JP2001046076A - Production of (r)-2-amino-1-phenylethanol or its halogen substitution compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain (R)-2-amino-1-phenylethanol or its halogen substitution compound useful as a raw material for various pharmaceuticals or the like in high yields by acting decarboxylase aldolase on the enantiomer mixture of DL-threo-3-phenylserine or its halogen substitution compound. SOLUTION: On the method of producing (R)-2-amino-1-phenylethanol or its halogen substitution compound represented by formula II from L-threo enantiomer by acting decarboxylase on the enantiomer mixture of DL-threo-3- phenylserine or its halogen substitution compound represented by formula I (X is H or a halogen), the objective compound represented by formula II is obtained by acting an aldolase, e.g. D-threonine aldolase or phenylserine aldolase, on the D-threo enantiomer constituting the above enantiomer mixture to decarboxylate. The objective compound is industrially important as a raw material for various medicines, particularly a raw material or the like for diabetes therapeutic drugs or antiobesity drugs.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing (R) -2-amino-1-phenylethanol represented by the formula (2) or a halogen-substituted product thereof. (R) -2-Amino-1-phenylethanol represented by the formula (2) or a halogen-substituted product thereof is industrially important as a raw material for various pharmaceuticals, particularly as a raw material for a therapeutic agent for diabetes or an antiobesity drug.

[0002]

Embedded image Equation (2)

[0003]

2. Description of the Related Art A method for producing (R) -2-amino-1-phenylethanol represented by the formula (2) or a halogen-substituted product thereof using a decarboxylase is known (WO95 / 2005).
23869). In this report, tyrosine decarboxylase, Enterococcus, Lactobacillus, and Providencia (P
rovidencia), Fusarium or Gibberella, acts on an enantiomeric mixture of threo-3-phenylserine represented by the formula (1) or a halogen-substituted derivative thereof, the formula (2) (R) -2-amino-1-phenylethanol represented by the formula (1) or a halogen-substituted product thereof is produced with almost 100% optical purity.
Threo represented by the formula (1) as a raw material in this reaction
-3-Phenylserine or a halogen-substituted product thereof is chemically synthesized, for example, based on a reaction represented by the formula (3).

[0004]

Formula (3)

[0005] Since this reaction has no optical specificity, the starting material obtained as shown in the formula (3) is an enantiomeric mixture containing D-form and L-form equally. In the previous report, an optically active (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof (formula 2) is produced by reacting a stereoselective decarboxylase with this enantiomeric mixture. Have been successful. However, in this reaction, D-form threo-3-phenylserine or a halogen-substituted form thereof is present in the raw material as an impurity. The mixture of the D-form, which accounts for about half of the raw materials, may hinder the purification of the reaction product, and may cause a decrease in the yield because it cannot be recovered as a final product. Therefore, in an enzymatic production method of optically active (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof (formula 2), a D-form compound (hereinafter referred to as D-threo) mixed as a contaminant in the raw material It is considered that a high yield can be achieved if a method is realized in which the compound (which may be described as an enantiomer) can be removed and effectively reused.

On the other hand, aldolase is known as an enzyme that decomposes a hydroxy amino acid into an amino acid and an aldehyde. Until now, threonine aldolase (JP-B-49-020)
514, Japanese Patent Publication 50-026638, Japanese Patent Publication 51-006239, Japanese Patent Publication 52-04
6313, JP-B-52-033915, JP-B-52-000105), D-threonine aldolase (JP-B-01-011279, JP-B-06-07550)
5), the existence of phenylserine aldolase (JP-A-09-238680) and the like have been elucidated. A method for synthesizing a hydroxyamino acid from an amino acid and an aldehyde using these known aldolases (JP-B-52-012273, JP-B-54-00395)
2, Tokiko 54-012554, Tokiko 01-011279, Tokiko 02-211
887, Japanese Patent Publication No. 02-207793), an optically active hydroxy amino acid obtained by optically resolving a racemic hydroxy amino acid (Japanese Unexamined Patent Publication No.
8-216691, Japanese Patent Publication No. 02-721536, JP-A 06-125790, JP-A
Methods for producing JP-A-04-330297, JP-A-04-346795, and JP-A-06-125787 are also known.

However, by applying these aldolases to an enzymatic production method of optically active (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof (formula 2), D-mixed in the starting compound can be obtained. -It is not known that the removal and reuse of the threo-enantiomer is possible.

[0008]

The present invention relates to an enzymatic production method of optically active (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof (formula 2). It is an object of the present invention to remove and reuse the D-threo enantiomer, and to achieve a high yield as a result. More specifically, it is an object of the present invention to provide a method capable of enzymatically removing the D-threo enantiomer.

[0009]

Means for Solving the Problems In order to solve the above problems, the present inventors have repeatedly studied a method for enzymatic removal of D-threo enantiomer which can be combined with a stereoselective decarboxylation reaction. As a result, they have found that the aldolase can achieve selective removal of the D-threo enantiomer, and have completed the present invention. That is, the present invention relates to a method for producing the following (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof.

[1] DL-threo represented by the formula (1)
A decarboxylase is allowed to act on an enantiomer mixture of 3-phenylserine or a halogen-substituted product thereof, and (R) -2-amino-1-phenylethanol represented by the formula (2) or a halogen-substituted product thereof represented by the formula (2) from the L-threo enantiomer Producing (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof,
A production method comprising a step of causing an aldolase to act on a D-threo enantiomer constituting the enantiomeric mixture to decompose it.

Embedded image Equation (1)

Embedded image Formula (2) (wherein X represents hydrogen or halogen) [2] The production method according to [1], wherein the aldolase is D-threonine aldolase or phenylserine aldolase. [3] D-threonine aldolase is an alkaline gene.
Zylooxidans (Alcaligenes xylosoxidans) IFO
It is derived from the 12669 strain. [4] In addition, D- by the action of aldolase
The production method according to [1], comprising a step of recovering an aldehyde and / or an amino acid generated by decomposition of the threo enantiomer.

[0011]

DETAILED DESCRIPTION OF THE INVENTION As described above, the enantiomeric mixture of DL-threo-3-phenylserine represented by the formula (1) or a halogen-substituted derivative thereof represented by the formula (1), which is a raw material for an enzymatic reaction in the present invention, is known. Reaction principle (KNF Shaw
& S. W. Fox, J.S. Amer. Chem. Soc., 75, 3421-3424,1
953). The reaction formula at this time is shown in formula (3). The specific operation is as described in Examples.

[0012]

Embedded image Formula (3) In the formula, X represents H or halogen. As the halogen, any element selected from the group consisting of F, Cl, Br, and I can be shown. When substituting with halogen, the substitution position may be any of o, m and p.

In the present invention, any aldolase that acts on the D-threo enantiomer can be used as long as it is an enzyme that decomposes D-threo-3-phenylserine or its halogen-substituted product into an aldehyde and an amino acid. The enzymatic reaction by threonine aldolase is shown below as formula (4).

[0014]

Embedded image Formula (4) In the formula, X means the aforementioned element.

Aldolases usable in the present invention include, for example, aldolases derived from the following microorganisms. Alcaligenes faecalis (IFO12669) Pseudomonas DK-2 (FERM-P6)
200) Arthrobacter DK-19
K-19, FERM-P6201) In the present specification, IF
O-numbered microorganisms are described in the 9th edition of the microorganism catalog published by the Institute for Fermentation (1992),
It can be obtained from the Institute. Microorganisms with NRIC numbers are the second strain catalog published by Tokyo University of Agriculture.
Edition (1992) and can be obtained from the university.

The enzymatic reaction of aldolase in the present invention is carried out by reacting the enantiomer mixture of DL-threo-3-phenylserine or its halogen-substituted form in which D-form and L-form are mixed in an arbitrary order. Can be. That is, it can act on the enantiomeric mixture before, after, or simultaneously with the action of a decarboxylase that catalyzes the decarboxylation reaction, which is the main reaction. At any stage, the D-threo enantiomer is decomposed into aldehydes and amino acids by the action of aldolase. If the aldehyde and amino acid produced as a result of the decomposition are recovered, the raw material DL-threo-3-
It can be used for chemical synthesis of phenylserine or a halogen-substituted product thereof. If a series of reactions constituting the present invention are comprehensively described including the synthesis step of DL-threo-3-phenylserine as a raw material or a halogen-substituted product thereof,
For example, they can be summarized as in the following equation (5).

Embedded image Equation (5)

In the present invention, an aldehyde generated from D-threo-3-phenylserine or a halogen-substituted product thereof by the action of an aldolase is recovered, for example, by the procedure shown in Examples. That is, benzaldehyde (or a halogen derivative thereof) can be extracted with ethyl acetate or the like by adjusting the pH of the reaction solution to sulfuric acid. Benzaldehyde (or its halogen derivative) recovered as a solid after desolvation of the ethyl acetate phase can be used again as a raw material for synthesizing the target DL-threo-3-phenylserine or its halogen-substituted product. . The amino acid can be crystallized by adding an alcohol such as ethanol after concentrating the reaction solution from which the aldehyde has been extracted. The precipitated and precipitated amino acids are
It can be recovered by filtration or the like.

On the other hand, the reaction by the decarboxylase, which is the main reaction, can be carried out, for example, according to a known method (WO95 / 23869) filed by the present inventors as a patent. For example, it can act on an enantiomeric mixture of threo-3-phenylserine or a halogen-substituted product thereof to produce (R) -2-amino-1-phenylethanol represented by the formula (2) or a halogen-substituted product thereof. The enzyme reaction represented by the formula (6) can be achieved by tyrosine decarboxylase.

[0019]

Embedded image In the formula, X represents the same element as described above. Equation (6)

The tyrosine decarboxylase used in the present invention can be of any origin. For example, Enterococcus faeca
lis) and Enterococcus hilae (Enterococcus h)
Enzymes derived from readily available microorganisms such as irae) can be said to be preferred enzymes. (Note that "Streptococcus faecalis" is an old name and is considered to be actually an enzyme derived from a microorganism belonging to the genus Enterococcus. After the classification was revised in 1981, the genus Enterococcus became independent from the genus Streptococcus. Bergey's Manual of Systematic Bacteriology
(1986), the former Streptococcus faecalis has been classified as Enterococcus faecalis. )

In the present invention, the decarboxylase reaction is carried out by threo.
Acts on an enantiomeric mixture of -3-phenylserine or a halogen-substituted form thereof, and is represented by the formula (2):
It can also be carried out using a microorganism capable of producing amino-1-phenylethanol or a halogen-substituted product thereof. Examples of microorganisms that can be used in the present invention include the following microorganisms. Enterococcus genus Lactobacillus genus Providencia genus Fusarium genus and Gibberella genus

As long as these microorganisms can catalyze the above reaction, any of these microorganisms, such as a wild-type strain, a mutant strain, or a recombinant strain derived by a genetic technique such as cell fusion or genetic manipulation, can be used. It can be used in the invention. The strains that are included in these genera, can be used in the present invention, and are easily available are shown below. Enterococcus
Enterococcus faecalis NRIC 1141, Enterococcus hirae IFO 318
1. Lactobacillus brevi
s) NRIC 1037, Providencia Stuarty (Prov
idencia stuatii) IFO 12930, Fusarium anguioides IFO 4467, Gibberella
Gibberella fujikuroi IFO 9977, IFO 30
336, IFO 30337, IFO 31251, NRIC 1240, Gibberella
Gibberella zeae IFO 7772, Gibberella lateritium IFO 7188, Gibberella acuminata IFO 30307
stock

The catalog of microorganisms, 9th edition, published by Fermentation Research Institute, pp. 312-320 (1992), and Shunichi Udagawa et al. 518-522, lower volume pp. Ten
55-1059, According to Kodansha (1978), the conidia of Gibberella fujikuroi are Fusarium moniliforme, and the conidia of Gibberella zeae are Fusarium graminearum. ), Gibberella lateritium conidia generation is Fusarium latium (Fusarium latium)
eritium). Thus, it is clear that the species names of these full generations and the corresponding conidium generation species names indicate the same species. That is, the present invention also includes a case where the present invention is implemented using Fusarium moniliform, Fusarium graminerum, or Fusarium laterium.

When an enzyme is used, the amounts of tyrosine decarboxylase and aldolase used in the reaction are as follows:
Usually, it is 0.005 to 5.0 units / ml. However, those skilled in the art can usually determine an appropriate amount of the enzyme to be used empirically according to the reaction conditions such as the concentration of the corresponding substrate, temperature, and pH. In the present invention, one unit of threonine aldolase means 30 ° C., pH 8.0.
Is defined as an enzyme activity capable of producing 1 μmol of acetaldehyde from threonine in one minute. One unit of tyrosine decarboxylase is 37 ° C., pH 5.5.
Is defined as an enzymatic activity capable of converting 1 μmol of substrate per minute.

As these enzymes, in addition to commercially available enzymes, those obtained by further purifying commercially available enzymes, or those obtained by purifying and obtaining from the above-mentioned enzyme-producing microorganism cells by a combination of known methods can be used. The enzyme reaction is carried out in a state where the purified enzyme is suspended or dissolved in the reaction solution, or may be brought into contact with the reaction solution as an immobilized enzyme immobilized on a carrier. Methods for immobilizing enzymes using carrageenan gel, alginate gel, polyacrylamide gel, cellulose, agar, or the like as a carrier are known.
The enzyme can also be reacted in a bioreactor equipped with an ultrafiltration membrane or the like.

When the microorganism itself is used as the enzyme, the culture is used as it is, and the cells are added to the culture with threo-3-phenylserine or an enantiomeric mixture of halogen-substituted products, by centrifugation or the like. Separation, after washing or washing, buffer,
There is a method in which threo-3-phenylserine or an enantiomeric mixture of a halogen-substituted product thereof is added to a suspension resuspended in water or the like and reacted. The microorganisms may be living cells, or may be those subjected to treatment such as crushed cells, acetone treatment, toluene treatment, and freeze-drying. Microorganisms (produced from viable cells or cells) are treated with carrageenan gel, alginate gel, polyacrylamide gel,
It is also possible to carry out the present invention by immobilizing it on cellulose, agar or the like by a known method, and it is also possible to react in a reactor using an ultrafiltration membrane or the like. The activity of the enzyme can be stably maintained by adding it to the reaction solution in an immobilized form, regardless of whether it is an enzyme or a cell, and the enzyme can be easily recovered and reused. The reaction solution contains the substrate Threo-3
Cetylpyridinium chloride, cetyltrimethylammonium bromide, Triton X, Tween (Twee)
When a surfactant such as n) is added in an amount of about 0.001 to 0.5%, good results may be obtained. Furthermore, when using microorganisms (viable cells or processed cells) and enzymes, the common matter is to replace the gas phase of the reaction solution with nitrogen, or seal the liquid surface with liquid paraffin to block oxygen. Good results may be obtained.

The enzymatic reaction with aldolase in the present invention can be expected to have a sufficient enzymatic activity when performed under the following conditions. That is, when, for example, an aldolase derived from the genus Alcaligenes is used as the aldolase of the present invention, the reaction temperature is 5 to 60 ° C, preferably 20 to 40 ° C. Desirable pH of the reaction solution is 4 to 10,
Preferably it is 6-8. 0.1 μM to 10 m for the reaction system
M, more preferably 1 μM to 1 mM pyridoxal phosphate, and if necessary, a divalent metal ion such as Mn is added to 0.1 μM to 10 mM, more preferably 1 μM to 1 mM, to achieve a high level of efficiency. Expression of enzyme activity can be expected.

On the other hand, the reaction with tyrosine decarboxylase is usually 5 to 70 ° C., preferably 20 to 40 ° C. for Lactobacillus and Providencia, 45 to 60 ° C. for Enterococcus, Gibberella and Fusarium.
Perform at 25-60 ° C for tyrosine decarboxylase. The reaction pH may be appropriately selected within the range in which the enzyme for decarboxylation reacts, but is usually at pH 4 to 11, preferably at pH 5 to 9, usually in a buffer or using a pH stat.
The reaction can be performed by standing, shaking, or stirring. The solvent used for the reaction is usually water, but an organic solvent such as an alcohol can be added as long as the reaction is not affected. The produced (R) -2-amino-1-phenylethanol or its halogen-substituted product is subjected to ultrafiltration, concentration,
It can be recovered and purified by combining ordinary methods such as column chromatography, extraction, and crystallization. The remaining optically active threo-3-phenylserine or its halogen-substituted product can be recovered and purified by the same operation.

The substrate, threo-3- represented by formula (1)
An enantiomeric mixture of phenylserine or a halogen-substituted form thereof is used in a concentration range in which substrate inhibition of the enzyme does not occur.
It may be added all at once, intermittently, or continuously, and is usually added in an amount of about 0.01 to 20% (w / w). The substrate is
It can be added as it is dissolved or dispersed in water as it is, dissolved in an organic solvent that does not dissolve in water or affect the reaction, or dispersed in a surfactant or the like.

The medium for culturing the microorganism used in the present invention is not particularly limited as long as the microorganism can grow. For example, as the carbon source, any of the above microorganisms can be used, and specifically, glucose,
Sugars such as fructose, sucrose and dextrin; alcohols such as sorbitol and glycerol;
Organic acids and salts thereof, such as fumaric acid, citric acid, acetic acid, and propionic acid, hydrocarbons such as paraffin, and mixtures thereof can be used. Examples of the nitrogen source include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, meat extract, yeast extract, corn steep liquor, and casein hydrolysis. , Urea, and other inorganic and organic nitrogen-containing compounds, or mixtures thereof. In addition, nutrients used in ordinary culture, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. Further, if necessary, a factor that promotes the growth of microorganisms, serine, tyrosine, valine, leucine, alanine, isoleucine, glycine, phenylalanine, tryptophan, threo-3-phenylserine or the like, which enhances the ability to produce the target compound of the present invention. Amino acid derivatives such as halogenated derivatives, pyridoxal-5'-
Factors such as phosphoric acid and vitamin B6 such as pyridoxal hydrochloride, or substances such as CaCO3 effective for maintaining the pH of the medium can also be added. For example, a bouillon medium for bacteria, an MRS medium, a GYP medium for lactic acid bacteria, a YM medium, and a potato-sucrose medium for mold cultivation are suitable (Microorganism Catalog No. 9 issued by the Institute of Fermentation, Japan). Edition (1992), pp 452-453, Tokyo University of Agriculture, strain catalog 2nd edition (1992), pp 51-52). As a culture method, the medium pH is 3.0 to 11.0, preferably 4 to 8, and the culture temperature is 20 to 45 ° C, preferably 25 to 37 ° C, under anaerobic or aerobic conditions suitable for the microorganism. The culture is performed for 120 hours, preferably for about 24-96 hours. Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

[0031]

EXAMPLES In the following examples, measurement of the optical purity of (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof and optically active threo-3-phenylserine or a halogen-substituted product thereof was carried out by the method described in "CROWNPAK". CR (+) "(manufactured by Daicel Chemical Industries, Ltd.) (column: CROWNPAK CR (+), inner diameter 4.6 × 150 mm, mobile phase: aqueous perchloric acid, pH 2.0 (pH 1.0), flow rate: 1.0) ml / min, temperature: 10 ° C. (5 ° C.), detection: UV 254 nm [() indicates measurement of optically active threo-3-phenylserine or its halogen-substituted product]). The quantification of threo-3-phenylserine or its halogen-substituted product, 2-amino-1-phenylethanol or its halogen-substituted product was performed by reverse-phase HPLC using an ODS column (column: Wakosil ODS II
HG (manufactured by Wako Pure Chemical Industries, Ltd.), inner diameter 4.6 x 250 mm, mobile phase: 50 mM
Potassium phosphate buffer, pH 2.5 / acetonitrile [9: 1, v
/ v], flow rate: 1.0 ml / min, temperature: 50 ° C, detection: 254 nm).

In the following examples, "for preparing bacterial cells"
"Medium 1A" "Bacteria preparation medium 1B" "Bacteria preparation medium"
2 "Bacteria preparation medium 3" "Bacteria preparation medium 4" "Bacteria
The preparation method of “Preparation medium 5” and “YM medium” is as follows.
You. (Cell culture medium 1A) 5 g glucose, yeast extract
East Pharmaceutical) 5g, Polypeptone (Nippon Pharmaceutical) 5g, MgSO _Four・ 7H_Two
O 1 g, DL-threo-3- (3-chlorophenyl) serine 0.2 g,
Mix 0.1 g of pyridoxal hydrochloride and deionize to total volume
Make up to 1000 ml and adjust to pH 7.0. (Cell culture medium 1B) 5 g glucose, yeast extract
East Pharmaceutical) 5g, Polypeptone (Nippon Pharmaceutical) 5g, MgSO _Four・ 7H_Two
O 1g, L-tyrosine 0.2g, pyridoxal hydrochloride 0.1g
Deionize to a total volume of 1000 ml and adjust to pH 7.0
You. (Cell culture medium 2) Glucose 5g, KH_TwoPO_Four0.7g, (N
H_Four)_TwoHPO_Four1.3g, MgSO_Four7H_TwoO 0.5g, yeast extract (Far East
Medicine) 3g, polypeptone (Nihon Pharmaceutical) 5g, meat extract (Far East)
Pharmaceutical) 3g, DL-threo-3- (3-chlorophenylserine) 0.
2g, make up to 1000ml total volume with deionized water, pH 7.2
Adjust to (Cell culture medium 3) 20 g of glycerin, yeast extract
East Pharmaceutical) 3g, Polypeptone (Nihon Pharmaceutical) 5g, Malt extract
(Farto Pharmaceutical) 3 g, pyridoxal hydrochloride 0.1 g, deionized
Mix 1000 ml of water and adjust to pH 7.0. (Cell culture medium 4) 5 g of glycerin, potato leachate (peel
Cut 200g into 1cm squares and add 1000ml of water
Boiled for 20 minutes and filtered with gauze) 200g
Minutes, deionized to a mixture of 0.1 g of pyridoxal hydrochloride
Water to make a total volume of 1000 ml and adjust to pH 5.6. (Cell culture medium 5) 24 g of glucose, yeast extract (A
19.2g, KH_TwoPO_Four 1.3 g, (NH_Four)_TwoSO_Four2.4g, M
gSO_Four・ 7H_TwoO 1.3g, FeSO_Four・ 7H_TwoO0.016g, ZnSO_Four・ 7H_TwoO
 0.016g, FS Antifoam 028 (manufactured by Dow Corning)
Mix 0.3 g, add deionized water to make total volume 1000 ml, pH
Adjust to 6.0. (YM medium) glucose 10g, yeast extract (Kyokuto Pharmaceutical) 3
g, malt extract (Kyokuto Pharmaceutical) 3 g, polypeptone (made in Japan)
5g), add deionized water to make total volume 1000ml, pH
 Adjust to 6.0.

Example 1 (Synthesis of threo-3- (3-chlorophenyl) serine) Sodium hydroxide (80.0 g, 2.0 mol) was placed in a 2 L separable flask.
l), glycine (100.0 g, 1.33 mol), and water (330 m
l) and cooled to 15 ° C. in an ice bath. m-Chlorobenzaldehyde (375.0 g, 2.66 mol) was added in one portion and stirred vigorously. A white solid gradually precipitated, and stirring became difficult in about 1 hour. The stirring was stopped and the reaction mixture was left at room temperature for 24 hours. After cooling to 15 ° C, concentrated sulfuric acid (36%, 202.6 g, 1
76 ml, 2.0 mol) was slowly added dropwise (about 1 hour). After completion of the dropwise addition, toluene (370 ml) was added and the mixture was stirred for about 1 hour. The mixture was subjected to suction filtration, and the obtained white solid was washed with toluene (about 500 ml). Isopropanol (1 L) was added to the obtained white solid, and the mixture was refluxed for 1 hour. After cooling, the mixture was subjected to suction filtration, and isopropanol (about 500 m
Washed well in l). The obtained white solid was dried under reduced pressure (50
C. for 2 days) to obtain 229.4 g of the target compound (yield: 79.9).
%). Melting point 169 ° C (decomposition) IR (KBr) 3094, 2896, 1634, 1600, 1574, 1479, 143
4,1401,1338,1200,1055,871,786,685cm ^-1 .

[Example 2] (Preparation of D-threonine aldolase) The D-threonine aldolase (D-TA) gene (dta) according to the present invention was obtained and analyzed by the following method. Alcaligenes xylosooxidans IFO by the usual method
Chromosomal DNA was prepared from the 12669 strain, and a DNA library was prepared using the BamHI site of cosmid pWE15 (Stratagene). Also, Alkagenes gyrooxidans
N-terminal amino acid sequence of enzyme purified from IFO 12669 (S
Primers A and B were designed based on QEVIRGIALPPAQPGDPLARVDTPS). Primer A 5'-CCGAAGCTTATGTCNCARGARGTNAT-3 '(N:
All bases, R: A or G) Plumer B 5'-GCCGAATTCGGIGTRTCIACICGNGC-3 '(I:
Inosine) Primer A had a restriction enzyme HindIII site, and primer B had a restriction enzyme BamHI site. Alkaligenes
Zylooxidans (Alcaligenes xylosooxidans) IF
Using a DNA isolated from the O12669 strain as a template, a gene fragment of about 90 bp was amplified by polymerase chain reaction (PCR) using the above primers. The resulting gene fragment was ligated to pUC119 vector, and labeled with ^alpha-32 P, it was used as a probe. As a result of searching from the above library using this probe, pWDTA containing a DNA fragment of about 32 kb was cloned. pWDTA is derived from the target gene dt
It became clear to include a. New primers C and D were designed based on this base sequence. Primer C 5′-GCCAAGCTTGGAGCGTCCCGAATGCCCAGG-3 ′ Primer D 5′-CCGAAGCTTTCTAAAGCCTGCGTGTGAAGC-3 ′ The restriction enzyme HindIII site was added to the primer. Using the above primers, polymerase chain reaction (P
CR) amplified a gene fragment of about 1.2 kb. The obtained gene fragment was ligated to a pUC119 vector, and the obtained vector was named pUDTA1. The vector was transformed into E. coli JM109 strain, and D-TA was produced using this transformant E. coli JM109 (pUDTA1). The gene sequence of dta is registered in Japan DNA Data Bank (DDBJ) accession number AB026892.

The production of D-TA by the transformant can be used under any conditions that allow the growth of the same bacterium, but is generally carried out as follows. 100 μg / ml of the antibiotic ampicillin and 0.2 mM IPTG (isopropyl-thio
-Galactoside) is inoculated into an LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl pH 7.0) and cultured with shaking at 37 ° C for 16 hours. In order to collect D-TA from the grown cells, the enzyme is first extracted from the cells because the present enzyme is present in the cells. That is, the culture is filtered or centrifuged to collect the cells, crushed by a mechanical method such as alumina, dynomill, or ultrasonic treatment, and extracted as a soluble enzyme.
Alternatively, a cell membrane obtained by crushing a cell membrane by treatment with an organic solvent such as acetone and drying under reduced pressure can be used as an enzyme-containing catalyst. From the culture obtained as above,
DHAK purified by a known method such as concentration, adsorption chromatography, ion exchange chromatography, gel filtration chromatography or the like can also be used. Furthermore, D-TA occupies about 40% of the soluble protein in the above-mentioned cells, so that a viable cell reaction can be carried out as long as the compound can cross the cell membrane.

Example 3 (Time-dependent change in resolution of DL-threo-3- (3-chlorophenyl) serine by purified D-TA) 20 mM DL-threo-3-
(3-chlorophenyl) serine, 0.2mM pyridoxal
0.5-ml reaction solution containing -6-phosphate, 0.1 mM MnCl ₂ , 5 units of D-TA purified enzyme solution and 100 mM acetate buffer (pH 5.5)
After reacting at 0 ° C. for 150 minutes, 10 mM D-threo-3-
(3-Chlorophenyl) serine was completely decomposed and 10 mM
L-threo-3- (3-chlorophenyl) serine is 100%
It remained (FIG. 1).

Example 4 20 mM DL-threo-3- (3-
Fusarium fujikur with chlorophenyl) serine, 0.2 mM pyridoxal-6-phosphate, 0.1 mM MnCl ₂ , live cells with 0.1 units of D-TA enzyme activity, 0.35 units of decarboxylase
Contains oi DC4986 cells and 100 mM acetate buffer (pH 5.5)
When 100 ml of the reaction solution was reacted at 30 ° C. for 6 hours, 10 mM D
-Threo-3- (3-chlorophenyl) serine was completely decomposed, producing 10 mM of (R) -2-amino-1-phenylethanol and about 10 mM of 3-chlorobenzaldehyde (FIG. 2). The enzymatic reaction is summarized in equation (7). In the following reaction formulas, the name of each compound means the following compound. D-threo-CHP: D-threo-3- (3-chlorophenyl)
Serine L-threo-CHP: L-threo-3- (3-chlorophenyl)
Serine RCPAE: (R) -2-amino-1- (3-chlorophenyl) ethanol

Embedded image Equation (7)

(Preparation of viable cells) Fusarium fujikuroi DC4
986 was cultured as described below to obtain viable cells. 1.2 L mini-jar (Marubishi Bienj) containing “Cell culture medium 3”
700 ml was added and sterilized at 121 ° C. for 15 minutes. After cooling, Fusariu
m fujikuroi DC4986 strain was shake-cultured at 30 ° C. for 24 hours in a “YM medium” (5 ml / inner diameter 21 mm test tube), and 1.4 ml was inoculated.
The cells were cultured at 600 rpm and 1.0 vvm for 40.5 hours. This culture 1
Centrifuge 00 ml and add 2 ml with 50 ml 100 mM acetate buffer (pH 5.5).
Washed twice.

Example 5 20 mM DL-threo-3- (3-
Chlorophenyl) serine, 0.2 mM pyridoxal-6-phosphate, 0.1 mM MnCl ₂ , live cells with 0.1 units of D-TA enzyme activity, 0.35 units of tyrosine decarboxylase (0.07U /
mg, Sigma, code number T-4379, indicated in the catalog as derived from Streptococcus faecalis) and 100 mM
100 ml of reaction solution containing acetate buffer (pH 5.5) at 30 ° C
After reacting for 6 hours, 10 mM of D-threo-3- (3-chlorophenyl) serine was completely decomposed, and 10 mM of (R) -2-amino-1-phenylethanol was produced.
mM of 3-chlorobenzaldehyde was produced.

Example 6 The same reaction as in Example 5 was performed except that tyrosine decarboxylase was purified from the disrupted cells of Enterococcus faecalis NR1C 1141 and used for the reaction. 10 mM (R)
-2-Amino-1-phenylethanol was produced, and about 10 mM of 3-chlorobenzaldehyde was produced.
Enterococcus faecal
is) Purification of tyrosine decarboxylase from crushed cells of NRlC 1141 was performed as follows. In the sterilized “Bacteria preparation medium 1A”, enterococcus faecalis NR
One platinum loop of IC 1141 was inoculated, and the cells were cultured with rotation and shaking at 37 ° C. for 24 hours, and the cells were collected by centrifugation. About 1 g of wet cells were suspended in 2.5 ml of 0.1 M acetate buffer (pH 5.5), and the cells were disrupted by ultrasonication. The supernatant was obtained by centrifugation, and then the nucleic acid was removed by treatment with 2% protamine sulfate. Centrifuge supernatant
After sufficient dialysis against a 1.0 M acetate buffer (pH 6.0), the solution was applied to a phenyl sepharose 4B column previously equilibrated with a 1.0 M acetate buffer (pH 6.0), and eluted with the same buffer.
The active fraction obtained as a single peak was collected, dialyzed against 0.1 M acetate buffer (pH 6.0), concentrated by an ultrafiltration membrane, and used for the reaction.

Example 7 The same reaction as in Example 5 was performed except that the viable cells prepared as described below were used. 10 each
mM (R) -2-amino-1-phenylethanol was produced and about 10 mM 3-chlorobenzaldehyde was produced. (Preparation of viable cells) “Cell culture medium 1A” was added to each of 30 cells.
Place each 100 ml Erlenmeyer flask in a 100 ml Erlenmeyer flask, sterilize, and enterococcus faecalis NRIC 11
41, Enterococcus hirae
IFO 3181, Lactobacillus
brevis) Inoculate NRIC 1037, Enterococcus faecalis NRIC 1141, Enterococcus hirae IFO 3181
At 37 ° C, Lactobacillus brevis NRIC 1037 at 30 ° C
For 24 hours. Subsequently, the cells were separated by centrifugation to obtain live cells.

Example 8 The same reaction as in Example 5 was performed except that the viable cells prepared as described below were used. 10 each
mM (R) -2-amino-1-phenylethanol was produced and about 10 mM 3-chlorobenzaldehyde was produced. (Preparation of live cells) Gibberella fujikuroi
ujikuroi) IFO 9977 shares, 30336 shares, 31251 shares, NRIC 1240
Strain, Gibberella acuminata
IFO 30307 strain, Gibberella zeae IF
O 7772, Gibberella laterium
itium) IFO 7188, Fusarium Anguioides
arium anguioides) IFO 4467, inoculated with a platinum loop into 5 ml of "Cell culture medium 3" for 48 hours for each strain of Gibberella fujikuroi, and for each strain of other species. The cells were cultured with shaking at 30 ° C. for 72 hours, and viable cells were obtained by centrifugation.

Example 9 600 mL of "Bacterial cell preparation medium 5" was placed in a 1.2 L mini jar (manufactured by Marubishi Biengi), and 121
The solution was sterilized at 15 ° C. for 15 minutes. After cooling, Gibberella fujikuroi IFO 30337 strain was added to the "YM medium" (2
5mL / Sakaguchi flask) at 30 ℃ for 24 hours
mL was inoculated and cultured at 30 ° C., 900 rotations and 1.0 vvm for 72 hours.
300 mL of this culture was centrifuged, washed twice with the same amount of deionized water, suspended in deionized water, and adjusted to 60 mL to prepare a cell suspension. Pre-deionized water 40
0 mL, crystals of DL-threo-3- (3-chlorophenyl) serine 21.
5 g, 10 mM pyridoxal-5'-phosphate solution (10 mL) was added and stirred and dissolved in the reaction solution.50 units of D-TA purified enzyme solution (30 mL) and the cell suspension (60 mL) were added, and nitrogen gas was added. The reaction was carried out for 200 hours at 30 ° C. for 10 hours while slightly passing through. The pH during the reaction was adjusted to 6.2 with 10% H ₂ SO ₄ . When the reaction-completed solution was analyzed by HPLC, 19.9 g / L of (R) -2-amino-1- (3-chlorophenyl) ethanol (yield 49.5%)
And 16.4 g / L of m-chlorobenzaldehyde were produced (yield 49.5%). Compared with the comparative example, the reaction time was shortened, and the production yield of (R) -2-amino-1- (3-chlorophenyl) ethanol was also improved.

[Comparative Example] 600 mL of "Bacteria preparation medium 5" was placed in a 1.2 L mini jar (manufactured by Marubishi Bienj) at 121 ° C.
Sterilized for 15 minutes. After cooling, Gibberella Fujikuroi (Gibb
erella fujikuroi) IFO 30337 strain was shake-cultured at 30 ° C. for 24 hours in “YM medium” (25 mL / Sakaguchi flask), and 12 mL was inoculated, and cultured at 30 ° C., 900 rpm, 1.0 vvm for 72 hours. 300 mL of this culture was centrifuged, washed twice with the same amount of deionized water, suspended in deionized water, and adjusted to 60 mL to prepare a cell suspension. 430 mL of deionized water, D
Crystals of L-threo-3- (3-chlorophenyl) serine 21.5 g, 10
Add 10 mL of mM pyridoxal-5'-phosphate solution and stir.
To the dissolved reaction solution, add 60 mL of the above cell suspension,
200 revolutions, 30 ° C, 48 with slight aeration of nitrogen gas
Reacted for hours. The pH during the reaction was adjusted to 6.2 with 10% H ₂ SO ₄ . When the reaction-completed solution was analyzed by HPLC, 19.5 g / L
(R) -2-amino-1- (3-chlorophenyl) ethanol was produced (48.5% yield), and 25.3 g / L of (+)-threo-3- (3-chlorophenyl) serine remained. (Residual rate: 49.9%) (liquid amount: 425 g).

Example 10 (Threo-3- (3 from recovered chlorobenzaldehyde)
Synthesis of -chlorophenyl) serine) After completion of the reaction of Example 5
425 g was adjusted to pH 3 with 10% H ₂ SO ₄ and the cells were removed by filtration, followed by extraction with 500 mL of ethyl acetate and removal of the solvent to recover m-chlorobenzaldehyde (5.6 g, 40 mmol). A 50 mL flask was charged with sodium hydroxide (1.6 g, 30.1 mmol), glycine (1.5 g, 19.9 mmol), and water (5 mL), and cooled to 15 ° C. in an ice bath. The m-chlorobenzaldehyde (5.6 g, 40 mmol) collected above was added in one portion and stirred vigorously. Gradually, a white solid precipitated, stirring was stopped, and the reaction mixture was left at room temperature for 24 hours. After cooling to 15 ° C., concentrated sulfuric acid (36%, 3.0 g, 30.1 mmol) was slowly added dropwise. After the addition was completed, toluene (10 mL) was added, and the mixture was stirred for about 1 hour.
The mixture was suction filtered and the resulting white solid was toluene (approx.
10 mL). Isopropanol (1 L) was added to the obtained white solid, and the mixture was refluxed for 1 hour. After cooling, the mixture was suction filtered and washed thoroughly with isopropanol (about 10 mL). The obtained white solid was dried under reduced pressure (50 ° C.) to obtain 3.5 g of threo-3- (3-chlorophenyl) serine (yield 79.
9%). Melting point 169 ° C (decomposition) IR (KBr) 3094, 2896, 1634, 1600, 1574, 1479, 1434,
1401, 1338, 1200, 1055, 871,786, 685cm ^-1 1H-NMR (D20-TMSPNa) δ7.52 (br s, 1H), 7.45-7.38
(m, 3H), 5.30 (d, J = 4.39Hz, 1H), 3.92 (d, J = 4.36Hz, 1
H)

[0046]

According to the present invention, (R) -2-
Amino-1-phenylethanol or a halogen-substituted product thereof can be synthesized. Aldehydes and amino acids generated by the aldolase are recovered and can be used again for chemical synthesis of the starting material, DL-threo-3-phenylserine or a halogen-substituted product thereof. In the present invention, since the D-enantiomer is decomposed by the enzyme aldolase, the enzymatic reaction can be performed in parallel under mild conditions similar to the main reaction of the decarboxylase. Therefore, a high yield can be achieved by a simple operation of adding aldolase to the reaction system.

[0047]

[Sequence List] SEQUENCE LISTING <110> DAICEL Chemical Industries LTD. <120> A manufacturing process of (R) -2-amino-1-phenylethanol or halogen derivatives of same. <130> D1-101 <140> <141> <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 1 ccgaagctta tgtcncarga rgtnat 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <220> <223> n indicates Inosine <400> 2 gccgaattcg gngtrtcnac ncgngc 26 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 3 gccaagcttg gagcgtcccg aatgcccagg 30 <210> 4 <211> 30 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 4 ccgaagcttt ctaaagcctg cgtgtgaagc 30

Claims (4)

[Claims] 1. A decarboxylase is allowed to act on an enantiomeric mixture of DL-threo-3-phenylserine or a halogen-substituted derivative thereof represented by the formula (1), and the L-threo enantiomer is represented by the formula (2). (R) -2-amino-1
In the method for producing (R) -2-amino-1-phenylethanol or a halogen-substituted product thereof, which produces -phenylethanol or a halogen-substituted product thereof, an aldolase is caused to act on the D-threo-enantiomer constituting the enantiomer mixture. A manufacturing method including a step of disassembling this. Embedded image Formula (1) Formula (2) (wherein X represents hydrogen or halogen) 2. The method according to claim 1, wherein the aldolase is D-threonine aldolase or phenylserine aldolase.
The production method described in 1. 3. The method according to claim 1, wherein the D-threonine aldolase is Alcaligenes xylosoxidan.
s) The method according to claim 2, wherein the method is derived from the IFO12669 strain. 4. The method according to claim 1, further comprising a step of recovering an aldehyde and / or an amino acid generated by the decomposition of the D-threo enantiomer by the action of the aldolase.