substituted glutaric acid monoamide compound.

This specification includes the contents described in the specification of Japanese Patent Application No. 2014-058189 which is the basis of the priority of the present application.

According to the present invention, an optically active 3-substituted glutaric acid monoamide can be produced by a reaction under mild conditions using a prochiral 3-substituted glutarimide as a raw material and an enzyme source.

Hereinafter, the present invention will be described in detail using embodiments. The present invention is not limited to these.

The 3-substituted glutarimide that is the raw material of the present invention is represented by the following formula (1);

In the formula (1), R is an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, and a substituent. An alkynyl group having 2 to 8 carbon atoms which may have a substituent, an aryl group having 4 to 20 carbon atoms which may have a substituent, or an aryl group having 5 to 20 carbon atoms which may have a substituent. An aralkyl group is shown.

The alkyl group having 1 to 8 carbon atoms may be linear or branched. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl Group, tert-butyl group, pentanyl group, hexanyl group, heptanyl group, octanyl group and the like.

Examples of the alkenyl group having 2 to 8 carbon atoms include ethenyl group, propenyl group, butenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group and the like.

Examples of the alkynyl group having 2 to 8 carbon atoms include ethynyl group, propynyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group and the like.

The aryl group having 4 to 20 carbon atoms may have a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. The number of heteroatoms contained in one aryl group is not particularly limited, but is usually 1 to 2. The heteroatom is particularly preferably a nitrogen atom. Examples of the aryl group having 4 to 20 carbon atoms include phenyl group, naphthyl group, anthranyl group, pyridyl group, pyrimidyl group, indanyl group, and indenyl group.

The aralkyl group having 5 to 20 carbon atoms may have a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. The number of heteroatoms contained in one aralkyl group is not particularly limited, but is usually 1 to 2. The heteroatom is particularly preferably a nitrogen atom. Examples of the aralkyl group having 5 to 20 carbon atoms include benzyl group, naphthylmethyl group, anthranylmethyl group, pyridylmethyl group, pyrimidylmethyl group, indanylmethyl group, and indenylmethyl group.

The alkyl group, alkenyl group, alkynyl group, aryl group and aralkyl group may have a substituent, and examples of the substituent include a halogen atom, a hydroxyl group, an amino group, and a nitro group. When the above group has a substituent, the number is not particularly limited, but is typically 1 to 2, preferably 1.

Specific examples of the 3-substituted glutarimide represented by the formula (1) include 3-isobutyl glutarimide, 3-propyl glutarimide, 3-phenyl glutarimide, 3- (4-chlorophenyl) glutarimide and the like.

The optically active 3-substituted glutaric monoamide that is the product of the present invention has the following formula (2):

In the formula (2), R is an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, and a substituent. An alkynyl group having 2 to 8 carbon atoms, an aryl group having 4 to 20 carbon atoms which may have a substituent, or an aryl group having 5 to 20 carbon atoms which may have a substituent An aralkyl group is shown.

R in the optically active 3-substituted glutaric acid monoamide of the formula (2) as the product is the same as R in the 3-substituted glutarimide of the formula (1) as the starting material. Details of R in formula (2) are as described in detail for R in formula (1).

The absolute configuration of the optically active 3-substituted glutaric monoamide represented by the formula (2) may be R or S. Specific examples of the optically active 3-substituted glutaric acid monoamide represented by the formula (2) include 3-isobutyl glutaric acid monoamide, 3-propyl glutaric acid monoamide, 3-phenylglutaric acid monoamide, whose absolute configuration is R, 3 -(4-chlorophenyl) glutaric acid monoamide, 3-isobutylglutaric acid monoamide having the absolute configuration S, 3-propylglutaric acid monoamide, 3-phenylglutaric acid monoamide, 3- (4-chlorophenyl) glutaric acid monoamide, etc. It is done. In the present invention, the optically active 3-substituted glutaric monoamide represented by the formula (2) has an optical purity (% ee) exceeding 0% ee, preferably 5% ee or more, more preferably 10% ee or more. More preferably 20% ee or more, more preferably 30% ee or more, more preferably 40% ee or more, more preferably 50% ee or more, more preferably 60% ee or more, more preferably 65% ee or more, more The optical purity (preferably 70% ee or higher, more preferably 75% ee or higher, more preferably 80% ee or higher, more preferably 85% ee or higher, more preferably 90% ee or higher, more preferably 95% ee or higher. % Ee). The definition of optical purity is as described in Examples.
Here, the three-dimensional structure of the optically active 3-substituted glutaric monoamide represented by the formula (2) whose absolute configuration is R is usually represented by the following formula.

(In the formula, details of R are as described in detail for R in formula (1)).
Here, the steric structure of the optically active 3-substituted glutaric monoamide represented by the formula (2) whose absolute configuration is S is usually represented by the following formula.

(In the formula, details of R are as described in detail for R in formula (1)).

In the present invention, the “enzyme source” includes an enzyme having an activity for asymmetric hydrolysis of the 3-substituted glutarimide of the formula (1) to the optically active 3-substituted glutaric acid monoamide of the formula (2). If it is, it will not specifically limit, The microorganisms which have the activity which asymmetrically hydrolyzes the said enzyme itself or the 3-substituted glutarimide of the said Formula (1) to the optically active 3-substituted glutaric acid monoamide of the said Formula (2) And the treated product thereof.

“Microbial cells of microorganisms” include, in addition to the cells themselves, a culture solution or cell suspension containing cells. In addition to wild-type strains, microbial cells include mutant strains that have gained advantageous properties, and further, DNA encoding the above-mentioned enzyme having asymmetric hydrolysis activity derived from the microorganism has been introduced. Transformed strains.

The “processed product” may be, for example, a crude extract, freeze-dried cells, acetone-dried cells, or a crushed product of these cells, and 3-substituted obtained from the cells. It may be an enzyme having an activity of asymmetric hydrolysis of glutaric imide. The degree of purification of the enzyme obtained from the cells is not particularly limited, and may be a purified enzyme or a partially purified enzyme.

When the enzyme source is the enzyme itself, the enzyme can be prepared by any means such as a cell-free protein synthesis system. The degree of enzyme purification is not particularly limited.

As an enzyme having an activity to asymmetrically hydrolyze 3-substituted glutarimide of the above formula (1) into an optically active 3-substituted glutaric acid monoamide of the above formula (2), contained in the above enzyme source or a processed product thereof For example, at least one selected from the group consisting of the following polypeptides (I) to (VI) can be preferably used.
(I) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(II) consisting of an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1, and the 3-substituted glutarimide of the formula (1) is represented by the formula (2) A polypeptide having an enzymatic activity for asymmetric hydrolysis to optically active 3-substituted glutaric acid monoamide;
(III) An amino acid sequence having 70% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the 3-substituted glutarimide of the formula (1) is substituted with the optically active 3-substituted group of the formula (2) A polypeptide having an enzymatic activity for asymmetric hydrolysis to glutaric monoamide;
(IV) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6;
(V) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 6, wherein the 3-substituted glutarimide of the formula (1) is converted to the formula (2) A polypeptide having an enzymatic activity for asymmetric hydrolysis to optically active 3-substituted glutaric acid monoamide;
(VI) consisting of an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 6, wherein the 3-substituted glutarimide of the formula (1) is substituted with the optically active 3-substitution of the formula (2) A polypeptide having an enzymatic activity that undergoes asymmetric hydrolysis to glutaric acid monoamide.

In the above (II) or (V), “one or more” relating to amino acid substitution, deletion, insertion or addition is, for example, 1 to 50, preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2. The amino acid substitution is preferably a conservative amino acid substitution. “Conservative amino acid substitution” refers to substitution between amino acids having similar physicochemical functions such as charge, side chain, polarity, and aromaticity. Amino acids with similar physicochemical functions include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (asparagine, glutamine, serine, threonine, cysteine, tyrosine), Classified into nonpolar amino acids (glycine, leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine) can do. The addition of one or more amino acids in the amino acid sequence described in SEQ ID NO: 1 or 6 is preferably 1 in total to at least one of the N-terminal and C-terminal of the amino acid sequence described in SEQ ID NO: 1 or 6. Or addition of multiple amino acids.

The identity of the polypeptide of (III) or (VI) to the amino acid sequence shown in SEQ ID NO: 1 or 6 is preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably Is 95% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more. In the present invention, the identity value of amino acid sequences indicates a value calculated with default settings using software (for example, FASTA, DANASYS, BLAST, Genetyx) that calculates identity between a plurality of amino acid sequences. The amino acid sequence identity value is calculated by calculating the number of matching amino acid residues when aligning a pair of amino acid sequences so that the degree of matching is maximized, and comparing the number of matching amino acid residues. Calculated as a percentage of the total number of amino acid residues in the sequence. Here, when there is a gap, the total number of amino acid residues is the number of amino acid residues obtained by counting one gap as one amino acid residue. For details on how to determine identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. MoI. Mol. Biol. 215, 403-410, 1990.

The similarity of the polypeptide of (III) or (VI) to the amino acid sequence of SEQ ID NO: 1 or 6 is preferably 75% or more, more preferably 80% or more, more preferably 85% or more, More preferably, it is 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more. The similarity value of amino acid sequences is calculated by adding the number of amino acid residues that match when aligning a pair of amino acid sequences so that the degree of coincidence is maximized, and the amino acid residues that have similar physicochemical functions. Then, it is calculated as a ratio of the total to the total number of amino acid residues of the compared amino acid sequences. Here, the amino acid sequence similarity can be calculated by a computer using the same software as described above with respect to amino acid sequence identity. The method for calculating the total number of amino acid residues is as described above for amino acid identity. The amino acid residues having similar physicochemical functions are as described above.

Asymmetric hydrolysis of the 3-substituted glutarimide of the formula (1) in the formula (II), (III), (V) or (VI) to the optically active 3-substituted glutaric acid monoamide of the formula (2) The enzyme activity can be confirmed according to the procedure described in Example 3 (1). The polypeptide preferably has a specific activity per 1 mg of total protein of 0.1 mU / mg or more, more preferably 1.0 mU / mg or more, more preferably 5.0 mU / mg or more, more preferably 10 mU / mg or more, Most preferably, it is 100 mU / mg or more, and the upper limit is not particularly limited, but for example, it can be added to the reaction system in the form of a protein mixture or purified product of 3000 mU / mg or less, typically 2000 mU / mg or less. The definition of the unit of enzyme activity and the measuring method are as described in Example 3 (1). The optical purity range of the optically active 3-substituted glutaric monoamide of formula (2) obtained by the enzyme activity is as described above.

The enzyme source can be used in a state immobilized by known means. Immobilization can be performed by methods well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.). The carrier for immobilizing the enzyme source is not particularly limited and can be appropriately selected and used by those skilled in the art.

Each microorganism described later in this specification can be obtained from a patent microorganism depositary or other research institution, and can also be separated from the natural world. For example, the microorganism identified by the NBRC number is the National Institute of Technology and Evaluation Biological Resource Center (Chiba, Japan), and the microorganism identified by the DSM number is the DSMZ-German microbial cell culture collection, AKU number. The microorganisms identified are available from the Fermentation Physiology and Brewing Science Laboratory (Kyoto, Japan), Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University.

Also, it can be separated from the natural world by enrichment culture using 3-substituted glutarimide or an inducer for inducing enzyme production.

The enzyme source having an asymmetric hydrolysis activity in the 3-substituted glutarimide is not particularly limited, and examples thereof include, for example, the genus Achromobacter, the genus Alcaligenes, the genus Burkholderia, the comamonas ( An enzyme source derived from a microorganism selected from the group consisting of the genus Comamonas, Delftia, and Pseudomonas.

Among the enzyme sources, examples of the enzyme source having the ability to produce 3-substituted glutaric monoamides whose absolute configuration is R include, for example, the genus Achromobacter, the genus Alcaligenes, and Burkholderia. ), An enzyme source derived from a microorganism selected from the group consisting of the genus Comamonas, the Delftia genus, and the Pseudomonas genus. Preferably, Achromobacter sp. Achromobacter xylosoxidans subspecies denitrificans, Alcaligenes faecalis, Burkholderia phytofirmans, coma Doboransu (Delftia acidovorans), Delftia species (Delftia sp.), Or Pseudomonas putida (Pseudomonas putida) enzyme source derived from microorganisms, and the like. More preferably, Achromobacter sp. A35 AKU 110, Achromobacter xylosoxidans sub sp. (Burkholderia phytofirmans) DSM 17436, Comamonas sp. I3-4, Delftia ovo acidovorans NBRC 14950, Delftia ovo sp.

In addition, the microorganism having the ability to produce a hydrolase derived from the microorganism may be either a wild strain or a mutant strain. Alternatively, microorganisms derived by genetic techniques such as cell fusion or gene manipulation can also be used. A microorganism that produces the genetically engineered enzyme is, for example, a process described in WO 98/35025 for isolating and / or purifying these enzymes to determine part or all of the amino acid sequence of the enzyme, this amino acid sequence A method comprising a step of obtaining a DNA sequence encoding an enzyme based on the above, a step of obtaining a recombinant microorganism by introducing this DNA into another microorganism, and a step of culturing the recombinant microorganism to obtain the enzyme, etc. Can be obtained.

Examples of the recombinant microorganism as described above include a transformed microorganism transformed with a plasmid having a DNA encoding the hydrolase. The host microorganism is preferably Escherichia coli.

The culture medium for the microorganism used as the enzyme source is not particularly limited as long as the microorganism can grow. For example, as a carbon source, carbohydrates such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid and esters thereof, oils such as rapeseed oil and soybean oil, and ammonium sulfate as a nitrogen source , Sodium nitrate, peptone, casamino acid, corn steep liquor, bran, yeast extract, etc. Inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium hydrogen phosphate, potassium dihydrogen phosphate and other malt sources, malt A normal liquid medium containing an extract, meat extract or the like can be used.

In addition, an inducer may be added to the medium in order to induce microbial enzyme production. Examples of the inducing agent include imide compounds. For example, examples of the imide compound include succinimide, 2-methylsuccinimide, glutarimide, maleimide, phthalimide, 3-isobutylglutarimide, 3-propylglutarimide, 3-phenylglutarimide, 3- (4-chlorophenyl) glutarimide, and the like. Can be mentioned.

The amount of these inducers added to the medium is 0.05% to 2.0%, preferably 0.1% to 1.0%. Here,% is% by weight. Cultivation is carried out aerobically. Usually, the cultivation time is about 1 to 5 days, the pH of the medium is 3 to 9, and the cultivation temperature is 10 to 50 ° C.

In the present invention, the stereoselective hydrolysis reaction of 3-substituted glutarimide (1) is carried out by adding 3-substituted glutarimide (1) as a raw material and the enzyme source to a suitable solvent, and adjusting the pH. This can be done by stirring.

Reaction conditions vary depending on the enzyme, microorganism or processed product, substrate concentration, etc. used. Usually, the substrate concentration is preferably about 0.1 to 99% by weight, more preferably 1 to 60% by weight. The reaction temperature is preferably 10 to 60 ° C, more preferably 20 to 50 ° C. The pH of the reaction is preferably 4-11, more preferably 6-9. The reaction time is preferably 0.5 to 120 hours, more preferably 1 to 120 hours, and particularly preferably 1 to 72 hours. The reaction can be carried out under atmospheric pressure conditions. The substrate can be added in batches, divided or added continuously. The reaction can be carried out batchwise or continuously.

In the optical resolution method disclosed in Patent Document 1 and Non-Patent Document 1, the target optically active substance can be obtained only from the racemic starting material in a yield of 50% at the maximum. In the method of the present invention, the optically active 3-substituted glutaric acid monoamide (2) can be obtained in a yield exceeding 50% from the 3-substituted glutaric imide (1) as a starting material. Is very advantageous.

Each of the optically active 3-substituted glutaric acid monoamides produced by the reaction can be isolated and purified by a conventional method. For example, a reaction liquid containing an optically active 3-substituted glutaric acid monoamide generated by hydrolysis reaction is extracted with an organic solvent such as ethyl acetate and toluene, and the organic solvent is distilled off under reduced pressure. Then, distillation, recrystallization, Alternatively, it can be isolated and purified by performing a treatment such as chromatography. Further, the filtrate from which the enzyme source has been removed from the reaction solution can be isolated and purified by neutralizing and crystallization using sulfuric acid or the like, and filtering out the precipitated target product.

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

[Example 1]
Stereoselective hydrolysis of 3- (4-chlorophenyl) glutarimide Each microorganism shown in Table 1 was sterilized in a test tube in 5 ml of medium (trypton 0.5 w / v% in water, yeast extract 0.5 w / v %, Glucose 0.1 w / v%, dipotassium hydrogen phosphate 0.1 w / v%, pH 7.0), and cultured with shaking at 30 ° C. for 65 hours. After completion of the culture, the cells were collected by centrifugation and suspended in 0.2 ml of 100 mM phosphate buffer (pH 7.0).

0.2 ml of this bacterial cell suspension and 1 mg of the substrate 3- (4-chlorophenyl) glutarimide synthesized by the method described in Reference Example 1 are mixed and shaken at 30 ° C. for 23 hours under atmospheric pressure. did. After completion of the reaction, the solid matter was removed by centrifugation, and the conversion rate (%) and optical purity (% ee) were determined by analyzing the substrate and product in the reaction solution by high performance liquid chromatography.
The results are shown in Table 1.

Conversion (%) = product amount / (base mass + product amount) × 100
Optical purity (% ee) = (A−B) / (A + B) × 100 (A and B represent the amount of the corresponding enantiomer, and A> B)

<High-performance liquid chromatography analysis conditions>
[Conversion rate analysis]
Column: 5C18-ARII (4.6 mmφ × 250 mm, manufactured by Nacalai Tesque)
Eluent: 20 mM phosphoric acid aqueous solution (pH 2.5) / acetonitrile = 6/4
Flow rate: 0.5 ml / min, column temperature: 30 ° C., measurement wavelength: 210 nm

[Analysis of optical purity]
Column: SUMICHIRAL OA-7000 (4.6 mmφ × 250 mm, manufactured by Sumika Chemical Analysis Co., Ltd.)
Eluent: 20 mM phosphoric acid aqueous solution (pH 2.5) / acetonitrile = 8/2
Flow rate: 0.5 ml / min, column temperature: room temperature, measurement wavelength: 210 nm

[Example 2]
Stereoselective hydrolysis of 3-isobutylglutarimide Each microorganism shown in Table 2 was sterilized in a test tube in 5 ml of medium (trypton 0.5 w / v% in water, yeast extract 0.5 w / v%, glucose 0 0.1 w / v%, dipotassium hydrogen phosphate 0.1 w / v%, pH 7.0), and cultured with shaking at 30 ° C. for 22 hours. After completion of the culture, the cells were collected by centrifugation and suspended in 0.2 ml of 100 mM phosphate buffer (pH 7.0).

0.2 ml of this bacterial cell suspension and 1 mg of the substrate 3-isobutylglutarimide synthesized by the method described in (Reference Example 2) were mixed and shaken at 30 ° C. for 24 hours under atmospheric pressure. After completion of the reaction, the solid matter was removed by centrifugation, and the conversion rate (%) was determined by analyzing the substrate and product in the reaction solution by high performance liquid chromatography.

Furthermore, the optical purity (% ee) was calculated | required by analyzing the derivative | guide_body obtained by derivatizing the product in a reaction liquid using phenacyl bromide by a high performance liquid chromatography.
The results are shown in Table 2.

[Analysis of optical purity]
Column: CHIRALPAK AD-RH (4.6 mmφ × 150 mm, manufactured by Daicel Chemical Industries)
Eluent: 20 mM phosphoric acid aqueous solution (pH 2.5) / acetonitrile = 1/1
Flow rate: 0.5 ml / min, column temperature: room temperature, measurement wavelength: 210 nm

Reference Example 1 Synthesis of 3- (4-chlorophenyl) glutarimide Urea (15.02 g, 250 mmol) was added to 3- (4-chlorophenyl) glutaric acid (24.27 g, 100 mmol) and stirred at 160 ° C. for 1 hour. did. After cooling the reaction solution, distilled water (83 ml) was added, and the mixture was stirred at 90 ° C. for 10 minutes and stirred at 25 ° C. for 1 hour. The precipitated solid was separated by filtration and dried under reduced pressure. Toluene (35 ml) was added to this solid and stirred at 90 ° C. for 10 minutes and then at 25 ° C. for 60 hours, and then 25% aqueous ammonia solution (10 ml) was added and stirred at 25 ° C. for 30 minutes. The precipitated solid was separated by filtration and dried under reduced pressure to obtain a white solid (17.87 g, 79.3 mmol) of 3- (4-chlorophenyl) glutarimide.
Title compound:
¹ H-NMR (400 MHz, CDCl ₃ ): δ (ppm) 2.72 (m, 2H), 2.91 (m, 2H), 3.41 (m, 1H), 7.16 (d, 2H) , 7.36 (d, 2H), 7.94 (br, 1H)

Reference Example 2 Synthesis of 3-isobutylglutarimide Urea (14.26 g, 238 mmol) was added to 3-isobutylglutaric acid (19.23 g, 95.0 mmol), and the mixture was stirred at 160 ° C. for 1 hour. After cooling the reaction solution, distilled water (79 ml) was added and stirred at 25 ° C. for 1 hour, and the precipitated solid was separated by filtration and dried under reduced pressure. A 25% aqueous ammonia solution (50 ml) was added to this solid and stirred at 25 ° C. for 1 hour, and the precipitated solid was filtered off. The solid separated by filtration was dried under reduced pressure to obtain a white solid (10.50 g, 61.9 mmol) of 3-isobutylglutarimide.
Title compound:
¹ H-NMR (400 MHz, CDCl ₃ ): δ (ppm) 0.91 (d, 6H), 1.26 (m, 2H), 1.67 (m, 1H), 2.23 (m, 3H) , 2.70 (m, 2H), 7.94 (br, 1H)

[Example 3]
Identification and production of imidase (1) Isolation and purification of imidase derived from Burkholderia phytofirmans strain DSM17436 Burkholderia phytofirmans strain DSM17436 was sterilized in vitro in 30 ml of TGY medium (5 g tryptone, 5 g yeast extract, 1 g glucose, deionized water). It was inoculated to 1 L, up to pH 7.0 before sterilization, and cultured with aerobic shaking at 28 ° C. for 22 hours. The whole amount of this culture solution was inoculated into 5 L of TGY medium sterilized in the flask, and cultured under aerobic shaking at 28 ° C. for 30 hours. After completion of the culture, the cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.5), disrupted by ultrasonic waves, and centrifuged. Ammonium sulfate was added to the obtained supernatant to a saturation concentration of 20%, and ammonium sulfate was further added to the supernatant obtained by centrifugation to a saturation concentration of 40%. The precipitate obtained by centrifuging this was suspended in 20 mM phosphate buffer (pH 7.5), and dialyzed with 20 mM Tris buffer (pH 7.5) using a cellulose tube as a dialysis membrane. Next, this was applied to HiPrep DEAE FF 16/10 (manufactured by GE Healthcare) and subjected to column chromatography, and a 0 mM to 1 M sodium chloride concentration gradient was applied with 20 mM Tris-HCl buffer (pH 7.5). Elute and collect active fractions. This active fraction was dialyzed with 20 mM Tris-HCl buffer (pH 7.5) using a cellulose tube as a dialysis membrane, applied to MonoQ 10/100 GL (manufactured by GE Healthcare), and subjected to column chromatography. Elution with a sodium chloride concentration gradient of 0M to 1M was performed with the same buffer. The obtained active fraction was concentrated using Amicon Ultra (centrifugal filter unit: manufactured by Merck Millipore), applied to Superdex 200 10/300 GL (manufactured by GE Healthcare), and subjected to gel filtration. Was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing sodium chloride. Ammonium sulfate was dissolved in the obtained active fraction to a final concentration of 2M, and then applied to RESOURCE PHE (manufactured by GE Healthcare) and subjected to column chromatography, and then with 20 mM Tris-HCl buffer (pH 7.5). Elution was performed with a gradient of 2M to 0M ammonium sulfate. The obtained active fraction was buffer-exchanged with 20 mM Tris-HCl buffer (pH 7.5) using Amicon Ultra, applied to TSK gel DEAE-5PW (manufactured by Tosoh Corporation), and subjected to column chromatography. The buffer was eluted with a gradient from 0M to 1M sodium chloride. The obtained active fraction was buffer-exchanged with 20 mM Tris-HCl buffer (pH 7.5) using Amicon Ultra, and this was applied to MonoQ 5/50 GL (manufactured by GE Healthcare) and subjected to column chromatography. The solution was eluted with a concentration gradient of 0 M to 1 M sodium chloride with the same buffer. The obtained active fraction was subjected to SDS-polyacrylamide electrophoresis, and the N-terminal amino acid sequence of the obtained band was determined with a protein sequencer. This enzyme was named BpIH.

<Method for measuring enzyme activity during enzyme purification>
5 μL of the active fraction was mixed with 55 μL of 100 mM phosphate buffer (pH 7.0) containing 0.1 w / v% of 3- (4-chlorophenyl) glutarimide, and reacted at 28 ° C. for 60 minutes. After the reaction, the reaction was stopped by diluting it twice with acetonitrile, the solid was removed by centrifugation, and the substrate and product in the reaction solution were analyzed by high performance liquid chromatography.

1 U is defined as the amount of enzyme that produces 1 μmol of 3- (4-chlorophenyl) glutaric acid monoamide per minute.

The total protein of each fraction was measured by the Bradford method.
<High-performance liquid chromatography analysis conditions>
Column: 5C18-ARII (4.6 mmφ × 250 mm, manufactured by Nacalai Tesque)
Eluent: 20 mM phosphoric acid aqueous solution (pH 2.5) / acetonitrile = 6/4
Flow rate: 0.5 ml / min, column temperature: 30 ° C., measurement wavelength: 210 nm

The enzyme activity was performed in the same procedure not only in this experiment but also in the following experiment.

(2) Determination of N-terminal amino acid sequence information and total amino acid sequence of BpIH The N-terminal amino acid sequence of imidase BpIH purified in (1) was examined using a protein sequencer (PPSQ-31B: manufactured by Shimadzu Corporation). The N-terminal sequence was determined to be PLDPNYPRDL.
When this N-terminal sequence was searched from the genome information of Burkholderia phytofirmans strain DSM17436 using Protein BLAST, it was 100% identical to the protein of GenBank accession No .: ACD16728.1 (SEQ ID NO: 1), and this protein is urate catabolism It was annotated as protein.

(3) Cloning of BpIH gene and production of BpIH in E. coli Genomic DNA was extracted from cells obtained by culturing Burkholderia phytofirmans DSM17436 strain as in (1) using DNeasy Blood & Tissue Kit (Qiagen) did. The full length of the BpIH gene is obtained by amplifying BpIH DNA by PCR using a DNA primer (SEQ ID NO: 2) having a sequence on the 5 ′ end side of the BpIH gene and a primer (SEQ ID NO: 3) having a sequence on the 3 ′ end side. A gene fragment containing (SEQ ID NO: 4) was obtained. The DNA obtained by PCR is confirmed by agarose gel electrophoresis, the size of the amplified fragment is confirmed, the target gene band is excised from the agarose gel, and the illustra GFX PCR DNA and Gel Band Purification Kit (manufactured by GE Healthcare) is used. And purified. PQE-60 treated with restriction enzymes HindIII and NcoI, and a DNA fragment encoding the full length of BpIH derived from Burkholderia phytofirmans DSM17436 strain obtained by the above procedure, Gibson Assembly Master Mix (manufactured by New England Biolabs) The recombinant vector pQE60-BpIH01 was constructed by ligating together.

E. coli using recombinant vector pQE60-BpIH01. E. coli JM109 competent cells were transformed. E. coli JM109 / pQE60-BpIH01 was obtained. The obtained transformant was inoculated into 2 mL of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) containing 50 μg / ml ampicillin and aerobic at 28 ° C. for 7 hours. After shaking culture, IPTG was added to the culture solution to a concentration of 1 mM, and shaking culture was further performed at 28 ° C. for 16 hours. The cells were collected by centrifugation, suspended in 500 μL of 100 mM phosphate buffer (pH 7.0) containing 0.1 w / v% of 3- (4-chlorophenyl) glutarimide, and the activity was confirmed by resting cell reaction.

(4) Cloning of imidase gene derived from Alcaligenes faecalis subsp. Faecalis NBRC 13111 and production in E. coli From known genomic sequence data of Alcaligenes faecalis subsp. Faecalis NBRC 13111, genes and amino acid sequences highly homologous to the BpIH gene and amino acid sequence (SEQ ID NOs: 5 and 6) could be obtained. According to the sequence comparison in the default setting of Genetyx software version 12 (Genetics Co., Ltd.), the base sequence of SEQ ID NO: 5 has 71% identity to the base sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 6 is It was calculated to have 75% identity to the amino acid sequence of SEQ ID NO: 1. Using this gene as the AfIH gene, using a DNA primer (SEQ ID NO: 7) having a sequence on the 5 ′ end side of the AfIH gene and a primer (SEQ ID NO: 8) having a sequence on the 3 ′ end side, the AfIH gene is used as in (3). A gene fragment containing the full length of the gene was obtained, agarose gel electrophoresed, excised, and purified. A recombinant vector pQE60-AfIH01 was constructed in the same manner as in (3). E. coli JM109 / pQE60-AfIH01 was obtained, cultured, and confirmed for activity by resting cell reaction.

(5) Expression and purification of recombinant BpIH and AfIH E. coli JM109 / pQE60-BpIH was inoculated into 2 ml of LB medium (tryptone 1%, yeast extract 0.5%, NaCl 0.5%, pH 7.0) containing 50 μg / ml ampicillin at 28 ° C. for 24 hours. Cultured with aerobic shaking. The total amount of this culture solution was inoculated into 500 ml of LB medium containing 50 μg / ml ampicillin, and after aerobic shaking culture at 28 ° C. for 7 hours, IPTG was added to the culture solution to a concentration of 1 mM. Further, shaking culture was performed for 17 hours. After completion of the culture, the cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.5), disrupted by ultrasonic waves, and centrifuged. The supernatant was dialyzed with 20 mM Tris buffer (pH 7.5) using a cellulose tube as a dialysis membrane. Subsequently, this was applied to MonoQ 10/100 GL (manufactured by GE Healthcare) and subjected to column chromatography, and eluted with a sodium chloride concentration gradient from 0 M to 1 M with the same buffer. The obtained active fraction was concentrated using Amicon Ultra (centrifugal filter unit: manufactured by Merck Millipore), applied to Superdex 200 10/300 GL (manufactured by GE Healthcare), and subjected to gel filtration. Was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing sodium chloride. When the obtained active fraction was subjected to SDS-polyacrylamide electrophoresis, BpIH was detected as a single band, confirming the purity of the purified enzyme.

Recombinant AfIH was expressed and purified in the same procedure.

(6) Evaluation of hydrolysis reaction of 3- (4-chlorophenyl) glutarimide and 3-isobutylglutarimide using recombinant purified BpIH or AfIH Each of the purified enzymes BpIH and AfIH obtained in (5) at an enzyme concentration of 100 μg / ml, and reacted in 500 μL of 100 mM phosphate buffer (pH 7.0) containing 3- (4-chlorophenyl) glutarimide 0.5 w / v% at 28 ° C. for 16 hours. After completion of the reaction, solids were removed by centrifugation, and the conversion rate (%) and optical purity (%) were determined by analyzing the substrate and product in the reaction solution by the high performance liquid chromatography described in Example 1. ee) was determined. The results are shown in Table 4.

The purified enzymes BpIH and AfIH obtained in (5) were each used at an enzyme concentration of 1.3 mg / ml, and 28 in 250 μL of 100 mM phosphate buffer (pH 7.0) containing 0.5 w / v% of 3-isobutylglutarimide. The reaction was carried out at 0 ° C. for 16 hours. After completion of the reaction, solids were removed by centrifugation, and the conversion rate (%) and optical purity (%) were determined by analyzing the substrate and product in the reaction solution by the high performance liquid chromatography described in Example 2. ee) was determined. The results are shown in Table 5.

(7) Substrate specificity and relative activity evaluation using recombinant purified BpIH or AfIH The reaction was performed in 100 μL with a certain amount of enzyme (purified BpIH or AfIH obtained with (5)) and 4 mM of various substrates [3- (4 -Chlorophenyl) glutarimide, 3-isobutylglutarimide, phthalimide, cis-1,2,3,6-tetrahydrophthalimide, allantoin, 2,4-thiazolidinedione, hydantoin, dihydrouracil, glutarimide, succinimide], 100 mM phosphoric acid The reaction was carried out at 28 ° C. for 30 minutes under conditions containing a buffer solution (pH 7.5).

After 30 minutes, 10 μL of 15% perchloric acid was immediately added to stop the reaction, followed by neutralization with 90 μL of 500 mM potassium phosphate buffer (KPB) (pH 7.0), and the supernatant obtained by centrifugation. Is subjected to high performance liquid chromatography analysis to analyze the decrease in substrate and increase in product, and the results of comparing the activity values are shown in Table 6. In Table 6, the activity value (U) for each substrate calculated in% when the activity value (U) for 3- (4-chlorophenyl) glutarimide is 100 is shown as “relative activity (%)”. Here, the activity value (U) for each substrate is defined as the activity value (U) of the fixed amount of purified protein used in the reaction when 1 U is defined as the amount of enzyme that produces 1 μmol of each product per minute. Point to.

Allantoin, hydantoin and dihydrouracil were analyzed under the following analytical conditions.
Column: 5C18-ARII (4.6 mmφ × 250 mm, manufactured by Nacalai Tesque)
Eluent: 250 mM aqueous potassium dihydrogen phosphate flow rate: 1.0 ml / min, column temperature: 40 ° C., measurement wavelength: 210 nm

Other substrates are the same as the high performance liquid chromatography analysis conditions of Example 1.

The method of the present invention can be used for the production of an optically active 3-substituted glutaric acid monoamide useful as a pharmaceutical intermediate.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Sequence numbers 2, 3, 7, 8: Primers

Claims

Following formula (1);

(In the formula, R may have an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, or an optionally substituted group. A preferable alkynyl group having 2 to 8 carbon atoms, an aryl group having 4 to 20 carbon atoms which may have a substituent, or an aralkyl group having 5 to 20 carbon atoms which may have a substituent. An enzyme source having asymmetric hydrolysis activity is allowed to act on a 3-substituted glutarimide represented by the following formula (2);

(Wherein, * represents an asymmetric carbon atom, R is the same as above), and a method for producing an optically active 3-substituted glutaric acid monoamide.
The enzyme source is selected from the group consisting of the genus Achromobacter, the genus Alcaligenes, the genus Burkholderia, the genus Comamonas, the Delftia, and the genus Pseudomonas The production method according to claim 1, which is a microbial cell of at least one microorganism and / or a processed product thereof.
The production method according to claim 2, wherein the optically active 3-substituted glutaric acid monoamide represented by the formula (2) having an absolute configuration of R is produced.
Enzyme sources are Achromobacter sp., Achromobacter xylosoxy dance sub-species Denitificans, Alcaligenes faecalis, Balk hortolia phytophyllia phytophyllia ), At least one microorganism selected from the group consisting of Comamonas sp., Delftia acidovorans, Delftia sp, and Pseudomonas putida. The manufacturing method of Claim 3 which is the processed material.
The production method according to any one of claims 1 to 4, wherein R is an isobutyl group, an n-propyl group, an isopropyl group, a phenyl group, or a 4-chlorophenyl group.
The enzyme source is a polypeptide of the following (I) to (VI):
(I) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(II) consisting of an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1, and the 3-substituted glutarimide of the formula (1) is represented by the formula (2) A polypeptide having an enzymatic activity for asymmetric hydrolysis to optically active 3-substituted glutaric acid monoamide;
(III) An amino acid sequence having 70% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the 3-substituted glutarimide of the formula (1) is substituted with the optically active 3-substituted group of the formula (2) A polypeptide having an enzymatic activity for asymmetric hydrolysis to glutaric monoamide;
(IV) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6;
(V) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 6, wherein the 3-substituted glutarimide of the formula (1) is converted to the formula (2) A polypeptide having an enzymatic activity for asymmetric hydrolysis into an optically active 3-substituted glutaric acid monoamide; and (VI) an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 6, At least one selected from the group consisting of polypeptides having an enzymatic activity for asymmetric hydrolysis of the 3-substituted glutarimide of the formula (1) into the optically active 3-substituted glutaric acid monoamide of the formula (2) The production method according to any one of claims 1 to 5, which is contained.