WO2006103995A1

WO2006103995A1 - PROCESS FOR PRODUCTION OF OPTICALLY ACTIVE α-AMINO ACID DERIVATIVE

Info

Publication number: WO2006103995A1
Application number: PCT/JP2006/305737
Authority: WO
Inventors: Makoto Ueda; Hirokazu Nanba
Original assignee: Kaneka Corporation
Priority date: 2005-03-25
Filing date: 2006-03-22
Publication date: 2006-10-05

Abstract

A process for producing an optically active 5-substituted hydantoin derivative and an optically active N-carbamoylamino acid derivative comprising cyclizing a racemic N-carbamoylamino acid derivative with a hydantoinase stereoselectively, the cyclization being performed in the presence of a divalent metal ion using such a hydantoinase that the hydrolysis of the 5-substituted hydantoin derivative into the N-carbamoylamino acid derivative (the reverse reaction) caused by the hydantoinase is inhibited by the metal ion. The method can produce an optically active 5-substituted hydantoin derivative and an optically active N-carbamoylamino acid derivative in a simple manner. These compounds can be used for readily producing an optically active α-amino acid derivative which is useful as an intermediate for a medicine or the like.

Description

Specification

Optically active a Method for producing a single amino acid derivative

Technical field

The present invention relates to a method for producing an optically active α-amino acid derivative that is useful as an intermediate for pharmaceuticals and the like. More specifically, in the presence of a divalent metal ion, the racemic 力 -strength rubamylamino acid derivative is allowed to produce a hydantoinase whose hydrolysis activity is inhibited by the metal ion. The present invention relates to a method for obtaining a hydantoin derivative and an optically active カル carbamyl amino acid derivative.

Background art

As a method for producing an optically active amino acid, various methods such as an extraction method, a chemical synthesis method, a fermentation method, and an enzymatic synthesis method are known. The extraction method requires large-scale purification equipment from protein hydrolysates. In the chemical synthesis method, the amino acid to be produced is usually a racemate, so that an expensive resolving agent, an asymmetric catalyst, etc. are required to produce an optically active substance. Fermentation methods have low product concentrations and require large-scale purification equipment, as with extraction methods. By utilizing an inexpensive biocatalyst, the enzymatic synthesis method can avoid these problems and provide an efficient production method at a lower cost.

[0003] As a method for synthesizing D-a amino acid by enzymatic synthesis, for example, a microorganism or enzyme having hydantoinase activity is used to selectively hydrolyze racemic 5-substituted hydantoin derivatives to form D Lubamil D—a Methods for producing amino acid derivatives are known (Patent Documents 1 and 2). In addition, it is generally known that there are examples in which divalent metal ions such as manganese and cobalt contribute to the improvement of enzyme activity in a hydrolysis reaction in which a hydantoinase is allowed to act on a 5-substituted hydantoin derivative to open the ring. (Non-patent documents 2 and 3).

[0004] On the other hand, a method of producing an optically active 5-substituted hydantoin derivative by reacting a racemic N-strength rubamyl amino acid derivative with a hydantoinase to produce a cyclization is also known (Patent Documents 3 and 4). There are also known examples of adding manganese, which is a divalent metal ion, to carry out a cyclization reaction (Patent Document 5). Patent Document 1: JP-A 53-44690

Patent Document 2: JP-A-53-91189

Patent Document 3: JP-A-60-180596

Patent Document 4: JP-A-1-124398

Patent Document 5: WO03Zl06689

Non-patent literature 1: Advances Biochemical EngmeermgZBiotechnoiogy, 41 卷, 29, 1990

Patent Document 2: Bioscience Biotechnology Biochemistry ^ 58 卷, 1621, 19 94

Non-Patent Document 3 Journal of Molecular Catalysis B: Enzymatic, 2 卷, 163, 1997

Disclosure of the invention

Problems to be solved by the invention

[0005] However, it is well known that the reaction of hydantoinase is a reversible reaction, and in the method of adding a divalent metal ion, the reverse reaction is not only the target hydrolysis reaction (or cyclization reaction). Is easily expected to be promoted. In order to efficiently produce the target optically active substance, a method of promoting only the target hydrolysis reaction or cyclization reaction, more preferably a method of promoting the target reaction and inhibiting the reverse reaction Is desired.

In view of the above, an object of the present invention is to provide a practical method for industrial production, for example, an optically active α-amino acid derivative useful as an intermediate of a pharmaceutical can be easily produced from a predetermined raw material. There is.

Means for solving the problem

As a result of intensive studies in view of the above, the present inventors have determined that optically active 5-substituted hydantoin derivatives can be obtained by stereoselectively cyclizing racemic and strong rubamyl amino acid derivatives using hydantoinase. In the case of carrying out the reaction for obtaining an optically active rubamoyl amino acid derivative 1, the divalent metal ion inhibits the hydrolysis activity, which is the reverse reaction of the above cyclization reaction. Found that the cyclization reaction was greatly accelerated. [0008] As a part of the subject of the present invention, for example, the following can be cited.

That is, the present invention relates to the general formula (2):

[0010] [Chemical 10]

[In the formula, R is an optionally substituted alkyl group having 1 to 20 carbon atoms, has a substituent !, may be an aralkyl group having 7 to 20 carbon atoms, or is substituted. An optically active 5-substituted dantoin derivative represented by the formula (1), or an aryl group having 6 to 20 carbon atoms, and * represents an asymmetric carbon atom. 3):

[0012] [Chemical 11]

[0013] (wherein, R and * are the same as those described above)

In the presence of a divalent metal ion, a hydantoinase that inhibits the activity of hydrolyzing a 5-substituted hydantoin derivative into N-strength rubamyl amino acid by the divalent metal ion is represented by the general formula (1):

[0014] [Chemical 12]

[0015] The present invention relates to a production method comprising stereoselectively cyclizing a high-strength rubamyl amino acid derivative represented by the formula (wherein R is as defined above).

[0016] The present invention also provides an optically active 5-substituted hydantoin derivative represented by the above formula (2), and

, An optical activity represented by the formula (3) Ν—a method for producing a powerful ruvamil amino acid derivative, wherein hydantoinase is used in the presence of cobalt ion or zinc ion, and the formula (1): The present invention relates to a production method characterized by stereoselectively cyclizing a strong rubamyl amino acid derivative.

The invention's effect

[0017] The present invention has the above-described configuration, and can efficiently produce an optically active 5-substituted hydantoin derivative and an optically active high-power rubamyl amino acid derivative.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] As described above, a general hydantoinase has an activity of hydrolyzing a 5-substituted hydantoin derivative to produce a strong rubamyl amino acid derivative, and the reverse reaction of the hydrolysis reaction. It is known to have both the activity of cyclizing strong rubamyl amino acid derivatives to form 5-substituted hydantoin derivatives. In addition, it is known that the enzyme activity of the above hydrolytic reaction of hydantoinase is improved by divalent metal ions.

[0019] On the other hand, as a result of examination by the present inventors, it was found that the D-form selective hydantoinase of Example has a property that the activity of the hydrolysis reaction is inhibited by a predetermined divalent metal ion ( (See Reference Examples 1 and 2 below.)

[0020] Generally, if a hydrolytic enzyme inhibits hydrolytic activity by a specific compound, the reverse reaction is expected to be inhibited by the compound. However, as a result of further studies by the present inventor, surprisingly, the D-form selective hydantoinase of Example It was proved that the cyclization reaction was promoted by divalent metal ions.

1 to 3).

Hereinafter, the present invention will be described in detail.

[0022] 1. Racemic N-powered rubamyl amino acid derivative

In the racemic N-strength rubamyl amino acid derivative (1) used in the present invention, R has a substituent, and may have an alkyl group having 1 to 20 carbon atoms, a substituent! However, it also has an aralkyl group having 7 carbon atoms or a aralkyl group having 20 substituents! Or may represent an aryl group having 6 to 20 carbon atoms.

[0023] The alkyl group having 1 to 20 carbon atoms is not particularly limited, and for example, a straight chain alkyl group such as a methyl group, an ethyl group, or an n-propyl group, an isopropyl group, an isobutyl group, or a t-butyl group. And branched alkyl groups such as a neopentyl group or a t-pentyl group. The aralkyl group having 7 to 20 carbon atoms which may have a substituent is not particularly limited, and examples thereof include a benzyl group, an indolylmethyl group, a 4-hydroxybenzyl group, and 3, 4-methylenedialkyl. And an oxybenzyl group. Examples of the aryl group having 6 to 20 carbon atoms which may have a substituent include a phenol group and a 4-hydroxyphenol group. The alkyl group, aryl group or aralkyl group may be unsubstituted or may have a substituent. Examples of the substituent include an amino group, a hydroxyl group, a phenol group, an aryl group, an alkanol group, an alkenyl group, an alkyl group, an alkoxyl group, and a halogen atom.

[0024] 2. Acquisition of hydantoinase

Hydantoinase is an enzyme having an activity of hydrolyzing a 5-substituted hydantoin derivative to produce an N-force rubamylamino acid derivative. In general, this enzyme is known to cyclize an N-strength rubamoyl amino acid derivative to produce a 5-substituted hydantoin derivative as the reverse reaction of the hydrolysis reaction. In addition, in the hydrolysis reaction that causes ring-opening by acting on 5-substituted hydantoin derivatives, it has been known that conventional hydantoinases have improved enzyme activity in the presence of divalent metal ions such as manganese and conoleto. It has been.

[0025] On the other hand, the hydantoinase used in the present invention has a property that its hydrolysis activity is inhibited by a predetermined divalent metal ion. Therefore, as the hydantoinase of the present invention, For example, hydantoinase derived from animals, plants, or microorganisms and having hydrolytic activity inhibited by divalent metal ions can be used. For industrial use, hydantoinase derived from microorganisms is preferred.

[0026] The microorganism from which hydantoinase is derived can be used as long as it is a microorganism having the ability to produce the enzyme. For example, the following known microorganisms having the ability to produce the enzyme can be mentioned. . In other words, bacteria include Aceto bacter, Achromobacter, Aerobacter, Agrobacterium, Alcaligenes, Arthrobacter, Bacillus ( Bacillus), Brevibacterium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Klebsiella, Microbacteria (Microbacterium), Micrococcus, Protaminobacter (Protaminobacter, Proteus no., Proteus no, No. ~~ Domonas for Pseudomonas (Sartina), Serratia, Serratia, Xanthomonas Genus (Xanthomonas), Aeromonas, Flavobataterium (Fla vobacterium), or a microorganism belonging to the genus Rhi zobium.

[0027] As actinomycetes, microorganisms belonging to the genus Actinomyces, Mycobacterium, Nocardia, Streptomyces, Actinoplanes or Rhodococcus Can be mentioned.

[0028] Examples of the fungi include microorganisms belonging to the genus Aspergillus, Paecilomyces, Penicillium, and the like.

[0029] Examples of the yeast include microorganisms belonging to the genus Candida, the genus Phichia, the genus Rhodo torula or the genus Torulopsis.

[0030] Preferably, enzymes derived from microorganisms belonging to the genus Agrobacterium, Bacillus, Pseudomonas or Rhizobium can be mentioned. More preferably, Agrobacterium sp. KNK712 (FERM BP—1900), Bacillus sp. KNK2 45 (FERM BP— 4863), Pseudomonas putida NBRC12 996, Pseudomonas sp. (Pseudomonas sp.) KNK003A (FERM BP— 3181) or Rhizobium sp. KNK1415 (FERM BP— 44) Is mentioned.

[0031] Above, Agrobacterium sp. KNK712 (FERM BP— 1900), Bacillus sp. KNK245 (FERM BP— 486 3), Pseudomonas sp. KNK003A (FERM BP-3 181) and Rhizobium sp. KNK1415 (FERM BP-441 9) are registered with the Patent Product Deposit Center (IPOD: 305- 8566 Deposited at Tsukuba Sakai Higashi, 1-chome, 1-chome, 1-center, 6), Ibaraki, Japan.

[Deposit date]

Agrobacterium sp. KNK712 (FERM BP— 1900): May 31, 1988

Bacillus sp. KNK245 (FERM BP— 4863): November 2, 1994

Pseudomonas sp. KNK003A (FERM BP— 3181): December 1, 1990

Rhizobium sp. KNK1415 (FERM BP- 4419): September 22, 1993.

[0032] The above-mentioned Pseudomonas putida NBRC12996 is an independent administrative agency, National Institute of Technology and Evaluation, Biotechnology Headquarters, Biogenetic Resource Division (NBRC: 292-0818, Chiba Prefecture Kisarazu Kazusa Kamashisa 2-5-8) It is stored and can be obtained from the same institution.

[0033] In order to obtain a highly active bacterium that efficiently produces hydantoinase, it is effective to produce a transforming microorganism as is well known. As a preparation method, for example, as described in WO96Z20275, a strain demonstrating hydantoinase activity also clones the hydantoinase gene, then creates a thread-replaceable plasmid with an appropriate vector, and uses this to prepare an appropriate host. Obtained by transforming bacteria. Recombinant DNA technology is well known in the art.

[0034] Any transformant obtained in this manner that produces a high yield of D-form selective hydantoinase can be used as long as the hydrolysis activity is inhibited by a divalent metal ion. As an example, the Escherichia coli HB 101 (pTH 104) (FERM BP— 4864) containing a hydantoinase gene derived from Bacillus sp. K NK245 (FERM BP-4863) described in WO96Z20275. Can be mentioned. The production of hydantoinase by these transformants, or the production of hydantoinase by a strain exhibiting the above-mentioned hydantoinase activity, for example, can be performed by culturing using a normal nutrient medium described in WO96 / 20275. In addition, treatment for enzyme induction can be performed.

[0035] The above-mentioned Escherichia coli HB 101 (pTHl 04) (FERM BP—486 4) is a patent biological deposit center (IPOD: 305) -8566 Deposited at Tsukuba Sakai Higashi, 1-chome, 1-chome, 1st, Central 6), Ibaraki, Japan (Deposit date: November 2, 1994).

[0036] In the present invention, the hydantoinase produced by the above-mentioned microorganism can be used not only as the enzyme itself, but also as a form of a microorganism having the enzyme activity or a processed product thereof. Here, the treated product of microorganisms means, for example, a crude extract, cultured cells, freeze-dried cells, acetone-dried cells, or a crushed product of these cells. Furthermore, the hydantoinase or a processed product thereof may be used as the enzyme itself or as a fixed enzyme obtained by fixing it with a known means in the form of cells. The immobilization may be carried out by a cross-linking method, a covalent bonding method, a physical adsorption method, a comprehensive method, etc., which are well known to those skilled in the art.

[0037] 3. Optical activity by hydantoinase 5 Oki Hydantoin Ken-conductor, and optical activity N synthetic method of rubamyl amino acid derivative

Next, the racemic N-strength rubamyl amino acid derivative (1) is stereoselectively cyclized with hydantoinase to synthesize an optically active 5-substituted hydantoin derivative (2) and an optically active N-force rubamyl amino acid derivative (3). How to do will be described. The enzyme reaction of the present invention can be carried out by the following method.

[0038] A racemic N-type rubamyl amino acid derivative represented by the general formula (1) is used as a substrate, and the reaction is carried out in an aqueous medium in the presence of the above-mentioned hydantoinase and a divalent metal ion. The concentration of the divalent metal ion is 0. OlmM or more, 1M or less, preferably 0. ImM or more, 1OmM or less, and the substrate concentration is 0.1% or more, 90% (wZv) or less, preferably 1% or less. Above, the reaction is performed in a dissolved or suspended state at 60% (wZv) or less. The reaction temperature is adjusted to an appropriate temperature of 10 ° C or higher and 80 ° C or lower, preferably 20 ° C or higher and 60 ° C or lower, and is kept at pH 4 or higher, 9 or lower, preferably pH 5 or higher, 8 or lower for a while. It may be left still or stirred. Further, the substrate may be added continuously. The reaction can be carried out batchwise or continuously. The reaction of the present invention may be performed using an immobilized enzyme, a membrane reactor, or the like.

[0039] Examples of the divalent metal ion include, but are not limited to, ions such as vanadium, chromium, manganese, iron, conoleto, nickel, copper, zinc, molybdenum, magnesium, or calcium. Cobalt, zinc, manganese or nickel ions are preferable, and cobalt or zinc ions are more preferable. These metal ions can be used alone or in combination of two or more metal ions.

[0040] Examples of the aqueous medium include water, a buffer solution, an aqueous medium containing a water-soluble organic solvent such as ethanol, or an organic solvent that is difficult to dissolve in water, such as ethyl acetate, butylacetate, toluene, A suitable solvent such as a two-layer system with an aqueous medium containing an organic solvent such as black mouth form and n-xane can be used. Furthermore, antioxidants, surfactants, coenzymes, metals, etc. can be added as necessary.

[0041] As a matter of fact, only one optically active form of the racemic N-force rubamyl amino acid derivative is cyclized by the stereoselective hydantoinase of the present invention to produce an optically active 5-substituted hydantoin derivative. However, an optically active N-force rubamyl amino acid derivative having the opposite steric structure remains. Isolation of the obtained optically active 5-substituted hydantoin derivative and optically active N-force rubamyl amino acid derivative can be performed by a conventional separation method such as extraction, concentration, crystallization, or column chromatography. Or they can be separated and purified by their combination.

[0042] Further, the obtained optically active 5-substituted hydantoin derivative and optically active N-carbamylamino acid derivative can be obtained by known chemical methods (for example, acid / alkali treatment) or enzymatic methods (for example, decarbamylase). Can be easily derived to the corresponding optically active amino acid by the weak ruby moisturizing treatment.

In the above description, as an example of the N-force rubamyl amino acid derivative of the present invention, the racemic N-force rubamyl amino acid derivative is exemplified. A conductor may be used. When an optically active N-force rubamyl amino acid derivative is used, the optical purity of, for example, the remaining optically active N-force rubamyl amino acid derivative or the optically active 5-substituted hydantoin derivative produced by the hydantoinase of the present invention. Can be improved.

Example

[0044] Specific examples of the present invention are shown below. However, the present invention is not limited to these examples.

[0045] (Participant Example 1) Synthesis of D—N—strength rubermoylleucine using hydantoinase

Escherichia coli HB101 (pTH104) (FERM BP-4864), a recombinant E. coli with D-selective hydantoinase activity, sterilized in a 500 ml Sakaguchi flask (tryptone 16 g, yeast extract 10 g, chloride) 5 g of sodium, 11 deionized water, 400 ppm of manganese chloride, pH 7.0 before sterilization, and filter sterilized ampicillin sodium added separately at a final concentration of lOO ppm) and shaken at 37 ° C for 24 hours. Cultured aerobically. The resulting culture is collected by centrifuging from 1 ml of the culture broth, suspended in 5 ml of 10 mM Tris-HCl buffer (pH 8.5), disrupted by sonication, and centrifuged to centrifuge the cells. The insoluble matter was removed, and a crude enzyme solution of D-form selective hydantoinase was obtained. To 0.1 ml of the obtained crude enzyme solution, 4 ml of 50 mM sodium carbonate Z sodium hydrogen carbonate buffer (pH 8.7) containing 30 mM D-5-isobutylhydantoin and various metal salts shown in Table 1 was mixed. After standing at 40 ° C for 15 minutes, the reaction was stopped by adding 1 ml of 5N sulfuric acid. Table 1 shows the results of analyzing the amount of D—N—force rubamoyl leucine produced using high performance liquid chromatography (HPLC).

[HPLC analysis conditions]

Column: COSMOSIL 5C18—AR (4.6 mm x 250 mm, manufactured by Nacalai Testa), eluent: 10 mM potassium phosphate buffer (pH 2) Z-acetonitrile = 5 Zl, flow rate: lmlZ min, column temperature: 30 ° C, measurement wavelength: 210nm

[0046] [Table 1] Additive metal salt; 辰 D—N—Strengthened rubermoyl leucine Relative activity

(mM) Production (U mol) (%) None-5.1 100.0 Cobalt chloride 0.1 3.7 72.6

1 1.9 38.0 Duplex sulphate 0.1 1.1 21.0

1 0.4 7.5

[0047] As shown in Table 1, in the case of the D-selective hydantoinase of Example, the formation reaction of D—N-powered rubamoyl leucine by hydrolysis of D-5 isobutylhydantoin is carried out with a predetermined metal ion (for example, cobalt and Divalent metal ions containing Z or zinc).

[0048] (Participant example 2) Synthesis of D—N-strength rubermoyl (4-hydroxyphenyl) glycine using hydantoinase

After culturing the cells from 1 ml of the culture solution obtained in Reference Example 1 by centrifugation, suspending the cells in 5 ml of 10 mM Tris-HCl buffer (pH 8.5), crushing the cells by ultrasound, and then centrifuging the cells by centrifugation. The insoluble matter derived from the body was removed and a crude enzyme solution of D-form selective hydantoinase was obtained. The obtained crude enzyme solution (0.1 ml) was added with 30 mM DL-5- (4 hydroxyphenol) hydantoin and 50 mM sodium carbonate Z sodium bicarbonate buffer (PH8. 7) 4ml was added and mixed. After standing at 40 ° C for 15 minutes, the reaction was stopped by adding 1 ml of 5 N sulfuric acid. Table 2 shows the results of analysis of the amount of D—N-force rubermoyl (4-hydroxyphenyl) glycine produced using HPLC.

[HPLC analysis conditions]

Column: COSMOSIL 5C18—AR (4.6 mm x 250 mm, manufactured by Nacalai Testa), eluent: 10 mM potassium phosphate buffer (pH 2) Z-acetonitrile = 19 Zl, flow rate: lmlZ min, column temperature: 30 ° C, measurement wavelength: 210nm

[0049] [Table 2] Addition metal salt Metal salt concentration D—N— (4-Hydroxyphenyl) glycine Relative activity

(mM) Production (/ i mol) (%) None-9.1 100.0 Cobalt chloride 0.1 2.8 31.4

1 1.0 10.9

[0050] As shown in Table 2, DL-5- (4 hydroxyphenol) hydantoin hydrolysis In the case of the D-selective hydantoinase of the example, the production reaction of D—N-force rubamoyl (4 hydroxyphenol) glycine was inhibited by a predetermined metal ion (for example, a divalent metal ion containing cobalt).

[Example 1] Synthesis of D-5 isoptylhydantoin using hydantoinase

Collect the cells by centrifuging the 5 ml culture solution obtained in Reference Example 1 and suspend the cells in 5 ml of 10 mM Tris-HCl buffer (pH 8.5). The insoluble matter derived from the bacterial cells was removed, and a crude enzyme solution of D-form selective hydantoinase was obtained. The obtained crude enzyme solution 0.1 ml contains 30 mM D—N—forced rubermoyl leucine and cobalt chloride or zinc sulfate at the concentrations shown in Table 3 50 mM 2— [4 (2- Hydroxy et hyl) 1 -piperazinyllethanesulfonic acid ( 4 ml of HEPES) -NaOH buffer (pH 7.0) was added and mixed. After standing at 40 ° C for 15 minutes, 4 ml of acetonitrile was added to stop the reaction. Table 3 shows the results of analysis of the amount of D-5 isoptylhydantoin produced in the same manner as in Reference Example 1.

[0052] [Table 3] Added metal salt <&> 辰 'D— 5-Isoptylhydantoin Relative activity

(mM) Production mol) (%) None-7.9 100.0 Cobalt chloride 0.1 15.6 198.2

1 15.7 200.0 Duplex sulfate 1 8.5 108.1

[0053] As shown in Table 3, the production of D-5 isobutyric hydantoin by the cyclization reaction of D—N—strength rubamoyl leucine is carried out in the case of the D-selective hydantoinase of the example in the case of a predetermined metal ion (E.g., divalent metal ions including cobalt or zinc).

[0054] (Example 2) D-5- (4-hydroxyphenyl) hydantoin using hydantoinase Culture medium used in Example 1 11. Bacteria were collected by centrifugation at 7 ml force, and 10 mM Tris —Suspend in 5 ml of hydrochloric acid buffer (pH 8.5) and crush the cells by ultrasound. Then, remove the insoluble matter derived from the cells by centrifugation and remove the crude enzyme solution of D-form selective hydantoinase. Got. 50 ml of HEPES-NaO containing 0.1 mM of the crude enzyme solution containing 30 mM D—N—strength rubermoyl (4-hydroxyphenyl) glycine and cobalt chloride at the concentrations shown in Table 4 4 ml of H buffer (pH 7.0) was added and mixed. After standing at 40 ° C for 15 minutes, the reaction was stopped by adding 4 ml of acetonitrile. Table 4 shows the results of analyzing the amount of D-5- (4-hydroxyphenyl) hydantoin produced in the same manner as in Reference Example 2.

[Table 4] Added metal salt Metal salt concentration D— 5--(4-hydroxyphenyl) hydantoin Relative activity

(mM) Production (ii mol) (%) None-10.0 100.0 Cobalt chloride 0.1 12.7 126.9

1 13.4 134.6

[0056] As shown in Table 4, the formation of D-5- (4-hydroxyphenol) hydantoin by the cyclization reaction of D-N-force rubamoyl (4-hydroxyphenol) glycine In the case of the body selective hydantoinase, it was promoted by certain metal ions (eg, divalent metal ions containing cobalt).

[0057] (Example 3) Synthesis of D-5 isoptylhydantoin using hydantoinase

The crude enzyme solution used in Example 1 was mixed with 0.05 ml of 1M HEPES-NaOH buffer (6.8) containing 10% (wZv) DL-N-strong rubermoyl leucine and ImM cobalt chloride (6.8). . After stirring at 40 ° C for 3 hours, the yield and optical purity of the produced D-5 isoptylhydantoin and the remaining LN strength rubermoyl leucine were analyzed using HPLC, and as a comparative example, Table 5 shows the results when no force was added.

[HPLC analysis conditions (optical purity)]

Column: Chirobiotic T (4.6 mm x 250 mm, manufactured by ASTEC), connected in two, 0.01% (V / v) Triethylamine acetate Η6.8) Ζ methanol = 9/1, flow rate: 0.7m Column temperature: 35 ° C, measurement wavelength: 210nm

[0058] [Table 5] Addition metal salt D-5-isobutylhydantoin L—N—strength rubamoylleucine Production amount (mol%) Optical purity J i <¼e.e) Residual amount (mol¾) Optical purity (¾e. e.) None 26.7 95.2 74.8 45.2 Cobalt chloride 43.7 _ 95.9 57.6 70.1

[0059] As shown in Table 5, the D-form selective H When Dantoinase is allowed to act, the D-selective cyclization reaction is promoted by certain metal ions (for example, divalent metal ions containing cobalt), resulting in the production of optically active D-5-isobutylhydantoin. However, the reverse optically active substance L—N—force rubermoyl leucine remained.

Claims

Claim General formula (2)

[Chemical 1]

(Wherein R has an optionally substituted alkyl group having 1 to 20 carbon atoms, has a substituent !, may have an aralkyl group having 7 to 20 carbon atoms, or has a substituent. / !, but represents an aryl group having 6 to 20 carbon atoms, and * represents an asymmetric carbon atom.) And an optically active 5-substituted hydantoin derivative represented by the general formula (3):

[Chemical 2]

(Wherein, R and * are the same as above), which is a method for producing an optically active N-force rubamyl amino acid derivative,

[Chemical 3]

(Wherein R is the same as defined above), N-force rubamyl amino acid derivative represented by the stereoselective cyclization.

[2] The production method according to claim 1, wherein the divalent metal ion is at least one metal ion selected from cobalt, zinc, manganese, and nickel.

[3] The method according to claim 1, wherein the divalent metal ion is a cobalt ion or a zinc ion.

[4] General formula (2):

[Chemical 4]

[Chemical 5]

(Wherein, R and * are the same as those mentioned above),

Using hydantoinase in the presence of cobalt ions or zinc ions, general formula (1):

5. The production method according to any one of claims 1 to 4, wherein the hydantoinase is derived from a microorganism belonging to the genus Bacillus.

[6] Hydantoinase is Bacillus sp. KNK245 (FERM BP— 4

The production method according to any one of claims 1 to 4, which is derived from 863).

[7] Hydantoinase is Escherichia coli HB101 (pTH104) (FERM

The production method according to any one of claims 1 to 4, which is derived from BP-4864).

[8] R may have a substituent, a phenyl group, a substituent, a benzyl group, or a substituent, which may have 1 carbon atom. The alkyl group of from 4 to!

The manufacturing method in any one of 1-7.

[9] The production method according to any one of claims 1 to 7, wherein the R force is a hydroxyphenol group or an isobutyl group.

10. The production method according to any one of claims 1 to 9, which is an optically active 5-substituted hydantoin derivative complex obtained.

[11] The production method according to any one of [1] to [10], wherein the optically active N-force rubamyl amino acid derivative is obtained.

[12] From an optically active N-force rubamyl amino acid derivative and Z or an optically active 5-substituted hydantoin derivative obtained by the production method according to any one of claims 1 to 11, A method for producing an optically active α-amino acid derivative to produce a corresponding optically active α-amino acid derivative.

The following general formula (1):

[Chemical 7]

(Wherein R has an optionally substituted alkyl group having 1 to 20 carbon atoms, has a substituent !, may have an aralkyl group having 7 to 20 carbon atoms, or has a substituent. N-force rubamyl amino acid derivative represented by the following formula (2): represents an aryl group having 6 to 20 carbon atoms).

(Wherein R represents the same as above, * represents an asymmetric carbon), and an optically active 5-tontoin derivative represented by the general formula (3):

[Chemical 9]

(Wherein R and * are the same as above), an optically active N-force rubamyl amino acid derivative A hydantoinase having an activity to produce

A hydantoinase characterized in that the cyclization reaction is promoted by a divalent metal ion, and the hydrolysis reaction from a 5-substituted hydantoin derivative to an N-force rubamyl amino acid derivative is inhibited.