Method for producing salt of amino acid or salt of peptide compound or solvate of either one of said salts comprising lithium salt precipitation step

Abstract

One aspect of the present invention relates to a method for producing a salt of an amino acid, a salt of a peptide compound, or a solvate of either one of the salts, the method comprising the following steps (A) and (B).　Step (A): a step for bringing a material to be purified which is a mixture of a component (i) that is a purification target substance and a component (ii) that is an impurity into contact with a lithium-containing substance, in which the component (i) is the amino acid having a protecting group at the N-terminal thereof or the peptide compound having a protecting group at the N-terminal thereof and the component (ii) is a compound other than the purification target substance. Step (B): a step for precipitating a lithium salt that is the purification target substance.

Description

Method for producing an amino acid salt, a peptide compound salt, or a solvate thereof, including a lithium salt precipitation step

[0001] The present invention relates to a method for producing a salt of an amino acid or a salt of a peptide compound, or a solvate thereof, which comprises a step of precipitating a lithium salt.

[0002] Salts of amino acids or peptide compounds, or solvates thereof, are important compounds that can be candidate molecules for pharmaceuticals or synthetic intermediates thereof. Known methods for purifying salts of amino acids or peptide compounds or solvates thereof include purifying them after protecting or modifying their terminals.

[0003] For example, Patent Document 1 discloses a method in which an optically active 3-chloroalanine derivative is used to produce an optically active aziridine-2-carboxylic acid derivative in which the amino group is protected with a benzenesulfonyl group substituted with a nitro group at the 2- and / or 4-position, or an optically active 3-haloalanine derivative in which the amino group is protected with a benzenesulfonyl group substituted with a nitro group at the 2- and / or 4-position.

[0004] Furthermore, Patent Document 2 discloses a method for producing a β-halogeno-α-aminocarboxylic acid or a salt thereof, which comprises treating a β-hydroxy-α-aminocarboxylic acid (wherein the basicity of the amino group at the α-position is not masked by the presence of a substituent on the amino group) or a salt thereof with an acid with a halogenating agent to halogenate the hydroxyl group.

[0005] Regarding the Fmoc group (9-fluorenylmethyloxycarbonyl group), which is widely used as a protecting group, Non-Patent Document 1 indicates that when a peptide compound is protected with the Fmoc group, Fmoc-β-alanine is generated as a by-product, and that Fmoc-β-alanine is derived from Fmoc-OSu (Fmoc N-hydroxysuccinimide ester). Fmoc-OSu is one of the raw materials commonly used when protecting an amino acid or a peptide compound with the Fmoc group. Even when a commonly used protecting group other than the Fmoc group (e.g., a tert-butoxycarbonyl group (Boc group), a benzyloxycarbonyl group (Cbz group), etc.) is used, β-alanine having that protecting group may be generated as an impurity. Therefore, there has been a need to improve the purity of candidate molecules for drug discovery or their synthetic intermediates by removing these impurities with high precision.

[0006] International Publication No. WO 2001 / 060795 International Publication No. WO 1999 / 033785

[0007] M. Obkircher et al., “Formation of Fmoc-β-alanine during Fmoc-protections with Fmoc-OSu”, Journal of Peptide Science, Volume14, Issue6, June 2008, Pages 763-766

[0008] However, it has been difficult to reduce the content of impurities such as β-alanine or its derivatives and to purify the target amino acid salt, peptide compound salt, or solvate thereof to a high purity. Therefore, an object of the present invention is to provide a method for obtaining a highly pure amino acid salt, peptide compound salt, or solvate thereof.

[0009] [1] A method for producing an amino acid salt or a peptide compound salt, or a solvate thereof, comprising the following steps (A) and (B): Step (A): A step of contacting a purification target, which is a mixture of the following purification target (i) and the following impurity (ii), with a lithium-containing substance: (i) the amino acid having a protecting group at the N-terminus or the peptide compound having a protecting group at the N-terminus (ii) a compound other than the purification target; Step (B): A step of precipitating a lithium salt of the purification target. [2] A method for removing impurities from a purification target, which comprises the following steps (A) and (B): Step (A): A step of contacting a purification target, which is a mixture of the following purification target (i) and the following impurity (ii), with a lithium-containing substance: (i) the amino acid having a protecting group at the N-terminus or the peptide compound having a protecting group at the N-terminus (ii) a compound other than the purification target; Step (B): A step of precipitating a lithium salt of the purification target. [3] The method according to [1] or [2], wherein the target product is a salt of an amino acid, a salt of a peptide compound, or a solvate thereof. [4] The method according to any one of [1] to [3], wherein the protecting group at the N-terminus of the target product is a carbamate protecting group. [5] The method according to any one of [1] to [4], wherein the target product has a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group as a protecting group. [6] The method according to any one of [1] to [5], wherein the impurity is an amino acid having a protecting group at the N-terminus or a peptide compound having a protecting group at the N-terminus other than the target product. [7] The method according to any one of [1] to [6], wherein the impurity is β-alanine having a protecting group at the N-terminus. [8] The method according to any one of [1] to [7], wherein the impurity has a carbamate protecting group.[9] The method according to any one of [1] to [8], wherein the impurity has a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group as a protecting group.

[10] The method according to any one of [1] to [9], wherein the target product and the impurity have the same protecting group.

[11] The method according to any one of [1] to

[10] , wherein the target product and the impurity have the same protecting group at the N-terminus.

[12] The method according to any one of [1] to

[11] , wherein a lithium salt of the target product is formed in step (A).

[13] The method according to any one of [1] to

[12] , wherein a lithium salt of the impurity is formed in step (A).

[14] The method according to any one of [1] to

[13] , wherein a lithium salt of the target product or a solvate thereof is precipitated as a solid in step (B).

[15] The method according to any one of [1] to

[14] , wherein in step (B), the lithium salt or solvate thereof of the target product for purification is precipitated as crystals.

[16] The method according to any one of [1] to

[15] , wherein step (A) is performed in the presence of a first organic solvent.

[17] The method according to

[16] , wherein the solubility of the target product for purification in the first organic solvent is 20 g / L or more.

[18] The method according to any one of

[16] or

[17] , wherein the solubility of the lithium salt of the target product for purification in the first organic solvent is 20 g / L or less.

[19] The method according to any one of

[16] to

[18] , wherein the solubility of the impurity in the first solvent is 20 g / L or more.

[20] The method according to any one of

[16] to

[19] , wherein the first organic solvent is at least one selected from the group consisting of nitriles, ethers, ketones, amides, ureas, sulfoxides, sulfones, alkanes, aromatic compounds, esters, and alkyl halides.

[21] The method according to any one of

[16] to

[20] , wherein the first organic solvent is a nitrile or an ether.

[22] The method according to any one of

[16] to

[21] , wherein the first organic solvent is at least one selected from the group consisting of acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, 1,2-dimethoxyethane, acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, sulfolane, heptane, methylcyclohexane, hexane, cyclohexane, pentane, benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, 1,4-dimethylbenzene, ethyl acetate, isopropyl acetate, dichloroethane, dichloromethane, chloroform, and carbon tetrachloride.

[23] The method according to any one of

[16] to

[22] , wherein the first organic solvent is acetonitrile or tetrahydrofuran.

[24] The method according to any one of

[16] to

[23] , wherein the first organic solvent consists of only one type.

[25] The method according to any one of [1] to

[24] , wherein step (A) is carried out in the presence of water.

[26] The method according to any one of

[16] to

[25] , wherein no organic solvent other than the first organic solvent is used in step (A).

[27] The method according to any one of [1] to

[26] , wherein step (B) is carried out in the presence of a second organic solvent.

[28] The method according to

[27] , wherein the solubility of the lithium salt of the purification target product in the second organic solvent is 10 g / L or less.

[29] The method according to

[27] or

[28] , wherein the second organic solvent is at least one selected from the group consisting of nitriles, alcohols, ethers, ketones, amides, ureas, sulfoxides, sulfones, alkanes, aromatic compounds, esters, and alkyl halides.

[30] The method according to any one of

[27] to

[29] , wherein the second organic solvent is a nitrile or an ether.

[31] The method according to any one of

[27] to

[30] , wherein the second organic solvent is at least one selected from the group consisting of acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, 1,2-dimethoxyethane, acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, sulfolane, heptane, methylcyclohexane, hexane, cyclohexane, pentane, benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, 1,4-dimethylbenzene, ethyl acetate, isopropyl acetate, dichloroethane, dichloromethane, chloroform, and carbon tetrachloride.

[32] The method according to any one of

[27] to

[31] , wherein the second organic solvent is acetonitrile or tetrahydrofuran.

[33] The method according to any one of

[27] to

[32] , wherein the second organic solvent consists of only one type.

[34] The method according to any one of [1] to

[33] , wherein step (B) is carried out in the presence of water.

[35] The method according to any one of

[13] to

[34] , wherein the solubility of the lithium salt of the impurity in water is 10 g / L or more.

[36] The method according to any one of

[27] to

[35] , wherein no organic solvent other than the second organic solvent is used in step (B).

[37] The method according to any one of

[27] to

[36] , wherein the sample obtained in step (A) is mixed with the second solvent in step (B).

[38] The method according to any one of

[27] to

[37] , wherein the second organic solvent is further added in step (B).

[39] The method according to

[37] or

[38] , wherein the lithium salt to be purified is precipitated by the step according to

[37] or

[38] .

[40] The method according to any one of

[27] to

[39] , wherein the second organic solvent is the same solvent as the first organic solvent.

[41] The method of any of [1] to

[40] , wherein steps (A) and (B) are carried out in the presence of a first organic solvent, and the volume of the first organic solvent per 1 g of the purification target is 1 ml to 40 ml throughout steps (A) and (B).

[42] The method of any of

[27] to

[41] , wherein the volume of the first organic solvent per 1 g of the purification target at the end of step (A) is 1 ml to 50 ml, if the proportion of the second organic solvent is increased in step (B).

[43] The method of any of

[27] to

[42] , wherein the volume of the first organic solvent per 1 g of the purification target at the end of step (A) is 1 ml to 100 ml, if the proportion of the second organic solvent is not increased in step (B).

[44] The method of any of [1] to

[43] , wherein steps (A) and (B) are carried out in the presence of a second organic solvent, and the volume of the second organic solvent per 1 g of the purification target is 0 ml to 100 ml throughout steps (A) and (B).

[45] The method of any of

[27] to

[44] , wherein the volume of the second organic solvent per 1 g of the purification target at the end of step (B) is 0 ml to 100 ml.

[46] The method of any of

[34] to

[45] , wherein the volume of the water per 1 g of the purification target is 0.1 ml to 50 ml throughout steps (A) and (B).

[47] The method of any of

[25] to

[46] , wherein the volume of the water per 1 g of the purification target at the end of step (A) is 0.1 ml to 50 ml.

[48] ​​The method according to any one of

[34] to

[47] , wherein the volume of water per 1 g of the purification target at the end of step (B) is 0.1 ml to 50 ml.

[49] The method according to any one of [1] to

[48] , wherein step (A) is carried out at a temperature range of -20°C to 100°C.

[50] The method according to any one of [1] to

[49] , wherein step (B) is carried out at a temperature range of -20°C to 100°C.

[51] The method according to any one of [1] to

[50] , wherein the amount of the lithium-containing substance in all liquid components at the end of step (A) is 0.5 equivalents to 2.0 equivalents based on the amount of the target substance to be purified.

[52] The method according to any one of [1] to

[51] , wherein the lithium-containing substance is at least one selected from the group consisting of lithium hydroxide, lithium carbonate, lithium hydride, trilithium phosphate, lithium methoxide, lithium ethoxide, lithium isopropoxide, lithium tert-butoxidemethyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and lithium tetramethylpiperidide.

[53] The method according to any one of

[27] to

[52] , wherein in step (A), the lithium-containing substance, the target substance to be purified, and the first organic solvent are simultaneously mixed.

[54] The method according to any one of

[27] to

[53] , wherein in the step (A), a mixed solution of the purification target product and the first organic solvent is mixed with the lithium-containing substance.

[55] The method according to any one of

[27] to

[54] , wherein in the step (A), a mixed solution of the lithium-containing substance and the first organic solvent is mixed with the purification target product.

[56] The method according to any one of [1] to

[55] , wherein the amino acid or the N-terminal amino acid constituting the peptide compound is represented by the following general formula (1): [In the above general formula (1), n ​​represents a number of 1 or more and 3 or less, R １ When there are multiple １ may be the same or different, R １ and R ２represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group or a heteroaryl group, or a substituent to which two or more groups selected from the group consisting of these substituents are bonded, and these groups may have a substituent, and these groups may be saturated hydrocarbon groups or unsaturated hydrocarbon groups; A represents a carbon atom; B represents a hydrogen atom or a bonding point with an amino acid or a peptide compound; Z represents a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group or a 2-(trimethylsilyl)ethoxycarbonyl group; R １ and R ２ may be bonded together with N and A to form a ring, in which case R １ and R ２ is a substituent having a structure in which one hydrogen atom is removed from the structure of a substituent not involved in ring formation, and when n is 2, R ２ and two R １At least one of the groups is not a hydrogen atom.

[57] The method according to any one of [1] to

[56] , wherein the N-terminal amino acid constituting the amino acid or peptide compound is at least one selected from the group consisting of (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid, 3-(tert-butoxycarbonylamino)propanoic acid, 3-(benzyloxycarbonylamino)propanoic acid, (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid, (S)-2-(benzyloxycarbonyl(methyl)amino)-3-phenylpropanoic acid, 3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid, (R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propanoic acid, and (S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid.

[58] The method according to any one of [1] to

[57] , wherein the amino acid or the N-terminal amino acid constituting the peptide compound is obtained by reductive amination using as a catalyst a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1.

[59] The method according to

[58] , wherein the polypeptide comprises an amino acid sequence in which one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified.

[60] The method according to

[58] or

[59] , wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 8.

[61] The method according to any one of

[58] to

[60] , wherein the polypeptide comprises, at either or both of the N-terminus and the C-terminus, an amino acid sequence other than a sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1 in which one amino acid residue has been modified.

[62] The method according to any one of

[58] to

[61] , wherein the polypeptide comprises a tag sequence at either or both of the N-terminus and the C-terminus.

[63] The method according to any one of

[58] to

[62] , wherein the polypeptide comprises at least one sequence selected from the group consisting of a streptavidin-binding peptide tag sequence and a His tag sequence at either or both of the N-terminus and the C-terminus.

[64] The method according to any one of

[58] to

[63] , wherein the number of amino acid residues in the polypeptide is 300 to 400.

[65] The method according to any one of [1] to

[64] , wherein the purity of the precipitate obtained in step (B) is 95 mol% or more.

[66] The method according to any one of [1] to

[65] , wherein the apparent purity of the precipitate obtained in step (B) is 95% or more.

[67] The method according to any one of [1] to

[66] , wherein the impurity content of the precipitate obtained in step (B) is 5 mol% or less.

[68] The method according to any one of [1] to

[67] , wherein the apparent impurity content of the precipitate obtained in step (B) is 5% or less.

[69] The method according to any one of [1] to

[68] , wherein the impurity content in the purification target is reduced.

[70] The method according to any one of [1] to

[69] , wherein the apparent impurity content in the purification target is reduced.

[71] The method according to any one of [1] to

[70] , wherein the salt of an amino acid or a salt of a peptide compound, or a solvate thereof, is a lithium salt of an amino acid or a lithium salt of a peptide compound, or a solvate thereof.

[72] The method according to any one of [1] to

[71] , further comprising a step of converting the lithium salt into a salt other than a lithium salt.

[73] A method for producing an amino acid or a peptide compound, or a salt thereof, or a solvate thereof, comprising the steps according to any one of [1] to

[72] .

[74] A method for producing a peptide compound, a salt thereof, or a solvate thereof, comprising the steps according to any one of [1] to

[73] .

[75] A method for producing a cyclic peptide compound, comprising the method according to any one of [1] to

[74] , and comprising a step of binding the amino acid or the peptide compound, or the solvate thereof, with an amino acid or a peptide compound.

[76] The method according to any one of

[73] to

[75] , further comprising a step of cyclizing the peptide compound or the peptide compound obtained by binding.

[77] A lithium salt of an amino acid or a lithium salt of a peptide compound, or a solvate thereof, wherein the lithium salt of the amino acid contains the lithium salt of an amino acid represented by the general formula (1) at a purity of 99 mol % or more or an apparent purity of 99% or more, and the lithium salt of the peptide compound contains the lithium salt of a peptide compound having a residue of the amino acid represented by the general formula (1) at a purity of 99 mol % or more or an apparent purity of 99%.

[78] The lithium salt of an amino acid or a lithium salt of a peptide compound, or a solvate thereof, according to

[77] , which is in a crystalline form.

[0010] According to the present invention, a method for obtaining a highly pure amino acid salt or peptide compound salt, or a solvate thereof, can be provided.

[0011] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means a number from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0012] In this specification, the term "to" indicating a range includes both ends of the range. For example, "A to B" means a range equal to or greater than A and equal to or less than B.

[0013] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus or minus 10% of that numerical value.

[0014] In the present invention, the meaning of the term "and / or" includes any combination of "and" and "or" appropriately combined. Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B, and C.

[0015] An embodiment of the present invention will be described below, however, the present invention is not limited to the following embodiment.

[0016] One embodiment of the present invention is a method for producing a salt of an amino acid or a salt of a peptide compound, or a solvate thereof, comprising the following steps (A) and (B): Step (A): A step of contacting a target for purification, which is a mixture of the following (i) as a target for purification and the following (ii) as an impurity, with a lithium-containing substance: (i) the amino acid or the peptide compound having a protecting group at the N-terminus, and (ii) a compound other than the target for purification; Step (B): A step of precipitating a lithium salt of the target for purification.

[0017] Another embodiment of the present invention is a method for removing impurities from a material to be purified, comprising the above steps (A) and (B).

[0018] Yet another embodiment of the present invention is a lithium salt of an amino acid or a lithium salt of a peptide compound, or a solvate thereof, wherein the lithium salt of the amino acid contains the lithium salt of an amino acid represented by the general formula (1) above at a purity of 99 mol % or more or an apparent purity of 99% or more, and the lithium salt of the peptide compound contains the lithium salt of a peptide compound having a residue of an amino acid represented by the general formula (1) above at a purity of 99 mol % or more or an apparent purity of 99%.

[0019] In the past, when purifying the above-mentioned purification target substances, it was difficult to purify them to a high purity. Generally, the purification target substances are used as raw materials for the synthesis of peptide compounds and proteins, etc. Obtaining highly pure peptide compounds and proteins is important for suppressing side effects caused by impurities when they are pharmaceuticals. In other words, it is important to purify the raw materials that constitute them to a high purity. As a result of extensive research, the present inventors surprisingly discovered that the purification target substances can be purified to a high purity by converting them into lithium salts, thereby improving the usefulness of the purification target substances and their usefulness in the synthesis of peptide compounds and proteins.

[0020] The amino acid salt or peptide compound salt, or solvate thereof, more preferably the amino acid salt or peptide compound salt, and even more preferably the lithium amino acid salt or the lithium peptide compound lithium salt, of the present invention can be obtained by treating a purification target through steps (A) and (B). The purification target of the present invention is a mixture of the purification target and the impurities. The purification target herein refers to the amino acid having a protecting group at the N-terminus or the peptide compound having a protecting group at the N-terminus. An example of the purification target is an amino acid represented by the general formula (1) above. Furthermore, the impurities herein refer to compounds contained in the purification target other than the purification target. Examples of the impurities include quasi-specific impurities (amino acids having a protecting group at the N-terminus or peptide compounds having a protecting group at the N-terminus other than the purification target). More specifically, β-alanine or a derivative thereof can be exemplified. As used herein, a derivative refers to a compound that has been modified, when a certain organic compound is considered as a parent, by introducing a functional group, oxidation, reduction, atom substitution, or other modifications that do not significantly change the structure or properties of the parent compound. An example of a β-alanine derivative is a specific impurity (β-alanine having a protecting group at the N-terminus). As used herein, "amino acid" includes natural amino acids and unnatural amino acids (sometimes referred to as amino acid derivatives). Furthermore, as used herein, "amino acid residue" includes natural amino acid residues and unnatural amino acid (amino acid derivative) residues.

[0021] Naturally occurring amino acids refer to glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), cysteine ​​(Cys), methionine (Met), lysine (Lys), arginine (Arg), and proline (Pro).

[0022] Examples of unnatural amino acids (amino acid derivatives) include, but are not limited to, β-amino acids, D-amino acids, N-substituted amino acids (excluding Pro), α,α-disubstituted amino acids, amino acids whose side chains differ from those of natural amino acids, hydroxycarboxylic acids, etc. As used herein, unnatural N-substituted amino acids refer to N-substituted amino acids other than Pro.

[0023] As used herein, amino acids are allowed to have any steric configuration. The side chain of an amino acid is not particularly limited, and can be freely selected from, in addition to a hydrogen atom, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group, a cycloalkyl group, or a spiro-linked cycloalkyl group. Each of these groups may be substituted, and the substituents are not limited. For example, one or more groups may be independently selected from any substituent containing a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom. Examples of such groups include optionally substituted alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., or oxo, aminocarbonyl, halogen atoms, etc. An amino acid according to one embodiment may be a compound having a carboxy group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).

[0024] Step (A) is a step of contacting a lithium-containing material with the target material for purification. Step (A) may form a lithium salt of the target material, or may form a lithium salt of the impurity. Step (B) is a step of precipitating the lithium salt of the target material for purification. Part of step (A) and part of step (B) may be performed overlapping in time. Prior to step (A), the target material for purification may be purified by a method other than that of the present invention. For example, the target material for purification may be purified by precipitating the target material in its free form without forming a lithium salt of the target material. At least one of steps (A) and (B) may be performed in the presence of a solvent, and in this specification, the generation of a solid from a solvent is referred to as "precipitation."

[0025] The protecting group of the purification target may be a carbamate group. The carbamate group refers to a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group, and may be a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, or may be a fluorenylmethoxycarbonyl group or a benzyloxycarbonyl group.

[0026] The protecting group of the impurity may be a carbamate group. The carbamate group refers to a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group, and may be a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, or may be a fluorenylmethoxycarbonyl group or a benzyloxycarbonyl group. These protecting groups may be protecting groups for the N-terminus of the impurity.

[0027] The protecting group of the product to be purified and the protecting group of the impurity may be different or the same, but it is preferred that the protecting group of the product to be purified and the protecting group of the impurity are the same.

[0028] Step (A) may be carried out in the presence of a first organic solvent. From the viewpoint of efficiently bringing the target product into contact with the lithium-containing material, the first organic solvent is a solvent capable of dissolving the target product. The solubility of the target product in the first organic solvent is preferably 20 g / L or more. The solubility of the lithium salt of the target product in the first organic solvent is preferably 20 g / L or less. The solubility of the impurities in the first organic solvent is preferably 20 g / L or more.

[0029] Examples of the first organic solvent include nitriles such as acetonitrile, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, and ethylene glycol, ethers such as tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, and 1,2-dimethoxyethane, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl- Examples of the first solvent include at least one selected from the group consisting of amides such as 2-pyrrolidone, ureas such as 1,3-dimethyl-2-imidazolidinone, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, alkanes such as heptane, methylcyclohexane, hexane, cyclohexane, and pentane, aromatic compounds such as benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, and 1,4-dimethylbenzene, esters such as ethyl acetate and isopropyl acetate, and alkyl halides such as dichloroethane, dichloromethane, chloroform, and carbon tetrachloride. The first solvent may be of multiple types, but is preferably of one type from the viewpoint of simplifying step (A).

[0030] Step (A) may be carried out in the presence of water.

[0031] Step (B) may be carried out in the presence of a second organic solvent. The second organic solvent is a solvent in which the lithium salt of the purification target has a low solubility, i.e., a solvent that functions as a poor solvent for the lithium salt of the purification target. The solubility of the lithium salt of the purification target in the second organic solvent is preferably 10 g / L or less.

[0032] Examples of the second organic solvent include nitriles such as acetonitrile, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, and ethylene glycol, ethers such as tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, and 1,2-dimethoxyethane, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl- Examples of the second solvent include at least one selected from the group consisting of amides such as 2-pyrrolidone, ureas such as 1,3-dimethyl-2-imidazolidinone, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, alkanes such as heptane, methylcyclohexane, hexane, cyclohexane, and pentane, aromatic compounds such as benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, and 1,4-dimethylbenzene, esters such as ethyl acetate and isopropyl acetate, and alkyl halides such as dichloroethane, dichloromethane, chloroform, and carbon tetrachloride. The second solvent may be of multiple types, but is preferably of one type from the viewpoint of simplifying step (B).

[0033] Step (B) may be carried out in the presence of water, and is preferably carried out in the presence of water from the viewpoint of dissolving the lithium salt of the impurity, the solubility of which in water is preferably 10 g / L or more.

[0034] The second organic solvent may be used together with the first organic solvent in step (A). In step (B), the lithium salt of the purification target product is precipitated. During the precipitation, the precipitation of the lithium salt of the purification target product can be promoted by adding the second organic solvent and increasing its proportion. When the proportion of the second organic solvent is increased, the second organic solvent may be added to the first organic solvent used in step (A).

[0035] Furthermore, the ratio of the first organic solvent or the ratio of the second organic solvent means a volume / mass ratio (ml / g, hereinafter also referred to as v / w) calculated by dividing the volume of the first organic solvent or the second organic solvent by the mass of the target product to be purified.

[0036] Furthermore, if the solubility of the lithium salt of the purification target in the first organic solvent used in step (A) is sufficiently low, the first organic solvent and the second organic solvent may be the same solvent. In this case, it is not necessary to add the second organic solvent in step (B). In this case, step (B) is started immediately after the purification target is contacted with the lithium-containing material in step (A), and the lithium salt of the purification target is precipitated.

[0037] The lithium salt to be purified is preferably precipitated as a solid. Here, the term "solid" includes crystalline and amorphous. It is more preferable to precipitate (crystallize) the lithium salt as a crystal. In this case, step (B) can also be said to be a step of crystallizing the lithium salt to be purified.

[0038] The volume of the first organic solvent per 1 g of the purification target may be 1 ml or more, 2 ml or more, or 3 ml or more, and may be 100 ml or less, 80 ml or less, 60 ml or less, or 40 ml or less throughout steps (A) and (B). The volume of the first organic solvent per 1 g of the purification target may be 1 ml or more and 100 ml or less, 1 ml or more and 80 ml or less, 2 ml or more and 60 ml or less, or 3 ml or more and 40 ml or less throughout steps (A) and (B).

[0039] The volume of the first organic solvent per gram of the purification target material at the end of step (A) (initial concentration of the first organic solvent) may be 1 ml or more, 2 ml or more, or 3 ml or more, and may be 50 ml or less, 40 ml or less, 30 ml or less, or 20 ml or less, when the proportion of the second organic solvent is increased in step (B). The initial concentration of the first organic solvent may be 1 ml or more and 50 ml or less, 1 ml or more and 40 ml or less, 2 ml or more and 3 ml or more and 20 ml or less.

[0040] The volume of the first organic solvent per gram of the purification target material at the end of step (A) (initial concentration of the first organic solvent) may be 1 ml or more, 10 ml or more, 20 ml or more, 30 ml or more, or 35 ml or more, and may be 100 ml or less, 75 ml or less, 50 ml or less, or 37 ml or less, when the proportion of the second organic solvent is not increased in step (B). The initial concentration of the first organic solvent may be 1 ml or more and 100 ml or less, 10 ml or more and 75 ml or less, 20 ml or more and 50 ml or less, 30 ml or more and 37 ml or less, or 35 ml or more and 37 ml or less.

[0041] The volume of the second organic solvent per 1 g of the target substance may be 0 ml or more, 40 ml or less, 60 ml or less, 80 ml or less, or 100 ml or less throughout steps (A) and (B). The volume of the second organic solvent per 1 g of the target substance may be 0 ml to 100 ml, 0 ml to 80 ml, 1 ml to 60 ml, or 1 ml to 40 ml throughout steps (A) and (B).

[0042] The volume of the second organic solvent per gram of the target substance at the end of step (B) may be 0 ml or more, 40 ml or less, 60 ml or less, 80 ml or less, or 100 ml or less. The volume of the second organic solvent per gram of the target substance at the end of step (B) may be 0 ml or more and 100 ml or less, 0 ml or more and 80 ml or less, 0 ml or more and 60 ml or less, or 0 ml or more and 40 ml or less.

[0043] The volume of water per 1 g of the purification target may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and may be 50 ml or less, 30 ml or less, 10 ml or less, or 5 ml or less throughout steps (A) and (B). The volume of water in the solvent may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 5 ml or less throughout steps (A) and (B).

[0044] The volume of water (initial concentration of water) per gram of the purification target at the end of step (A) may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and may be 50 ml or less, 30 ml or less, 10 ml or less, or 4 ml or less. The initial concentration of water may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 4 ml or less.

[0045] At the end of step (B), the volume of water (initial concentration of water) per gram of the purification target may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and may be 50 ml or less, 30 ml or less, 10 ml or less, or 4 ml or less. The initial concentration of water may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 4 ml or less.

[0046] Step (A) and step (B) may be carried out in the presence of a solvent other than the first organic solvent and the second organic solvent, such as a phosphate buffer solution, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane), or bicine (N,N-di(2-hydroxyethyl)glycine).

[0047] The temperature at which step (A) is carried out may be -20°C or higher, 0°C or higher, 10°C or higher, or 20°C or higher. The temperature at which step (A) is carried out may be 100°C or lower, 80°C or lower, 60°C or lower, 40°C or lower, or 30°C or lower. The temperature at which step (A) is carried out may be -20 to 100°C, 0 to 80°C, 10 to 60°C, 10 to 40°C, or 20 to 30°C.

[0048] The temperature at which step (B) is carried out may be -20°C or higher, 0°C or higher, 10°C or higher, 20°C or higher, 30°C or higher, or 35°C or higher. The temperature at which step (A) is carried out may be 100°C or lower, 80°C or lower, 60°C or lower, 40°C or lower, or 30°C or lower. The temperature at which step (A) is carried out may be -20 to 100°C, 0 to 80°C, 10 to 60°C, 10 to 40°C, or 20 to 30°C.

[0049] The conditions in step (A) and step (B), such as temperature and the concentrations of substances contained in the various solutions, may be the same or different. When these conditions are different between the two, conditions selected from the various conditions for step (A) described above may be combined. Furthermore, the amount of the lithium-containing substance at the end of step (A) may be 0.5 equivalents or more, 0.8 equivalents or more, or 1.0 equivalents or more, based on the amount of substance of the target product to be purified, and may be 2.0 equivalents or less, 1.5 equivalents or less, or 1.1 equivalents or less. The content of the lithium-containing substance based on the mass of the reactants may be 0.5 equivalents or more, 0.8 equivalents or more, 1.5 equivalents or more, or 1.0 equivalents or more, 1.1 equivalents or less.

[0050] The lithium-containing substance in this specification refers to a substance containing lithium to be brought into contact with the purification target product in step (A). Examples of the lithium-containing substance used in step (A) include at least one selected from the group consisting of lithium hydroxide, lithium tert-butoxide, lithium hydroxide, lithium carbonate, lithium hydride, trilithium phosphate, lithium methoxide, lithium ethoxide, lithium isopropoxide, lithium tert-butoxidemethyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and lithium tetramethylpiperidide.

[0051] Although there is no limitation on the method for contacting the target material with the lithium-containing substance, the target material and the lithium-containing substance can be contacted in the presence of the first organic solvent. In this case, the target material, the lithium-containing substance, and the first organic solvent may be mixed in the same container from the beginning, or the target material and the first organic solvent may be mixed and the lithium-containing substance may be added to the resulting mixture, or the lithium-containing substance and the first organic solvent may be mixed and the target material may be added to the resulting mixture.

[0052] The content of the target substance in the target substance, which serves as a basis for determining the amounts of the first organic solvent, the second organic solvent, and the lithium-containing substance, may be an actually measured value, an estimated value, or a calculated value, preferably an actually measured value or a calculated value, and most preferably an actually measured value.

[0053] Examples of the amino acid having a protecting group at the N-terminus or the amino acid constituting a peptide compound having a protecting group at the N-terminus contained in the purification target include the amino acid represented by the above general formula (1) (also referred to as amino acid (1)), hydrophobic amino acids, aliphatic amino acids, and aromatic amino acids.

[0054] n represents a number of 1 or more and 3 or less, and may be an integer of 1 or more and 3 or less. n may be 1, 2, or 3. When n is 2, R ２ and two R １ At least one of R is not a hydrogen atom, ２ and two R １ Two or more of R may not be hydrogen atoms, ２ and two R １ All three of these do not have to be hydrogen atoms.

[0055] R １ and R ２ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, or a substituent to which two or more selected from the group consisting of these substituents are bonded (for example, a benzyl group, etc.), and these groups may have a substituent and may be saturated or unsaturated.

[0056] A represents a carbon atom, and B represents a hydrogen atom or a point of attachment to an amino acid or peptide compound.

[0057] Z represents a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group.

[0058] R １ and R ２ may be bonded together with N and A to form a ring. In that case, R １ and R ２ is a substituent having a structure in which one hydrogen atom is removed from the structure of the substituent when it is not involved in ring formation.

[0059] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyl includes not only linear but also branched chain alkyls. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C １ ~C ２０ , hereinafter referred to as “C ｐ ~C ｑ " means that the number of carbon atoms is p to q), and preferably C １ ~C １０ Alkyl, more preferably C １ ~C ６Specific examples of the alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, and 2-ethylbutyl.

[0060] As used herein, "alkylene" refers to a divalent group derived from the above-mentioned "alkyl" by further removing one optional hydrogen atom, and C ４ ~C ８ Alkylene is preferred. Specific examples of alkylene include —CH ２ -, -(CH ２ ) ２ -, -(CH ２ ) ３ -, -CH(CH ３ ) CH ２ -, -C(CH ３ ) ２ -, -(CH ２ ) ４ -, -CH(CH ３ ) CH ２ CH ２ -, -C(CH ３ ) ２ CH ２ -, -CH ２ CH (CH ３ ) CH ２ -, -CH ２ C(CH ３ ) ２ -, -CH ２ CH ２ CH (CH ３ ) -, -(CH ２ ) ５ -, -(CH ２ )６ -, -(CH ２ ) ７ -, -(CH ２ ) ８ - and others.

[0061] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent SP ２ Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be in the Entgegen (E) or Zusammen (Z), cis or trans configuration. Alkenyl includes not only straight chains but also branched chains. C is preferred as alkenyl. ２ ~C １０ alkenyl, more preferably C ２ ~C ６ Examples include alkenyl, specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, and the like.

[0062] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. C ２ ~C １０ Alkynyl, more preferably C ２ ~C ６ Specific examples include alkynyl, such as ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, and 3-methyl-(5-phenyl)-4-pentynyl.

[0063] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. ３ ~C ８Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and the like.

[0064] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C ６ ~C １０ Specific examples of the aryl include phenyl and naphthyl (for example, 1-naphthyl and 2-naphthyl).

[0065] As used herein, the term "heterocyclyl" refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclyl may have a double and / or triple bond in the ring, and a carbon atom in the ring may be oxidized to form a carbonyl. The heterocyclyl may be a monocyclic or fused ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), more preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, and isothiazolidinyl. nyl, 1,2-thiazinane, thiadiazolidinyl, azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, 2-oxaspiro[3.3]heptyl, and the like.

[0066] As used herein, "heteroaryl" refers to an aromatic, cyclic, monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a monocyclic ring or a condensed ring with another ring, and may be partially saturated. The number of atoms constituting the ring may be 5 to 12 (5- to 12-membered heteroaryl), 6 to 10 (5- to 12-membered heteroaryl), or 6 to 7 (6- to 7-membered heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0067] In this specification, "optionally substituted" means "may have a substituent," meaning that a certain group may be substituted with any substituent. Examples of the substituent include substituents containing a halogen atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, a phosphorus atom, etc. More specifically, examples of the substituent include an alkyl group, an alkoxy group, a fluoroalkyl group, a fluoroalkoxy group, an oxo group, an aminocarbonyl group, an alkylsulfonyl group, an alkylsulfonylamino group, a cycloalkyl group, an aryl group, a heteroaryl group, a heterocyclyl group, an arylalkyl group, a heteroarylalkyl group, a halogen group, a nitro group, an amino group, a monoalkylamino group, a dialkylamino group, a cyano group, a carboxyl group, an alkoxycarbonyl group, a formyl group, etc.

[0068] The N-terminal amino acid constituting the amino acid or peptide compound can be obtained by reductive amination using a polypeptide containing an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 as a catalyst.

[0069] The identity to the amino acid sequence represented by SEQ ID NO: 1 may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0070] The identity with the amino acid sequence represented by SEQ ID NO: 1 can be determined by the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7). Based on this algorithm, a program called BLASTN or BLASTX has been developed (Altschul et al., J. Mol. Biol. (1990) 215:403-10). When analyzing an amino acid sequence using BLAST-based BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. For specific techniques for these analysis methods, information on the BLAST (Basic Local Alignment Search Tool) website of the National Center for Biotechnology Information (NCBI) is available.

[0071] The polypeptide according to this embodiment may be a polypeptide in which one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may have been modified. The number of modified amino acid residues may be 3 or less, 2 or less, or 1.

[0072] The modification may be one or more selected from the group consisting of substitution, deletion, and insertion, and may be a substitution. The modification may be a conservative modification. A conservative modification means a modification of an amino acid residue that does not reduce the desired catalytic activity compared to the polypeptide before modification.

[0073] The modification may be a substitution with a natural amino acid different from the amino acid residue before modification. Furthermore, the natural amino acid used for the substitution may be one or more selected from the group consisting of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, asparagine, glutamic acid, aspartic acid, cysteine, methionine, lysine, arginine, and proline.

[0074] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the histidine residue at position 44, the phenylalanine residue at position 117, the methionine residue at position 141, the threonine residue at position 156, the histidine residue at position 182, the glutamine residue at position 186, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 have been modified. Since this further improves the catalytic activity for reductive amination, the polypeptide may be a polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the methionine residue at position 141, the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified. Furthermore, the polypeptide may be a polypeptide comprising a sequence in which amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of the methionine residue at position 141, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified.

[0075] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue located at a position corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than histidine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. The amino acid residue represented by X in SEQ ID NO: 2 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0076] Since catalytic activity for reductive amination is further improved, the polypeptide may contain a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 44 is substituted with a methionine residue. That is, the polypeptide of this embodiment may contain an amino acid sequence (H44M) in which the amino acid residue at the site corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue.

[0077] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue at the position corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than phenylalanine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3. The amino acid residue represented by X in SEQ ID NO: 3 is an alanine residue, aspartic acid residue, glutamic acid residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0078] Since the catalytic activity for reductive amination is further improved, the polypeptide may contain a sequence in which the amino acid residue at the position corresponding to the phenylalanine residue at position 117 is substituted with a leucine residue. That is, the polypeptide of this embodiment may contain an amino acid sequence (F117L) in which the amino acid residue at the position corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue.

[0079] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than methionine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4. The amino acid residue represented by X in SEQ ID NO: 4 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0080] Since the catalytic activity for the reductive amination reaction is further improved, the amino acid residue located at the site corresponding to the methionine residue at position 141 may comprise a sequence substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, valine residues, threonine residues, serine residues, arginine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues, and alanine residues. That is, the polypeptide according to this embodiment may comprise an amino acid sequence (M141Y, M141W, M141V, M141T, M141S, M141R, M141L, M141K, M141I, M141H, M141F, or M141A) in which the amino acid residue at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, tryptophan residue, valine residue, threonine residue, serine residue, arginine residue, leucine residue, lysine residue, isoleucine residue, histidine residue, phenylalanine residue, or alanine residue. Furthermore, the polypeptide may comprise a sequence in which the amino acid residue at the site corresponding to the methionine residue at position 141 is substituted with one or more amino acid residues selected from the group consisting of tyrosine residue, tryptophan residue, valine residue, lysine residue, isoleucine residue, phenylalanine residue, and alanine residue. That is, the polypeptide of this embodiment may include an amino acid sequence (M141Y, M141W, M141V, M141K, M141I, M141H, M141F, or M141A) in which the amino acid residue located at the position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, tryptophan residue, valine residue, lysine residue, isoleucine residue, histidine residue, phenylalanine residue, or alanine residue.

[0081] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue at a position corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than threonine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 5. The amino acid residue represented by X in SEQ ID NO: 5 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0082] Since the catalytic activity for reductive amination is further improved, the polypeptide may contain a sequence in which the amino acid residue at the position corresponding to the threonine residue at position 156 is substituted with a serine residue. That is, the polypeptide of this embodiment may contain an amino acid sequence (T156S) in which the amino acid residue at the position corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a serine residue.

[0083] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than histidine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6. The amino acid residue represented by X in SEQ ID NO: 6 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0084] Since catalytic activity in reductive amination reactions is further improved, the polypeptide may include a sequence in which the amino acid residue at the position corresponding to the histidine residue at position 182 is substituted with one or more amino acid residues selected from the group consisting of tyrosine, glutamine, methionine, leucine, glycine, phenylalanine, and alanine. That is, the polypeptide of this embodiment may include an amino acid sequence in which the amino acid residue at the position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine, glutamine, methionine, leucine, glycine, phenylalanine, or alanine residue (H182Y, H182Q, H182M, H182L, H182G, H182F, or H182A). Furthermore, the polypeptide may include a sequence in which the amino acid residue at the position corresponding to the histidine residue at position 182 is substituted with one or more amino acid residues selected from the group consisting of methionine, leucine, and phenylalanine. That is, the polypeptide of this embodiment may include an amino acid sequence (H182M, H182L, or H182F) in which the amino acid residue located at a position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue, a leucine residue, or a phenylalanine residue.

[0085] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than glutamine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 7. The amino acid residue represented by X in SEQ ID NO: 7 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0086] Since this further improves the catalytic activity for reductive amination, the polypeptide may comprise a sequence in which the amino acid residue at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one or more amino acid residues selected from the group consisting of methionine residues and glutamic acid residues. That is, the polypeptide of this embodiment may comprise an amino acid sequence (Q186M or Q186E) in which the amino acid residue at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue or a glutamic acid residue.

[0087] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than a tryptophan residue. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8. The amino acid residue represented by X in SEQ ID NO: 8 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, or tyrosine residue.

[0088] Since this further improves the catalytic activity for reductive amination reactions, the amino acid residue located at the site corresponding to the tryptophan residue at position 253 may comprise a sequence in which the amino acid residue is substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, valine residues, threonine residues, serine residues, arginine residues, glutamine residues, proline residues, asparagine residues, methionine residues, leucine residues, lysine residues, isoleucine residues, histidine residues, phenylalanine residues, and alanine residues. That is, the polypeptide of this embodiment may comprise an amino acid sequence (W253Y, W253V, W253T, W253S, W253R, W253Q, W253P, W253N, W253M, W253L, W253K, W253I, W253H, W253F, or W253A) in which the amino acid residue located at a position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, a valine residue, a threonine residue, a serine residue, an arginine residue, a glutamine residue, a proline residue, an asparagine residue, a methionine residue, a leucine residue, a lysine residue, an isoleucine residue, a histidine residue, a phenylalanine residue, or an alanine residue. Furthermore, the polypeptide may comprise a sequence in which the amino acid residue at the position corresponding to the tryptophan residue at position 253 is substituted with one or more amino acid residues selected from the group consisting of leucine residues, isoleucine residues, and histidine residues. That is, the polypeptide of this embodiment may comprise an amino acid sequence (W253L, W253I, or W253H) in which the amino acid residue at the position corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue, an isoleucine residue, or a histidine residue.

[0089] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which the amino acid residue located at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than lysine. The polypeptide may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9. The amino acid residue represented by X in SEQ ID NO: 9 is an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, leucine residue, methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.

[0090] Since the catalytic activity for reductive amino reaction is further improved, the amino acid residue located at the site corresponding to the lysine residue at position 260 may be substituted with one or more amino acid residues selected from the group consisting of tyrosine residues, tryptophan residues, threonine residues, serine residues, arginine residues, glutamine residues, asparagine residues, methionine residues, leucine residues, histidine residues, glycine residues, phenylalanine residues, glutamic acid residues, and alanine residues. That is, the polypeptide according to this embodiment may have an amino acid sequence in which the amino acid residue located at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tyrosine residue, tryptophan residue, threonine residue, serine residue, arginine residue, glutamine residue, asparagine residue, methionine residue, leucine residue, histidine residue, glycine residue, phenylalanine residue, glutamic acid residue, or alanine residue (K260Y, K260W, K260T, K260S, K260R, K260Q, K260N, K260M, K260L, K260H, K260G, K260F, K260E, or K260A). Furthermore, the polypeptide may include a sequence in which the amino acid residue located at the site corresponding to the lysine residue at position 260 is substituted with one or more amino acid residues selected from the group consisting of glutamine residue, methionine residue, glutamic acid residue, and asparagine residue. That is, the polypeptide of this embodiment may have an amino acid sequence (K260Q, K260M, K260E, or K260N) in which the amino acid residue located at a position corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glutamine residue, a methionine residue, a glutamic acid residue, or an asparagine residue.

[0091] The polypeptide according to this embodiment may include an amino acid sequence a1 (mutation: M141V) represented by SEQ ID NO: 4, in which the amino acid residue represented by X is a valine residue; an amino acid sequence a2 (mutation: M141Y) represented by SEQ ID NO: 4, in which the amino acid residue represented by X is a tyrosine residue; an amino acid sequence a3 (mutation: H182L) represented by SEQ ID NO: 6, in which the amino acid residue represented by X is a leucine residue; an amino acid sequence a4 (mutation: W253H) represented by SEQ ID NO: 8, in which the amino acid residue represented by X is a histidine residue; or an amino acid sequence a5 (mutation: K260E) represented by SEQ ID NO: 9, in which the amino acid residue represented by X is a glutamic acid residue.

[0092] The polypeptide according to this embodiment may comprise a sequence having 90% or more sequence identity with amino acid sequence a1, a2, a3, a4, or a5. The sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%.

[0093] The polypeptide according to this embodiment may be a polypeptide comprising a sequence in which one or more amino acid residues in the amino acid sequence a1, a2, a3, a4, or a5 have been modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may be modified. The number of modified amino acid residues may be 3 or less, 2 or less, or 1.

[0094] The polypeptide according to this embodiment may be fused with another polypeptide or protein. That is, the polypeptide according to this embodiment may have, at either or both of the N-terminus and C-terminus, an amino acid sequence (another amino acid sequence) other than the sequence having 90% or more sequence identity with the sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified. The other amino acid sequence may be, for example, a tag sequence.

[0095] Examples of polypeptides or proteins having such other amino acid sequences include His tags (6XHis, 10XHis, etc.) that are tags consisting of several (e.g., 6, 10, etc.) His (histidine) residues, streptavidin-binding peptide tags (SBP tags) that contain an amino acid sequence capable of binding to the biotin-binding site of streptavidin, GST (Glutathione S-transferase), HA (influenza agglutinin), immunoglobulin constant regions, B-galactosidase, MBP (maltose-binding protein), FLAG (Hopp, T.P. et al., J. Immunol. Chem. Soc. 1999, 123:111-112, 2001), and the like. al., BioTechnology (1988) 6, 1204-1210), influenza agglutinin (HA), a fragment of human c-myc, a fragment of VSV-GP, a fragment of p18HIV, T7-tag, HSV-tag, E-tag, a fragment of SV40 T antigen, Ick tag, a fragment of α-tubulin, B-tag, a fragment of Protein C, Tag, StrepTag, HaloTag, etc. The His tag may be, for example, the amino acid sequence represented by SEQ ID NO: 11. The SBP tag may be, for example, the amino acid sequence represented by SEQ ID NO: 12.

[0096] The polypeptide according to this embodiment may contain one or more sequences selected from the group consisting of a streptavidin-binding peptide tag sequence and a His tag sequence at either or both of the N-terminus and the C-terminus, and may contain a streptavidin-binding peptide tag sequence (SBP tag) and a His tag sequence at the C-terminus.

[0097] The polypeptide according to this embodiment may have an amino acid sequence a1 (mutation: M141V) represented by SEQ ID NO: 4, in which the amino acid residue represented by X is a valine residue; an amino acid sequence a2 (mutation: M141Y) represented by SEQ ID NO: 4, in which the amino acid residue represented by X is a tyrosine residue; an amino acid sequence a3 (mutation: H182L) represented by SEQ ID NO: 6, in which the amino acid residue represented by X is a leucine residue; an amino acid sequence a4 (mutation: W253H) represented by SEQ ID NO: 8, in which the amino acid residue represented by X is a histidine residue; or an amino acid sequence a5 (mutation: K260E) represented by SEQ ID NO: 9, in which the amino acid residue represented by X is a glutamic acid residue, and a tag sequence. The polypeptide according to this embodiment may have, in addition to the amino acid sequence a1, a2, a3, a4, or a5 and the tag sequence, a linker sequence connecting the amino acid sequence a1, a2, a3, a4, or a5 to the tag sequence. The linker sequence may have, for example, an amino acid sequence represented by GGSS or GGS.

[0098] A polypeptide according to one embodiment may be polypeptide X1 comprising an amino acid sequence a1, a2, a3, a4, or a5, a linker sequence having the amino acid sequence GGSS and bound to the C-terminus of amino acid sequence a1, a2, a3, a4, or a5, and a His tag having the amino acid sequence SEQ ID NO: 11 and bound to the linker sequence. One or more amino acid residues in polypeptide X1 may be modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may be modified. The number of modified amino acid residues in polypeptide X1 may be 3 or less, 2 or less, or 1.

[0099] A polypeptide according to one embodiment may be polypeptide X2 comprising the amino acid sequence a1, a2, a3, a4, or a5, an SBP tag represented by SEQ ID NO: 12 attached to the C-terminus of the amino acid sequence a1, a2, a3, a4, or a5, a His tag represented by SEQ ID NO: 11, and a linker sequence linked between the His tag and the SBP tag and represented by the amino acid sequence GGS. One or more amino acid residues in polypeptide X2 may be modified, and 1 to 20, 1 to 15, 1 to 10, 1 to 7, or 1 to 5 amino acid residues may be modified. The number of modified amino acid residues in polypeptide X2 may be 3 or less, 2 or less, or 1.

[0100] The polypeptide according to the present embodiment may be used as a mixture with other polypeptides, or may be used in an isolated and purified state. When used as a mixture with other polypeptides, the target amino acid can be obtained through a process of isolating and purifying the target product.

[0101] When the polypeptide according to this embodiment exists as a monomer, the number of amino acid residues may be 300 or more, 310 or more, 320 or more, 325 or more, or 330. Alternatively, the number may be 400 or less, 390 or less, 380 or less, or 375 or less. The number of amino acid residues of the polypeptide may be 300 or more and 400 or less.

[0102] Furthermore, when the other amino acid sequence is not added to either the N-terminus or the C-terminus, the number of amino acid residues of the polypeptide according to this embodiment may be 300 or more, 310 or more, 320 or more, 325 or more, or 330. Alternatively, it may be 360 ​​or less, 350 or less, 340 or less, or 335 or less.

[0103] Furthermore, when the other amino acid sequence is added to either or both of the N-terminus and the C-terminus, the number of amino acid residues of the polypeptide according to this embodiment may be 340 or more, 350 or more, 360 or more, or 370 or more, or may be 374. Alternatively, it may be 400 or less, 390 or less, 380 or less, or 375 or less.

[0104] The polypeptide according to this embodiment may be used as a monomer, or in a form in which two or more monomers are associated with each other.Furthermore, the polypeptide according to this embodiment may be a homodimer.

[0105] When the polypeptide according to this embodiment is a homodimer, the number of amino acid residues is twice that when it exists as a monomer.

[0106] The peptide compound having a protecting group at the N-terminus may be a peptide compound consisting of 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer, or 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acid residues. Furthermore, the peptide compound having a protecting group at the N-terminus may be a peptide compound consisting of 2 to 10, 2 to 8, 2 to 6, or 2 to 4 amino acid residues. The peptide compound having a protecting group at the N-terminus may be a peptide compound consisting of 5 to 30, 7 to 25, 8 to 15, 9 to 14, 10 to 13, or 11 amino acid residues, etc. Also included are peptide compounds containing a residue of amino acid (1) at the N-terminus.

[0107] In step (B), the purified product may be precipitated so that the content of quasi-specific impurities contained in the precipitate (purified product) obtained in step (B) is reduced. In step (B), the purified product may be precipitated so that the purity of the purified product is 95 mol% or more. The purity is more preferably 96 mol% or more, even more preferably 97 mol% or more, even more preferably 98 mol% or more, particularly preferably 99 mol% or more, and most preferably 100 mol%. In this specification, "purity" broadly means the proportion of the target substance contained in the substance whose purity is to be measured, and narrowly means the proportion of the amount of substance of the lithium salt of the target substance based on the total amount of substance of the lithium salt of the target substance and the lithium salt of the specific impurity contained in the purified product.

[0108] In step (B), the purified product may be precipitated so that the impurity content in the purified product is 5 mol% or less. The impurity content is preferably 4 mol% or less, more preferably 3 mol% or less, even more preferably 2 mol% or less, particularly preferably 1 mol% or less, and most preferably 0 mol%. In this specification, the "impurity content" refers to the amount of the lithium salt of the specific impurity contained in the purified product based on the total amount of the lithium salt of the target product and the lithium salt of the specific impurity.

[0109] The purity or impurity content can be measured, for example, by a nuclear magnetic resonance spectrometer, gas chromatography, or high performance liquid chromatography. When high performance liquid chromatography is used, the purity or impurity content can be calculated from the area ratio of UV absorption peaks. The following formula 1a shows a formula for calculating the purity, and the following formula 2a shows a formula for calculating the impurity content.

[0110] Here, the amount of substance per unit UV absorption peak area is a value obtained by dividing the UV absorption peak area of ​​the target product or specific impurity by the amount of substance.

[0111] In step (B), the purified product may be precipitated so that the apparent purity of the purified product is 95% or more. The apparent purity is more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, particularly preferably 99% or more, and most preferably 100%. As used herein, "apparent purity" refers to the ratio of the UV absorption peak area of ​​the target product at a specific wavelength to the sum of the UV absorption peak areas of the target product and specific impurities at a specific wavelength, as measured after separating the purified product into the target product and specific impurities by high performance liquid chromatography.

[0112] In step (B), the purified product may be precipitated so that the apparent impurity content of the purified product is 5% or less. The apparent impurity content is preferably 4% or less, more preferably 3% or less, even more preferably 2% or less, particularly preferably 1% or less, and most preferably 0%. As used herein, the term "apparent impurity content" refers to the ratio of the UV absorption peak area of ​​a specific impurity at a specific wavelength to the sum of the UV absorption peak areas of the target product and the specific impurity at a specific wavelength, as measured after separating the purified product into the target product and the specific impurity by high performance liquid chromatography.

[0113] The following formula 1b shows a formula for calculating the apparent purity, and the following formula 2b shows a formula for calculating the apparent impurity content.

[0114] The specific wavelength in this specification refers to a wavelength within 10% of the absorption maximum wavelength exhibited by the protecting group of the product to be purified. Furthermore, when there are multiple absorption maximum wavelengths, any absorption maximum wavelength may be selected as the absorption maximum wavelength as long as it exhibits an absorption intensity of 30% or more based on the absorption intensity at the maximum absorption wavelength among them. Furthermore, the influence of noise can be reduced by selecting a longer wavelength among the multiple absorption maximum wavelengths. Furthermore, the sample concentration can be adjusted according to the absorption intensity of the selected absorption maximum wavelength. During measurement, the absorption maximum wavelength that is most preferable from the standpoint of ease of sample concentration adjustment and noise reduction can be selected.

[0115] For example, the specific wavelength of UV light can be 254 nm for an Fmoc group, 197 nm for a Boc group, or 210 nm for a Cbz group.

[0116] In the production method according to one embodiment, the content of β-alanine or a derivative thereof in the object to be purified can be reduced by obtaining a lithium salt. In other words, the production method according to this embodiment may include a step of reducing the content of β-alanine or a derivative thereof.

[0117] The production method according to this embodiment may further include a step of converting the amino acid or peptide compound into a salt other than the lithium salt. Specifically, for example, the lithium salt obtained by the production method according to one embodiment may be desalted to obtain a free amino acid or a free peptide compound. Desalting may be performed by a known method. Furthermore, a salt other than the lithium salt may be produced from the obtained free amino acid or peptide compound. Examples of salts other than the lithium salt include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; salts with alkali metals (sodium or potassium); salts with alkaline earth metals such as calcium and magnesium; and ammonium salts.

[0118] Furthermore, in one embodiment of the production method, the obtained free amino acid or peptide compound can be linked to an amino acid or peptide compound by a conventional method to produce a peptide compound with a desired number of amino acid residues, for example, a peptide compound having a cyclic portion (cyclic peptide compound).

[0119] The number of amino acid residues in the cyclic peptide compound according to this embodiment may be 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Furthermore, the cyclic peptide compound according to this embodiment may be a peptide compound consisting of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acids. Furthermore, the number of amino acid residues in the cyclic peptide compound according to this embodiment may be 2 or more and 10 or less, 2 or more and 8 or less, 2 or more and 6 or less, or 2 or more and 4 or less. Furthermore, the number of amino acid residues in the cyclic peptide compound according to this embodiment may be 5 or more and 30 or less, 7 or more and 25 or less, 8 or more and 15 or less, 9 or more and 14 or less, 10 or more and 13 or less, or 11.

[0120] The "cyclic portion" of a cyclic peptide compound refers to a cyclic portion formed by linking two or more amino acid residues. The number of amino acid residues constituting the cyclic portion of the cyclic peptide compound may be 5 to 14, 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 5 to 13, 6 to 13, 7 to 13, 8 to 13, 9 to 13, 10 to 13, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 5 to 11, 6 to 11, 7 to 11, 8 to 11, 9 to 11, 10 to 11, or 11. The cyclic moiety is preferably formed via a covalent bond such as an amide bond, a carbon-carbon bond-forming reaction, an S-S bond, a thioether bond, or a triazole bond, and can be formed by bonding a group at the N-terminus and a group at the C-terminus of a linear peptide compound to which amino acid residues are linked. Specifically, for example, it can be formed by bonding an amino group at the N-terminus and a carboxyl group at the C-terminus of a linear peptide compound to which amino acid residues are linked. The cyclization may be performed in any manner, such as via a carbon-nitrogen bond such as an amide bond, via a carbon-oxygen bond such as an ester bond or an ether bond, via a carbon-sulfur bond such as a thioether bond, via a carbon-carbon bond, via a sulfur-sulfur bond, or via a heterocyclic ring structure. Among these, cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxy group in a side chain and an amino group in the main chain is more preferred. The position of the carboxy group and amino group used for cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position that allows cyclization.

[0121] The cyclic peptide compound according to this embodiment may have 15 to 46 ring atoms. As used herein, the term "number of ring atoms" refers to the number of atoms (ring atoms) in a cyclic compound, including the innermost ring. When a compound has multiple rings, the term is defined as the number of atoms in the ring with the largest number of atoms. When two rings share some atoms, the ring with the fewer shared atoms is used to calculate the number of ring atoms in each ring. To further illustrate the "number of ring atoms" with a specific example, using this method, tetrahydrofuran (THF) has 5 ring atoms, and tacrolimus (FK506) has 21 ring atoms.

[0122] The number of ring atoms in the cyclic peptide compound according to this embodiment may be, for example, 34 to 46, 34 to 43, 34 to 40, 34 to 37, 34 to 36, or 34. The ring atoms used to calculate the number of ring atoms may be selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, sulfur atoms, phosphorus atoms, and silicon atoms, or may be selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, and oxygen atoms.

[0123] The cyclic peptide compound according to this embodiment may have a linear portion in addition to a cyclic portion. Specific embodiments of the number of amino acid residues in the cyclic peptide compound are the same as those of the peptide compound described above. When the cyclic peptide compound has a linear portion, it is preferable that the total number of amino acid residues in the cyclic portion and the linear portion be within the same range. Furthermore, when the cyclic peptide compound has a linear portion, the number of amino acid residues constituting the cyclic portion is preferably 5 to 15, 6 to 15, 6 to 14, 7 to 14, 8 to 14, or 7 to 13, more preferably 7 to 12, or 8 to 11, even more preferably 9 to 11, and particularly preferably 10 or 11. The number of amino acid residues constituting the linear portion is preferably 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3.

[0124] The molecular weight of the cyclic peptide compound according to this embodiment is not particularly limited, but may be, for example, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, or 950 or more, with 1000 or more, 1100 or more, 1200 or more, 1300 or more, or 1400 or more being preferred. The upper limit of the molecular weight of the peptide compound according to this embodiment is not particularly limited, but is preferably 5000 or less, 4000 or less, 3000 or less, 2500 or less, or 2000 or less. The molecular weight herein refers to the sum of the atomic weights of the atoms constituting the compound molecule (unit: "g / mol") and is obtained by calculating the sum of the atomic weights of the atoms contained in the molecular structure (unit: "g / mol"). In this specification, the molecular weight unit may be omitted. The molecular weight of the peptide compound can be measured by liquid chromatography mass spectrometry (LC / MS) as described in the Examples. The specific aspects of the peptide compound described above can be applied to (i) the peptide compound containing one or more N-substituted amino acid residues according to this embodiment without any restrictions, as long as it is a peptide compound containing one or more N-substituted amino acid residues.

[0125] The cyclic peptide compound according to this embodiment may contain one or more N-substituted amino acid residues, preferably at least three N-substituted amino acid residues, more preferably at least four N-substituted amino acid residues, and even more preferably at least five N-substituted amino acid residues. The N-substituted amino acid residues may be present consecutively or discontinuously in the N-substituted cyclic peptide compound.

[0126] The present invention is further illustrated by, but not limited to, the following examples. Tetrahydrofuran, acetonitrile, aqueous lithium hydroxide solution, lithium tert-butoxide tetrahydrofuran solution, and starting materials for amino acid derivatives, such as (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid, used in the practice of the present invention were obtained from commercial suppliers and used without purification, unless otherwise specified. The abbreviations listed in Tables 1 and 2 are used in the examples.

[0127] High-performance liquid chromatography conditions 1 Apparatus: Waters AQUITY H-class / QDa Column: (I.D. x length (mm), particle size (μm)): ACQUITY UPLC HSS T3 (2.1 x 50, 1.8) Mobile phase: A) 0.05% TFA HO, B) 0.05% TFA MeCN Gradient (A / B): 100 / 0 to 2 / 98 (5.0 min) ⇒ 2 / 98 (1.0 min) ⇒ 100 / 0 (0.01 min) ⇒ 100 / 0 (2.0 min) Flow rate (ml / min): 0.5 Column temperature: 30°C

[0128] High-performance liquid chromatography conditions 2 Apparatus: Waters AQUITY H-class Column: (I.D. x length (mm), particle size (μm)): Ascentis Express RP-Amide (2.1 x 50, 2.7) Mobile phase: A) 0.05% TFA HO, B) 0.05% TFA MeCN Gradient (A / B): 95 / 5 to 0 / 100 (4.0 min) ⇒ 0 / 100 (0.5 min) ⇒ 95 / 5 (0.1 min) ⇒ 95 / 5 (1.4 min) Flow rate (ml / min): 0.5 Column temperature: 35°C

[0129] High-performance liquid chromatography condition 3 Apparatus: Waters AQUITY H-class / QDa Column: (I.D. x length (mm), particle size (μm)): Ascentis Express C18 (3.0 x 50, 2.7) Mobile phase: A) 0.05% TFA HO, B) 0.05% TFA MeCN Gradient (A / B): 95 / 5 to 0 / 100 (5.0 min) ⇒ 0 / 100 (1.0 min) ⇒ 95 / 5 (0.01 min) ⇒ 95 / 5 (2.0 min) Flow rate (ml / min): 0.5 Column temperature: 30°C

[0130] High-performance liquid chromatography conditions 4 Apparatus: Waters AQUITY H-class / QDa Column (I.D. x length (mm), particle size (μm)): DAICEL CHIRALPAK IC-3 (4.6 x 150, 3) Mobile phase: A) 0.05% TFA HO, B) 0.05% TFA MeCN Gradient (A / B): 95 / 5 to 40 / 60 (20.0 min) ⇒ 95 / 5 (0.1 min) ⇒ 95 / 5 (4.9 min) Flow rate (ml / min): 1.0 Column temperature: 30°C

[0131] Preparation Example 1: Preparation of W253H-SBP-His A gene was synthesized in which a streptavidin-binding peptide tag sequence (GTDEKTTGWRGGHVVEGLAGEELEQLRARLEHHPQ), a linker sequence (GGS), and a His tag sequence (HHHHHH) were added to the C-terminus of the amino acid sequence represented by SEQ ID NO: 8, where X is His, and the gene was cloned into an E. coli expression vector. This expression vector was introduced into the BL21(DE3) E. coli strain (Novagen), and the cells were cultured at 18°C ​​for 2 days using Overnight Express Instant TB Medium (Novagen) to express the target protein (SEQ ID NO: 10, W253H), which was the final product.

[0132] Synthesis Example 1: Synthesis of crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid

[0133] 2-Cyclopentyl-2-oxo-acetic acid sodium salt (3.9 g, 24 mmol), methylamine hydrochloride (8.1 g, 120 mmol), D-glucose (8.7 g, 48 mmol), and N,N-di(2-hydroxyethyl)glycine (7.8 g, 48 mmol) were dissolved in distilled water (50 mL), and the external temperature of the reaction vessel was set to 25 °C. A 5 M aqueous solution of sodium hydroxide (6.0 mL) was added, and the pH was adjusted to 8.8. NADP+ (0.18 g, 0.24 mmol), D-glucose dehydrogenase (3.8 mg, 960 U, 250 U / mg), and NMAADH modified enzyme solution (final preparation, 1.6 mL, equivalent to 43 mg of enzyme, 1.1% by mass) were added, and the reaction vessel was stirred for 24 hours at an external temperature of 25 °C. Concentrated hydrochloric acid (3.0 mL) was added to adjust the pH to 1.9, and the mixture was stirred for 1 hour. The insoluble matter was filtered off through a Celite pad, and the Celite pad was washed with distilled water (20 mL) to give a solution of (S)-2-cyclopentyl-2-(methylamino)acetic acid (120 g).

[0134] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. The retention time was 1.5 minutes, m / z = 158.1 (M+H) ＋ The peak was confirmed, and it was confirmed that the desired (S)-2-cyclopentyl-2-(methylamino)acetic acid solution was obtained.

[0135] A 10 M aqueous solution of sodium hydroxide (10 mL) was added to a solution of (S)-2-cyclopentyl-2-(methylamino)acetic acid (60 g, 12 mmol) and the pH was adjusted to 12.0. The resulting solution was concentrated to dryness under reduced pressure at an external temperature of 40°C. Distilled water (30 mL) was added, and concentrated hydrochloric acid (0.75 mL) was added to adjust the pH to 9.7. Acetonitrile (10 mL), N-carbobenzoxysuccinimide (4.2 g, 17 mmol), and methyl tert-butyl ether (10 mL) were added and stirred for 1 hour. A 50 w / v% aqueous solution of sodium hydroxide (1.5 mL) was added and the mixture was stirred for 3 hours. N-carbobenzoxysuccinimide (1.2 g, 4.8 mmol) was added and the mixture was stirred for 90 minutes. A 50 w / v% aqueous solution of sodium hydroxide (0.5 mL) was added and the mixture was stirred for 18 hours. Concentrated hydrochloric acid (8.0 mL) was added, and the target product was extracted into the organic layer. The resulting organic layer was concentrated to dryness under reduced pressure at an external temperature of 40°C in the reaction vessel. Methyl tert-butyl ether (15 mL) was added, and the organic layer was washed twice with 5% aqueous disodium hydrogen phosphate (10 mL x 2) and once with 2M aqueous sodium hydroxide (10 mL), and the target product was extracted into the aqueous layer. The resulting aqueous layers were combined, and methyl tert-butyl ether (20 mL) and concentrated hydrochloric acid (1.5 mL) were added to re-extract the target product into the organic layer. The resulting organic layer was concentrated to dryness under reduced pressure at an external temperature of 40°C in the reaction vessel. Methyl tert-butyl ether (30 mL) and 1M aqueous sodium hydroxide (30 mL) were added, and the target product was extracted into the aqueous layer. The resulting aqueous layer was washed with methyl tert-butyl ether (30 mL). Toluene (30 mL) and concentrated hydrochloric acid (3.0 mL) were added, and the target product was re-extracted into the organic layer. The resulting organic layer was concentrated to dryness under reduced pressure at an external temperature of 40° C. to give crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (2.8 g).

[0136] Synthesis Example 2: Synthesis of crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid

[0137] 2-Cyclopentyl-2-oxo-acetic acid sodium salt (5.3 g, 33 mmol), methylamine hydrochloride (11 g, 160 mmol), D-glucose (12 g, 65 mmol), and N,N-di(2-hydroxyethyl)glycine (11 g, 65 mmol) were dissolved in distilled water (69 mL), and the external temperature of the reaction vessel was set to 25 °C. A 50 w / v% aqueous sodium hydroxide solution (4.8 mL) was added, and the pH was adjusted to 9.2. NADP+ (0.25 g, 0.33 mmol), D-glucose dehydrogenase (26 mg, 1300 U, 50 U / mg), and NMAADH modified enzyme solution (final preparation, 2.0 mL, equivalent to 53 mg of enzyme, 1.0% by mass) were added, and the reaction vessel was stirred at an external temperature of 25 °C for 21 hours (reaction conversion rate of 99% or more). Concentrated hydrochloric acid (7.5 mL) was added, and the pH was adjusted to 1.9, and the mixture was stirred for 1 hour. The insoluble matter was filtered off through a Celite pad, and the Celite pad was washed with distilled water (11 mL) to give a solution of (S)-2-cyclopentyl-2-(methylamino)acetic acid (130 g).

[0138] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. The retention time was 1.4 minutes, m / z = 158.1 (M+H) ＋ The peak was confirmed, and it was confirmed that the desired (S)-2-cyclopentyl-2-(methylamino)acetic acid solution was obtained.

[0139] A 50 w / v% aqueous sodium hydroxide solution (3.2 mL) was added to the (S)-2-cyclopentyl-2-(methylamino)acetic acid solution (27 g, equivalent to 6.5 mmol), and the pH was adjusted to 12.3. The resulting solution was concentrated to dryness at an external temperature of 40°C. Distilled water (24 mL) and concentrated hydrochloric acid (0.53 mL) were added to the reaction vessel at an external temperature of 25°C, and the pH was adjusted to 9.2. Methyl tert-butyl ether (5.3 mL), acetonitrile (2.7 mL), and N-carbobenzoxysuccinimide (1.8 g, 7.2 mmol) were added, and the mixture was stirred for 105 minutes. Acetonitrile (1.6 mL) was added, and the mixture was stirred for an additional 30 minutes. A 50 w / v% aqueous sodium hydroxide solution (0.8 mL) was added, and the mixture was adjusted to pH 9.3, and the mixture was stirred for 105 minutes. N-carbobenzoxysuccinimide (1.0 g, 3.9 mmol) was added, and the mixture was stirred for 105 minutes. After adjusting the pH to 9.2 with 50 w / v% aqueous sodium hydroxide (0.5 mL), N-carbobenzoxysuccinimide (1.1 g, 4.6 mmol) was added and the mixture was stirred for 13 hours. After adjusting the pH to 0.4 with concentrated hydrochloric acid (4.3 mL), methyl tert-butyl ether (3.0 mL) was added, and the target substance was extracted into the organic layer. Methyl tert-butyl ether (5.3 mL) was added to the resulting aqueous layer, and the target substance was extracted into the organic layer. The resulting organic layers were combined and made basic by the addition of 2 M aqueous sodium hydroxide (11 mL), and methyl tert-butyl ether (5.3 mL) was added to extract the target substance into the aqueous layer. The aqueous layer was washed with methyl tert-butyl ether (11 mL). Methyl tert-butyl ether (11 mL) and concentrated hydrochloric acid (2.1 mL) were added to the aqueous layer to adjust the pH to 0.0, and the target substance was extracted into the organic layer to obtain a solution of crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (14 g).

[0140] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. The retention time was 3.9 minutes, m / z = 292.1 (M+H). ＋ The peak was confirmed, confirming that the target crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid was obtained.

[0141] Synthesis Example 3: Synthesis of (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt seed crystals (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (1.0 g, 3.4 mmol, commercially available) was dissolved in tetrahydrofuran (5.0 ml, 5.0 v / w) and stirred at an external temperature of 25°C in a reaction vessel. Lithium tert-butoxide (0.30 g, 3.8 mmol) was added, and precipitation of crystals was confirmed. Tetrahydrofuran (20 ml, 20 v / w) was added, and the mixture was stirred for 1 hour. The crystals were filtered and washed with tetrahydrofuran (5.0 ml, 5.0 v / w). The resulting crystals were dried under reduced pressure at an external temperature of 40°C in a reaction vessel for 3 hours to obtain (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt (0.90 g, yield 88%).

[0142] Comparative Example 1 and Example 1: Purification of Amino Acid Derivatives in Free Form and in Lithium Salt Purification of amino acid derivatives in free form (carboxylic acid) and in lithium salt was compared. Amino acid derivatives containing impurities were isolated as free forms or lithium salts, and the apparent purities were compared. The apparent purity or apparent impurity content was calculated from the UV absorption peak area ratio by high performance liquid chromatography. The apparent purity was calculated using Equation 1b, and the apparent impurity content was calculated using Equation 2b.

[0143] Comparative Example 1: Purification of (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (free form) The crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid obtained in Synthesis Example 1 (1.4 g, corresponding to a theoretical yield of 1.8 g, 6.0 mmol as (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid) was dissolved in toluene (3.0 mL, 1.7 v / w), and the mixture was stirred using a stirring blade at an external temperature of 25°C in a reaction vessel. Heptane (1.0 mL, 0.6 v / w) and seed crystals (crystals of (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (free form), commercially available product, 5.0 mg) were added, and the mixture was stirred for 15 minutes, and precipitation of crystals was confirmed. Heptane (5.5 mL, 3.1 v / w) was added dropwise over 1 hour, and the mixture was stirred for an additional 30 minutes. The mixture was stirred for an additional hour at an external temperature of 0°C. The crystals were filtered and washed with heptane / toluene (v / v) = 4 / 1 (4.0 mL, 2.3 v / w). The crystals were dried under reduced pressure at an external temperature of 40°C for 1 hour to obtain (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (1.3 g, yield 73% (calculated based on 2-cyclopentyl-2-oxo-acetic acid sodium salt of Synthesis Example 1)).

[0144] The structure was confirmed by mass spectrometry (MS(ESI)) using high performance liquid chromatography condition 3. The m / z value of Cbz-β-Ala-OH, one of the impurities, was 224.1 (M+H) at a retention time of 2.6 minutes. ＋ A peak of m / z=292.1 (M+H) belonging to Cbz-MeGly(cPent)-OH was confirmed at a retention time of 3.9 minutes. ＋A peak of the formula was confirmed. The UV absorption peak areas of Cbz-MeGly(cPent)-OH and Cbz-β-Ala-OH were measured at a measurement wavelength of 210 nm, and the apparent purity and apparent impurity content were calculated using the above-mentioned formulas 1b and 2b. The calculations of the apparent purity and apparent impurity content were performed for both the crude (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (before purification) obtained in Synthesis Example 1 and the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (after purification) obtained in Comparative Example 1. The results are shown in Table 3.

[0145] Example 1-1: Purification of (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid with lithium salt

[0146] (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (500 mg, 1.7 mmol) obtained in Comparative Example 1 was dissolved in acetonitrile (2.5 ml, 5.0 v / w) and stirred using a stirring blade at an external temperature of 25°C. A 4 M aqueous solution of lithium hydroxide (0.47 ml, 1.9 mmol) and distilled water (0.50 ml, 1.0 v / w) were added and the reaction vessel was stirred at an external temperature of 40°C for 20 minutes. Acetonitrile (5.0 ml, 10 v / w) was added over 10 minutes, and seed crystals (synthesized according to Synthesis Example 3, 5.0 mg) were added and the mixture was stirred for 40 minutes, confirming the precipitation of crystals. The external temperature of the reaction vessel was set to 25°C and the mixture was stirred for 3 hours. The external temperature of the reaction vessel was set to 0°C and the mixture was stirred for 30 minutes, after which acetonitrile (2.5 ml, 5.0 v / w) was added over 5 minutes and the mixture was stirred for an additional 30 minutes. Acetonitrile (5.0 ml, 10 v / w) was added over 5 minutes, and the mixture was stirred for 30 minutes. The crystals were filtered, and the resulting crystals were washed with distilled water / acetonitrile (v / v) = 5 / 95 (2.0 mL, 4.0 v / w). The crystals were dried under reduced pressure at an external temperature of 40°C for 3 hours to obtain lithium (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid salt (360 mg, yield 71%, calculated based on the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid obtained in Comparative Example 1).

[0147] Mass spectrometry (MS(ESI)) and apparent purity analysis based thereon were performed using high-performance liquid chromatography condition 3. A peak corresponding to Cbz-β-Ala-OH, one of the impurities, was confirmed at a retention time of 2.6 minutes. A peak corresponding to Cbz-MeGly(cPent)-OH (m / z=292.1 (M+H)) was observed at a retention time of 3.9 minutes. ＋ The peaks were confirmed. The UV absorption peak areas of Cbz-MeGly(cPent)-OH and Cbz-β-Ala-OH were measured at a measurement wavelength of 210 nm, and the apparent purity and apparent impurity content were calculated using Equation 1b and Equation 2b. The calculations of the apparent purity and apparent impurity content were performed for both the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (before purification) obtained in Comparative Example 1 and the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt (after purification) obtained in Example 1-1. The results are shown in Table 4.

[0148] Example 1-2: Purification with (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt

[0149] The (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentylacetic acid crude product solution obtained in Synthesis Example 2 (7.0 g, corresponding to a theoretical yield of 0.95 g, 3.3 mmol as (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentylacetic acid) was concentrated to dryness at an external temperature of 40°C. Acetonitrile (3.2 mL, 3.4 v / w) was added, and the mixture was stirred using a stirring blade at an external temperature of 25°C. Distilled water (0.53 mL, 0.6 v / w) and a 4 M aqueous lithium hydroxide solution (0.90 mL, 3.6 mmol) were added, and the mixture was stirred for 20 minutes. Acetonitrile (2.7 mL, 2.8 v / w) was added over 10 minutes, and seed crystals (synthesized according to Synthesis Example 3, 5.0 mg) were added, followed by stirring for 15 minutes. Acetonitrile (5.3 mL, 5.6 v / w) was added over 20 minutes, and the mixture was stirred for 17 hours. Acetonitrile (5.3 mL, 5.6 v / w) was added over 30 minutes, and the mixture was stirred for 1 hour. Acetonitrile (5.3 mL, 5.6 v / w) was added over 30 minutes, and the mixture was stirred for 1 hour. The crystals were filtered, washed with distilled water / acetonitrile (v / v) = 5 / 95 (3.2 mL, 3.4 v / w), and dried under reduced pressure for 2.5 hours at an external temperature of 40°C in the reaction vessel to obtain (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt (yield: 620 mg, yield: 64%, calculated based on 2-cyclopentyl-2-oxo-acetic acid sodium salt in Synthesis Example 2).

[0150] Mass spectrometry (MS(ESI)) and apparent purity analysis based thereon were performed using high-performance liquid chromatography condition 1. A peak corresponding to Cbz-β-Ala-OH, one of the impurities, was observed at a retention time of 2.8 minutes. A peak corresponding to Cbz-MeGly(cPent)-OH (m / z=292.1 (M+H)) was observed at a retention time of 3.9 minutes. ＋A peak of the formula was confirmed. The UV absorption peak areas of Cbz-MeGly(cPent)-OH and Cbz-β-Ala-OH were measured at a measurement wavelength of 210 nm, and the apparent purity and apparent impurity content were calculated using the above-mentioned formula 1b and formula 2b. The calculations of the apparent purity and apparent impurity content were performed for both the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid crude product solution (before purification) obtained in Synthesis Example 2 and the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt (after purification) obtained in Example 1-2. The results are shown in Table 5.

[0151] Chiral analysis was carried out using high performance liquid chromatography conditions 4. The measurement wavelength was 210 nm. Analysis of a standard sample (purchased from Amatek) confirmed that the retention times for the (S) and (R) isomers were 16.7 minutes for the (S) isomer and 17.3 minutes for the (R) isomer. As a result of the chiral analysis, no (R) isomer was detected in the (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid lithium salt (purified) obtained in Example 1-2, confirming that the optical purity was 99.9% ee or higher.

[0152] Example 2: Purification of amino acid derivatives by lithium chloride The purification effect of amino acid derivatives by lithium chloride was evaluated. 3-aminopropanoic acid (β-alanine) having a specific protecting group was added as an impurity to an amino acid derivative having the same protecting group to prepare a mixture. The mixture was isolated and purified by lithium chloride, and the purification effect was verified by comparing the apparent purity of the mixture and the isolated compound. The apparent impurity content was calculated using the same method as described in Example 1.

[0153] Example 2-1: Synthesis of (R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propanoic acid lithium salt

[0154] (R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propanoic acid (500 mg, 1.3 mmol), 3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (20 mg, 0.065 mmol), tetrahydrofuran (18 ml, 36 v / w), and distilled water (2.0 ml, 4.0 v / w) were added to the reaction vessel and stirred at an external temperature of 25 °C using a stirring blade. 4 M aqueous lithium hydroxide solution (0.081 ml, 0.32 mmol) was added over 3 minutes and stirred for 10 minutes. 4 M aqueous lithium hydroxide solution (0.081 ml, 0.32 mmol) was added over 3 minutes and stirred for 1 hour, and crystal precipitation was confirmed. Furthermore, 4 M aqueous lithium hydroxide solution (0.081 ml, 0.32 mmol) was added over 3 minutes and stirred for 1 hour. This operation was repeated twice. The crystals were filtered, washed with distilled water / tetrahydrofuran (v / v) = 10 / 90 (5.0 mL, 10 v / w), and dried under reduced pressure at an external temperature of 40°C for 2 hours to obtain lithium (R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoate (430 mg, yield 84%).

[0155] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. At a retention time of 4.2 minutes, Fmoc-β-Phg-OH had an m / z of 388.2 (M+H). ＋ A peak was observed. Apparent purity analysis was performed using high performance liquid chromatography condition 2. A peak for the impurity Fmoc-β-Ala-OH was observed at a retention time of 2.4 minutes. A peak for Fmoc-β-Phg-OH was observed at a retention time of 2.8 minutes. The UV absorption peak areas of Fmoc-β-Phg-OH and Fmoc-β-Ala-OH were measured at a measurement wavelength of 254 nm, and the apparent purity and apparent impurity content were calculated using the above formulas 1b and 2b. The calculations of the apparent purity and apparent impurity content were performed before and after Example 2-1 (before and after purification). The results are shown in Table 6.

[0156] Example 2-2: Synthesis of (S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid lithium salt

[0157] (S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid (500 mg, 1.5 mmol) and 3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (23 mg, 0.074 mmol) were added to tetrahydrofuran (6.8 ml, 14 v / w) and distilled water (0.75 ml, 1.5 v / w), and the mixture was stirred using a stirring blade at an external temperature of 25°C in a reaction vessel. 4 M aqueous lithium hydroxide solution (0.37 ml, 1.5 mmol) was added over 2 minutes and stirred for 1 hour. Tetrahydrofuran (5.0 ml, 10 v / w) was added over 10 minutes and stirred for 20 minutes, and crystal precipitation was confirmed. Tetrahydrofuran (7.5 ml, 15 v / w) was added over 15 minutes and stirred for 20 minutes. Tetrahydrofuran (5.0 ml, 10 v / w) was added over 10 minutes and stirred for 40 minutes. The crystals were filtered, washed with distilled water / tetrahydrofuran (v / v) = 5 / 95 (7.5 mL, 15 v / w), and dried under reduced pressure at an external temperature of 40°C for 2 hours to obtain lithium (S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylate (400 mg, yield 79%).

[0158] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. The retention time was 3.9 minutes, and the m / z of Fmoc-Pro-OH was 338.1 (M+H). ＋ A peak was observed. Apparent purity analysis was performed using high performance liquid chromatography condition 2. A peak for the impurity Fmoc-β-Ala-OH was observed at a retention time of 2.4 minutes. A peak for Fmoc-Pro-OH was observed at a retention time of 2.6 minutes. The UV absorption peak areas of Fmoc-Pro-OH and Fmoc-β-Ala-OH were measured at a measurement wavelength of 254 nm, and the apparent purity and apparent impurity content were calculated using the above formulas 1b and 2b. The calculations of the apparent purity and apparent impurity content were performed before and after Example 2-2 (before and after purification). The results are shown in Table 7.

[0159] Example 2-3: Synthesis of (S)-2-(benzyloxycarbonyl(methyl)amino)-3-phenylpropanoic acid lithium salt

[0160] Acetonitrile (6.8 ml, 14 v / w) and distilled water (0.75 ml, 1.5 v / w) were added to (S)-2-(benzyloxycarbonyl(methyl)amino)-3-phenylpropanoic acid (500 mg, 1.6 mmol) and 3-(benzyloxycarbonylamino)propanoic acid (18 mg, 0.080 mmol), and the mixture was stirred using a stirring blade at an external temperature of 25 °C in a reaction vessel. 4 M aqueous lithium hydroxide solution (0.44 ml, 1.8 mmol) was added over 2 minutes, stirred for 10 minutes, and the precipitation of crystals was confirmed. Acetonitrile (13 ml, 25 v / w) was added over 30 minutes and stirred for 90 minutes. The crystals were filtered and washed with distilled water / acetonitrile (v / v) = 5 / 95 (5.0 mL, 10.0 v / w). The reaction vessel was dried under reduced pressure for 3 hours at an external temperature of 40° C. to obtain lithium (S)-2-(benzyloxycarbonyl(methyl)amino)-3-phenylpropanoate (460 mg, yield 90%).

[0161] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. The retention time was 3.9 minutes, and the m / z of Cbz-MePhe-OH was 314.1 (M+H). ＋ A peak was observed. Apparent purity analysis was performed using high performance liquid chromatography condition 2. A peak for the impurity Cbz-β-Ala-OH was observed at a retention time of 1.6 minutes. A peak for Cbz-MePhe-OH was observed at a retention time of 2.5 minutes. The UV absorption peak areas of Cbz-MePhe-OH and Cbz-β-Ala-OH were measured at a measurement wavelength of 210 nm, and the apparent purity and apparent impurity content were calculated using Equation 1b and Equation 2b. The calculations of the apparent purity and apparent impurity content were performed before and after Example 2-3 (before and after purification). The results are shown in Table 8.

[0162] Example 2-4: Synthesis of (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid lithium salt

[0163] Acetonitrile (9.8 ml, 20 v / w) and distilled water (0.25 ml, 0.5 v / w) were added to (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid (500 mg, 1.9 mmol) and 3-(tert-butoxycarbonylamino)propanoic acid (18 mg, 0.094 mmol), and the mixture was stirred using a stirring blade at an external temperature of 25°C in a reaction vessel. A 1M solution of lithium tert-butoxide in tetrahydrofuran (1.0 ml, 1.0 mmol) was added over 5 minutes and the mixture was stirred for 30 minutes. A 1M solution of lithium tert-butoxide in tetrahydrofuran (1.0 ml, 1.0 mmol) was then added over 5 minutes and the mixture was stirred for 30 minutes. Acetonitrile (10 ml, 20 v / w) was added over 1 hour, and the mixture was stirred for 2 hours. The crystals were filtered, washed with distilled water / acetonitrile (v / v) = 1 / 99 (5.0 mL, 10 v / w), and dried under reduced pressure at an external temperature of 40°C for 2 hours to obtain lithium (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoate (360 mg, yield 71%).

[0164] Mass spectrometry (MS(ESI)) was performed using high performance liquid chromatography condition 1. The m / z of Boc-Phe-OH was 166.0 (M-Boc) at a retention time of 3.5 minutes. ＋ A peak of Boc-β-Ala-OH was observed at a retention time of 2.5 minutes. A peak of Boc-Phe-OH was observed at a retention time of 3.5 minutes. The UV absorption peak areas of Boc-Phe-OH and Boc-β-Ala-OH were measured at a measurement wavelength of 197 nm, and the apparent purity and apparent impurity content were calculated using Equation 1b and Equation 2b. The calculations of the apparent purity and apparent impurity content were performed before and after Example 2-4 (before and after purification). The results are shown in Table 9.

Claims

1. A method for producing an amino acid salt or a peptide compound salt or a solvate thereof, comprising the following steps (A) and (B): Step (A): A step of contacting a lithium-containing substance with a product to be purified, which is a mixture of the following (i), which is the product to be purified, and the following (ii), which is an impurity. (i) The amino acid having a protecting group at the N-terminus or the peptide compound having a protecting group at the N-terminus (ii) Compounds other than the product to be purified, Step (B): A step of precipitating the lithium salt of the product to be purified.

2. A method for removing impurities from a product to be purified, comprising the following steps (A) and (B): Step (A): A step of contacting a lithium-containing substance with a product to be purified, which is a mixture of the following (i), which is the product to be purified, and the following (ii), which is an impurity. (i) Amino acids or peptide compounds having a protecting group at the N-terminus (ii) Compounds other than the product to be purified, Step (B): A step of precipitating the lithium salt of the product to be purified.

3. The method according to claim 1 or 2, wherein the protecting group at the N-terminus of the product to be purified is a carbamate protecting group.

4. The method according to claim 1 or 2, wherein the product to be purified has a protecting group consisting of a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group.

5. The method according to claim 1 or 2, wherein the impurity is an amino acid having a protecting group at its N-terminus or a peptide compound having a protecting group at its N-terminus, other than the product to be purified.

6. The method according to claim 1 or 2, wherein the impurity is β-alanine having a protecting group at its N-terminus.

7. The method according to claim 1 or 2, wherein the lithium salt of the product to be purified is formed in step (A) above.

8. The method according to claim 1 or 2, wherein in step (B), the lithium salt or solvate thereof of the product to be purified is precipitated as a solid.

9. The method according to claim 1 or 2, wherein in step (B), the lithium salt or solvate thereof of the product to be purified is precipitated as crystals.

10. The method according to claim 1 or 2, wherein step (A) is carried out in the presence of a first organic solvent.

11. The method according to claim 1 or 2, wherein step (B) is carried out in the presence of a second organic solvent.

12. The method according to claim 1 or 2, wherein step (B) is carried out in the presence of water.

13. The method according to claim 1 or 2, wherein the N-terminal amino acid constituting the amino acid or peptide compound is represented by the following general formula (1). 【Chemistry 1】 [In the above general formula (1), n represents a number between 1 and 3, R １ When there are multiple instances, R １ They may be the same or different from each other. R １ and R ２ represents a substituent consisting of two or more atoms selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may have substituents, and these groups may be saturated hydrocarbon groups or unsaturated hydrocarbon groups. A represents a carbon atom, B indicates a bond site with a hydrogen atom or an amino acid or peptide compound. Z represents a fluorenyl methoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group. R １ and R ２ R may be bonded together with N and A to form a ring, in which case R １ and R ２ This substituent has a structure in which one hydrogen atom has been removed from the substituent structure when it is not involved in ring formation. When n is 2, R ２ and two R's that exist １ any one or more of which is not a hydrogen atom.]]

14. The method according to claim 1 or 2, wherein the purity of the precipitate obtained in step (B) is 95 mol% or more.

15. Lithium salts of amino acids or peptide compounds, or solvates thereof, The lithium salt of the aforementioned amino acid contains a lithium salt of an amino acid represented by the following general formula (1) in a purity of 99 mol% or more or an apparent purity of 99% or more. The lithium salt of the peptide compound contains a lithium salt of an amino acid or a lithium salt of a peptide compound or a solvate thereof, with a purity of 99 mol% or more or an apparent purity of 99% or more of a lithium salt of a peptide compound having an amino acid residue represented by the following general formula (1). 【Chemistry 2】 [In the above general formula (1), n represents a number between 1 and 3, R １ When there are multiple instances, R １ They may be the same or different from each other. R １ and R ２ represents a substituent consisting of two or more atoms selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, and these groups may have substituents, and these groups may be saturated hydrocarbon groups or unsaturated hydrocarbon groups. A represents a carbon atom, B indicates a bond site with a hydrogen atom or an amino acid or peptide compound. Z represents a fluorenyl methoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group. R １ and R ２ R may be bonded together with N and A to form a ring, in which case R １ and R ２ This substituent has a structure in which one hydrogen atom has been removed from the substituent structure when it is not involved in ring formation. If n is 2, R ２ and the two R １ At least one of them is not a hydrogen atom.

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