WO2003014057A1 - Processes for producing halogenophenylpyruvic acid and optically active halogenophenylalanine - Google Patents

Abstract

A process for producing a halogenophenylpyruvic acid, characterized by hydrolyzing a 4-(halogenobenzylidene)-2-(lower alkyl)-5-oxazolone with an alkali metal hydroxide and hydrolyzing the resultant 2-(lower alkanoylamino)-3-(halogenophenyl)propenoic acid with an acid in a mixed solvent comprising water and an ether; and a process for producing an optically active halogenophenylalanine, characterized by treating the halogenophenylpyruvic acid with transaminase in the presence of an amino donor.

Description

 Description Process for producing optically active halognophenylalanine, halognophenylpyruvate Technical field

 The present invention relates to an industrially advantageous and improved process for producing halognophenylpyruvic acid. The present invention also relates to an improved method for producing optically active halogenophenylalanine. Background art

_Α -Keto acids, such as phenylvirbic acid, are extremely important compounds not only in organic synthetic chemistry but also in biochemistry, and are used in food additives and agrochemicals with the advancement of biotechnology in recent years. ο; —It has become an important starting material for amino acids. In particular, halognophenylpyruvic acid has become increasingly important in recent years as a raw material for halogenogenphenylalanine used in the synthesis of agrochemicals.

 Methods for producing halognophenylpyruvic acid include a method of hydrolyzing 5- (substituted arylidene) hydantoin (Japanese Patent Application Laid-Open No. Sho 61-22042), and a method of producing 4- (substituted benzylidene). It is known to hydrolyze a 15-oxazolone compound with acetone-water and then hydrolyze the resulting 2- (acetylamino) 13- (halogenophenyl) propenoic acid in hydrochloric acid (Matsumura et al.

Enzymatic Synthesis of Fluorine-containing Amino acids (Kukhar such as editing, Fluorine-containing Amino Acids:. Synthesis and Properties N 2 4 5 the pane, John Wiley & Sons published, 1995)). The hydrolysis with acetone-water and the hydrolysis with hydrochloric acid described in this method have the disadvantage that the reaction is slow and the yield is low (about 20%). Therefore, there has been a demand for an industrially and economically more advantageous production method.

In addition, regarding the method for synthesizing optically active halogenofuluralanine from halogenophenirbirubinic acid, a method using a microbial transaminase is described in Matsumura et al., And Uchida et al., Journal of Organic Synthetic Chemistry, 46 Volume, 9 7 7 Page (1988). In Uchida et al.'S literature, microorganisms belonging to the genera Previbacterium, Pseudomonas, and Alcaligenes are listed as microorganisms that produce a transaminase that synthesizes optically active halogenophenylalanine from halogenophenylpyruvic acid. · It states that Hue squirrel is preferable. However, this reaction is slow and requires a long time to synthesize with a yield of 50% or more. For example, in a reaction using a transaminase derived from Alcaligenes' eucalys, even after 20 hours, only 5 O mM of promophenyl pyruvate produces only 1 O mM of promofuer-laryaune. Disclosure of the invention

 An object of the present invention is to provide an improved method for synthesizing halogenophenyl rubic acid. Another object of the present invention is to provide a method for efficiently obtaining optically active halognoferalene from the halogen-substituted phenylpyruvic acid thus obtained. '

 The present inventors have sought to correct the drawbacks of the prior art and to develop a method for producing halogonoful pyruvic acid in a high yield economically and industrially advantageously. 1- (Halogenobenzylidene) 1-5-year-old xazolone compound is hydrolyzed with metal hydroxide hydroxide, and the obtained 2- (lower alcoholylylamino) 1-3-1 (halognophenyl) propenoic acid is soluble in water. It has been found that the above object can be achieved by hydrolysis in a mixed solvent of a suitable ether solvent and water. In addition, the inventors of the present invention have prepared a halognophenylpyruvic acid obtained by adding a recombinant plasmid containing a DNA encoding a transaminase derived from a microorganism of another genus to a host microorganism. The present inventors have found that an optically active halogenophenylalanine can be obtained in high yield by treating with a transaminase produced by a living organism, thereby completing the present invention.

That is, according to the present invention, general formula (III)

(However, X represents a halogen atom, and R represents a lower alkyl group.)

Is obtained by hydrolyzing a 4- (halogenobenzylidene) -15-oxazolone compound represented by the formula (II)

(However, the symbols have the same meaning as described above.)

2- (lower alkanoylamino) 1-3- (halognophenyl) propanoic acid compound or a salt thereof represented by the following formula, and then acid in a mixed solvent of water-soluble ether solvent and water By hydrolysis, and further conversion to a salt if desired

(I)

(However, the symbols have the same meaning as described above.)

And the salt thereof represented by the formula: can be produced. Further, according to the present invention, a DNA encoding a transaminase derived from a microorganism belonging to another genus is incorporated in the host microorganism into the halognophenylpyruvic acid (I) or a salt thereof obtained as described above. By reacting the recombinant microorganism containing the recombinant plasmid or a processed product of the microorganism in the presence of an amino donor, the general formula

(V)

( _V)

(However, the symbols have the same meaning as described above.) The optically active halogenophenylalanine represented by or a salt thereof can be produced. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the production method of the present invention will be described in more detail.

 In the production method of the present invention, the compounds used in the reaction are inorganic salts such as hydrochloride, hydrobromide, sulfate, and nitrate, for example, acetate, tartrate, quencher, and the like, as long as the reaction is not hindered. Organic salts such as acid, fumarate and maleate salts; metal salts such as sodium, potassium, calcium and aluminum salts; salts with bases such as ethylamine, guanidine and ammonium salts May be used in the form of a salt. When the compound to be used in the reaction is obtained in a free form, it can be converted to a salt thereof by a conventional method. When the compound to be used in the reaction is obtained as a salt, the salt can be converted to a free form by a conventional method.

 In the present invention, as the “halogen atom”, any of these atoms such as a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom can be suitably used, but a bromine atom and an iodine atom are more preferable, and a bromine atom is more preferable. Is most preferred.

 Further, in the present description, the term "lower alkyl group" means a linear or branched anoalkyl group having 1 to 6 carbon atoms, and specifically, a methyl group, an ethyl group, a propyl group, an isopropyl group, and a butyl group. Group, isobutyl group, sec-butyl group, tert-butyl group, pentynole group, isopenpentyl group, neopentynole group, tert-pentynole group, hexinole group, and isohexyl group. Preferably, it refers to an alkyl group having 1 to 4 carbon atoms, and specific examples thereof include a methyl group, an ethyl group, a propynole group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. More preferably, it is an alkyl group having 1 or 2 carbon atoms (that is, a methyl group or an ethyl group), and particularly preferably a methyl group.

Furthermore, in the present invention, the term "lower alkanoyl" means a straight-chain or branched-chain alkanoyl group having 2 to 7 carbon atoms, specifically, an acetyl group, a propioyl group, a ptyryl group, an isoptyryl group, a valeryl group. And an isovaleryl group. It is preferably an alkanoyl group having 2 to 5 carbon atoms, and more preferably an alkanoyl group. It is a tinole group or a propionyl group, particularly preferably an acetyl group.

 According to the present invention, first, 4- (halogenobenzylidene) -15-oxazolone compound (III) is hydrolyzed with an alkali metal hydroxide to give 2- (lower / recanylamino) 13- (halogenophenyl). Propenoic acid compound (II) is produced.

 Alkali metals such as sodium hydroxide, hydroxide hydroxide and lithium hydroxide have no difference in their effects, but sodium heptaoxide is inexpensive and industrially preferable.

 The amount of the alkali metal hydroxide used is in the range of 1 molar equivalent to 3 molar equivalents to compound (III), preferably in the range of 1.1 molar equivalents to 2 molar equivalents. It is better to use 1.5 molar equivalents.

 The reaction is usually performed at room temperature. For this reason, the reaction proceeds under milder conditions as compared with the conventional method requiring heating, which is industrially preferable. If necessary, the reaction can be carried out under cooling or heating.

 Water is usually used as the solvent, but other organic solvents that do not affect the reaction, for example, ethers such as tetrahydrofuran and dioxane, and alcohols such as methanolic ethanol and ethanol can also be used. It is. It is also possible to use a mixed solvent of these organic solvents and water.

 After hydrolysis with an alkali metal hydroxide, a post-treatment with an acid is carried out to obtain the compound (II) which is a carboxylic acid. It is also possible to obtain as. The alkali metal salt of compound (II) can be isolated and purified by a conventional method. Further, an alkali metal salt can be used in the next step.

 Next, the 2- (lower alkanoylamino) -13- (halogenophenyl) probenic acid compound (11) or a salt thereof is hydrolyzed with an acid and, if desired, converted to a salt to give halogenophenol-rubic acid ( I) or a salt thereof As the acid, a mineral acid other than hydrogen halide, such as nitric acid or sulfuric acid, which usually uses hydrogen halide such as hydrogen chloride or hydrogen bromide, can also be used.

As a solvent, a mixed solvent with water-soluble ether solvent and water is used. in water As the soluble ether solvent, tetrahydrofuran, dioxane and the like are preferable. The ether-based solvent is involved in promoting the reaction and improving the yield. If the amount of the ether-based solvent is small, the progress of the reaction is slow, and a long-time reaction is inevitably required. If the reaction time is prolonged, the produced halognophenylpyruvic acid is decomposed, and the yield decreases, which is not preferable. The ratio of water to the ether-based solvent is in the range of water: ether-based solvent = 10: 1 to 1:10, but it is particularly preferable to perform the operation in the ratio of water: ether-based solvent = 5: 1.

 The reaction can be carried out from room temperature to under heating, but is preferably carried out at a temperature in the range of 50 to 150 ° C, particularly preferably at around 100 ° C. Further, this reaction can be carried out under normal pressure or under calo pressure.

 Examples of the salt of halognophenylpyruvic acid include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and amine salts such as ammonium salt and methylamine salt. Conversion to these salts and isolation / purification can be carried out by a conventional method.

Further, according to the present invention, a recombinant plasmid comprising a DNA encoding a transaminase derived from a microorganism belonging to another genus in a host microorganism, to the halogonofelpyruvate or a salt thereof obtained as described above. Is treated with an amino donor to form a treated product thereof, and further converted to a salt thereof, if desired, to obtain a compound represented by the general formula (V):

(However, the symbols have the same meaning as described above.)

The optically active halogenophenylalanine represented by or a salt thereof can be produced.

 The transaminase may be any as long as it can produce halognophenylalanine (V) from halogenophenylenolepinolevic acid (I) in the presence of an amino donor, and aromatic amino acid 'transaminase (that is, Aromatic-araino ~ acid

aminotransferase), pheninolealanine 'transaminase (ie phenylalanine aminotransferase) Force S Drilled as an example.

 The microorganism serving as a source of DNA encoding transaminase is not particularly limited as long as it is a microorganism capable of producing transaminase, and examples thereof include microorganisms belonging to the genus Paracoccus. Paracoccus denitrificans IF012442 (Paracoccus denitrificans

 IF012442) can be suitably used.

 As a DNA encoding a transaminase, for example, a translation region encoding a phenylalanine transaminase in a phenylalanine transaminase gene derived from a microorganism of the genus Paracoccus can be used.

Hue from Paracoccus microorganisms - Ruaranin 'transaminase gene, for example, the literature (Takagi et _al., Biotechnology and Applied Biochemistry Λ 13 Certificates, a 112- page 119, 1991) in analogy to the method described by shotgun claw Jung It can be isolated from a microorganism of the genus Paracoccus as follows.

 First, a chromosomal DNA is prepared from a microorganism of the genus Paracoccus. After treatment with an appropriate restriction enzyme (such as partial digestion with Sau3AI), the downstream of the appropriate vector plasmid promoter (BamHI cleavage site of pLG339, multicloning of UC18) Site). Next, host Escherichia coli is transformed using the obtained recombinant plasmid.

 By culturing the transformed cells and measuring the transaminase activity using the obtained cells, it can be confirmed that the target gene is contained on the recombinant plasmid. The transaminase activity 1 "is obtained by using microbial cells as an enzyme source in a reaction solution containing phenylinolepinoleic acid, 2-oxo-14-phenylbutyric acid, etc. as substrates and L-glutamic acid, L-aspartic acid, etc. as an amino donor. And can be measured by known methods.

 Further, a Phenylalanine 'transaminase gene derived from a microorganism of the genus Paracoccus can also be obtained by using known nucleotide sequence information.

For example, the literature by Odze et al. (J. Biochem., Vol. 121, pp. 161-171, 1997) states that the phenylalanine transaminase gene isolated from Paracoccus' denitrififans IFO 12442. Base sequence of the chromosome fragment containing The amino acid sequence of heninolealanine 'transaminase is disclosed.

 Design primers and probes based on the disclosed nucleotide sequence information, and use them.Apply PCR (Polymerase chain reaction), colony hybridization, etc. From the rally, a Phenylalanine 'transaminase gene derived from a microorganism of the genus Paracoccus can be obtained.

 The DNA library can be prepared using the chromosome DNA of a microorganism belonging to the genus Paracoccus, for example, by the method described in "Molecular Cloning" (Sambrook et al., Cold Spring Harbor Laboratory Press, 1998).

 The nucleotide sequence of the Phenylalanine 'transaminase gene derived from the microorganism of the genus Paracoccus isolated in this way can be determined, and the translation region can be identified. A DNA fragment containing this translation region can be cut out and used as a DNA encoding phenylalanine transaminase.

The DNA encoding fenylalanine transaminase can use the translation region of the fenylalanine transaminase gene that exists in nature, but it has been obtained by modifying a part of its base sequence. One to six codons encoding one amino acid are known, and when modifying the nucleotide sequence, it is usually designed so that the encoded amino acid sequence does not change. DN a with sequence linked chemically synthesized DN a, fragmentation and bind the DN a, part mutagenesis Iriho (site specific mutagenesis) (Proceedings of National Academy of Sciences ^ ⁸ Volume 1, Chapter 5662～ 5666 page, 1984>.

 Plasmid for expressing transaminase (hereinafter referred to as transaminase expression plasmid) can be obtained by ligating the DNA encoding transaminase downstream of the motor in an appropriate vector plasmid.

The vector plasmid is not particularly limited as long as it is a plasmid that can be replicated in a host microorganism. When Escherichia coli is used as a host microorganism, such vector plasmids include, for example, pBluescript SK (+) (Stratagene), LG339 (Gene, Vol. 18, pp. 335, 1). 9 1982, ATCC 37131), UC 18 (Gene, Vol. 33, p. 103, 1985, ATCC 37253) and the like. Of these, pUC18 is particularly preferred.

 In the transaminase expression plasmid, the DNA encoding the transaminase is operably linked downstream of the promoter, and the transaminase is preferably expressed under the control of the promoter.

 The promoter may be any promoter that can function in the host microorganism, and is not particularly limited. Such a promoter includes, for example, the 1 ac promoter (E. coli lactose operon promoter).

 The nucleotide sequence between the promoter and the translation initiation codon of the transaminase is DNA fragmentation of the DNA encoding transaminase, such as by restriction enzyme treatment, binding of fragmented DNA, and ligation of chemically synthesized linker DNA. It can be constructed by appropriately combining site-directed mutagenesis, PCR and the like.

 A recombinant microorganism can be obtained by introducing the transaminase expression plasmid into a host microorganism by a usual transformation method. Such host microorganisms are not particularly limited, but Escherichia coli can be preferably used. Specifically, Escherichia coli DH5 strain, Escherichia coli JM109 strain, Escherichia coli HB101 strain (Journal of molecular biology, vol. 41, p. 459, 1969, ATCC 33694), Escherichia coli JM105 strain (protein / nucleic acid enzyme, vol. 29, p. 294, 1981) and the like. Among these, Escherichia coli HB101 strain is preferred.

 The recombinant microorganism (transformant) obtained by encapsulation can be used in the present invention. The recombinant plasmid into which DNA encoding a transaminase derived from a microorganism belonging to the genus Paracoccus is integrated is Escherichia coli. It is preferable to use a hybrid organism contained in a host microorganism. In particular, Escherichia coli containing a recombinant plasmid into which a DNA encoding a transaminase derived from Paracoccus' denitrififans is incorporated is more preferable.

Specifically, a deoxyribonucleic acid carrying the genetic information of phenalanalanin transaminase obtained by digesting DNA derived from the chromosome of Paracoccus ■ detririfans with the restriction enzyme Sau3AI, described in JP-A-11-153084, is disclosed. plus The restriction enzyme BamHI is inserted into the restriction enzyme BamHI cleavage site of pLG339, and the resulting hybrid plasmid is digested with the restriction enzymes EcoRV and Sail. Hybrid plasmid integrated into the Smal and Sail cleavage sites

 It is preferable to use a microorganism containing (PPAP142) in Escherichia coli HB101 strain.

 Further, described in JP-A-2000-270882, in the DNA fragment derived from the chromosome of Paracoccus' denitrificans, the original promoter region of the gene source and the region encoding the oral daniase-like protein were deleted from pPAP142, A transformant obtained by introducing the hybrid plasmid PPAP243, which retains only the region necessary for transaminase expression in Escherichia coli, into Escherichia coli HB101 strain

Preferably, HB101 (pPAP243) is used.

 Plasmid PPAP243 is constructed by transcribing the approximately 300 bp DNA fragment containing the N-terminal side of the transaminase translation region (177 bp) in pPAP142 and a part of the upstream untranslated region (95 bp) into the vector plasmid pB1uescript SK (+ ) (Manufactured by Stratagene), inserted into the Eco RV cleavage site, cut the resulting plasmid with Eco RI, and integrated the resulting DNA fragment into the Eco RI cleavage site of PPAP142.

In the production method of the present invention, a transaminase-expressing plasmid pPAP243 derived from Paracoccus ■ denitrificans described in JP-A-2000-270882, which is contained in Escherichia coli HB101 strain, is transformed into HB101 ( p PAP 243) is particularly preferred. . Microorganisms (live cells, cultures, etc.) used in the enzyme reaction and their processed products (washed cells, dried cells, culture supernatant, crushed cells, self-erasing germs, cell extracts) Etc.) may be any as long as it has transaminase activity, and its form is not particularly limited. Cultivation of the microorganism can be performed by a conventional method. For example, the pH of a normal nutrient medium containing a carbon source, a nitrogen source, organic nutrients, inorganic salts, etc. is adjusted to 5.0 to 9.0, and after inoculating microorganisms, the temperature is 10 to 45 ° C, preferably 28 to 37 ° C. Aerobic culture may be performed at ° C. Transaminase is constructed to be expressed under the control of 1 ac promoter In this case, it is desirable to add an enzyme-inducing substance such as ratatose, isopropyl-11-thiol] 3-D-galactoside to the medium in order to enhance the expression of transaminase.

 Viable cells can be obtained from the culture of the microorganism by centrifugation or filtration. Washed cells can be obtained by washing the viable cells with physiological saline or the like, and dried cells can be obtained by freeze-drying or acetone-drying the viable cells / washed cells. . In addition, live cells, washed cells, etc., are treated by various physicochemical methods (eg, ultrasonic treatment, French press, osmotic pressure, freeze-thawing, alumina destruction, lytic enzymes, surfactants, organic solvents, etc.). By the treatment, a crushed cell can be obtained, and an extract of the cell can be obtained by removing a solid substance from the crushed cell of the cell by filtration or centrifugation. .

 In addition, from the viewpoint of economical use of the catalyst, the obtained enzyme, bacterial cell extract, etc. are immobilized on a suitable solid support and prepared as a reusable immobilized enzyme for a bioreactor. Is also good. Methods for immobilizing such enzymes are known in the art. The halognophenylpyruvic acid and the amino donor as substrates can be supplied to the reaction system in a free form or in a salt form.

 Examples of the amino donor include L-aspartic acid and L-glutamic acid, and L-aspartic acid is preferred. The amino donor is usually used in an amount of usually from 1 to 3 mol, particularly preferably from 1.3 to 1.5 mol, per mol of halognophenylpyruvic acid.

The enzymatic reaction is preferably carried out in consideration of the stability of the transaminase in less than 4 0 ° C is carried out at a good better Iga, especially 28 to 37 ° C, also becomes the _P H is 7-9 It is preferable to adjust it. During the enzymatic reaction, a surfactant such as cetyltrimethylammonium bromide or cetylpyridinium bromide is added to the reaction solution in an amount of about 0.001 to 0.1%. This can promote the enzymatic reaction.

Separation and purification of the desired halogenophenylarayun produced in the reaction solution can be easily carried out by using a conventional ion exchange resin method or other known methods alone or in combination. The starting material, 4- (halogenobenzylidene) -15-oxazolone compound (III) has the general formula (IV)

(However, the symbols have the same meaning as described above.)

And a halogenobenzaldehyde represented by the general formula (VI)

 0

R to N COOH (VI)

 H

(However, the symbols have the same meaning as described above.)

Can be obtained by condensing an N-lower alkenylglycine represented by The condensation reaction between the compound (IV) and the compound (VI) is carried out by a conventional method, for example, in the presence of a base and a detaurant in a suitable solvent or without a solvent. As the base, sodium acetate can be suitably used. Acetic anhydride can be suitably used as the dehydrating agent. When an appropriate amount of acetic anhydride is used, for example, when acetic anhydride is used in an amount of 2 molar equivalents or more with respect to compound (IV), it is not necessary to use a solvent. If a solvent is used, there is no particular limitation as long as the solvent does not affect the reaction. This reaction is carried out at room temperature to under heating, preferably at 100 to 150 ° C.

 Hereinafter, the present invention will be described in detail with reference to Examples, but these do not limit the present invention in any way.

Example 1

(1) 138.3 g of 4- (4-bromobenzylidene) _2-methyl-5-oxazolone obtained in Reference Example 1 was added to 382 mL of a 2N aqueous sodium hydroxide solution, followed by stirring at room temperature for 4 hours. After dilution with water, Norite was added and left at room temperature for 1 B. After filtering the insolubles, the filtrate was washed with ethyl acetate. The filtrate was acidified with concentrated hydrochloric acid and cooled on ice. The precipitated crystals are collected by filtration, washed with water, and blow-dried to give 2- (acetylamine). No.) 13- (4-Promophenyl) 133.6 g of propenoic acid was obtained. Melting point: 22 1 222 ° C

 Purity was measured by high performance liquid chromatography (HPLC), and the purity was 89% and the yield was calculated to be 81%.

HP LC conditions

Column: CHIRALCEL 0J (manufactured by Daicel Chemical Industries, Ltd.), elution solvent: hexane / ethanol Z trifluoroacetic acid = 92Z8Z0.5, flow rate: 1.0 ml / min, temperature: 40 ° C, detection: 254 nm.

 (2) 2- (Acetylamino) _3- (4-bromophenol) A mixture of 40.Og of propenoic acid, 80Om1 of 2N hydrochloric acid and 16Om1 of dioxane was stirred at 100 ° C for 4 hours. After allowing to cool, the liquid volume was concentrated to about half and cooled with ice. The precipitated crystals were collected by filtration, washed with water, and blow-dried to obtain 32.7 g of (4-promophenyl) pyruvic acid. Melting point: 179—181 ° C

 When the purity was measured under the above-mentioned HPLC conditions, the purity was 88% and the yield was calculated to be 95%.

Example 2

 Production medium (1% lactose, 0.2% pudose, 0.5% L-sodium glutamate, 2% corn steep liquor, 2% mist N, 0.3% rhodium phosphate, 0% Medium containing 7% dibasic potassium phosphate, 0.1% ammonium sulfate, 0.025% magnesium sulfate .Hydrate, 0.03% caraline (pH 7.0) 1

E. coli transformed HB101 (pPAP 243) was inoculated in a loop of 0 Om1 and cultured at 37 ° C. for 24 hours. The culture was centrifuged at 8000 X g to remove the supernatant. Purified water was added to the precipitated cells so that the amount of the culture broth became the initial amount of the culture solution, and an enzyme solution was obtained. 9.64 g of (4-bromophenyl) pyruvic acid obtained in Example 1 and 9.09 g of L-aspartic acid monosodium salt monohydrate were added to 700 ml of the enzyme solution thus obtained in 2 ml of 2N aqueous solution. The pH was adjusted to 8.6 with an aqueous sodium oxide solution, and the mixture was stirred at 30 ° C. to perform an enzyme reaction. Two hours after the start of the reaction, 2.89 g of (4-bromopheninole) pinolevic acid and 2.73 g of L-aspartic acid monosodium monohydrate were added at 30-minute intervals until 2 hours later. At that time, 2N sodium hydroxide The pH was adjusted to 8.6 with an aqueous solution. Then, at hourly intervals until 5 hours later, 2.89 g of (4-promophenyl) pyruvic acid and 2.73 g of L-aspartic acid monosodium salt monohydrate were added (with 2N aqueous sodium hydroxide solution). adjusted to pH 8.6). Seven hours after the start of the reaction, 200 ml of the enzyme solution was added, the pH was adjusted to 8.6 with a 2N aqueous sodium hydroxide solution, and the mixture was stirred for 21 hours, and then measured under the following HPLC conditions (1). The conversion rate to L-4-promofenilalanine in the reaction solution was 92.2%. After the completion of the reaction, a 30% aqueous sodium hydroxide solution was added to make the mixture basic, and the mixture was centrifuged at 8000 X g to collect a supernatant. The supernatant was weakly acidified by adding concentrated hydrochloric acid, and then concentrated under reduced pressure until the liquid volume was about 1 to 8 to 19. After heating to 50 ° C, the mixture was cooled and the precipitated crystals were collected by filtration. The obtained crystals were washed and dried to obtain 20.5 g of L-4-promopheniralanine. Melting point: 213—214 ° C

 When the optical purity was measured under the HPLC condition (2) described below, the optical purity was 100%.

HPLC conditions (1)

Column: Nucleocil ₁₀ C ₁₈ (MACHEREY- NAGEL Inc.), _{eluent: 4 OmM KH 2 P0 4 -} H 3 P0 4 (pH 2.5) Z Asetonitoriru = 9Zl, flow rate: 1.0 ml / min, temperature: 50 ° C, Detection: 254 nm.

HPLC conditions (2)

Column: Crownpak CR (+) (4.0 X 250 ram) ( manufactured by Daicel Chemical Industries, Ltd.), elution Solvent: HC10 _{4 (P} H2.0) / methanol = 85Z15, flow rate: 0.4 MLZ min, temperature: 2 5 ° C , Detection: 254 nm.

Reference example 1

 After dissolving 110 g of 4-bromobenzaldehyde, 69.6 g of N-acetinoleglysine, 36 g of sodium acetate, and 151.7 g of acetic anhydride at 80-100 ° C, the mixture was stirred at 140 ° C for 1 hour. . The mixture was left overnight at room temperature, and the precipitated crystals were collected by filtration, washed with water, and dried to obtain 140.4 g of 4- (4-bromobenzylidene) -12-methyl-5-oxazolone.

Reference example 2 (1) Preparation of expression plasmid pPAP142

 According to the method described in Takagi et al. (Biotechnology and Applied Biochemistry, Vol. 13, pp. 112-119, 1991) or JP-A-1-1530084, ぺ Lacoccus denitrificans (Paracoccus denitrificans IF012442) A DNA fragment containing the transaminase gene was isolated from the chromosomal DNA of the above, and a recombinant expression plasmid PPAP142 containing this was obtained.

 PPAP142 is linked downstream of the 1 ac promoter of the vector plasmid pUC18, a force of a paracoccus' denitrifinance chromosomal DNA fragment (about 2.2 kb) containing the transaminase gene. In this chromosomal DNA fragment, a promoter region and a region encoding a rhodaace-like protein derived from Paracoccus' denitrificans exist upstream of the transaminase gene translation region.

 (2) Preparation of expression plasmid pPAP243

 Plasmid p PAP142 obtained in the above (1), which is obtained by deleting a promoter region and a region encoding a rodinase-like protein derived from Paracoccus ■ denitrificans, is disclosed in Japanese Patent Application Laid-Open No. 2000-270882. It was constructed according to the method described in the gazette. First, PCR (polymerase chain)

A fragment containing the N-terminal side of the transaminase translation region (177 bp) and part of the upstream untranslated region (95 bp) in pPAP142 was amplified by the reaction). As primers for PCR, synthetic oligonucleotides were used as sense primers and antisense primers. The sequence of the sense primer was designed so that the N-terminal side of the upstream untranslated region partial sequence was partially modified to generate a Kp η I recognition site. In addition, the sequence of the antisense primer was designed based on the partial distribution in the transaminase translation region. P CR is plasmid _P PAP142 of 5μ 1 (0.09 μ g), primers each 4 Micromax, 1.0 units of DNA polymerase, 10 fold buffer 5 [mu] 1, Dokishi NT P and 30.5Myu 1 of each 5 [mu] 1 (2 mM) 9 using a mixture of water

The process of 30 for 4 seconds, 30 seconds at 55 ° C, and 1 minute at 72 was repeated 30 times. After the reaction, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment (approximately 300 bp) of the target PCR product was recovered from the phenol. The obtained DNA fragment was subjected to Eco-coupling of the vector plasmid pB1uescript SK (+) (Stratagene). RV was inserted into the cleavage site to obtain plasmid pBSKl. The plasmid pBSKl is further digested with EcoRI, and the obtained DNA fragment of about 150 bp is digested with! ) The recombinant expression plasmid PPAP243 was obtained by ligating with the EcoRI digested fragment of PAP142 (about 3800 bp: including the ampicillin resistance gene, the 1 ac promoter and the 3 terminal end of the transaminase gene).

 (3) Acquisition of Escherichia coli transformant containing expression plasmid P PAP243

 The expression plasmid ρ PAP 243 obtained in the above section (2) was introduced into Escherichia coli (Escherichia coli) HB101 strain by a conventional method to obtain a transformed original HB101 (pPAP243). Industrial applicability

 The production method of the present invention is an industrially advantageous method which eliminates the drawbacks of requiring a low yield of halognophenylpyruvic acid and a long reaction time in the conventional method. Further, by the transamination method of the present invention, optically active halogenophenylalanine can be obtained in a relatively short time and in good yield.

Claims

The scope of the claims 1. General formula (II)

(However, X represents a halogen atom, and R represents a lower alkyl group.) Wherein the compound represented by the formula (I) is hydrolyzed with an acid in a mixed solvent of water-soluble ether solvent and water with an acid, and then converted into a salt, if desired.

(However, the symbols have the same meaning as described above.) A method for producing halognofenirvirbic acid or a salt thereof represented by the formula: 2. —General formula (III)

(However, X represents a halogen atom, and R represents a lower alkyl group.) Is hydrolyzed with an alkali metal hydroxide to give a compound of the general formula (II)  (II)

(However, the symbols have the same meaning as described above.) A compound represented by the formula: or a salt thereof, is hydrolyzed with an acid in a mixed solvent of water-soluble ether solvent and water with an acid, and further converted to a salt, if desired. I)

(However, the symbols have the same meaning as described above.) A method for producing halognophenylpyruvic acid or a salt thereof represented by the formula: 3. General formula (IV)

(However, X represents a halogen atom.) And a compound represented by the general formula (VI)  0  J eight  Rz, Ν '"COOH  H (However, R represents a lower alkyl group.) Condensing the compound represented by the general formula (I I I) (Hi)

(However, the symbols have the same meaning as described above.) After preparing a compound represented by the formula, the compound is hydrolyzed with an alkali metal hydroxide to obtain a compound represented by the general formula (II):

(However, the symbols have the same meaning as described above.) Or a salt thereof, and then hydrolyzing with an acid in a mixed solvent of a water-soluble ether solvent and water with an acid and, if desired, converting the compound into a salt. )

(However, the symbols have the same meaning as described above.) The method for producing halognofluorbirubic acid or a salt thereof represented by the formula: 4. General formula (I) obtained by the method according to claim 1, 2 or 3.

(However, X represents a halogen atom.) A hybrid organism or a microorganism thereof containing a recombinant plasmid containing a DNA encoding a transaminase derived from a microorganism of another genus in a host microorganism, in the halogenophenol-rubyric acid or a salt thereof represented by the following formula: Wherein the treated product is treated in the presence of an amino donor, and further converted to a salt if desired.

(However, the symbols have the same meaning as described above.) A method for producing an optically active halognophenylalanine or a salt thereof represented by the formula: 5. The host organism contains a recombinant plasmid containing a DNA encoding a transaminase derived from a microorganism belonging to another genus, and the recombinant microorganism has a DNA encoding a transaminase derived from a microorganism belonging to the genus Paracoccus. A recombinant microorganism that contains a recombinant plasmid in a host microorganism that is Escherichia coli. The manufacturing method according to claim 4, wherein  6. The method according to claim 5, wherein the transaminase derived from a microorganism belonging to the genus Paracoccus is a transaminase derived from Paracoccus denitrififans. 7. Phenylalanine obtained by digestion of DNA derived from a chromosome of Paracoccus' denitrificans with a restriction enzyme Sau3AI by a transfection plasmid incorporating a DNA encoding a transaminase derived from a microorganism belonging to the genus Paracoccus. genetic the Dokishiripo nucleic carrying information embedded in restriction enzyme BaraHI cleavage site of the plasmid _P LG339, de responsible for genetic information obtained by digesting the resulting plasmid further with the restriction enzymes EcoRV and Sail phenylene Ruaranin transaminase Okishiribo nucleic acid Which is a hybrid plasmid obtained by incorporating the plasmid pUC18 into the restriction enzymes Smal and Sail cleavage sites of the plasmid pUC18, and the native promoter of the gene source in the DNA fragment derived from the chromosome of Paracoccus' dutrififans from the hybrid plasmid. Domains and Roda Youth Quality is Haipuri' de plasmid was deleted region encoding, method according to claim 5, wherein.  8. The method according to any one of claims 1 to 7, wherein X is a bromine atom or an iodine atom.  9. The method according to claim 8, wherein X is a bromine atom.