CH699014B1 - Method for recovery of salt of L-biphenyl alanine compound, involves preparing L-biphenyl alanine compound, separating aqueous layer and organic layer, and adding inorganic salt and organic solvent to aqueous layer - Google Patents

Abstract

A L-biphenyl alanine compound (2') is prepared by hydrolyzing biphenyl alanine ester compound (1) with protease derived from microorganism belonging to Bacillusin mixed solvent of organic solvent and water at pH of 6-13. An aqueous layer containing L-biphenyl alanine compound is separated from an organic layer containing unreacted D-biphenyl alanine ester compound (3). Then, L-biphenyl alanine compound is extracted from the aqueous layer by adding inorganic salt and organic solvent to the aqueous layer, to recover L-biphenyl alanine compound. A L-biphenyl alanine compound of formula (2') is prepared by hydrolyzing biphenyl alanine ester compound of formula (1) with protease derived from microorganism belonging to Bacillusin mixed solvent of organic solvent and water at pH of 6-13. An aqueous layer containing L-biphenyl alanine compound is separated from an organic layer containing unreacted D-biphenyl alanine ester compound of formula (3). Then, L-biphenyl alanine compound is extracted from the aqueous layer by adding inorganic salt and organic solvent to the aqueous layer, to recover L-biphenyl alanine compound. R 1>aralkyl, aryl, alkyl, haloalkyl, alkenyl, cycloalkyl, or substituent; R 2>protective group of amino group;and M : alkali metal atom or alkaline-earth metal atom. An independent claim is included for recovering L-N-Boc-biphenyl alanine ester compound. [Image].

Description

Technical area

The present invention relates to a method for obtaining an L-biphenylalanine compound salt produced in producing D-biphenylalanine compounds useful as intermediates for drugs and the like, and a method for obtaining biphenylalanine ester compounds who employ the same.

State of the art

D-biphenylalanine is useful as an intermediate for drugs such as neutral endopeptidase inhibitors (see Patent Document 1). [Patent Document 1] Japanese Unexamined Patent Publication HEI No. 6-228187.

Disclosure of the invention

Problems to be Solved by the Invention

Methods for producing D-biphenylalanine include hydrolysis methods of biphenylalanine esters using enzymes (hereinafter also referred to as enzyme hydrolysis) (production of D-biphenylalanine ester and L-biphenylalanine salts). However, the L-biphenylalanine salts must either be discarded or recovered as free acids for effective use by a complicated recovery process which involves separation and crystallization in an acidic environment.

An object of the present invention is to provide a recovery process and effective use of L-biphenylalanine salts produced by enzyme hydrolysis of biphenylalanine esters using a simple method.

Means of solving the problem

As a result of careful research aimed at solving the above problem, the inventors completed this invention after discovering that L-biphenylalanine compound salts can be recovered in the form of salts by a simple process wherein the reaction mixture obtained after enzyme hydrolysis of a biphenylalanine ester is separated into an aqueous layer and an organic layer, and an inorganic salt and an organic solvent are added to the separated aqueous layer for extraction. The inventors further found that, when the recovered L-biphenylalanine compound salt is esterified and then racemized, it can be converted into a biphenylalanine ester, making it possible to recover the starting material of enzyme hydrolysis by a simple process.

In particular, the invention provides the following. [1] A recovery method for an L-biphenylalanine compound salt (2 '), which comprises: using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical Formula 1]

(where R1 represents an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R2 represents a protective group for the amino group) (hereinafter also referred to as “biphenylalanine ester compound (1)”) in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the aqueous layer produced by the following formula (2 ́) reproduced L-biphenylalanine compound salt contains: [Chemical formula 2]

(where M represents an alkali metal atom or 1⁄2 alkaline earth metal atom and R2 has the same definition as above), (hereinafter also referred to as "L-biphenylalanine compound salt (2 ')") from the organic layer containing the unreacted D-biphenylalanine ester compound represented by the following formula (3): [Chemical formula 3]

(the symbols have the same definitions as above), (hereinafter also referred to as “D-biphenylalanine ester compound (3)”); and adding an inorganic salt and an organic solvent to the aqueous layer to extract the L-biphenylalanine compound salt (2 ') in the aqueous layer. [2] The recovery method according to [1] above, wherein the organic solvent for hydrolysis is at least one selected from methyl tert-butyl ether and toluene. [3] The recovery method according to [1] above, wherein the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene. [4] A recovery method for a biphenylalanine ester compound (1), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolyzing a biphenylalanine ester compound (1) in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the aqueous ones Layer containing the generated L-biphenylalanine compound salt (2 ') and the organic layer containing the unreacted D-biphenylalanine ester compound (3); Adding an inorganic salt and an organic solvent to the aqueous layer to extract the L-biphenylalanine compound salt (2 ') in the aqueous layer; Esterifying the extracted L-biphenylalanine compound salt (2 ') to obtain an L-biphenylalanine ester compound represented by the following formula (2) (hereinafter also referred to as "L-biphenylalanine ester compound (2)"): [Chemical formula 4]

(the symbols have the same definitions as above); and racemizing the L-biphenylalanine ester compound (2) with a base to obtain the biphenylalanine ester compound (1). [5] The recovery method according to [4] above, wherein the organic solvent for hydrolysis is at least one selected from methyl tert-butyl ether and toluene. [6] The recovery method according to [4] above, wherein the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene. [7] The recovery method according to [4] above, wherein the esterification is carried out by reacting with the corresponding sulfuric acid ester or halide in the presence of a base. [8] Recovery method according to [4] above, wherein R1 is methyl. [9] The recovery method according to [8] above, wherein the esterification is carried out by reacting with dimethylsulfuric acid in the presence of a base. [10] Recovery process according to the above [4], the base used for the racemization being selected from alkali metal carbonates and alkali metal alcoholates. [11] Recovery method according to [4] above, wherein the base used for the racemization is an alkali metal alcoholate. [12] Recovery process according to [4] above, wherein the base used for the racemization is sodium methylate. [13] The recovery method according to [4] above, wherein R1 is an alkyl group and R2 is a group represented by the formula -CO2R3 (where R3 is an alkyl, an optionally substituted aryl, optionally substituted aralkyl, or a 9-fluorenemethyl group). [14] The recovery method according to [13] above, wherein the racemization is carried out after isolating the L-biphenylalanine ester compound (2) as a solid. [15] Recovery process for DL-N-Boc-biphenylalanine methyl ester (1a), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a DL-N-Boc-biphenylalanine methyl ester represented by the following formula (1a): [Chemical formula 5]

(hereinafter also referred to as “DL-N-Boc-biphenylalanine methyl ester (1a)”) in a mixed solvent comprising methyl tert-butyl ether and water in a pH range of 6 to 13 with an aqueous solution of taurine and potassium hydroxide , and separating the aqueous layer containing the generated LN-Boc-biphenylalanine potassium salt represented by the following formula (2 ́a): [Chemical formula 6]

(hereinafter also referred to as "L-N-Boc-biphenylalanine potassium salt (2 ́a)") and the organic layer containing the unreacted D-N-Boc-biphenylalanine methyl ester represented by the following formula (3a): [Chemical formula 7]

(hereinafter also referred to as “D-N-Boc-biphenylalanine methyl ester (3a)”); Adding an inorganic salt and methyl tert-butyl ether to the aqueous layer to extract the L-N-Boc-biphenylalanine potassium salt (2 ́a) in the aqueous layer; Esterification of the extracted L-N-Boc-biphenylalanine potassium salt (2 ́a) with dimethylsulfuric acid in the presence of a base to obtain the L-N-Boc-biphenylalanine methyl ester represented by the following formula (2a): [Chemical formula 8]

(hereinafter also referred to as “L-N-Boc-biphenylalanine methyl ester (2a)”); and Racemizing the L-N-Boc-biphenylalanine methyl ester (2a) with sodium methylate after its isolation as a solid, to obtain the DL-N-Boc-biphenylalanine methyl ester (1a). [16] A method for producing a D-biphenylalanine ester compound (3), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a biphenylalanine ester compound (1) obtained by a recovery method according to the above [4] in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the organic layer containing the unreacted D-biphenylalanine ester compound (3); and purifying the D-biphenylalanine ester compound (3). [17] A method for producing D-N-Boc-biphenylalanine methyl ester (3a), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of DL-N-Boc-biphenylalanine methyl ester (1a) obtained by a recovery method according to the above [15] in a mixed solvent containing an organic solvent and water comprises, in a pH range of 6 to 13, and separating the organic layer containing the unreacted DN-Boc-biphenylalanine methyl ester (3a); and Purify the D-N-Boc-biphenylalanine methyl ester (3a).

Effects of the invention

According to the method of the invention, it is possible to recover L-biphenylalanine compound salts in the form of salts from reaction mixtures obtained after enzyme hydrolysis of biphenylalanine ester compounds using a simple method. In addition, since conversion to biphenylalanine ester compounds is possible by esterifying and racemizing the recovered L-biphenylalanine compound salts, the starting materials of enzyme hydrolysis can be recovered by a simple process. Therefore, the L-biphenylalanine compounds which are not the target of enzyme hydrolysis can be effectively used for more efficient production of D-biphenylalanine ester compounds, and hence the process of the invention is economically advantageous.

Best Mode for Carrying Out the Invention

[0008] The definitions of the substituents used in the present specification will now be explained. As “alkyl groups”, C1-C6, straight-chain or branched alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl can be mentioned, methyl or ethyl being preferred and methyl is more preferred.

As "alkenyl groups", C2-C6 straight-chain or branched alkenyl groups can be listed, such as allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and 3-butenyl, allyl being preferred.

As “cycloalkyl groups”, C3-C8 cycloalkyl groups can be cited, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, with cyclopentyl or cyclohexyl being preferred.

Fluorine, chlorine, bromine and iodine can be cited as "halogen atoms", fluorine, chlorine or bromine being preferred. As “haloalkyl groups”, “alkyl groups” according to the definition given above, which are substituted by halogen atoms, can be cited. The number of halogen atom substituents is not particularly limited, but is preferably 1 to 3. Examples of "haloalkyl groups" include chloromethyl, bromomethyl, fluoromethyl, dichloromethyl, dibromomethyl, difluoromethyl, trichloromethyl, tribromomethyl, trifluoromethyl, 2,2-dichloroethyl and 2 , 2,2-trichloroethyl, with trifluoromethyl being preferred.

As "alkoxy groups" C1-C6 straight-chain or branched alkoxy groups can be listed, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy and hexyloxy, methoxy or Ethoxy are preferred.

As “aryl groups” for the “optionally substituted aryl group”, C6-C14 aryl groups can be cited, such as phenyl, 1-naphthyl and 2-naphthyl, with phenyl being preferred.

Such aryl groups may have substituents at substitutable positions where the substituents are halogen atoms (examples thereof are mentioned above), alkyl groups (examples thereof are mentioned above), haloalkyl groups (examples thereof are mentioned above), hydroxy, alkoxy groups (examples thereof are mentioned above), cyano, nitro and the like. The number of the substituents is not particularly limited, but is preferably 1 to 3. When the number of the substituents is two or more, the substituents may be the same or different.

The “aralkyl groups” for the “optionally substituted aralkyl group” can be the aforementioned “alkyl groups” which are substituted with the aforementioned “aryl groups”. The number of aryl group substituents is not particularly limited, but is preferably 1 to 3. Examples of "aralkyl groups" include benzyl, phenethyl, 1-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylmethyl, 2 -Naphthylmethyl, benzhydril and trityl can be listed, benzyl being preferred.

Such aralkyl groups may have substituents at substitutable positions where the substituents are halogen atoms (examples thereof are mentioned above), alkyl groups (examples thereof are mentioned above), haloalkyl groups (examples thereof are mentioned above), hydroxy, alkoxy groups (examples thereof are mentioned above), cyano, nitro and the like. The number of the substituents is not particularly limited, but is preferably 1 to 3. When the number of the substituents is two or more, the substituents may be the same or different.

The "amino group protecting group" represented by R2 may be any protecting group known as an amino group protecting group without any particular limitation. Such protecting groups can be -CO2R3 (where R3 represents an alkyl, an optionally substituted aryl, optionally substituted aralkyl or a 9-fluorene methyl group), -COR4 (where R4 is an alkyl, haloalkyl, alkenyl, an optionally substituted aryl group or optionally substituted aralkyl group reproduces), and optionally substituted aralkyl groups are listed. As examples of the “protective group for the amino group”, methoxycarbonyl, ethoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl (Boc), allyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 9-fluorenemethyloxycarbonyl, benzoyl, benzyl, benzyloxycarbonyl, and trityyl are given.

[0018] R1 is preferably an alkyl group, more preferably a C1-C4 alkyl group, even more preferably methyl or ethyl, and most preferably methyl. R2 is preferably -CO2R3 (where R3 has the same definition as above), and more preferably tert-butoxycarbonyl (Boc).

Potassium, sodium and lithium can be cited as "alkali metal atoms". Calcium and magnesium can be cited as «alkaline earth metal atoms». M is preferably an alkali metal atom, more preferably potassium or sodium, and most preferably potassium. The biphenylalanine ester compound (1) used as a starting material in the process of the invention can be produced, for example, by the following process.

[Chemical formula 9]

(The symbols in the formulas have the same definitions as above).

Step 1 The compound (4) and hydantoin can be reacted in the presence of a base to obtain the compound (5). step 2 The compound (5) can be reduced to obtain the compound (6). The reduction is preferably carried out by catalytic hydrogenation. step 3 The compound (6) can be hydrolyzed to obtain the compound (7). Step 4 The compound (7) can be added for protection of the amino group and the esterification reaction with the carboxyl group to obtain the biphenylalanine ester compound (1). The protection of the amino group and the esterification of the carboxyl group can be carried out by conventional methods. There is no particular limitation on the order of protecting the amino group and esterifying with the carboxyl group, and these can be carried out in any desired order.

The biphenylalanine ester compound (1) includes two optical isomers (L- and D-isomers) having an asymmetric center on the carbon atom at the α-position, and the biphenylalanine ester compound (1) used in the process of the invention can be a racemic mixture containing equal amounts of the optical isomers, or a mixture containing either of the two optical isomers in excess (in any desired proportion). It is preferably a racemic mixture.

The biphenylalanine ester compound (1) obtained in the above-described manner is hydrolyzed using a protease from a microorganism belonging to the genus Bacillus. Using the protease for hydrolysis results in preferential hydrolysis of the L-isomer.

The proteases from the microorganisms belonging to the genus Bacillus are preferably proteases from Bacillus licheniformis because of their excellent enantioselectivity. As specific examples of proteases from Bacillus licheniformis, a protease from Bacillus licheniformis containing subtilisin can be given, among which ALCALASE (registered trademark of Novozymes) is preferred and ALCALASE 2.4 L (registered trademark of Novozymes) is particularly preferred.

There are no particular restrictions on the purity and form of the protease, and various forms can be used including purified enzymes, crude enzymes, microbial cultures, cells or treated products of the foregoing. A treated product can be, for example, lyophilized cells, minced cells, or a cell extract. An enzyme of any purity and in any of the various forms mentioned above, immobilized on an inorganic support material such as silica gel or ceramic, or on cellulose, an ion exchange resin, or the like, can be used.

The amount of the protease used is not particularly limited, but it is usually 0.001-0.5 g, and preferably 0.001-0.1 g, such as purified enzyme, per one gram of the biphenylalanine ester compound (1).

The hydrolysis with the protease is carried out in a pH range of 6 to 13, preferably pH 6 to 10, and more preferably 6.0 to 9.5, depending on the type of protease. Hydrolysis within this pH range can give a product with high optical purity.

The pH can be kept within the aforementioned range by appropriately using an alkaline and / or buffering agent. As examples of buffering agents, phosphate buffer, acetate buffer, amino acid buffer, aminosulfonate buffer and the like can be given.

As the phosphate buffer, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and the like can be given. As the acetate buffer, sodium acetate, potassium acetate and the like can be cited.

The amino acids in the amino acid buffers are organic compounds that have both an amino group (including substituted amino groups and cyclically substituted amino groups) and a carboxyl group in one molecule (these include imino acids, a hydrogen of the amino group having a cyclic structure with part of the Side chain), and they include not only α-amino acids, but also β-amino acids, γ-amino acids and δ-amino acids. As specific examples of α-amino acids, neutral amino acids such as glycine, alanine, α-aminobutyric acid, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, methionine, cysteine, threonine, serine, proline, hydroxyproline, asparagine and glutamine can be given; acidic amino acids such as aspartic acid and glutamic acid; and basic amino acids such as lysine, tryptophan, arginine, ornithine, histidine and hydroxylysine. As specific examples of β-amino acids, β-alanine and β-aminobutyric acid can be given. As a specific example of a γ-amino acid, γ-aminobutyric acid can be given. As a specific example of a δ-amino acid, 5-aminovaleric acid can be given. Glycine is preferred as the amino acid.

An aminosulfonic acid for an aminosulfonate buffer is an organic compound having a sulfur group in place of the carboxyl group in any of the aforementioned amino acids, and as specific examples, taurine, N-methyltaurine and 2- (4-morpholinyl) ethanesulfonic acid can be given, with taurine being preferred.

[0033] Taurine or phosphate buffers are preferred as buffering agents, with taurine being particularly preferred.

The concentration of the buffer agent is usually 0.05-0.8 M, and preferably 0.1-0.8 M, and more preferably 0.1-0.5 M. A concentration of the buffer solution within this range can give a product with high optical purity.

The amount of the buffering agent used depends on the concentration, but it is usually 0.1-100 ml, and preferably 0.4-10 ml, based on 1 g of the biphenylalanine ester compound (1). Using the buffer solution within this range helps ensure even conversion.

When a base is used, it can be used as a solid or an aqueous solution, although an aqueous solution is preferred because of handleability. As aqueous basic solutions, aqueous solutions of alkali metal hydroxide (sodium hydroxide, potassium hydroxide), aqueous solutions of alkaline earth metal hydroxide (calcium hydroxide, magnesium hydroxide), aqueous solutions of alkali metal carbonate (sodium carbonate, potassium carbonate), aqueous solutions of alkaline earth metal carbonate (calcium carbonate), aqueous solutions of alkaline earth metal carbonate (calcium carbonate), Sodium hydrogen carbonate, potassium hydrogen carbonate), aqueous solutions of alkaline earth metal hydrogen carbonate (calcium hydrogen carbonate, magnesium hydrogen carbonate) and the like. Preferred among these are aqueous solutions of sodium hydroxide and potassium hydroxide, with an aqueous solution of potassium hydroxide being particularly preferred.

The amount of base used may be an amount for adjusting the pH to 6-13.

The pH adjustment is preferably carried out using taurine and an aqueous solution of potassium hydroxide or a phosphate buffer and an aqueous solution of potassium hydroxide, and most preferably it is carried out using taurine and an aqueous solution of potassium hydroxide.

The hydrolysis is carried out in a mixed solvent comprising an organic solvent and water. As the organic solvent for the hydrolysis, hydrophobic organic solvents and hydrophilic organic solvents can be mentioned. Ethers such as methyl tert-butyl ether (MTBE) and diisopropyl ether and hydrocarbons such as toluene, hexane, cyclohexane and heptane can be mentioned as hydrophobic organic solvents. As the hydrophilic organic solvent, ethers such as tetrahydrofuran (THF); Alcohols such as methanol, ethanol, isopropanol, n-butanol and tert-butanol; Sulfoxides such as dimethyl sulfoxide; Ketones such as acetone; and nitriles such as acetonitrile can be cited. These organic solvents can be used alone or in combinations of two or more. MTBE and toluene are preferred as organic solvents, MTBE being particularly preferred.

The amount of the organic solvent used is usually 1-50 ml, and preferably 1-5 ml based on 1 g of the biphenylalanine ester compound (1). Using the organic solvent within this range contributes to smooth conversion.

The water in the buffer agent and in the aqueous alkaline solution can also serve as a solvent.

The hydrolysis is carried out by mixing the biphenylalanine ester compound (1), the protease from a microorganism belonging to the genus Bacillus, a solvent, the base or its aqueous solution and / or a buffer agent. There is no particular restriction on the order of addition, and the method may include, for example: (i) adding the biphenylalanine ester compound (1) dissolved in an organic solvent to a base and / or a buffering agent and then adding the protease, (ii) adding a base and / or a buffering agent to the biphenylalanine ester compound (1) dissolved in an organic solvent and then adding the protease, (iii) adding the protease (if necessary with water) to the biphenylalanine ester compound (1) dissolved in an organic solvent and then adding a base and / or a buffering agent (preferably by dropwise addition), or (iv) adding the protease and the buffering agent (with water if necessary) to the biphenylalanine ester compound (1) dissolved in an organic solvent, and then adding a base (preferably by dropwise addition).

Additional protease can, if necessary, be added during the reaction. If the pH of the reaction mixture falls below the aforesaid range during the reaction, a base or its aqueous solution is used to adjust the pH of the reaction mixture back to the range.

The reaction temperature for the hydrolysis is preferably 30-60 ° C, more preferably 35-55 ° C, and most preferably 40-45 ° C in terms of enzyme stability and conversion rate. The reaction time for the hydrolysis is usually 3-24 hours, and preferably 4-15 hours.

The liquid separation of the reaction mixture into an aqueous and an organic layer after the hydrolysis allows the separation of the L-biphenylalanine compound salt (2 ') generated by the hydrolysis and the unreacted D-biphenylalanine ester compound (3). The L-biphenylalanine compound salt (2 ') is contained in the aqueous layer, and the D-biphenylalanine ester compound (3) is contained in the organic layer. The D-biphenylalanine ester compound can be purified from the organic layer by a known method.

According to the invention, an inorganic salt and an organic solvent are added to the aqueous layer containing the L-biphenylalanine compound salt (2 ') to extract the L-biphenylalanine compound salt (2') with the organic solvent. This enables the L-biphenylalanine compound salt (2 ́) to be obtained directly from the aqueous layer in the form of a salt.

As the inorganic salts for this step, sodium chloride, potassium chloride, ammonium chloride and the like can be cited, of which potassium chloride and sodium chloride are preferred from the viewpoint of economy and solubility of the L-biphenylalanine compound salt (2 '). The amount of the inorganic salt used is usually 6 g or more, and preferably 6-9 g based on 100 g of the aqueous solution containing the L-biphenylalanine compound salt (2 ') from the viewpoint of a satisfactory extraction rate. The inorganic salt can also be added to the aqueous layer in a supersaturation amount.

The organic solvent for extraction may be a solvent such as MTBE, toluene, ethyl acetate or the like, of which MTBE or toluene is preferred, and MTBE is particularly preferred. The amount of the organic solvent used is usually 20-100 g, and preferably 25-50 g based on 100 g of the aqueous solution containing the L-biphenylalanine compound salt (2A). The extraction can, if necessary, be carried out twice or more than once. The extraction is usually carried out at 10-50 ° C.

The extract containing the L-biphenylalanine compound salt (2 ') can be washed with an aqueous alkaline solution. The aqueous alkaline solution may be an aqueous solution of potassium hydroxide, an aqueous solution of sodium hydroxide, or the like, usually at a concentration of 5-15% (w / w). The amount of the aqueous alkaline solution used is 0.95-1.01 mol based on 1 mol of the L-biphenylalanine compound salt (2 '). When the extract is washed with the aqueous alkaline solution, an inorganic salt such as potassium chloride, potassium bromide or sodium chloride can be added to the aqueous solution for an improved yield. The amount of the inorganic salt used may be an amount for an inorganic salt concentration of at least 5% (w / w) in the aqueous alkaline solution.

Therefore, according to the method of the invention, the L-biphenylalanine compound salt (2 ') can be directly recovered in the salt form without being converted into a free acid.

The recovered L-biphenylalanine compound salt (2 ') can be esterified and then racemized for conversion to the biphenylalanine ester compound (1) for recovery of the starting material of enzyme hydrolysis.

On the other hand, the D-biphenylalanine ester compound (3) is useful as an intermediate for producing drugs such as inhibitors of neutral endopeptidase. For example, the method described in Japanese Unexamined Patent Publication HEI No. 6-228187 can be used for preparing the N-phosphonomethylbiphenyl substituted dipeptide derivative from the D-biphenylalanine ester compound (3) described in the same publication.

A method for esterifying and racemizing the L-biphenylalanine compound salt (2 ') for conversion into the biphenylalanine ester compound (1) will now be described.

First, the L-biphenylalanine compound salt (2 ') is esterified to obtain an L-biphenylalanine ester compound (2). The esterification is usually carried out in a solvent, but the extract containing the L-biphenylalanine compound salt (2 ') is preferably used directly. If necessary, a solvent can also be added. MTBE and toluene are preferred as organic solvents, with MTBE being more preferred.

The esterification can be carried out by a conventional method such as a method in which an acid and an alcohol such as methanol or ethanol are used, a method in which a base and a sulfuric acid ester ((R1O) 2SO2), such as dimethylsulfuric acid or diethylsulfuric acid; a method in which R1OH and a condensing agent such as DCC are used; or a method in which a base and a halide (R1X, X: halogen atom) such as methyl iodide, bromoethane or benzyl chloride are used; in these processes, the use of bases and sulfuric acid esters is preferred with regard to the lower by-products and greater expediency.

A method in which a base and a sulfuric acid ester are used will now be explained. As bases, there can be mentioned inorganic bases such as sodium hydrogen carbonate, potassium hydrogen carbonate, potassium carbonate, sodium hydroxide and potassium hydroxide, and organic bases such as diisopropylethylamine, 2,6-dimethylpyridine, triethylamine and pyridine, with sodium hydrogen carbonate being preferred. The amount of the base used is usually 0.3-1.2 moles, and preferably 0.8-1.2 moles based on 1 mole of the L-biphenylalanine compound salt (2 ').

The sulfuric acid ester is selected according to the desired L-biphenylalanine ester compound (2) product, but is preferably a methyl ester in the invention, and therefore dimethylsulfuric acid is preferably used. The amount of the sulfuric acid ester used is usually 1.2-2.5 moles, and preferably 1.6-2.0 moles based on 1 mole of the L-biphenylalanine compound salt (2 ').

In terms of reactivity, the method of esterification preferably includes adding dropwise the sulfuric acid ester to a solution containing the base and the L-biphenylalanine compound salt (2%).

The temperature for the esterification is usually 30-50 ° C. The reaction time varies depending on the amount of reagents and the reaction temperature, but it is usually 1-10 hours. Completion of the reaction can be confirmed by HPLC analysis.

After the reaction has ended, an amine such as triethylamine can be added and the mixture stirred at a temperature of 30-50 ° C for 2 to 5 hours in order to degrade the remaining sulfuric acid ester. The amount of amine used can be approximately 10 mol%, based on the sulfuric acid ester.

The separated aqueous layer is then removed by liquid separation and the organic layer is dehydrated. The dehydration can be carried out using a dehydrating agent such as anhydrous magnesium sulfate, anhydrous sodium sulfate, or molecular sieving, but in terms of operability, the method preferably includes azeotropic dehydrogenation with a solvent azeotropic with water (for example, toluene). The solvent azeotropic with water can be used in an amount that enables sufficient dehydration, and is usually 50-100% by weight with respect to the solution.

The subsequent racemization can be carried out on the dehydrated solution without isolating the L-biphenylalanine ester compound (2), or the racemization can be carried out after it is first isolated as a solid such as a crystalline form or an oil.

In the former case, the water content of the solution is preferably not higher than 500 ppm, and therefore sufficient dehydration is preferred.

In the latter case, when the L-biphenylalanine ester compound (2) is, for example, one in which R1 is an alkyl group and R2 is -CO2R3, the dehydrated solution can be concentrated and crystallization from methanol and water can be carried out. Isolation in the form of a solid such as a crystal or an oil in this way can prevent the consumption of the base by the esterification reaction residue (for example, sulfuric ester-derived compounds), which enables the racemization to be carried out with a smaller amount of the base and also prevents the hydrolysis of the L-biphenylalanine ester compound (2).

A specific preferred example of crystallization from methanol and water will now be described. The amount of methanol used is usually 380-520 g, and preferably 450-490 g based on 100 g of the L-biphenylalanine ester compound (2).

[0066] Water is added dropwise to a methanol-dissolved solution at a temperature of about 45-55 ° C. The amount of water added is usually 160-220 g, and preferably 190-210 g based on 100 g of the L-biphenylalanine ester compound (2). After adding the water, a small amount of a seed crystal is added and the mixture is stirred at the same temperature. After confirming the deposition of crystals, water is added at the same temperature for sufficient crystal deposition. The amount of water in this step is usually 0-60 g, and preferably 40-60 g based on 100 g of the L-biphenylalanine ester compound (2). The mixture is then cooled to about 5 ° C and the crystals are filtered and washed with an about 70% aqueous methanol solution. The crystals of L-biphenylalanine ester compound (2) can be obtained by drying under reduced pressure.

The L-biphenylalanine ester compound (2) obtained by the esterification is then racemized to be converted into a biphenylalanine ester compound (1).

In this racemization step, the L-biphenylalanine ester compound (2) can be used directly as the dehydrated solution obtained in the esterification step, or the L-biphenylalanine ester compound (2) can be isolated first. In the latter case, the L-biphenylalanine ester compound (2) is dissolved in a solvent. MTBE and toluene are preferred as solvents in this case, with MTBE being more preferred. The amount of the solvent used is usually 150-230 g, and preferably 180-220 g based on 100 g of the L-biphenylalanine ester compound (2).

The racemization is carried out using a base. As bases, organic bases such as triethylamine can be cited; Alkali metal hydrogen carbonates such as potassium hydrogen carbonate and sodium hydrogen carbonate; Alkali metal carbonates such as potassium carbonate and sodium carbonate; Alkali metal alcoholates such as sodium methylate, sodium ethylate and potassium tert-butyrate; Alkali metal hydrides such as sodium hydride and potassium hydride; and mixtures of the foregoing, among which alkali metal alcoholates, alkali metal carbonates, and mixtures thereof are preferred, with alkali metal alcoholates being most preferred in terms of handleability, and sodium methylate being particularly preferred in terms of economy. The base can be added dropwise as a solution.

When the L-biphenylalanine ester compound (2) is used directly as the dehydrated solution obtained from the esterification step, the amount of the base used is usually 20-120 mol%, and preferably 50-100 mol%, based on the L-biphenylalanine ester compound (2).

When the L-biphenylalanine ester compound (2) is used after it has been isolated, the amount is usually 5-50 mol%, and preferably 10-50 mol% for an alkali metal carbonate. For an alkali metal alcoholate, the amount is usually 1-5 mol%, and preferably 2-4 mol%.

As mentioned above, the use of the isolated L-biphenylalanine ester compound (2) can avoid the consumption of the base by the esterification residue, enabling the racemization to be carried out with a smaller amount of the base. This can also prevent the hydrolysis of the L-biphenylalanine ester compound (2).

From the viewpoint of promoting racemization, it is preferable to add an alcohol such as methanol, ethanol or isopropanol. The amount of alcohol used is usually 10-120% by weight, and preferably 10-30% by weight based on the L-biphenylalanine ester compound (2).

The reaction temperature for racemization is usually 30-70 ° C, preferably 30-60 ° C, and more preferably 30-40 ° C. The reaction time depends on the amount of reagents used and the temperature, but is usually 10 minutes to 25 hours, and preferably 10 minutes to 6 hours. The completion of the racemization reaction can be confirmed by HPLC.

After the racemization is complete, it is preferred to add an acid such as acetic acid to the reaction mixture in order to minimize the degradation of the ester. The amount of acid used is usually 1.1-1.3 molar equivalents, based on the base used for the racemization. Post-treatment after the completion of the racemization can be carried out according to conventional methods.

The solution of the biphenylalanine ester compound (1) obtained in the manner described above can be fed directly for enzyme hydrolysis, or it can be fed for enzyme hydrolysis after being concentrated and dissolved in another organic solvent.

Examples

The present invention will now be explained in more detail using the following preparation examples and working examples, provided that the invention is not limited to these examples.

The optical purities (enantiomeric excess rates) and the racemization of the optically active compounds were determined by high performance liquid chromatography (HPLC) analysis. HPLC analysis conditions: Column: CHIRALPAK AD-RH (product of Daicel Chemical Industries, Ltd) (4.5 mm ϕ × 15 cm, 5 µm) Mobile phase: Solution A: 0.1% aqueous phosphoric acid solution Solution B: acetonitrile Elution conditions: Solution B 40% (15 min.) - 30 min. -80% (0 min) gradient Column temperature: 40 ° C Flow rate: 1.0 ml / min Detector: UV (254 nm) Retention time: LN-Boc-biphenylalanine: <sep> 10 minutes DN-Boc-biphenylalanine: <sep> 13 minutes LN-Boc-biphenylalanine methyl ester: <sep> 27 minutes DN-Boc-biphenylalanine methyl ester: <sep > 30 minutes (Boc is the abbreviation for tert-butoxycarbonyl) <sep>

The esterification was analyzed under the following conditions. HPLC analysis conditions: Column: SUMIPAX A212 ODS (product of Sumika Chemical Analysis Service, Ltd) (ϕ: 6 mm × L: 15 cm) Mobile phase: Solution A: 25 mM aqueous dipotassium hydrogen phosphate solution (prepared with a pH of 6.8 with phosphoric acid) Solution B: acetonitrile Elution conditions: solution B 40% (15 min.) - 20 min. -80% (5 min) gradient Column temperature: 40 ° C Flow rate: 1.0 ml / min Detector: UV (254 nm) Retention time: L-N-Boc-biphenylalanine: 7 minutes L-N-Boc-biphenylalanine methyl ester: 24 minutes

[Chemical formula 10]

Production Example 1 (Production of DL-Biphenylalanine (7) A mixture of 4-biphenylaldehyde (1) (100.0 g, 0.549 mol), hydantoin (82.4 g, 0.823 mol) and ammonium acetate (63.5 g, 0.824 mol) was dissolved in acetic acid ( 360 ml) heated. After adding water (360 ml), the mixture was cooled to room temperature, and the precipitated crystals were filtered out and washed with isopropanol-water (1: 1, 400 ml) to obtain hydantoin compound (5) (143.14 g, 98.7 % Yield) washed. To the mixture of hydantoin compound (5), (60.2 g), THF (540 ml) and water (60 ml) was added 5% palladium-carbon (50% water content, 2.7 g), and the resulting mixture was at 60 ° C for 3 hours under a hydrogen atmosphere at 0.5 MPa. After removing the catalyst by filtration, the filtrate was concentrated to obtain the reduced form (6) (60.63 g, 100% yield). To the mixture of the reduced form (6) (59.7 g, 0.224 mol), ethylene glycol (300 ml) and water (10 ml) was added sodium hydroxide (36.65 g), and the mixture was heated at 130-140 ° C stirred for 5 hours. After cooling to room temperature, water (130 ml) was added, an aqueous hydrochloric acid solution comprising concentrated hydrochloric acid (85 g) and water (99 g) was further added, and the pH of the mixture was adjusted to 6.9. The precipitated crystals were filtered off and washed with water (300 ml) and then with methanol (300 ml). The crystals were dried to obtain DL-biphenylalanine (7) (53.25 g, 98.4% yield).

Preparation Example 2 (preparation of DL-N-Boc-biphenylalanine methyl ester (1a)) DL-biphenylalanine (7) (20.0 g, 0.0829 mol) was added to a 10% aqueous sodium hydroxide solution (116 g, 0.29 mol). Then, after THF (50 ml) was added, a THF solution (20 ml) containing di-tert-butyl dicarbonate (23.5 ml, 0.108 mol) was added dropwise over a period of one hour at 30 ° C. Tetrabutylammonium bromide (0.20 g, 0.62 mmol) was further added, and dimethylsulfuric acid (12.5 g, 0.099 mol) was added dropwise at 30 ° C. The mixture was stirred at room temperature for 16 hours and then dimethylsulfuric acid (5.4 g, 0.0428 mol) was added and stirring was continued at 35 ° C for 4.5 hours. Dimethylsulfuric acid (2.93 g, 0.0232 mol) was further added and stirring was continued at 35 ° C for 2.5 hours. Then, after MTBE (40 ml) and water (100 ml) were added, the mixture was separated and the aqueous layer was removed to obtain 104.1 g of MTBE solution containing DL-N-Boc-biphenylalanine methyl ester (1a). As a result of quantification by HPLC, the DL-N-Boc-biphenylalanine methyl ester (1a) content was 29.4 g, showing a 99.8% yield of DL-biphenylalanine (7). A 78.1 g portion of the MTBE solution of DL-N-Boc-biphenylalanine methyl ester (1a) thus obtained was concentrated and dried. The residue was recrystallized from isopropanol (9 ml) and heptane (80 ml) to obtain DL-N-Boc-biphenylalanine methyl ester (1a) (19.1 g) as colorless crystals. (Recrystallization yield: 86.6%) <1> H-NMR (DCD13); 1.42 (9H, s), 3.09 (1H, dd, J = 5, 14 Hz), 3.16 (1H, dd, J = 5, 14 Hz), 3.74 (3H, s), 4.55-4.70 (1H, m), 4.90-5.08 (1H, m), 7.20 (2H, d, J = 8Hz), 7.33 (1H, t, J = 8 Hz), 7.43 (2H, t, J = 8Hz), 7.52 (2H, d, J = 8Hz), 7.57 (2H, d, J = 8Hz).

Preparation example 3 (enzyme hydrolysis of DL-N-Boc-biphenylalanine methyl ester (1a)) To a solution of 25.0 g (70.3 mmol) of DL-N-biphenylalanine methyl ester (1a) in 41.14 g of MTBE were added 11.25 g of water, 1.76 g (14.1 mmol) of taurine and 4.50 g g ALCALASE 2.4 L FG (from Bacillus licheniformis) (Novozymes) and the mixture was stirred at 40 ° C. The mixture was stirred at 40 ° C. for 17 hours while 47.28 g (40.67 mmol) of a 5% aqueous potassium hydroxide solution was added dropwise. The pH of the reaction mixture was adjusted to a range of 6.30-8.16. Analysis of the reaction mixture showed an optical purity of 99.4% ee (enantiomeric excess) for the DN-Boc-biphenylalanine methyl ester (3a) and an optical purity of 99.9% ee for the LN-Boc-biphenylalanine potassium salt (2 ́A). The reaction mixture was allowed to stand for 5 minutes for liquid separation, to obtain 35.72 g of the organic layer A and 83.46 g of the aqueous layer A. After adding 37.5 g of toluene to the aqueous layer, the mixture was stirred at 40 ° C. for 30 minutes. It was then allowed to stand for 5 minutes for liquid separation, to obtain 42.80 g of the organic B layer and 76.80 g of the aqueous B layer. The organic layer A and the organic layer B were combined, 58 g of water and 1.49 g of sodium carbonate were added, and the mixture was stirred at 40 ° C. for 30 minutes. It was then allowed to stand for 5 minutes for liquid separation, to obtain 76.07 g of organic layer C. The 76.06 g of the organic layer C was concentrated while heating to 50 ° C, and 63.2 g was distilled off. To the 12.87 g of the concentration residue, 75 ml of methanol and 18.75 g of water were added while heating to 40 ° C. Next, 2 mg of D-N-Boc-biphenylalanine methyl ester (3a) seed crystals were added, and the mixture was stirred at 40 ° C for 30 minutes. Then, 12.5 g of water was added dropwise over a period of 30 minutes, and the mixture was heated to 40 ° C for one hour. It was then cooled to 20 ° C and filtered. The crystals were washed with a mixed solution containing 8.75 g of methanol and 3.75 g of water. The crystals were dried under reduced pressure to obtain 10.94 g of D-N-Boc-biphenylalanine methyl ester (3a) as white crystals. The D-N-Boc-biphenylalanine methyl ester (3a) yield was 43.8% based on the DL-N-Boc-biphenylalanine methyl ester (1a).

example 1

(i) Obtaining L-N-Boc-biphenylalanine potassium salt (2 ́a)

To 328.54 g of an aqueous solution containing 55.4 g (0.146 mol) of LN-Boc-biphenylalanine potassium salt (2 ́a), which was obtained in the same manner as in Preparation Example 3 (aqueous layer B), 50 g of toluene and 37.5 g of sodium chloride were added, and the mixture was stirred at 40 ° C. for 20 minutes. It was then left to stand for 5 minutes for liquid separation, to obtain 156.23 g of an organic layer.

(ii) Preparation of L-N-Boc-biphenylalanine methyl ester (2a)

To the aforementioned organic layer, 12.27 g (0.146 mol) of sodium hydrogen carbonate was added, and the mixture was stirred at 40 ° C. After 33.15 g (0.263 mol) of dimethylsulfuric acid was added dropwise over a period of two hours, the mixture was stirred at 40 ° C. for one hour. After analyzing the reaction mixture by HPLC, it was found that the starting materials are below the detection limit. Next, 2.95 g (0.029 mol) of triethylamine was added to the reaction mixture, and the mixture was stirred at 40 ° C for three hours. It was then allowed to stand for 5 minutes, after which the aqueous layer was separated and removed to obtain 114.04 g of an organic layer. To this, 86.72 g of toluene was added to concentrate while it was heated to 50 ° C., and 57.92 g of toluene was distilled off. Further, 7.39 g of toluene was added to obtain 150.23 g of a toluene solution. The quantification of the toluene solution obtained showed an L-N-Boc-biphenylalanine methyl ester (2a) yield of 96.2%, based on the L-N-Boc-biphenylalanine potassium salt (2 ́a). After measurement with a Karl Fischer moisture meter, the moisture content of the resulting toluene solution was determined to be 204 ppm. L-N-Boc-biphenylalanine methyl ester (2a) <1> H-NMR (CDCl3); 1.42 (9H, s), 3.09 (1H, dd, J = 6, 12 Hz), 3.17 (1H, dd, J = 6, 12 Hz), 3.73 (3H, s), 4.60-4.65 (1H, m), 5.01 (1H, d, J = 8 Hz), 7.20 (2H, d, J = 8 Hz), 7.33 (1H, t, J = 8 Hz), 7.43 (2H, t, J = 8Hz), 7.52 (2H, d, J = 8Hz), 7.57 (2H, d, J = 8Hz).

(iii) Racemization of L-N-Boc-biphenylalanine methyl ester (2a)

To 150.23 g of a toluene solution containing 50 g (0.141 mol) of L-N-Boc-biphenylalanine methyl ester (2a) was added 50 g of methanol, and the mixture was stirred at 40 ° C. After 27.20 g (0.141 mol) of a 28% sodium methylate-methanol solution was further added dropwise over a period of one hour, the mixture was stirred at 40 ° C. for one hour. Analysis of the reaction mixture showed an optical purity of 0.02% ee for the L-N-Boc-biphenylalanine methyl ester (2a) and an HPLC area percentage of 89.3% for the DL-N-Boc-biphenylalanine methyl ester (1a). After 10.16 g (0.169 mol) of acetic acid was added dropwise to the reaction mixture over a period of 15 minutes, the mixture was stirred at 40 ° C. for 30 minutes. Next, 50 g of water was added and stirring was continued at 40 ° C for 5 minutes. The mixture was allowed to stand for 5 minutes for liquid separation, to obtain 151.90 g of an organic layer. After 45 g of water and 2.37 g (0.028 mol) of sodium hydrogen carbonate were added to the organic layer, the mixture was stirred at 40 ° C for 50 minutes. It was then left to stand for 5 minutes for liquid separation, to obtain 147.67 g of an organic layer. The quantification of the solution obtained by HPLC gave a DL-N-Boc-biphenylalanine methyl ester (1a) yield of 89.7%, based on the L-N-Boc-biphenylalanine methyl ester (2a). The solution was concentrated while warming to 54-56 ° C and 62.67 g was distilled off. Next, 75 g of MTBE was added, the mixture was concentrated while heated to 52 ° C, and 74.23 g was distilled off. After 75 g of MTBE was further added, the mixture was concentrated while being heated to 52 ° C. and 84.71 g was distilled off. To the residue was added 51.46 g of MTBE to obtain 127.52 g of a solution. The quantification of the solution obtained by HPLC gave a DL-N-Boc-biphenylalanine methyl ester (1a) yield of 89.5%, based on the L-N-Boc-biphenylalanine methyl ester (2a). The toluene content of the MBTE solution was 18.5%.

[Example 2

(i) Recovery of L-N-Boc-Biphenylalanine potassium salt (2 ́a)

To 227.19 g of an aqueous solution containing 50.0 g (0.132 mol) of LN-Boc-biphenylalanine potassium salt (2 ́a), 70.0 g of MTBE and 25.0 g of potassium chloride were added, and the Mixture was stirred at 40 ° C for 30 minutes. The mixture was allowed to stand for 5 minutes for liquid separation, to obtain 204.97 g of an organic layer. After further adding 73.9 g (0.132 mol) of a 10% aqueous potassium hydroxide solution and 10 g of potassium chloride to the organic layer, the mixture was allowed to stand at 40 ° C. for 15 minutes. It was then allowed to stand for 5 minutes for liquid separation to obtain 190.18 g of an organic layer.

(ii) Preparation of L-N-Boc-biphenylalanine ester (2a)

5.55 g (0.066 mol) of sodium hydrogen carbonate were then added to 95.1 g of the organic layer obtained (corresponding to 22.5 g of LN-Boc-Bi-phenylalanine potassium salt), and the mixture was stirred at 35.degree . Next, 15.0 g (0.119 mol) of dimethylsulfuric acid was added dropwise over a period of 30 minutes, and the mixture was stirred at 35 ° C for 5 hours. After analysis of the reaction mixture by HPLC, the unreacted L-N-Boc-biphenylalanine potassium salt (2 ́a) was determined to be 0.19%. Next, 1.33 g (0.013 mol) of triethylamine and 35 g of water were added to the reaction mixture, and the mixture was stirred at 35 ° C for three hours. It was then allowed to stand for 5 minutes for liquid separation to obtain an aqueous layer (78.16 g). To the organic layer was added 41.9 g (0.010 mol) of a 2.5% by weight aqueous sodium carbonate solution, and the mixture was stirred for 1 hour and 20 minutes. It was then allowed to stand for 5 minutes for liquid separation, to obtain 60.74 g of an organic layer. Quantification of the organic layer gave an L-N-Boc-biphenylalanine methyl ester (2a) yield of 92.3%, based on the L-N-Boc-biphenylalanine potassium salt (2 ́a).

(iii) Crystallization of the L-N-Boc-biphenylalanine methyl ester (2a)

A 408.12 g portion of the MTBE solution containing the LN-Boc-diphenylalanine methyl ester (2a) obtained by the above-described method was reduced under reduced pressure to obtain 130.3 g of an oily one Concentrate. To this, 617.4 g of methanol was added, and the mixture was stirred at 40 ° C. to obtain 747.65 g of a methanol solution (equivalent to 130.3 g of L-N-Boc-biphenylalanine methyl ester (2a)). A 373.82 g portion of the methanol solution obtained (corresponds to 65.1 g of L-N-Boc-biphenylalanine methyl ester (2a)) was heated to 50 ° C. while 130.3 g of water were added. Next, 1 mg of L-N-Boc-biphenylalanine methyl ester seeds was added and the mixture was stirred for one hour. Then 32.6 g of water was added dropwise over 30 minutes and the mixture was heated to 50 ° C for one hour. It was then cooled to 5 ° C and the precipitated crystals were filtered. The crystals were washed with a mixed solution containing 45.6 g of methanol and 19.5 g of water. The crystals were dried under reduced pressure to obtain 60.77 g of white L-N-Boc-biphenylalanine methyl ester (2a) crystals. The L-N-Boc-biphenylalanine methyl ester (2a) yield was 93.3% based on the L-N-Boc-biphenylalanine potassium salt (2 ́a).

Example 3

(Racemization of the L-N-Boc-biphenylalanine methyl ester (2a))

20 g (56.3 mmol) of L-N-Boc-biphenylalanine methyl ester (2a) were dissolved in 40 g of MTBE and the mixture was warmed to 30 ° C. To this, a mixed solution containing 4.0 g of methanol and 0.22 g (1.1 mmol) of a 28% sodium methylate-methanol solution was added, and the resulting mixture was stirred at 30 ° C for two hours. Analysis of the reaction mixture showed an optical purity of 0.44% ee for the L-N-Boc-biphenylalanine methyl ester (2a) and an HPLC area percentage of 98.9% for the DL-N-Boc-biphenylalanine methyl ester (1a). After adding 0.07 g (1.2 mmol) of acetic acid to the reaction mixture, the mixture was stirred at 30 ° C. for 5 minutes. Next, 20 g of water was added and stirring was continued at 30 ° C for 5 minutes. The mixture was then allowed to stand for 5 minutes for liquid separation, to obtain 61.56 g of an organic layer (upper layer). To this, 18 g of water and 0.94 g (11.3 mmol) of sodium hydrogen carbonate were added, and the mixture was stirred at 30 ° C. for 30 minutes. The mixture was then left to stand for 5 minutes for liquid separation, to obtain 58.80 g of an organic layer. Quantification of the obtained organic layer by HPLC showed a DL-N-Boc-biphenylalanine methyl ester (2a) yield of 100% based on the L-N-Boc-biphenylalanine methyl ester (1a). The value by HPLC quantification was 101.6%.

Example 4

(Racemization of the L-N-Boc-biphenylalanine methyl ester (2a))

2.0 g (5.63 mmol, 81.25% ee) LN-Boc-biphenylalanine methyl ester (2a) was dissolved in 2.0 g MTBE and 12.0 g methanol and the mixture was heated to 60 ° C . To this, 0.15 g (1.13 mmol) of potassium carbonate was added, and stirring was continued at 60 ° C. for 8 hours. Analysis of the reaction mixture showed an optical purity of 2.25% ee for the L-N-Boc-biphenylalanine methyl ester (2a) and an HPLC area percentage of 81.3% for the DL-N-Boc-biphenylalanine methyl ester (1a).

Example 5

(Racemization of the L-N-Boc-biphenylalanine methyl ester (2a))

2.0 g (5.63 mmol, 81.25% ee) of LN-Boc-biphenylalanine methyl ester (2a) was dissolved in 2.0 g of MTBE and 12.0 g of methanol, and the mixture was heated to 60 ° C warmed up. To this, 0.12 g (1.13 mmol) of sodium carbonate was added and stirring was continued at 60 ° C for 25 hours. Analysis of the reaction mixture showed an optical purity of 6.02% ee for the L-N-Boc-biphenylalanine methyl ester (2a) and an HPLC area percentage of 90.0% for the DL-N-Boc-biphenylalanine methyl ester (1a).

Industrial applicability

According to the method of the invention, it is possible to recover L-biphenylalanine compound salts in the form of salts from reaction mixtures obtained after enzyme hydrolysis of biphenylalanine ester compounds using a simple method. In addition, since conversion into biphenylalanine ester compounds is possible by esterifying and racemizing the recovered L-biphenylalanine compound salts, the starting materials of enzyme hydrolysis can be recovered. Therefore, L-biphenylalanine compounds which are not the target of enzyme hydrolysis can be effectively used, and the process of the invention is therefore economically advantageous.

Claims (17)

1. A recovery method for an L-biphenylalanine compound salt represented by formula (2 '), which comprises: using a protease from a microorganism belonging to the genus Bacillus to hydrolyze a biphenylalanine ester compound represented by the following formula (1): [Chemical Formula 1] where R1 represents an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R2 represents a protective group for the amino group in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the aqueous layer containing the produced L-biphenylalanine compound salt represented by the following formula (2 '): [Chemical formula 2] where M represents an alkali metal atom or 1⁄2 alkaline earth metal atom and R2 has the same definition as above, from the organic layer containing the unreacted D-biphenylalanine ester compound represented by the following formula (3): [Chemical formula 3] the symbols having the same definitions as above; and adding an inorganic salt and an organic solvent to the aqueous layer to extract the L-biphenylalanine compound salt represented by formula (2 ') in the aqueous layer. 2. The recovery method according to claim 1, wherein the organic solvent for hydrolysis is at least one selected from methyl tert-butyl ether and toluene. 3. The recovery method according to claim 1, wherein the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene. 4. A recovery method for a biphenylalanine ester compound represented by formula (1), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical formula 4] where R1 represents an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R2 represents a protective group for the amino group, in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the aqueous layer containing the produced L-biphenylalanine compound salt represented by the following formula (2 '): [Chemical formula 5] where M represents an alkali metal atom or 1⁄2 alkaline earth metal atom and R2 has the same definition as above, from the organic layer containing the unreacted D-biphenylalanine ester compound represented by the following formula (3): [Chemical formula 6] the symbols having the same definitions as above; Adding an inorganic salt and an organic solvent to the aqueous layer to extract the L-biphenylalanine compound salt represented by formula (2 ') in the aqueous layer; Esterifying the extracted L-biphenylalanine compound salt represented by formula (2 ') to obtain an L-biphenylalanine ester compound represented by the following formula (2): [Chemical formula 7] the symbols having the same definitions as above; and Racemizing the L-biphenylalanine ester compound represented by formula (2) with a base to obtain the biphenylalanine ester compound represented by formula (1). The recovery method according to claim 4, wherein the organic solvent for hydrolysis is at least one selected from methyl tert-butyl ether and toluene. 6. The recovery method according to claim 4, wherein the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene. 7. The recovery process according to claim 4, wherein the esterification is carried out by reacting with the corresponding sulfuric acid ester or halide in the presence of a base. 8. The recovery process of claim 4, wherein R1 is methyl. 9. The recovery process according to claim 8, wherein the esterification is carried out by reacting with dimethylsulfuric acid in the presence of a base. 10. The recovery process according to claim 4, wherein the base used for the racemization is selected from alkali metal carbonates and alkali metal alcoholates. 11. The recovery process according to claim 4, wherein the base used for the racemization is an alkali metal alcoholate. 12. The recovery process according to claim 4, wherein the base used for the racemization is sodium methylate. The recovery method of claim 4, wherein R1 is an alkyl group and R2 is a group represented by the formula -CO2R3, where R3 is an alkyl, an optionally substituted aryl, optionally substituted aralkyl, or a 9-fluorene methyl group. 14. The recovery method according to claim 13, wherein the racemization is carried out after isolating the L-biphenylalanine ester compound represented by formula (2) as a solid. 15. A recovery method for a DL-N-Boc-biphenylalanine methyl ester represented by formula (1a), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a DL-N-Boc-biphenylalanine methyl ester represented by the following formula (1a): [Chemical formula 8] in a mixed solvent comprising methyl tert-butyl ether and water in a pH range of 6 to 13 with an aqueous solution of taurine and potassium hydroxide, and separating the aqueous layer that is produced by the following formula (2 ́a) reproduced LN-Boc-Biphenylalanine Potassium Salt contains: [Chemical formula 9] and the organic layer containing the unreacted D-N-Boc-biphenylalanine methyl ester represented by the following formula (3a): [Chemical formula 10] Adding an inorganic salt and methyl tert-butyl ether to the aqueous layer to extract the L-N-Boc-biphenylalanine potassium salt represented by the formula (2 ́a) in the aqueous layer; Esterification of the extracted L-N-Boc-biphenylalanine potassium salt represented by the formula (2 ́a) with dimethylsulfuric acid in the presence of a base to obtain the L-N-Boc-biphenylalanine methyl ester represented by the following formula (2a): [Chemical formula 11] Racemizing the L-N-Boc-biphenylalanine methyl ester represented by the formula (2 ́a) with sodium methylate after its isolation as a solid, to obtain the DL-N-Boc-biphenylalanine methyl ester represented by the formula (1a). 16. A method for producing a D-biphenylalanine ester compound represented by the formula (3), which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical formula 12] the symbols having the same definitions as above; obtained by a recovery process according to claim 4 in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the organic layer containing the unreacted D represented by the following formula (3) Biphenylalanine Ester Compound contains: [Chemical formula 13] the symbols having the same definitions as above; and purifying the D-biphenylalanine ester compound. 17. A method for making D-N-Boc-biphenylalanine methyl ester, which comprises: Using a protease from a microorganism belonging to the genus Bacillus for hydrolysis of a DL-N-Boc-biphenylalanine methyl ester represented by the following formula (1a): [Chemical formula 14] obtained by a recovery method according to claim 15 in a mixed solvent comprising an organic solvent and water, in a pH range of 6 to 13, and separating the organic layer having the unreacted DN represented by the following formula (3a) -Boc-biphenylalanine methyl ester contains: [Chemical formula 15] Purify the D-N-Boc-biphenylalanine methyl ester.