Method for producing amino acid and peptide compound, using enzyme and additive

Abstract

One aspect of the present invention pertains to a method for producing an amino acid, the method comprising a step for conducting a reaction of (i) or (ii), in the presence of a compound D represented by formula (1), a reducing agent, and a polypeptide having an amino acid sequence that has an identity of 90% or more with respect to the amino acid sequence represented by SEQ ID NO. 1.　(i) An intermolecular reductive amination reaction between: a compound A selected from the group consisting of compounds having an amino group and salts thereof; and a compound B selected from the group consisting of compounds having a carbonyl group and salts thereof. (ii) An intramolecular reductive amination reaction of a compound C selected from the group consisting of compounds having an amino group and a carbonyl group, and salts thereof.　

Description

Method for producing amino acids and peptide compounds using enzymes and additives

[0001] The present invention relates to a method for producing amino acids and peptide compounds using enzymes and additives.

[0002] Methods for synthesizing amino acids from amines and keto acids using enzymes that promote reductive amination reactions (hereinafter referred to as reductive amination enzymes) have been known. For example, Patent Documents 1 to 4 disclose techniques for synthesizing various amino acids using reductive amination enzymes.

[0003] JP 2003-319788 A JP 2005-095167 A JP 2012-080878 A JP 2012-080879 A

[0004] However, the reaction efficiency of the reaction to obtain amino acids using reductive amination enzymes is not necessarily sufficient, and improvement has been desired.

[0005] An object of the present invention is to provide a method for efficiently producing amino acids.

[0006] The present invention relates to, for example, the following inventions: [1] A method for producing an amino acid, comprising the step of carrying out the following reaction (i) or (ii) in the presence of a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1, a reducing agent, and a compound D represented by the following formula (1): (i) an intermolecular reductive amination reaction between compound A selected from the group consisting of compounds having an amino group and salts thereof, and compound B selected from the group consisting of compounds having a carbonyl group and salts thereof, or (ii) an intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof. [In formula (1), v and w each independently represent 0 or 1, at least one of v and w represents 1, T represents a carbon atom, a phosphorus atom, or a sulfur atom, and a compound represented by the following formula (1a): The functional group represented by the formula (1a) represents ═O, —ORd, or a hydroxy group; when v and w are both 1, two functional groups represented by the formula (1a) may be the same or different; Ra, Rb, and Rc each independently represent a hydrogen atom, C １ ~C ３ Alkyl group, alkylamino group or -CH ２ any two or more of Ra, Rb, and Rc may be bonded to each other together with T to form a ring structure, and Rd represents C １ ~C ３represents an alkyl group, d, e, and f each independently represent 0 or 1, any one or more of d, e, and f represent 1, when v and w are both 1, any one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc do not combine with each other to form a ring structure with T, when any one or more of Ra, Rb, and Rc are methylamino groups, Ra, Rb, and Rc do not combine with each other to form a ring structure with T, when the functional group represented by formula (1a) is a hydroxy group and T is a carbon atom, v is 1, w is 0, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.] [2] The production method according to [1], wherein the polypeptide comprises an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1. [3] The production method according to [1] or [2], wherein the polypeptide comprises an amino acid sequence having 98% or more identity with the amino acid sequence represented by SEQ ID NO: 1. [4] The method according to any one of [1] to [3], wherein in formula (1), T is a phosphorus atom or a sulfur atom, the functional group represented by formula (1a) is ═O, and Ra, Rb, and Rc are all methyl groups. [5] The method according to any one of [1] to [3], wherein compound D is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, dimethoxyethane, trimethylphosphine oxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide. [6] The method according to any one of [1] to [5], wherein compound D is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide. [7] The method according to any one of [1] to [6], wherein compound D is dimethyl sulfoxide.[8] A method for producing an amino acid, comprising the steps of: carrying out an intermolecular reductive amination reaction between compound A selected from the group consisting of compounds having an amino group and salts thereof and compound B selected from the group consisting of compounds having a carbonyl group and salts thereof; or an intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof; in the presence of a polypeptide having catalytic activity under at least one reaction condition for an intermolecular reductive amination reaction between compound A and compound B; or an intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof; and compound D'; wherein compound D' is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethylphosphine oxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide. [9] The method for producing an amino acid according to [8], wherein compound D' is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide.

[10] The method according to [8] or [9], wherein the compound D' is dimethyl sulfoxide.

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

[10] , wherein the reducing agent is one or more compounds selected from the group consisting of reduced nicotinamide adenine dinucleotide phosphate (NADPH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide (NADH), and oxidized nicotinamide adenine dinucleotide (NAD+).

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

[11] , wherein the reducing agent is reduced nicotinamide adenine dinucleotide phosphate.

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

[12] , wherein the polypeptide has catalytic activity for the intermolecular reductive amination reaction of compound A with compound B or the intramolecular reductive amination reaction of compound C under at least one reaction condition.

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

[13] , 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.

[15] The method for producing the polypeptide according to any one of [1] to

[14] , wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 8, where the amino acid residue represented by X is a histidine residue.

[16] The method for producing the polypeptide according to any one of [1] to

[15] , 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 with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified.

[17] The method for producing the polypeptide according to any one of [1] to

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

[18] The method for producing the polypeptide according to any one of [1] to

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

[19] The method for producing the polypeptide according to any one of [1] to

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

[20] The production method according to any one of [1] to

[19] , wherein the step is a step of carrying out an intermolecular reductive amination reaction between the compound A and the compound B.

[21] The production method according to

[20] , wherein the compound A is one or more compounds selected from the group consisting of compounds represented by the following formula (2) and salts thereof: [In formula (2), R １ and R ２ each independently 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, and these groups may be substituted; R １ or R ２ At least one of R is a hydrogen atom. １ is a hydrogen atom, and R ２

[23] The method according to

[21] , wherein R １ is a hydrogen atom, and R ２is a hydrogen atom, a methyl group, or an ethyl group.

[24] The method according to any one of

[20] to

[23] , wherein compound B is one or more compounds selected from the group consisting of compounds represented by the following formula (3) and salts thereof: [In formula (3), X represents a carbon atom, Y represents a hydrogen atom or a group represented by formula (4), n represents an integer of 0 to 2, and R ６ represents a hydrogen atom, an optionally substituted C １ ~C ６ alkyl group, optionally substituted C １ ~C ６ an aryl group, a heteroaryl group having 5 to 12 atoms constituting a ring which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom, and in formula (4), represents a bonding point with X, m represents an integer of 0 to 6, p is 0 or 1, q is 0 or 1, r is 0 or 1, and Z １ represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms; when m is an integer of 2 or more, a plurality of Z １ may be the same or different, Z ２ represents a carbon atom, and R ３ , R ４ and R ５ are each independently a hydrogen atom, an optionally substituted C １ ~C ６ alkyl group, optionally substituted C ５ ~C １２ R represents an aryl group, a heteroaryl group having 5 to 12 atoms constituting a ring that may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom; ３ , R ４ and R ５ Any two or more of these are connected to each other to form Z ２ and R may form a ring structure together, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted; ３ , R ４ , and R５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２ When n is 0 and R is bonded to R, at least one of p, q, and r is 0.]

[25] In the formula (3), ６ is a hydrogen atom, Y is C ３ ~C ８ a cycloalkyl group, or C ６ ~C ９ The aralkyl group is C １ ~C ３

[26] The method according to

[24] , wherein n is 0, R ６ is a hydrogen atom, and Y is a (2-chlorophenyl)ethyl group, a phenylmethyl group, or a cyclopentyl group. １ is a hydrogen atom, and R ２ is a hydrogen atom or a methyl group, in the formula (3), n and m are both 0, Y is a group represented by the formula (4), and R ３ is a (2-chlorophenyl)methyl group, and R ４ , R ５ and R ６

[28] The method according to any one of

[24] to

[26] , wherein each of R is a hydrogen atom, and p, q, and r are each 1. １ is a hydrogen atom, and R ２ is an ethyl group, in the formula (3), n and m are both 0, Y is a group represented by the formula (4), and R ３ is a phenyl group, and R ４ , R ５ and R ６

[29] The method according to any one of

[24] to

[26] , wherein each of R is a hydrogen atom, and p, q, and r are each 1. １ is a hydrogen atom, and R ２is a methyl group, in the formula (3), n and m are both 0, Y is a group represented by the formula (4), and R ３ and R ４ are connected to each other to form Z ２ together with R to form a cyclopentane ring, ５ and R ６

[30] The method according to any one of

[24] to

[26] , wherein each of R is a hydrogen atom, and p, q, and r are each 1. ３ , R ４ and R ５

[31] The production method according to any one of [1] to

[20] , wherein compound A is one or more compounds selected from the group consisting of ammonia, methylamine, ethylamine, and salts thereof.

[32] The production method according to any one of [1] to

[20] or

[31] , wherein compound B is one or more compounds selected from the group consisting of 4-(2-chlorophenyl)-2-oxobutanoic acid, phenylpyruvic acid, 2-cyclopentyl-2-oxo-acetic acid, and salts thereof.

[33] The production method according to any one of [1] to

[20] or

[31] or

[32] , wherein compound A is ammonia or a salt thereof, or methylamine or a salt thereof, and compound B is 4-(2-chlorophenyl)-2-oxobutanoic acid or a salt thereof, or phenylpyruvic acid or a salt thereof.

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

[20] , or

[31] or

[32] , wherein compound A is ethylamine or a salt thereof, and compound B is phenylpyruvic acid or a salt thereof.

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

[20] , or

[31] or

[32] , wherein compound A is methylamine or a salt thereof, and compound B is 2-cyclopentyl-2-oxo-acetic acid or a salt thereof.

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

[35] , wherein in the step, the concentration of compound A in the total amount of reaction solution at the start of the reaction is 100 mM or more and 3000 mM or less.

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

[36] , wherein in the step, the concentration of compound B in the total amount of reaction solution at the start of the reaction is 0.001 mM or more and 1000 mM or less.

[38] The production method according to any one of [1] to

[37] , wherein the step is carried out under conditions where the ratio of the concentration of compound A to the concentration of compound B in the reaction solution is 1 or more.

[39] The production method according to any one of [1] to

[19] , wherein the step is a step of carrying out an intramolecular reductive amination reaction of compound C.

[40] The production method according to

[39] , wherein compound C is a compound represented by the following formula (5): [In formula (5), n′ represents an integer of 0 to 2, and R７ represents an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, an arylene group, a heterocyclylene group, or a heteroarylene group, which may be substituted; R ８ represents a hydrogen atom, an optionally substituted C １ ~C ６ alkyl group, optionally substituted C １ ~C ６It represents an aryl group, a heteroaryl group having 5 to 12 atoms constituting an optionally substituted ring, a group containing a nitrogen atom, or a group containing an oxygen atom.]

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

[40] , wherein in the step, the concentration of compound C in the total amount of the reaction solution at the start of the reaction is 0.001 mM to 1000 mM.

[42] The production method according to any of [1] to

[41] , wherein in the step, glucose and glucose dehydrogenase are present in the reaction solution.

[43] The production method according to any of [1] to

[42] , wherein the step is carried out under appropriate reaction conditions.

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

[43] , wherein the step is carried out under a temperature condition of 0°C to 50°C.

[45] The production method according to any of [1] to

[44] , wherein the step is carried out under a pH condition of 7 to 11.

[46] The production method according to any one of [1] to

[45] , wherein the step is carried out under conditions where, if Compound D or Compound D' is a liquid at 25°C and 1 atmosphere, the concentration of Compound D or Compound D' in the reaction solution is 1 w / v% to 60 v / v%, or if Compound D or Compound D' is a solid at 25°C and 1 atmosphere, the concentration of Compound D or Compound D' in the reaction solution is 1 w / v% to 60 v / v%.

[47] The production method according to any one of [1] to

[46] , wherein the step is carried out under conditions where the concentration of the polypeptide in the reaction solution is 0.1 μM to 10 μM.

[48] The production method according to any one of [1] to

[47] , wherein the step is carried out under conditions where the concentration of the reducing agent in the reaction solution is 0.1 mM to 100 mM.

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

[48] , wherein the yield of the amino acid produced by the reaction in the presence of compound D or compound D' is 1.2 times or more the yield of the amino acid produced by the reaction in the absence of compound D or compound D'.

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

[49] , wherein the yield of the amino acid produced in the step is 50% or more.

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

[50] , wherein the yield of the amino acid produced in the step is measured under the following conditions: The reaction solution contains 50 mM phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, or 2-cyclopentyl-2-oxo-acetic acid, 100 mM D(+)-glucose, 500 mM ammonia, methylamine, or ethylamine, 100 mM phosphate buffer, 1 mM NADPH, 0.002 unit / μL GDH solution, and 2.5 μM polypeptide at a concentration; when Compound D or Compound D' is used, Compound D or Compound D' is 20 v / v % if Compound D or Compound D' is liquid under conditions of 25°C and 1 atmosphere, or 20 w / v % if Compound D or Compound D' is solid under conditions of 25°C and 1 atmosphere; and the reaction is initiated under conditions of 37°C, pH 8 to 9, and the yield of the amino acid produced by the reductive amination reaction is determined after 19 hours have elapsed.

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

[51] , wherein the catalytic activity of the polypeptide is evaluated under the following conditions: a reaction solution containing 50 mM sodium 4-(2-chlorophenyl)-2-oxobutanoate, sodium phenylpyruvate, or sodium 2-cyclopentyl-2-oxoacetate, 100 mM D(+)-glucose, 500 mM methylamine or ethylamine, 100 mM phosphate buffer, 1 mM NADPH, 0.002 unit / μL GDH solution, 2.5 μM of the polypeptide to be evaluated, and 20 v / v % or 20 w / v % Compound D or Compound D', the reaction is initiated under conditions of 25° C. or 37° C., pH 8 to 9, and the yield of amino acid produced by the reductive amination reaction is determined after 3 hours or 23 hours.

[53] A method for producing a peptide compound, comprising the steps of: (1) A step of producing an amino acid by the production method according to any one of [1] to

[51] ; and (2) a step of linking the amino acid to one or more selected from the group consisting of other amino acids and other peptides to produce a peptide compound.

[54] A reductive amination reaction accelerator represented by the following formula (1), which accelerates the following reaction (i) or (ii): (i) an intermolecular reductive amination reaction between a compound A selected from the group consisting of compounds having an amino group and salts thereof, and a compound B selected from the group consisting of compounds having a carbonyl group and salts thereof, or (ii) an intramolecular reductive amination reaction of a compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof. [In formula (1), v and w each independently represent 0 or 1, at least one of v and w represents 1, T represents a carbon atom, a phosphorus atom, or a sulfur atom, and a compound represented by the following formula (1a): The functional group represented by the formula (1a) represents ═O, —ORd, or a hydroxy group; when v and w are both 1, two functional groups represented by the formula (1a) may be the same or different; Ra, Rb, and Rc each independently represent a hydrogen atom, C １ ~C ３ Alkyl group, alkylamino group or -CH ２ any two or more of Ra, Rb, and Rc may be bonded to each other together with T to form a ring structure, and Rd represents C １ ~C ３represents an alkyl group, d, e, and f each independently represent 0 or 1, any one or more of d, e, and f represent 1, when v and w are both 1, any one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc do not bond to each other to form a ring structure together with T, when any one or more of Ra, Rb, and Rc are methylamino groups, Ra, Rb, and Rc do not bond to each other to form a ring structure together with T, when the functional group represented by formula (1a) is a hydroxy group, and T is a carbon atom, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.]

[55] The reductive amination reaction accelerator according to

[54] , wherein, in formula (1), T is a phosphorus atom or a sulfur atom, the functional group represented by formula (1a) is ═O, and Ra, Rb, and Rc are all methyl groups.

[56] The reductive amination reaction accelerator according to

[54] or

[55] , which is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, dimethoxyethane, trimethylphosphine oxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide.

[57] The reductive amination reaction accelerator according to any of

[54] to

[56] , which is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide.

[58] The reductive amination reaction accelerator according to any of

[54] to

[57] , which is dimethyl sulfoxide.

[0007] According to the present invention, a method for efficiently producing amino acids can be provided.

[0008] FIG. 1 shows the compound (2S,3S)-2-amino-3-phenyl-butanoic acid (I) obtained in the reaction in the examples, the purchased standard compound (2S,3R)-2-amino-3-phenyl-butanoic acid hydrochloride (II), and (2S,3S)-2-amino-3-phenyl-butanoic acid hydrochloride (III). １2 shows the chiral HPLC analysis data of the compound (2S,3S)-2-amino-3-phenyl-butanoic acid (I) obtained in the reaction in the examples, and a mixture (V) prepared by mixing purchased authentic compounds (III) and (IV) in a ratio of (III):(IV)=7:3.

[0009] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0010] 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.

[0011] 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.

[0012] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus and minus 10% of that numerical value.

[0013] In this specification, 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.

[0014] In this specification, the term "alkyl group" 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. The alkyl group includes not only linear ones but also branched ones. The alkyl group preferably has 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 group, more preferably C １ ~C ６ alkyl group, more preferably C １ ~C ３ Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an s-butyl group, a t-butyl group, an isobutyl (2-methylpropyl) group, an n-pentyl group, an s-pentyl (1-methylbutyl) group, a t-pentyl (1,1-dimethylpropyl) group, a neopentyl (2,2-dimethylpropyl) group, an isopentyl (3-methylbutyl) group, a 3-pentyl (1-ethylpropyl) group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, an n-hexyl group, a 1,1,2-trimethylpropyl group, a 1,2,2-trimethylpropyl group, a 1,1,2,2-tetramethylpropyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1-ethylbutyl group, and a 2-ethylbutyl group.

[0015] As used herein, the term "alkenyl group" refers to an alkenyl group having at least one double bond (two adjacent SP ２Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl groups include not only straight chain but also branched chain. The alkenyl group is preferably C ２ ~C １０ Alkenyl groups, more preferably C ２ ~C ６ Examples of alkenyl groups include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, and hexenyl groups.

[0016] As used herein, the term "alkynyl group" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). The alkynyl group includes not only linear but also branched groups. The preferred alkynyl group is C ２ ~C １０ Alkynyl groups, more preferably C ２ ~C ６ Specific examples include alkynyl groups, 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.

[0017] As used herein, the term "cycloalkyl group" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group. The cycloalkyl group includes a monocyclic, bicyclic, or spirocyclic ring. The cycloalkyl group is preferably a C ３ ~C ８ Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and spiro[3.3]heptyl groups.

[0018] In the present specification, the term "aryl group" refers to a monovalent aromatic hydrocarbon ring. The aryl group is preferably C ６ ~C １０ Examples of the aryl group include a phenyl group and a naphthyl group (for example, 1-naphthyl, 2-naphthyl).

[0019] As used herein, the term "heterocyclyl group" refers to a non-aromatic, cyclic, monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclyl group 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 group may be a monocyclic or fused ring. In the case of a fused ring, an aromatic ring such as a benzene ring, a pyridine ring, or a pyrimidine ring may be fused with a saturated alicyclic ring such as a cyclopentane ring or a cyclohexane ring, or with a saturated heterocyclic ring such as a tetrahydropyran ring, a dioxane ring, or a pyrrolidine ring to form a fused ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl group), and more preferably 4 to 7 (4- to 7-membered heterocyclyl group). Specific examples of the heterocyclyl group include an azetidinyl group, an oxiranyl group, an oxetanyl group, an azetidinyl group, a dihydrofuryl group, a tetrahydrofuryl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydropyridyl group, a tetrahydropyrimidyl group, a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a pyrazolidinyl group, an imidazolinyl group, an imidazolidinyl group, an oxazolidinyl group, an isoxazolidinyl group, a thiazolidinyl group, an isothiazolidinyl group, and an isothiazolidinyl group. a zolidinyl group, a 1,2-thiazinane group, a thiadiazolidinyl group, an azetidinyl group, an oxazolidone group, a benzodioxanyl group, a benzoxazolyl group, a dioxolanyl group, a dioxanyl group, a tetrahydropyrrolo[1,2-c]imidazole group, a thietanyl group, a 3,6-diazabicyclo[3.1.1]heptanyl group, a 2,5-diazabicyclo[2.2.1]heptanyl group, a 3-oxa-8-azabicyclo[3.2.1]octanyl group, a sultam group, and a 2-oxaspiro[3.3]heptyl group.

[0020] As used herein, the term "protected heterocyclyl group" refers to a group in which one or more functional groups, such as amino groups, included in the above-defined "heterocyclyl group" are protected with any protecting group. Preferred examples of the protected heterocyclyl include protected 4- to 7-membered heterocyclyl groups. Specific examples of the protecting group include Boc, Fmoc, Cbz, Troc, and Alloc. Specific examples of the protected heterocyclyl include Boc-protected azetidine.

[0021] As used herein, the term "heterocyclidene group" refers to a divalent group formed by removing two hydrogen atoms from one carbon atom present in a non-aromatic ring containing 1 to 5 heteroatoms in addition to carbon atoms. The free valence in the heterocyclidene group may or may not be part of a double bond. The non-aromatic ring containing 1 to 5 heteroatoms in addition to carbon atoms may be the same as the ring described for the "heterocyclyl group." Preferred examples of the heterocyclidene group include 4- to 7-membered heterocyclidene groups, and specific examples include a tetrahydropyran-4-ylidene group and an azetidin-3-ylidene group.

[0022] As used herein, the term "protected heterocyclidene group" refers to a group in which one or more functional groups included in the above-defined "heterocyclidene group," such as an amino group, are protected with any protecting group. Preferred examples of the protected heterocyclidene group include protected 4- to 7-membered heterocyclidene groups. Specific examples of the protecting group include Boc, Fmoc, Cbz, Troc, and Alloc. Specific examples of the protected heterocyclidene group include a Boc-protected azetidin-3-ylidene group.

[0023] As used herein, the term "heteroaryl group" 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 group), 6 to 10 (6- to 10-membered heteroaryl group), or 6 to 7 (6- to 7-membered heteroaryl group). Specific examples of heteroaryl groups 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 groups.

[0024] As used herein, the term "alkoxy group" refers to an oxy group to which the above-defined "alkyl" is bonded. The alkoxy group is preferably C １ ~C ６ Examples of the alkoxy include a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, an n-butoxy group, an i-butoxy group, an s-butoxy group, a t-butoxy group, a pentyloxy group, and a 3-methylbutoxy group.

[0025] As used herein, the term "alkylthio group" refers to a thio group to which the above-defined "alkyl group" is bonded. The alkylthio group is preferably C １ ~C ６ Specific examples of the alkylthio group include a methylthio group, an ethylthio group, a 1-propylthio group, a 2-propylthio group, an n-butylthio group, an i-butylthio group, an s-butylthio group, and a t-butylthio group.

[0026] As used herein, the term "alkenyloxy group" refers to an oxy group to which the above-defined "alkenyl group" is bonded. The alkenyloxy group is preferably C ２ ~C ６ Specific examples of the alkenyloxy group include a vinyloxy group, an allyloxy group, a 1-propenyloxy group, a 2-propenyloxy group, a 1-butenyloxy group, a 2-butenyloxy group (including cis and trans), a 3-butenyloxy group, a pentenyloxy group, and a hexenyloxy group.

[0027] As used herein, the term "cycloalkoxy group" refers to an oxy group to which the above-defined "cycloalkyl group" is bonded. ３ ~C ８ Examples of the cycloalkoxy group include a cyclopropoxy group, a cyclobutoxy group, and a cyclopentyloxy group.

[0028] As used herein, the term "aryloxy group" refers to an oxy group to which the above-defined "aryl group" is bonded. The aryloxy group is preferably C ６ ~C １０ Examples of the aryloxy group include a phenoxy group, a 1-naphthyloxy group, and a 2-naphthyloxy group.

[0029] In the present specification, the term "amino group" means, in a narrow sense, -NH ２ and in a broader sense, -NRR', where R and R' are independently selected from a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, or R and R' together with the nitrogen atom to which they are attached form a ring. ２ , Mono C １ ~C ６ Alkylamino group, diC １ ~C ６Examples include alkylamino groups and 4- to 8-membered cyclic amino groups.

[0030] In the present specification, the term "monoalkylamino group" refers to an "amino group" as defined above, in which R is a hydrogen atom and R' is an "alkyl group" as defined above. The monoalkylamino group is preferably a mono-C １ ~C ６ Alkylamino groups, more preferably mono C １ ~C ３ Specific examples of the monoalkylamino group include a methylamino group, an ethylamino group, an n-propylamino group, an i-propylamino group, an n-butylamino group, an s-butylamino group, and a t-butylamino group.

[0031] As used herein, the term "dialkylamino group" refers to an "amino group" as defined above, in which R and R' are independently an "alkyl group" as defined above. The dialkylamino group is preferably a diC １ ~C ６ Alkylamino groups, more preferably diC １ ~C ３ Examples of dialkylamino groups include alkylamino groups. Specific examples of dialkylamino groups include dimethylamino groups and diethylamino groups. In this specification, monoalkylamino groups and dialkylamino groups may be collectively referred to as "alkylamino groups." For example, the term "methylamino group" may include monomethylamino groups and dimethylamino groups.

[0032] As used herein, the term "cyclic amino group" refers to the above-defined "amino group" in which R and R' form a ring together with the nitrogen atom to which they are bonded. Preferred examples of the cyclic amino group include 4- to 8-membered cyclic amino groups. Specific examples of the cyclic amino group include a 1-azetidyl group, a 1-pyrrolidyl group, a 1-piperidyl group, a 1-piperazyl group, a 4-morpholinyl group, a 3-oxazolidyl group, a 1,1-dioxidethiomorpholinyl-4-yl group, and a 3-oxa-8-azabicyclo[3.2.1]octan-8-yl group.

[0033] As used herein, the term "protected amino group" refers to an amino group protected with any protecting group. Specific examples of the protected amino include amino groups protected with protecting groups such as Boc, Fmoc, Cbz, Troc, and Alloc.

[0034] As used herein, the term "aminocarbonyl group" refers to a carbonyl group to which the above-defined "amino group" is bonded. The aminocarbonyl group is preferably -CONH ２ , Mono C １ ~C ６ Alkylaminocarbonyl group, diC １ ~C ６ Examples of the aminocarbonyl group include an alkylaminocarbonyl group and a 4- to 8-membered cyclic aminocarbonyl group. ２ , dimethylaminocarbonyl group, 1-azetidinylcarbonyl group, 1-pyrrolidinylcarbonyl group, 1-piperidinylcarbonyl group, 1-piperazinylcarbonyl group, 4-morpholinylcarbonyl group, 3-oxazolidinylcarbonyl group, 1,1-dioxidethiomorpholinyl-4-ylcarbonyl group, 3-oxa-8-azabicyclo[3.2.1]octan-8-ylcarbonyl group, and the like.

[0035] As used herein, the term "alkenyloxycarbonyl group" refers to a carbonyl group to which the above-defined "alkenyloxy group" is bonded. The alkenyloxycarbonyl is preferably C ２ ~C ６ Specific examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group, an allyloxycarbonyl group, a 1-propenyloxycarbonyl group, a 2-propenyloxycarbonyl group, a 1-butenyloxycarbonyl group, a 2-butenyloxycarbonyl group (including cis and trans), a 3-butenyloxycarbonyl group, a pentenyloxycarbonyl group, and a hexenyloxycarbonyl group.

[0036] As used herein, the term "alkylsulfonyl group" refers to a sulfonyl group to which the above-defined "alkyl group" is bonded. １ ~C ６ Examples of the alkylsulfonyl group include a methylsulfonyl group.

[0037] The term "hydroxyalkyl group" as used herein means a group in which one or more hydrogen atoms of the "alkyl group" defined above have been substituted with a hydroxyl group. １ ~C ６ Preferred are alkyl groups. Specific examples of hydroxyalkyl groups include a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 2-hydroxy-2-methylpropyl group, and a 5-hydroxypentyl group.

[0038] As used herein, the term "haloalkyl group" refers to a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted with halogen. As the haloalkyl group, haloC １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Specific examples of haloalkyl groups include a difluoromethyl group, a trifluoromethyl group, a 2,2-difluoroethyl group, a 2,2,2-trifluoroethyl group, a 3,3-difluoropropyl group, a 4,4-difluorobutyl group, and a 5,5-difluoropentyl group.

[0039] The term "cyanoalkyl group" as used herein refers to a group in which one or more hydrogen atoms of the above-defined "alkyl group" are substituted with a cyano group. １ ~C ６ An alkyl group is preferred. Specific examples of the cyanoalkyl group include a cyanomethyl group and a 2-cyanoethyl group.

[0040] The term "aminoalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "amino group" as defined above. １ ~C ６ Alkyl groups are preferred. Specific examples of aminoalkyl groups include 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, and 4-aminobutyl groups.

[0041] The term "carboxyalkyl group" as used herein refers to a group in which one or more hydrogen atoms of the "alkyl group" defined above have been substituted with a carboxy group. ２ ~C ６ Carboxyalkyl groups are preferred. Specific examples of carboxyalkyl groups include carboxymethyl groups.

[0042] The term "alkenyloxycarbonylalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkenyloxycarbonyl group" as defined above. ２ ~C ６ Alkenyloxycarbonyl C １ ~C ６ Alkyl groups are preferred, and C ２ ~C ６ Alkenyloxycarbonyl C １ ~C ２ Alkyl groups are more preferred. Specific examples of alkenyloxycarbonylalkyl groups include an allyloxycarbonylmethyl group and a 2-(allyloxycarbonyl)ethyl group.

[0043] The term "alkoxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkoxy group" as defined above. １ ~C ６ Alkoxy C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６Alkoxy C １ ~C ２ Specific examples of the alkoxyalkyl group include a methoxymethyl group, an ethoxymethyl group, a 1-propoxymethyl group, a 2-propoxymethyl group, an n-butoxymethyl group, an i-butoxymethyl group, an s-butoxymethyl group, a t-butoxymethyl group, a pentyloxymethyl group, a 3-methylbutoxymethyl group, a 1-methoxyethyl group, a 2-methoxyethyl group, and a 2-ethoxyethyl group.

[0044] The term "alkylthioalkyl group" as used herein refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkylthio group" as defined above. １ ~C ６ Alkylthio C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkylthio C １ ~C ２ Specific examples of the alkylthioalkyl group include a methylthiomethyl group, an ethylthiomethyl group, a 1-propylthiomethyl group, a 2-propylthiomethyl group, an n-butylthiomethyl group, an i-butylthiomethyl group, an s-butylthiomethyl group, and a t-butylthiomethyl group.

[0045] The term "alkenyloxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkenyloxy group" as defined above. ２ ~C ６ Alkenyloxy C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkenyloxy C １ ~C ２ Alkyl groups are more preferred. Specific examples of alkenyloxyalkyl groups include vinyloxymethyl and allyloxymethyl groups.

[0046] The term "cycloalkylalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "cycloalkyl group" as defined above. ３ ~C ８ Cycloalkyl C １ ~C ６ Alkyl groups are preferred, and C ３ ~C ６ Cycloalkyl C １ ~C ２ An alkyl group is more preferred. Specific examples of the cycloalkylalkyl group include a cyclopropylmethyl group, a cyclobutylmethyl group, a cyclopentylmethyl group, and a cyclohexylmethyl group.

[0047] As used herein, the term "cycloalkoxyalkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "cycloalkoxy group" as defined above. ３ ~C ８ Cycloalkoxy C １ ~C ６ Alkyl groups are preferred, and C ３ ~C ６ Cycloalkoxy C １ ~C ２ An alkyl group is more preferred. Specific examples of the cycloalkoxyalkyl group include a cyclopropoxymethyl group and a cyclobutoxymethyl group.

[0048] As used herein, the term "heterocyclylalkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "heterocyclyl group" as defined above. The heterocyclylalkyl group includes a 4- to 7-membered heterocyclyl C １ ~C ６ Alkyl groups are preferred, and 4- to 7-membered heterocyclyl C １ ~C ２ An alkyl group is more preferred. Specific examples of heterocyclylalkyl groups include a 2-(tetrahydro-2H-pyran-4-yl)ethyl group and a 2-(azetidin-3-yl)ethyl group.

[0049] The term "alkylsulfonylalkyl group" as used herein refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "alkylsulfonyl group" as defined above. １ ~C ６ Alkylsulfonyl C １ ~C ６ Alkyl groups are preferred, and C １ ~C ６ Alkylsulfonyl C １ ~C ２ An alkyl group is more preferred. Specific examples of the alkylsulfonylalkyl group include a methylsulfonylmethyl group and a 2-(methylsulfonyl)ethyl group.

[0050] The term "aminocarbonylalkyl group" as used herein refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "aminocarbonyl group" as defined above. １ ~C ６ Alkyl groups are preferred, and aminocarbonyl C １ ~C ４ Specific examples of the aminocarbonylalkyl group include a methylaminocarbonylmethyl group, a dimethylaminocarbonylmethyl group, a t-butylaminocarbonylmethyl group, a 1-azetidinylcarbonylmethyl group, a 1-pyrrolidinylcarbonylmethyl group, a 1-piperidinylcarbonylmethyl group, a 4-morpholinylcarbonylmethyl group, a 2-(methylaminocarbonyl)ethyl group, a 2-(dimethylaminocarbonyl)ethyl group, a 2-(1-azetidinylcarbonyl)ethyl group, a 2-(1-pyrrolidinylcarbonyl)ethyl group, a 2-(4-morpholinylcarbonyl)ethyl group, a 3-(dimethylaminocarbonyl)propyl group, and a 4-(dimethylaminocarbonyl)butyl group.

[0051] The term "aryloxyalkyl group" as used herein means a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "aryloxy group" as defined above. ６ ~C１０ Aryloxy C １ ~C ６ Alkyl groups are preferred, and C ６ ~C １０ Aryloxy C １ ~C ２ An alkyl group is more preferred. Specific examples of the aryloxyalkyl group include a phenoxymethyl group and a 2-phenoxyethyl group.

[0052] As used herein, the term "aralkyl (arylalkyl) group" refers to a group in which at least one hydrogen atom of an "alkyl group" as defined above is substituted with an "aryl group" as defined above. ７ ~C １４ An aralkyl group is preferred, and C ７ ~C １０ An aralkyl group is more preferred. Specific examples of the aralkyl group include a benzyl group, a phenethyl group, and a 3-phenylpropyl group.

[0053] As used herein, the term "aralkoxy group" refers to an oxy group to which the above-defined "aralkyl group" is bonded. ７ ~C １４ An aralkoxy group is preferred, and C ７ ~C １０ An aralkoxy group is more preferred. Specific examples of the aralkoxy group include a benzyloxy group, a phenethyloxy group, and a 3-phenylpropoxy group.

[0054] As used herein, the term "aralkoxyalkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with an "aralkoxy group" as defined above. ７ ~C １４ Aralkoxy C １ ~C ６ Alkyl groups are preferred, and C ７ ~C １４ Aralkoxy C １ ~C ２An alkyl group is more preferred. Specific examples of the aralkoxyalkyl group include a benzyloxymethyl group and a 1-(benzyloxy)ethyl group.

[0055] As used herein, the term "heteroarylalkyl group" refers to a group in which at least one hydrogen atom of an "alkyl group" as defined above is substituted with a "heteroaryl group" as defined above. The heteroarylalkyl group is preferably a 5- to 10-membered heteroaryl C １ ~C ６ Alkyl groups are preferred, and 5- to 10-membered heteroaryl C １ ~C ２ Specific examples of heteroarylalkyl groups include a 3-thienylmethyl group, a 4-thiazolylmethyl group, a 2-pyridylmethyl group, a 3-pyridylmethyl group, a 4-pyridylmethyl group, a 2-(2-pyridyl)ethyl group, a 2-(3-pyridyl)ethyl group, a 2-(4-pyridyl)ethyl group, a 2-(6-quinolyl)ethyl group, a 2-(7-quinolyl)ethyl group, a 2-(6-indolyl)ethyl group, a 2-(5-indolyl)ethyl group, and a 2-(5-benzofuranyl)ethyl group.

[0056] As used herein, the term "heteroarylalkoxy group" refers to an oxy group to which the above-defined "heteroarylalkyl group" is bonded. The heteroarylalkoxy group is preferably a 5- to 10-membered heteroaryl C １ ~C ６ Alkoxy groups are preferred, and 5- to 10-membered heteroaryl C １ ~C ２ An alkoxy group is more preferred. Specific examples of the heteroarylalkoxy group include a 3-thienylmethoxy group and a 3-pyridylmethoxy group.

[0057] As used herein, the term "heteroarylalkoxyalkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "heteroarylalkoxy group" as defined above. The heteroarylalkoxyalkyl group includes a 5- to 10-membered heteroaryl C １ ~C ６ Alkoxy C １ ~C ６Alkyl groups are preferred, and 5- to 10-membered heteroaryl C １ ~C ２ Alkoxy C １ ~C ２ An alkyl group is more preferred. Specific examples of heteroarylalkoxyalkyl groups include a 3-pyridylmethoxymethyl group.

[0058] As used herein, the term "heterocyclidenealkyl group" refers to a group in which one or more hydrogen atoms of an "alkyl group" as defined above are substituted with a "heterocyclidene group" as defined above. The heterocyclidenealkyl group includes a 4- to 7-membered heterocyclidene C １ ~C ６ Alkyl groups are preferred, and 4- to 7-membered heterocyclidene C １ ~C ２ An alkyl group is more preferred. Specific examples of heterocyclidenealkyl groups include a tetrahydro-4H-pyran-4-ylidenemethyl group and an azetidin-3-ylidenemethyl group.

[0059] As used herein, the term "alkoxyalkenyl group" refers to an "alkenyl group" defined above in which one or more hydrogen atoms have been substituted with an "alkoxy group" defined above. １ ~C ６ Alkoxy C ２ ~C ６ An alkenyl group is preferred. Specific examples of the alkoxyalkenyl group include an (E)-4-methoxybut-2-en-1-yl group.

[0060] The term "aminocarbonylalkenyl group" as used herein refers to an "alkenyl group" defined above in which one or more hydrogen atoms have been substituted with an "aminocarbonyl group" defined above. ２ ~C ６ An alkenyl group is preferred. Specific examples of aminocarbonylalkenyl groups include (E)-3-(dimethylaminocarbonylcarbonyl)-prop-2-en-1-yl groups.

[0061] As used herein, the term "haloalkoxy group" refers to an "alkoxy group" defined above in which one or more hydrogen atoms have been substituted with halogen atoms. １ ~C ６ A haloalkoxy group is preferred. Specific examples of the haloalkoxy group include a difluoromethoxy group, a trifluoromethoxy group, a 2,2-difluoroethoxy group, and a 2,2,2-trifluoroethoxy group.

[0062] As used herein, the term "alkylene group" refers to a divalent group derived by further removing one arbitrary hydrogen atom from the above-mentioned "alkyl group." ４ ~C ８ An alkylene group is preferred. Specific examples of the alkylene group 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.

[0063] As used herein, the term "cycloalkylene group" refers to a divalent group derived by further removing any one hydrogen atom from the "cycloalkyl group". ３ ~C ８ A cycloalkylene group is preferred, and specific examples of the cycloalkylene group include a cyclopropane-1,2-diyl group, a cyclobutane-1,2-diyl group, a cyclopentane-1,2-diyl group, and a cyclohexane-1,2-diyl group.

[0064] As used herein, the term "alkenylene group" refers to a divalent group derived from the aforementioned "alkenyl group" by further removing any one hydrogen atom. Depending on the configuration of the double bond and substituents (if any), the geometry of the double bond can be entgegen (E) or zusammen (Z), cis or trans. The alkenylene group includes linear and branched groups, and can be C ２ ~C １０ Alkenylene groups are preferred, C ２ ~C ６ An alkenylene group is more preferred.

[0065] As used herein, the term "alkynylene group" refers to a divalent group derived from the above-mentioned "alkynyl group" by further removing one arbitrary hydrogen atom. The alkynylene group includes linear and branched groups, and is C ２ ~C １０ Alkynylene groups are preferred, C ２ ~C ６ An alkynylene group is more preferred.

[0066] As used herein, the term "arylene group" refers to a divalent group derived by further removing any one hydrogen atom from the aforementioned "aryl group." The arylene group may be a single ring or a fused ring. The number of atoms constituting the ring is not particularly limited, but is preferably 6 to 10 (C6-10 arylene). Specific examples of arylene include a 1,2-phenylene group, a 1,3-phenylene group, a 1,4-phenylene group, a 1,2-naphthylene group, a 1,3-naphthylene group, and a 1,4-naphthylene group.

[0067] As used herein, the term "heterocyclylene group" refers to a divalent group derived by further removing one arbitrary hydrogen atom from the above-mentioned "heterocyclyl group."

[0068] In the present specification, the term "heteroarylene group" refers to a divalent group derived by further removing one arbitrary hydrogen atom from the above-mentioned "heteroaryl group".

[0069] As used herein, the term "spirocycloalkyl group" refers to a group formed by sharing one carbon atom constituting a cycloalkane ring with a carbon atom in a group to which it is bound. Preferred spirocycloalkyl groups include C ３ ~C ８ Examples include spirocycloalkyl groups, and specific examples include a spirocyclopropyl group, a spirocyclobutyl group, a spirocyclopentyl group, a spirocyclohexyl group, a spirocycloheptyl group, and a spirocyclooctyl group.

[0070] As used herein, the term "spiroheterocyclyl group" refers to a group in which one or more carbon atoms in the above-mentioned "spirocycloalkyl group" are replaced by heteroatoms. Preferred examples of the heterospirocycloalkyl group include 4- to 10-membered spiroheterocyclyl groups.

[0071] As used herein, "alicyclic ring" refers to a non-aromatic hydrocarbon ring. The alicyclic ring may have an unsaturated bond within the ring, or may be a polycyclic ring having two or more rings. Furthermore, the carbon atoms constituting the ring may be oxidized to form a carbonyl. Preferred examples of the alicyclic ring include 3- to 8-membered alicyclic rings, and specific examples include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, and a bicyclo[2.2.1]heptane ring.

[0072] As used herein, the term "heterocycle" refers to a non-aromatic heterocycle containing preferably 1 to 5, more preferably 1 to 3, heteroatoms among the atoms constituting the ring. The heterocycle 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, and may be a monocyclic, fused, or spirocyclic ring. The number of atoms constituting the ring is preferably 3 to 12 (3- to 12-membered heterocycle), more preferably 4 to 8 (4- to 8-membered heterocycle). Specific examples of the heterocyclic ring include an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, an oxazolidone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, or an azocane ring, as well as rings in which one or more single bonds in these saturated heterocyclic rings have been replaced with double bonds or triple bonds.

[0073] As used herein, the term "saturated heterocycle" refers to a non-aromatic heterocycle containing 1 to 5 heteroatoms in addition to carbon atoms and containing no double and / or triple bonds within the ring. The saturated heterocycle may be a monocycle or may form a condensed ring with another ring, for example, an aromatic ring such as a benzene ring. When a saturated heterocycle forms a fused ring, the saturated heterocycle is preferably a 4- to 7-membered saturated heterocycle, and specific examples thereof include an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, an oxazolidone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, an indoline ring, and an azepane ring.

[0074] [Peptide Compound] As used herein, the term "peptide compound" is not particularly limited as long as it is a peptide compound in which amino acid residues are linked by amide bonds or ester bonds. The number of amino acid residues in a peptide compound is not particularly limited, and may be 5 or more, 7 or more, 8 or more, or 9 or more. The number of amino acid residues in a peptide compound may be 30 or less, 25 or less, 15 or less, or 13 or less. The number of amino acid residues in a peptide compound may be, for example, 5 to 30, 7 to 25, 8 to 15, or 9 to 13, or even 11. The peptide compound may have a branched structure.

[0075] As used herein, "amino acid" includes natural amino acids and unnatural amino acids. As used herein, "natural amino acids" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Unnatural amino acids are not particularly limited, and examples thereof include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids whose side chains differ from those of natural amino acids. As used herein, unnatural N-substituted amino acids refer to N-substituted amino acids other than Pro. As used herein, amino acids may have any configuration. The side chain of the amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group, and one or two non-adjacent methylene groups in these groups may be replaced by an oxygen atom, a carbonyl group (—CO—), or a sulfonyl group (—SO ２-). Each of these may be substituted with a substituent, and the substituents are not limited, and may be independently selected from any substituents containing a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom. That is, examples include optionally substituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. In a non-limiting embodiment, the amino acid herein 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).

[0076] The main chain amino group of the amino acid is unsubstituted (NH ２ group), and may be substituted (i.e., -NHR group: R represents an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group which may have a substituent, and one or two non-adjacent methylene groups in these groups are replaced by an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO ２ -), or the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline. The substituent of R is selected in the same manner as the substituent in the amino acid side chain described above. When the main chain amino group is substituted, R is included in the "amino acid side chain" in this specification. An amino acid in which such a main chain amino group is substituted is referred to as an "N-substituted amino acid" in this specification. As used herein, the "N-substituted amino acid" is preferably an N-alkyl amino acid, an N-C １ ~C ６ Alkyl amino acids, N-C １ ~C ４ Examples include, but are not limited to, alkyl amino acids, N-methyl amino acids, and N-ethyl amino acids.

[0077] As used herein, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (the sum of the number of protons and neutrons), in an abundance ratio different from the natural abundance ratio. Examples of isotopes included in "amino acids" as used herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is ２ H. ３ H. １３ C. １４ C. １５ N. １７ O. １８ O. ３１ P. ３２ P. ３５ S. １８ F. ３６ Cl and the like are included.

[0078] In this specification, examples of the substituent containing a halogen atom include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and the like, each of which has a halogen atom as a substituent, and more specific examples thereof include a fluoroalkyl, a difluoroalkyl, a trifluoroalkyl, and the like.

[0079] In this specification, examples of the substituent containing an oxygen atom include a hydroxy group (—OH), an oxy group (—OR), a carbonyl group (—C(═O)—R), a carboxy group (—CO ２ H), oxycarbonyl group (-C(=O)-OR), carbonyloxy group (-O-C(=O)-R), thiocarbonyl group (-C(=O)-SR), carbonylthio group (-S-C(=O)-R), aminocarbonyl group (-C(=O)-NHR), carbonylamino group (-NH-C(=O)-R), oxycarbonylamino group (-NH-C(=O)-OR), sulfonylamino group (-NH-SO ２ -R), aminosulfonyl group (-SO ２ -NHR), sulfamoylamino group (-NH-SO ２ -NHR), thiocarboxy group (-C(=O)-SH), carboxycarbonyl group (-C(=O)-CO ２H).

[0080] Examples of oxy (—OR) include an alkoxy group, a cycloalkoxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group, a heteroaryloxy group, and an aralkyloxy group. １ ~C ４ Alkoxy group, C １ ~C ２ An alkoxy group is preferred, and among these, a methoxy group or an ethoxy group is more preferred.

[0081] Examples of the carbonyl group (-C(=O)-R) include a formyl group (-C(=O)-H), an alkylcarbonyl group, a cycloalkylcarbonyl group, an alkenylcarbonyl group, an alkynylcarbonyl group, an arylcarbonyl group, a heteroarylcarbonyl group, and an aralkylcarbonyl group.

[0082] Examples of the oxycarbonyl group (—C(═O)—OR) include an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, and an aralkyloxycarbonyl group.

[0083] Examples of the carbonyloxy group (—O—C(═O)—R) include an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group, and an aralkylcarbonyloxy group.

[0084] Examples of the thiocarbonyl group (-C(=O)-SR) include an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group, and an aralkylthiocarbonyl group.

[0085] Examples of the carbonylthio group (—S—C(═O)—R) include an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group, and an aralkylcarbonylthio group.

[0086] Examples of aminocarbonyl groups (—C(═O)—NHR) include alkylaminocarbonyl groups (e.g., C １ ~C ６ or C １ ~C ４ Examples include alkylaminocarbonyl groups, particularly ethylaminocarbonyl groups and methylaminocarbonyl groups. Also included are cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. In addition to these, examples include compounds in which the H atom bonded to the N atom in —C(═O)—NHR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0087] Examples of carbonylamino groups (-NH-C(=O)-R) include alkylcarbonylamino groups, cycloalkylcarbonylamino groups, alkenylcarbonylamino groups, alkynylcarbonylamino groups, arylcarbonylamino groups, heteroarylcarbonylamino groups, and aralkylcarbonylamino groups. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group are also included.

[0088] Examples of oxycarbonylamino groups (-NH-C(=O)-OR) include alkoxycarbonylamino groups, cycloalkoxycarbonylamino groups, alkenyloxycarbonylamino groups, alkynyloxycarbonylamino groups, aryloxycarbonylamino groups, heteroaryloxycarbonylamino groups, and aralkyloxycarbonylamino groups. In addition to these, examples include compounds in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0089] Sulfonylamino group (-NH-SO ２ Examples of —R) include alkylsulfonylamino groups, cycloalkylsulfonylamino groups, alkenylsulfonylamino groups, alkynylsulfonylamino groups, arylsulfonylamino groups, heteroarylsulfonylamino groups, and aralkylsulfonylamino groups. ２ Examples include compounds in which the H atom bonded to the N atom in —R is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0090] Aminosulfonyl group (-SO ２ Examples of —NHR) include alkylaminosulfonyl groups, cycloalkylaminosulfonyl groups, alkenylaminosulfonyl groups, alkynylaminosulfonyl groups, arylaminosulfonyl groups, heteroarylaminosulfonyl groups, and aralkylaminosulfonyl groups. ２ Examples include compounds in which the H atom bonded to the N atom in —NHR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0091] Sulfamoylamino group (-NH-SO ２Examples of —NHR) include alkylsulfamoylamino groups, cycloalkylsulfamoylamino groups, alkenylsulfamoylamino groups, alkynylsulfamoylamino groups, arylsulfamoylamino groups, heteroarylsulfamoylamino groups, and aralkylsulfamoylamino groups. ２ The two H atoms bonded to the N atom in -NHR may be substituted with substituents independently selected from the group consisting of an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, and these two substituents may form a ring.

[0092] Examples of the substituent containing an S atom include a thiol group (-SH), a thio group (-S-R), a sulfinyl group (-S(=O)-R), and a sulfonyl group (-SO ２ -R), sulfo group (-SO ３ H).

[0093] Examples of the thio group (-S-R) are selected from alkylthio groups, cycloalkylthio groups, alkenylthio groups, alkynylthio groups, arylthio groups, heteroarylthio groups, aralkylthio groups, and the like.

[0094] Sulfonyl group (-SO ２ Examples of —R) include alkylsulfonyl groups, cycloalkylsulfonyl groups, alkenylsulfonyl groups, alkynylsulfonyl groups, arylsulfonyl groups, heteroarylsulfonyl groups, and aralkylsulfonyl groups.

[0095] As used herein, examples of the nitrogen-containing substituent include an azide group (—N ３ ), cyano group (-CN), primary amino group (-NH ２ ), secondary amino group (-NH-R; also called mono-substituted amino group), tertiary amino group (-NR(R'); also called di-substituted amino group), amidino group (-C(=NH)-NH ２ ), a substituted amidino group (—C(═NR)—NR′R″), a guanidino group (—NH—C(═NH)—NH ２), substituted guanidino group (—NR—C(═NR′″)—NR′R″), aminocarbonylamino group (—NR—CO—NR′R″), pyridyl group, piperidino group, morpholino group, azetidinyl group, and the like.

[0096] Examples of secondary amino groups (—NH—R; monosubstituted amino groups) include alkylamino groups, cycloalkylamino groups, alkenylamino groups, alkynylamino groups, arylamino groups, heteroarylamino groups, and aralkylamino groups. The alkylamino group is preferably C １ ~C ６ alkyl group, more preferably C １ ~C ４ Alkyl groups are preferred, and methyl and ethyl groups are more preferred.

[0097] Examples of tertiary amino groups (-NR(R'); disubstituted amino groups) include alkyl(aralkyl)amino groups and other amino groups having any two substituents independently selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and the like, and these two substituents may form a ring together with the nitrogen atom to which they are bonded. Specifically, dialkylamino groups, particularly C １ -C ６ Dialkylamino group, C １ -C ４ Examples include a dialkylamino group, a dimethylamino group, and a diethylamino group. ｐ -C ｑ "Dialkylamino group" means an amino group having C ｐ -C ｑ It means a group substituted with two alkyl groups, and both C ｐ -C ｑ The alkyl groups may be the same or different.

[0098] Examples of substituted amidino groups (-C(=NR)-NR'R") include groups in which the three substituents R, R', and R" on the N atom are each independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, such as an alkyl(aralkyl)(aryl)amidino group.

[0099] Examples of the substituted guanidino group (-NR-C(=NR'")-NR'R") include groups in which R, R', R", and R'" are each independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, or groups in which these groups form a ring.

[0100] Examples of aminocarbonylamino groups (—NR—CO—NR′R″) include groups in which R, R′, and R″ are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, or groups in which these groups form a ring.

[0101] In this specification, "peptide residue" and "amino acid residue" may be simply referred to as "peptide" and "amino acid", respectively.

[0102] As used herein, the term "corresponding site" can be used to characterize amino acid residues in the amino acid sequence of the peptide according to this embodiment by reference to the amino acid sequence represented by SEQ ID NO: 1. Alignment to determine corresponding sites can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0103] The salt of the compound used herein may preferably be a chemically or pharmaceutically acceptable salt. The salt may also be a solvate thereof, preferably a chemically or pharmaceutically acceptable solvate thereof. Examples of the salt of the compound include hydrochloride, hydrobromide, hydroiodide, phosphate, phosphonate, sulfate, sulfonate such as methanesulfonate and p-toluenesulfonate, carboxylate such as acetate, citrate, malate, tartrate, succinate, and salicylate, alkali metal salt such as lithium salt, sodium salt, and potassium salt, alkaline earth metal salt such as magnesium salt and calcium salt, and ammonium salt such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, and tetraalkylammonium salt.

[0104] As used herein, the term "solvate" refers to a compound that forms a single molecular group together with a solvent, and when the solvent is water, it is referred to as a hydrate. As a solvate of a salt of a compound of the present disclosure, a hydrate is preferred, and specific examples of such a hydrate include mono- to decahydrates, preferably mono- to pentahydrates, and more preferably mono- to trihydrates. Solvates of the compound of the present disclosure include not only solvates with a single solvent such as water, alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), and dimethylformamide, but also solvates with multiple solvents.

[0105] [Method for Producing Amino Acid] A method for producing an amino acid according to one embodiment includes a step (reaction step) of carrying out the following reaction (i) or (ii) in the presence of a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1, a reducing agent, and compound D represented by the following formula (1): (i) an intermolecular reductive amination reaction between compound A selected from the group consisting of compounds having an amino group and salts thereof, and compound B selected from the group consisting of compounds having a carbonyl group and salts thereof; (ii) an intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof. Here, an amino acid having a substituted or unsubstituted amino group is formed by the intermolecular reductive amination reaction between the amino group of compound A and the carbonyl group of compound B, or by the intramolecular reductive amination reaction of the amino group and the carbonyl group in compound C.

[0106] According to another embodiment, the method for producing an amino acid includes a step (reaction step) of carrying out the following reaction (i) or (ii) in the presence of a polypeptide having catalytic activity under at least one reaction condition for an intermolecular reductive amination reaction between compound A selected from the group consisting of compounds having an amino group and salts thereof and compound B selected from the group consisting of compounds having a carbonyl group and salts thereof, or an intramolecular reductive amination reaction between compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof, a reducing agent, and compound D': (i) an intermolecular reductive amination reaction between compound A selected from the group consisting of compounds having an amino group and salts thereof and compound B selected from the group consisting of compounds having a carbonyl group and salts thereof; or (ii) an intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof. In the production method, compound D' is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethylphosphine oxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide.

[0107] <Compound D and Compound D'> The reaction step of carrying out the reductive amination reaction is carried out in the presence of Compound D represented by the following formula (1).

[0108] In formula (1), v and w each independently represent 0 or 1, provided that at least one of v and w represents 1, and T represents a carbon atom, a phosphorus atom, or a sulfur atom.

[0109] In formula (1), the following formula (1a) The functional group represented by formula (1a) represents ═O, —ORd, or a hydroxy group (—OH). Here, when the functional group represented by formula (1a) is ═O, it means that T is bonded to an oxygen atom via a double bond. That is, compound D, in which the functional group represented by formula (1a) is ═O, has a structure represented by T═O. When the functional group represented by formula (1a) is —ORd, it means that T is bonded to ORd via a single bond. That is, compound D, in which the functional group represented by formula (1a) is —ORd, has a structure represented by T-ORd. When the functional group represented by formula (1a) is a hydroxy group, it means that T and the hydroxy group are directly bonded. That is, compound D, in which the functional group represented by formula (1a) is —OH, has a structure represented by T-OH.

[0110] When v and w are both 1, two functional groups represented by formula (1a) present in plural may be the same or different, and Ra, Rb, and Rc each independently represent a hydrogen atom, C １ ~C ３ Alkyl group, alkylamino group or -CH ２ -ORd, and any two or more of Ra, Rb, and Rc may be bonded to each other together with T to form a ring structure, and Rd is C １ ~C ３ represents an alkyl group; d, e, and f each independently represent 0 or 1, and at least one of d, e, and f represents 1;

[0111] When v and w are both 1, any one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc are not bonded to each other to form a ring structure together with T. For example, when v and w are both 1, T is a sulfur atom, all functional groups represented by formula (1a) are ═O, f is 0, and at least one of Ra and Rb may be a methyl group.

[0112] When any one or more of Ra, Rb, and Rc is a methylamino group, Ra, Rb, and Rc do not bond to each other to form a ring structure with T. When formula (1a) is a hydroxy group and T is a carbon atom, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.

[0113] Compound D may be a compound represented by the following formula (1-1), in which T in formula (1) is a carbon atom.

[0114] The compound represented by formula (1-1) may be a compound represented by the following formula (1-1a) in which v is 1 and w is 0.

[0115] In formula (1-1a), when the functional group represented by formula (1a) is ═O, two of d, e, and f are 1, and the rest are 0. When the functional group represented by formula (1a) is ═O, the compound represented by formula (1-1a) may be a compound represented by the following formula (1-1b) in which d and e are 1 and f is 0.

[0116] In the compound represented by formula (1-1b), one of Ra and Rb is a hydrogen atom or C １ ~C ３ The compound represented by formula (1-1b) may be a compound in which one group is an alkyl group and the other is an alkylamino group. The alkylamino group may be a monomethylamino group or a dimethylamino group. For example, the compound represented by formula (1-1b) may be N,N-dimethylformamide, N,N-dimethylacetamide, or N-methylformamide.

[0117] In formula (1-1a), when the functional group represented by formula (1a) is a hydroxy group, d, e, and f are all 1. When the functional group represented by formula (1a) is a hydroxy group, the compound represented by formula (1-1a) may be a compound represented by the following formula (1-1c) in which d, e, and f are all 1.

[0118] Ra, Rb and Rc each independently represent a hydrogen atom or C １ ~C ３ The compound represented by formula (1-1c) may be methanol in which Ra, Rb and Rc are all hydrogen atoms.

[0119] In formula (1-1a), when the functional group represented by formula (1a) is -ORd, d, e, and f may all be 1, and Rb and Rc may all be hydrogen atoms. When the functional group represented by formula (1a) is -ORd, the compound represented by formula (1-1a) may be a compound represented by the following formula (1-1d) in which d, e, and f are all 1, and Rb and Rc are all hydrogen atoms. In the compound represented by formula (1-1d) below, Ra is -CH ２ In the compound represented by the following formula (1-1d), Ra may be —CH ２ -ORd, and each Rd is -CH ３ It may be dimethoxyethane,

[0120] Compound D may be a compound represented by the following formula (1-2), in which T in formula (1) is a phosphorus atom.

[0121] The compound represented by formula (1-2) may be a compound represented by the following formula (1-2a) in which v is 1, w is 0, and the functional group represented by formula (1a) is ═O.

[0122] In formula (1-2a), Ra, Rb and Rc are all C １ ~C ３The compound represented by formula (1-2a) may be trimethylphosphine oxide in which Ra, Rb and Rc are all methyl groups.

[0123] Compound D may be a compound represented by the following formula (1-3), in which T in formula (1) is a sulfur atom.

[0124] The compound represented by formula (1-3) may be a compound represented by the following formula (1-3a), in which v is 1, w is 0, the functional group represented by formula (1a) is ═O, d and e are both 1, and f is 0.

[0125] In formula (1-3a), Ra and Rb are both C １ ~C ３ They may be alkyl groups, and Ra and Rb may be linked to each other to form a ring structure together with the sulfur atom (S).

[0126] In the compound represented by formula (1-3a), Ra and Rb may each independently be a methyl group or an ethyl group. In the compound represented by formula (1-3a), Ra and Rb may each independently be a methyl group, such as dimethyl sulfoxide, or Ra and Rb may each independently be an ethyl group, such as diethyl sulfoxide. In the compound represented by formula (1-3a), Ra and Rb may each independently be C １ ~C ３ It may be tetramethylene sulfoxide, which is an alkyl group in which the alkyl groups are linked to each other to form a ring structure together with S.

[0127] The compound represented by formula (1-3) may be a compound represented by the following formula (1-3b), in which v and w are both 1, the functional group represented by formula (1a) is ═O, d and e are both 1, and f is 0.

[0128] In the compound represented by formula (1-3b), at least one of Ra and Rb is a methyl group. The compound represented by formula (1-3b) may be dimethyl sulfone in which both Ra and Rb are methyl groups.

[0129] Compound D may be compound D', which is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethylphosphine oxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide, because compound D allows amino acids to be produced in higher yields. Compound D or compound D' may be one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide, or may be dimethyl sulfoxide, because compound D allows amino acids to be produced in higher yields.

[0130] Compound D or Compound D' may be liquid or solid under conditions of 25°C and 1 atmospheric pressure, and may be liquid under conditions of 25°C and 1 atmospheric pressure, since this further improves the production efficiency of the amino acid. Examples of Compound D or Compound D' that are liquid under conditions of 25°C and 1 atmospheric pressure include dimethyl sulfoxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, and N-methylformamide.

[0131] <Compound A> Compound A selected from the group consisting of compounds having an amino group and salts thereof may be one or more selected from the group consisting of compounds represented by the following formula (2) and salts thereof:

[0132] In formula (2), R １ and R ２ each independently 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, and these groups may be substituted; R １ or R ２ Any one or more of is a hydrogen atom.

[0133] In formula (2), R １ may be a hydrogen atom, and R ２ is a hydrogen atom or C １ ~C ６ may be an alkyl group, a hydrogen atom or a C １ ~C ３It may be an alkyl group, and may be a hydrogen atom, a methyl group, or an ethyl group.

[0134] Compound A is a compound represented by formula (2), １ and R ２ The alkylamine or a salt thereof may be one or more selected from the group consisting of methylamine, ethylamine, and salts thereof. １ and R ２ and (b) may be ammonia, in which both are hydrogen atoms. Compound A may be one or more selected from the group consisting of ammonia, methylamine, ethylamine, and salts thereof.

[0135] <Compound B> Compound B selected from the group consisting of compounds having a carbonyl group and salts thereof may be one or more selected from the group consisting of compounds represented by the following formula (3) and salts thereof:

[0136] In formula (3), X represents a carbon atom, and Y represents a hydrogen atom or a group represented by formula (4) above.

[0137] n represents an integer of 0 to 2. n may be 0 or 1, or may be 0.

[0138] R ６ represents a hydrogen atom, an optionally substituted C １ ~C ６ alkyl group, optionally substituted C ５ ~C １２ It represents an aryl group, a heteroaryl group having 5 to 12 atoms constituting a ring which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom.

[0139] In formula (4), indicates the point of attachment to X.

[0140] m represents an integer of 0 or more and 6 or less. The lower limit of m is 0 or more, and may be 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more. The upper limit of m is 6 or less, and may be 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

[0141] p represents 0 or 1, q represents 0 or 1, and r represents 0 or 1.

[0142] Z １ represents an alkylene group which may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms. When m is an integer of 2 or more, a plurality of Z １ may be the same or different. Examples of the "ether bond-containing group" include an alkyl group substituted with an alkoxy group, an allyloxy group, etc. Specific examples of the ether bond-containing group having from 1 to 6 carbon atoms include a methoxymethyl group, an ethoxymethyl group, an allyloxymethyl group, an allyloxyethyl group, and an allyloxypropyl group.

[0143] Z ２ indicates a carbon atom.

[0144] R ３ , R ４ and R ５ are each independently a hydrogen atom, an optionally substituted C １ ~C ６ alkyl group, optionally substituted C ５ ~C １２ It represents an aryl group, a heteroaryl group having 5 to 12 atoms constituting a ring which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom.

[0145] R ３ , R ４ and R ５ Any two or more of these are connected to each other to form Z ２ together may form a ring structure, which may be a cycloalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, which may be substituted.

[0146] R ３ , R ４ , and R ５ Is Z ２ may form a double bond or a triple bond with R ３ , R ４ , and R ５ is connected to Z by a double bond or a triple bond. ２When p is bonded to q, any one or more of p, q, and r is 0.

[0147] In formula (4), R ３ , R ４ and R ５ At least one of these may not be methyl.

[0148] Compound B may be a compound represented by the following formula (3').

[0149] In formula (3′), Y′ is C ３ ~C ８ a cycloalkyl group, or C ７ ~C １０ represents an aralkyl group, and the aralkyl group is C １ ~C ３ It may be substituted with an alkyl group or a halogen.

[0150] The compound represented by formula (3') or a salt thereof may be one or more selected from the group consisting of 4-(2-chlorophenyl)-2-oxobutanoic acid, phenylpyruvic acid, 2-cyclopentyl-2-oxo-acetic acid, 3-(4,4-difluorocyclohexyl)-2-oxopropanoic acid, 2-oxo-3-phenylbutanoic acid, 3,3-dimethyl-2-oxobutyric acid, and salts thereof. Specific examples of the salt include sodium salts, magnesium salts, potassium salts, and calcium salts.

[0151] The combination of compound A and compound B is represented by the formula (2), １ and R ２ Compound A (ammonia) in which both are hydrogen atoms, or R １ is a hydrogen atom, and R ２ is a methyl group, and in formula (3), n and m are both 0, Y is a group represented by formula (4), and R ３ is a (2-chlorophenyl)methyl group, and R ４ , R ５ and R ６are all hydrogen atoms, and p, q, and r are all 1; or in formula (3), n and m are all 0, Y is a group represented by formula (4), and R ３ is a phenyl group, and R ４ , R ５ and R ６ are all hydrogen atoms, and p, q, and r are all 1, and in formula (2), R １ is a hydrogen atom, and R ２ is an ethyl group, and a compound A (ethylamine) in which n and m are both 0, Y is a group represented by formula (4), and R ３ is a phenyl group, and R ４ , R ５ and R ６ are all hydrogen atoms, and p, q, and r are all 1, and in formula (2), R １ is a hydrogen atom, and R ２ is a methyl group, and in formula (3), n and m are both 0, Y is a group represented by formula (4), and R ３ , R ４ and Z ２ are linked together to form a cyclopentane ring, and R ５ and R ６ are all hydrogen atoms, and p, q and r are all 1.

[0152] The combination of compound A and compound B is represented by the formula (2), １ is a hydrogen atom, and R ２ is a methyl group, and in formula (3), n and m are both 0, Y is a group represented by formula (4), and R ３ is a 4,4-difluorocyclohexyl group, and R ４ , R ５ and R ６are all hydrogen atoms, and p, q, and r are all 1. The combination of compound A and compound B may be a combination of compound A and compound B (3-(4,4-difluorocyclohexyl)-2-oxopropanoic acid) in which R １ and R ２ Compound A (ammonia) in which both n and m are hydrogen atoms, and in formula (3), n and m are both 0, Y is a group represented by formula (4), and R ３ is a methyl group, and R ４ is a phenyl group, and R ５ and R ６ are all hydrogen atoms, and p, q, and r are all 1. The combination of Compound A and Compound B may be a combination of Compound A and Compound B (2-oxo-3-phenylbutanoic acid) in which R １ is a hydrogen atom, and R ２ is a methyl group, and in formula (3), n and m are both 0, Y is a group represented by formula (4), and R ３ , R ４ and R ５ are all methyl groups, and R ６ is a hydrogen atom, and p, q, and r are all 1.

[0153] <Compound C> Compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof may be a compound represented by the following formula (5).

[0154] n′ represents an integer of 0 to 2. n may be 0 or 1, and may be 0.

[0155] R ７ represents an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, an arylene group, a heterocyclylene group, or a heteroarylene group, and these groups may be substituted.

[0156] R ８ represents a hydrogen atom, an optionally substituted C １ ~C ６alkyl group, optionally substituted C １ ~C ６ It represents an aryl group, a heteroaryl group having 5 to 12 atoms constituting a ring which may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom.

[0157] <Polypeptide> The polypeptide comprises an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1. 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%.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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%.

[0181] 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.

[0182] The polypeptide according to this embodiment may have two or more alterations in the amino acid sequence represented by SEQ ID NO: 1. Preferred embodiments of the polypeptide having two or more alterations will be described below.

[0183] The polypeptide preferably comprises a sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the methionine residue at position 141, the histidine residue at position 182, and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as the first modification site, and an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1, but different from the amino acid residue modified as the first modification site, has been modified as the second modification site.

[0184] The polypeptide preferably comprises: a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141 and the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141 and the tryptophan residue at position 253 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141 and the lysine residue at position 260 have been modified; a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182 and the tryptophan residue at position 253 have been modified; a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182 and the lysine residue at position 260 have been modified; or a sequence in which the amino acid residues located at positions corresponding to the tryptophan residue at position 253 and the lysine residue at position 260 have been modified; More preferably, the amino acid sequence comprises: a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182 and the lysine residue at position 260 have been altered; or a sequence in which the amino acid residues located at positions corresponding to the tryptophan residue at position 253 and the lysine residue at position 260 have been altered.

[0185] The polypeptide preferably comprises a sequence in which the amino acid residue located at a position corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with one amino acid residue selected from the group consisting of tyrosine residues, tryptophan residues, valine residues, threonine residues, arginine residues, lysine residues, isoleucine residues, and alanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with one amino acid residue selected from the group consisting of tyrosine residues, valine residues, and alanine residues.

[0186] The polypeptide preferably comprises 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 one amino acid residue selected from the group consisting of tyrosine residues, methionine residues, leucine residues, and phenylalanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with one amino acid residue selected from the group consisting of methionine residues and leucine residues.

[0187] The polypeptide preferably comprises a sequence 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 one amino acid residue selected from the group consisting of tyrosine residues, threonine residues, serine residues, asparagine residues, methionine residues, histidine residues, and alanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with a histidine residue.

[0188] The polypeptide preferably comprises 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 one amino acid residue selected from the group consisting of serine residues, glutamine residues, asparagine residues, leucine residues, isoleucine residues, histidine residues, glycine residues, phenylalanine residues, glutamic acid residues, and alanine residues, and more preferably comprises a sequence in which the amino acid residue is substituted with one amino acid residue selected from the group consisting of glutamine residues and glutamic acid residues.

[0189] In the amino acid sequence represented by SEQ ID NO: 1, m１ Amino acid residue X at position １ The amino acid residue located at the position corresponding to １ Other amino acid residues other than x １ and m ２ Amino acid residue X at position ２ The amino acid residue located at the position corresponding to ２ Other amino acid residues other than x ２ The sequence replaced by X １ m １ x １ _X ２ m ２ x ２ It is written as follows.

[0190] The sequence in which two amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified may be, for example, an amino acid sequence selected from the group consisting of Groups a1 to a4 consisting of the amino acid sequences shown below, preferably an amino acid sequence selected from the group consisting of Groups a1 to a3, more preferably an amino acid sequence selected from the group consisting of Groups a1 and a2, and most preferably an amino acid sequence selected from Group a1. Group a1: M141Y_K260E, H182L_K260Q, M141A_K260E, H182M_K260Q, M141A_K260Q, H182M_K260E, M141V_K260E, W253H_K260E, and H182L_K260E. Group a2: M141V_K260Q, W253H_K260Q, W253M_K260E, M141Y_H182M, M141Y_K260Q, H182F_K260Q, M141V_ K260F, M141V_H182L, M141V_K260L, M141Y_K260G, H182L_W253H, H182F_K260E, and M141T_K260Q Group a3: M141Y_H182L, W253M_K260Q, M141V_W253M, M141V_K260S, M141V_K260A, M141A_K260F, M141A_K260H, W253A_K260E, M141V_W253A, M141V_W253H, M141K_K260L, M141V_K260G, and M141A_K260L. Group a4: M141W_K260A, M141V_W253Y, M141K_K260G, M141A_K260G, M141A_K260S, M141A_K260N, M141Y_W253H, M141A_K260A, M141K_K260S, W253A_K260Q, M141V_H182M, M141A_K260I, M141T_K260G, W253M_K260L, M141I_W253A, W253T_K260L, M141R_K260G, W253A_K260L, M141K_W253S, M141A_H182L, W253N_K260L, M141K_K260A, M141K_W253M, H182Y_W253A, H182M_W253H, M141R_K260A, H182M_W253A and M141K_W253A.

[0191] The polypeptide may, for example, comprise a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of groups a1 to a4, preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of groups a1 to a3, more preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of groups a1 and a2, and most preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from group a1.

[0192] The polypeptide may comprise an amino acid sequence selected from the group consisting of groups a1 to a4, preferably comprises an amino acid sequence selected from the group consisting of groups a1 to a3, more preferably comprises an amino acid sequence selected from the group consisting of groups a1 and a2, and most preferably comprises an amino acid sequence selected from group a1.

[0193] The polypeptide according to this embodiment may have, for example, three or more alterations in the amino acid sequence represented by SEQ ID NO: 1. Preferred embodiments of the polypeptide having three or more alterations will be described below.

[0194] The polypeptide preferably comprises a sequence in which an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the methionine residue at position 141 and the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 has been modified as the first modification site; an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the histidine residue at position 182 and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residue modified as the first modification site has been modified as the second modification site; and an amino acid residue located at a position corresponding to one amino acid residue selected from the group consisting of the tryptophan residue at position 253 and the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 and different from the amino acid residues modified as the first and second modification sites has been modified as the third modification site.

[0195] The polypeptide preferably comprises: a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141, the histidine residue at position 182 and the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141, the histidine residue at position 182 and the lysine residue at position 260 have been modified; a sequence in which the amino acid residues located at positions corresponding to the methionine residue at position 141, the tryptophan residue at position 253 and the lysine residue at position 260 have been modified; a sequence in which the amino acid residues located at positions corresponding to the histidine residue at position 182, 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; More preferably, the sequence comprises: a sequence in which amino acid residues located at positions corresponding to the methionine residue at position 141, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified; or a sequence in which amino acid residues located at positions corresponding to the histidine residue at position 182, the tryptophan residue at position 253, and the lysine residue at position 260 have been modified.

[0196] The polypeptide preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises 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 one amino acid residue selected from the group consisting of tyrosine residue, valine residue, threonine residue, serine residue, leucine residue, lysine residue, isoleucine residue, and alanine residue; and more preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises 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 one amino acid residue selected from the group consisting of tyrosine residue, valine residue, and isoleucine residue.

[0197] The polypeptide preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue located 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 one amino acid residue selected from the group consisting of methionine residues, leucine residues, and phenylalanine residues; and more preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence in which the amino acid residue located 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 one amino acid residue selected from the group consisting of methionine residues and leucine residues.

[0198] The polypeptide preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence 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 one amino acid residue selected from the group consisting of valine residue, threonine residue, serine residue, glutamine residue, methionine residue, leucine residue, histidine residue, phenylalanine residue, and glutamic acid; and more preferably has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and comprises a sequence 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 one amino acid residue selected from the group consisting of glutamine residue, methionine residue, leucine residue, and histidine residue.

[0199] It is preferable that the polypeptide has three alterations in the amino acid sequence represented by SEQ ID NO: 1, and contains a 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 one amino acid residue selected from the group consisting of glutamine residues and glutamic acid residues.

[0200] In the amino acid sequence represented by SEQ ID NO: 1, m １ Amino acid residue X at position １ The amino acid residue located at the position corresponding to１ Other amino acid residues other than x １ is replaced by m ２ Amino acid residue X at position ２ The amino acid residue located at the position corresponding to ２ Other amino acid residues other than x ２ and m ３ Amino acid residue X at position ３ The amino acid residue located at the position corresponding to ３ Other amino acid residues other than x ３ The sequence replaced by X １ m １ x １ _X ２ m ２ x ２ _X ３ m ３ x ３ It is written as follows.

[0201] The sequence in which three amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified may be, for example, an amino acid sequence selected from the group consisting of Groups b1 to b4 consisting of the amino acid sequences shown below, preferably an amino acid sequence selected from the group consisting of Groups b1 to b3, more preferably an amino acid sequence selected from the group consisting of Groups b1 and b2, and most preferably an amino acid sequence selected from Group b1. Group b1: M141Y_H182M_K260E, M141V_W253M_K260E, H182L_W253H_K260E, M141Y_H182M_K260Q, M141V_H18 2M_K260E, M141V_H182L_K260Q, M141Y_H182L_K260Q, M141V_W253Q_K260E, H182L_W253Q_K260E, M 141I_W253H_K260E, M141V_H182L_K260E, M141Y_H182L_K260E, H182L_W253M_K260E, M141V_W253 Q_K260Q, M141V_W253H_K260Q, M141V_W253L_K260Q, M141I_W253H_K260Q, and M141V_W253M_K260Q. Group b2: H182M_W253H_K260E, M141V_W253V_K260Q, H182L_W253H_K260Q, M141V_W253H_K260E, H182L_W253Q_K260Q, M141L_W25 3H_K260E, H182M_W253Q_K260E, M141V_W253F_K260Q, M141V_W253L_K260E, M141V_W253T_K260Q, M141Y_H182F_K260E, H 182M_W253M_K260E, H182L_W253M_K260Q, M141Y_W253H_K260E, M141Y_W253Q_K260E, M141V_H182M_K260Q, H182M_W253L_K260E, H182F_W253H_K260E, M141A_H182F_K260Q, M141Y_W253M_K260E, H182M_W253Q_K260Q, and H182M_W253M_K260Q.Group b3: H182L_W253T_K260Q, M141Y_W253S_K260E, M141V_H182F_K260E, M141A_H182M_K260E, M141T_H182L_K260Q, M141Y_W25 3T_K260E, H182M_W253L_K260Q, M141V_H182F_K260Q, M141S_W253H_K260E, M141T_W253H_K260E, H182M_W253H_K260Q, M 141A_H182L_K260Q, H182M_W253F_K260Q, M141A_H182M_K260Q, M141A_W253Q_K260E, M141A_H182L_K260E, M141A_W253H_K260E, M141V_W253E_K260Q, M141A_W253M_K260E, M141A_W253H_K260Q, M141K_H182L_W253H, and M141T_H182M_K260Q. Group b4: M141A_W253M_K260Q, and M141A_H182F_K260E.

[0202] The polypeptide may, for example, comprise a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of Groups b1 to b4, preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of Groups b1 to b3, more preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of Groups b1 and b2, and most preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from Group b1.

[0203] The polypeptide may comprise an amino acid sequence selected from the group consisting of Groups b1 to b4, preferably comprises an amino acid sequence selected from the group consisting of Groups b1 to b3, more preferably comprises an amino acid sequence selected from the group consisting of Groups b1 and b2, and most preferably comprises an amino acid sequence selected from Group b1.

[0204] 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.

[0205] 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: 12. The SBP tag may be, for example, the amino acid sequence represented by SEQ ID NO: 13.

[0206] 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.

[0207] 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.

[0208] 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: 12 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.

[0209] 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: 13 attached to the C-terminus of the amino acid sequence a1, a2, a3, a4, or a5, a His tag represented by SEQ ID NO: 12, 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] In one embodiment, the polypeptide has catalytic activity for an intermolecular reductive amination reaction between compound A and compound B, or an intramolecular reductive amination reaction between compound C, under at least one reaction condition.

[0217] In measuring catalytic activity, a combination of methylamine as compound A and sodium 4-(2-chlorophenyl)-2-oxobutanoate as compound B can be used. In this case, the catalytic activity of the polypeptide can be evaluated using MeHph(2-Cl) synthesis activity as an indicator.

[0218] In another embodiment, the catalytic activity can be measured using a combination of ethylamine as compound A and sodium phenylpyruvate as compound B. In this case, the catalytic activity of the polypeptide can be evaluated using EtPhe synthesis activity as an index.

[0219] In another embodiment, the catalytic activity can be measured using a combination of methylamine as compound A and sodium 2-cyclopentyl-2-oxoacetate as compound B. In this case, the catalytic activity of the polypeptide can be evaluated using MeGly(cPent) synthesis activity as an index.

[0220] In another embodiment, the catalytic activity can be measured using a combination of ammonia as compound A and phenylpyruvic acid as compound B. In this case, the catalytic activity of the polypeptide can be evaluated using Phe synthesis activity as an index.

[0221] In one embodiment, the catalytic activity of a polypeptide can be evaluated using the reaction conditions described in the Examples. For example, the concentrations of each compound in the reaction solution at the start of the reaction can be: compound B or a salt thereof 50 mM, D(+)-glucose 100 mM, compound A or a salt thereof 500 mM, phosphate buffer 100 mM, NADPH 1 mM, GDH solution 0.002 unit / μL, polypeptide to be evaluated 2.5 μM, and compound D or compound D′ 20 v / v % or 20 w / v %.

[0222] In one embodiment, catalytic activity can be evaluated by determining the yield of amino acid produced by the reductive amination reaction after 3 hours or 23 hours have passed, starting the reaction under conditions of 25°C or 37°C, pH 8 to 9, and containing 50 mM sodium 4-(2-chlorophenyl)-2-oxobutanoate, sodium phenylpyruvate, or sodium 2-cyclopentyl-2-oxoacetate, 100 mM D(+)-glucose, 500 mM methylamine or ethylamine, 100 mM phosphate buffer, 1 mM NADPH, 0.002 unit / µL GDH solution, 2.5 µM polypeptide to be evaluated, and 20 v / v % or 20 w / v % compound D or compound D' in a reaction solution.

[0223] The catalytic activity is calculated based on the amount of amino acid (hereinafter, sometimes referred to as "target product") produced by the reductive amination reaction. The amount of target product can be measured using a liquid chromatograph mass spectrometer (LCMS). Specifically, the measurement can be performed under the conditions described in the Examples below.

[0224] The polypeptide according to this embodiment can be produced, for example, by a method comprising culturing a recombinant cell that is a transformant according to a conventional method and isolating or purifying the polypeptide of interest from the culture obtained by culturing. The produced polypeptide can also be used without isolation or purification from the culture obtained by culturing, or after only crude purification.

[0225] The target polypeptide can be obtained from the culture by any commonly used isolation and purification method, including, for example, methods that utilize solubility such as salting out and solvent precipitation, methods that utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, methods that utilize charge such as ion exchange chromatography and hydroxylapatite chromatography, methods that utilize specific affinity such as affinity chromatography, methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography, and methods that utilize differences in isoelectric point such as isoelectric focusing.

[0226] When the target polypeptide is present in the periplasm or cytoplasm of cultured recombinant cells (such as E. coli), the culture is subjected to a conventional method such as filtration or centrifugation to collect the bacterial bodies or cells, which are then suspended in an appropriate buffer solution. The cell walls and / or cell membranes of the cells are disrupted by, for example, ultrasonic waves, lysozyme, or freeze-thawing, and a fraction containing the target polypeptide is obtained by centrifugation, filtration, or the like. The fraction is then purified by Triton X-ray diffraction. ＴＭ The target polypeptide can be isolated and purified from the crude solution by conventional methods such as those described above.

[0227] The polypeptide according to this embodiment can be produced using known gene recombination techniques as described above, but can generally be prepared as follows: First, a plasmid containing a wild-type polypeptide gene is used as a template, and site-specific mutations are introduced into specific positions and specific amino acids by PCR using primers from the template. The template plasmid is then digested with a restriction enzyme and transformed into Escherichia coli or the like, and the desired mutation-introduced plasmid is cloned.

[0228] To introduce a second amino acid mutation, the plasmid containing the mutation at a specific position is used as a template, and site-directed mutagenesis using a primer is repeated in the same manner as above to construct a plasmid DNA encoding a two-amino acid substitution.

[0229] The prepared DNA is transformed into Escherichia coli BL21 strain or the like together with a plasmid such as pREP4 encoding the lac repressor (Laci), and the resulting transformant is isolated and cultured, followed by induction of expression with IPTG. The resulting strain is then disrupted, and the target polypeptide is purified by passing the supernatant through an affinity column utilizing a His tag, for example.

[0230] Alternatively, the polypeptide can be prepared by the following method: a nucleotide sequence encoding the desired polypeptide is gene-synthesized, transferred into an expression vector, and then the protein is expressed and purified using an affinity column utilizing various purification tags.

[0231] The method for producing the polypeptide according to this embodiment is not limited to the above-described method, and various gene manipulation techniques can be used, such as well-known point mutation techniques, gene synthesis techniques, and methods for introducing modified fragments using restriction enzymes. Expression is not limited to E. coli, and animal cells and cell-free translation systems can also be used. Furthermore, the purification method is not limited to affinity columns using polyhistidine, and various peptide tags and purification columns can be used.

[0232] <Reducing Agent> The reducing agent may be one or more selected from the group consisting of reduced nicotinamide adenine dinucleotide phosphate (NADPH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide (NADH), and oxidized nicotinamide adenine dinucleotide (NAD+). In one embodiment, the reducing agent may be NADPH.

[0233] Furthermore, additives that reduce NADP+ and NAD+ to NADPH and NADH, respectively, such as glucose dehydrogenase (GDH) and glucose, formate dehydrogenase and formic acid, alcohol dehydrogenase and alcohol, amino acid dehydrogenase and amino acid, and organic acid dehydrogenase (such as malate dehydrogenase) and organic acid, can also be added. That is, various additives may be further present in the reaction step, and glucose and glucose dehydrogenase may also be present.

[0234] For example, when NADPH is used as a reducing agent, the amount of NADPH used can be reduced, and therefore a reactant that reduces NADP+ (oxidized nicotinamide adenine dinucleotide phosphate) that may be generated in the reaction solution to NADPH may be used. An example of such a reactant is a combination of glucose and GDH (glucose dehydrogenase). When glucose and GDH are used together with NADPH, the amount of NADPH used can be reduced to a catalytic amount.

[0235] <Reaction Step> The reaction step is carried out in a reaction solution containing a polypeptide, a reducing agent, compound D, and compounds A and B, or compound C. The reaction solution may contain other components such as a solvent (excluding compounds corresponding to compound D). The solvent may be water or a buffer solution. Examples of buffer solutions include phosphate buffer, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane), and bicine (N,N-di(2-hydroxyethyl)glycine). The reaction step may be carried out under appropriate reaction conditions. "Appropriate reaction conditions" means conditions under which the polypeptide can exhibit catalytic activity.

[0236] The reaction step may be carried out under temperature conditions of 0°C or higher and 50°C or lower. The lower limit of the temperature may be 0°C or higher, 10°C or higher, or 20°C or higher. The upper limit of the temperature may be 50°C or lower, 40°C or lower, or 30°C or lower. The temperature may be, for example, 0°C or higher and 50°C or lower, 10°C or higher and 40°C or lower, or 20°C or higher and 30°C or lower. In one aspect, the temperature may be 25°C or 16°C. The lower limit of the temperature may be 30°C or higher. The upper limit of the temperature may be 40°C or lower. The temperature may be, for example, 30°C or higher and 40°C or lower. The temperature may be 37°C. The reaction temperature may be changed stepwise or continuously.

[0237] The reaction step may be carried out under a pH condition of 7 or more and 11 or less. The lower limit of the pH may be 7 or more, 7.5 or more, 8 or more, or 8.5 or more. The upper limit of the pH may be 11 or less, 10 or less, 9.5 or less, or 9 or less. The pH may be, for example, 7 or more and 11 or less, 8 or more and 10 or less, or 8.5 or more and 9.5 or less. In one aspect, the pH may be 8 or 9. Note that the pH here refers to the pH in the reaction solution at the start of the reaction, and pH fluctuations during the reaction are allowed. The pH fluctuation during the reaction may be within 2, within 1.5, or within 1.

[0238] In the reaction step, the concentration of compound A in the total reaction solution at the start of the reaction may be 10 mM or more and 3000 mM or less. The lower limit of the concentration of compound A in the total reaction solution may be 10 mM or more, 100 mM or more, 300 mM or more, 500 mM or more, 700 mM or more, 900 mM or more, 1100 mM or more, 1300 mM or more, 1500 mM or more, or 1700 mM or more. The upper limit of the concentration of compound A in the total reaction solution may be 3000 mM or less, 2500 mM or less, or 2000 mM or less. The concentration of compound A in the total reaction solution may be 10 mM or more and 3000 mM or less, or 500 mM or more and 2000 mM or less. In one aspect, the concentration of compound A in the total reaction solution may be 500 mM or 1750 mM.

[0239] In the reaction step, the concentration of compound B or compound C in the total amount of the reaction solution at the start of the reaction may be 0.001 mM or more and 1000 mM or less.

[0240] The concentration of compound B in the total reaction solution at the start of the reaction may be 10 mM or more and 500 mM or less. The lower limit of the concentration of compound B in the total reaction solution at the start of the reaction may be 10 mM or more, 30 mM or more, 50 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 250 mM or more, 300 mM or more, or 330 mM or more. The upper limit of the concentration of compound B in the total reaction solution at the start of the reaction may be 500 mM or less, 450 mM or less, 400 mM or less, or 380 mM or less. The concentration of compound B in the total reaction solution at the start of the reaction may be 10 mM or more and 500 mM or less, or 300 mM or more and 400 mM or less. In one aspect, the concentration of compound B in the total reaction solution at the start of the reaction may be 50 mM or 350 mM.

[0241] The concentration of compound C in the total reaction solution at the start of the reaction may be 10 mM or more and 500 mM or less. The lower limit of the concentration of compound C in the total reaction solution at the start of the reaction may be 10 mM or more, 30 mM or more, 50 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 250 mM or more, 300 mM or more, or 330 mM or more. The upper limit of the concentration of compound C in the total reaction solution at the start of the reaction may be 500 mM or less, 450 mM or less, 400 mM or less, or 380 mM or less. The concentration of compound C in the total reaction solution at the start of the reaction may be 10 mM or more and 500 mM or less, or 300 mM or more and 400 mM or less. In one aspect, the concentration of compound C in the total reaction solution at the start of the reaction may be 50 mM or 350 mM.

[0242] In the reaction step, the ratio of the number of moles of compound A to the number of moles of compound B (number of moles of compound A / number of moles of compound B) in the total amount of reaction solution at the start of the reaction may be 1 or more. The lower limit of the ratio of the number of moles of compound A to the number of moles of compound B in the total amount of reaction solution at the start of the reaction may be 1 or more, 3 or more, 5 or more, 8 or more, or 9 or more. The upper limit of the ratio of the number of moles of compound A to the number of moles of compound B in the total amount of reaction solution at the start of the reaction may be 100 or less, 50 or less, 30 or less, 20 or less, or 15 or less. The ratio of the number of moles of compound A to the number of moles of compound B in the total amount of reaction solution at the start of the reaction may be 5 or 10.

[0243] When compound D or compound D' is a liquid under conditions of 25°C and 1 atmosphere, the reaction step may be carried out under conditions where the concentration of compound D or compound D' in the reaction solution is 1 v / v% or more and 60 v / v% or less. The lower limit of the concentration of compound D or compound D' in the reaction solution may be 1 v / v% or more, 5 v / v% or more, 10 v / v% or more, 20 v / v% or more, 30 v / v% or more, or 40 v / v% or more, since this can further improve the yield. The upper limit of the concentration of compound D or compound D' in the reaction solution may be 60 v / v% or less, 50 v / v% or less, or 40 v / v% or less, since this can further improve the yield. In one embodiment, the concentration of compound D or compound D' in the reaction solution may be 20 v / v% or 10 v / v%. The concentration of compound D or compound D' in the reaction solution means the concentration of compound D or compound D' in the reaction solution at the start of the reaction.

[0244] When compound D or compound D' is solid under conditions of 25°C and 1 atmosphere, the reaction step may be carried out under conditions where the concentration of compound D or compound D' in the reaction solution is 1 w / v% or more and 60 w / v% or less. The lower limit of the concentration of compound D or compound D' in the reaction solution may be 1 w / v% or more, 5 w / v% or more, 10 w / v% or more, 20 w / v% or more, 30 w / v% or more, or 40 w / v% or more, since this can further improve the yield. The upper limit of the concentration of compound D or compound D' in the reaction solution may be 60 w / v% or less, 50 w / v% or less, or 40 w / v% or less, since this can further improve the yield. In one aspect, the concentration of compound D or compound D' in the reaction solution may be 20 w / v% or 10 w / v%. The concentration of compound D or compound D' in the reaction solution means the concentration of compound D or compound D' in the reaction solution at the start of the reaction.

[0245] The reaction step may be carried out under conditions in which the concentration of the polypeptide in the reaction solution is 0.1 μM or more and 10 μM or less. The lower limit of the polypeptide concentration in the reaction solution may be 0.5 μM or more, 1.0 μM or more, 1.5 μM or more, 2.0 μM or more, 2.3 μM or more, or 2.4 μM or more. The upper limit of the polypeptide concentration in the reaction solution may be 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2.7 μM or less, or 2.6 μM or less. In one aspect, the concentration of the polypeptide in the reaction solution may be 2.5 μM.

[0246] The reaction step may be carried out under conditions in which the concentration of the reducing agent in the reaction solution is 0.1 mM or more and 100 mM or less. The lower limit of the concentration of the reducing agent in the reaction solution may be 0.1 mM or more, 0.2 mM or more, 0.4 mM or more, 0.6 mM or more, 0.8 mM or more, or 1 mM or more. The upper limit of the concentration of the reducing agent in the reaction solution may be 100 mM or less, 80 mM or less, 60 mM or less, 40 mM or less, 20 mM or less, or 10 mM or less. In one aspect, the concentration of the reducing agent in the reaction solution may be 1 mM or 2 mM.

[0247] The yield of the amino acid produced by the reaction in the presence of compound D or compound D' may be 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, or 1.8 times or more, and may be 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less, 5 times or less, 4 times or less, 3 times or less, or 2 times or less, of the yield of the amino acid produced by the reaction in the presence of compound D or compound D' compared to the yield of the amino acid produced in the reaction step in the absence of compound D or compound D'. The yield of the amino acid produced by the reaction in the presence of compound D or compound D' can be measured at any reaction temperature and any reaction time, and can be determined, for example, by calculating the ratio of the yield of the target product under both conditions at 25°C or 37°C, 1 hour, 3 hours, or 23 hours after the start of the reaction. Specifically, the yield of the amino acid produced by the reaction in the presence of Compound D or Compound D' can be determined by dividing the yield of the amino acid produced in the reaction step in the absence of Compound D or Compound D'. Preferably, the yield is calculated from the yield at 25°C after 3 hours from the start of the reaction.

[0248] The yield of the amino acid produced in the reaction step may be 15% or more, 30% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 93% or more, or 95% or more, or may be 100% or less, 99% or less, 98% or less, 97% or less, or 96% or less.

[0249] The yield of the amino acid produced in the reaction step can be measured under the following conditions. The reaction solution contains 50 mM phenylpyruvic acid, 2-oxo-3-(p-tolyl)propanoic acid, or 2-cyclopentyl-2-oxo-acetic acid, 100 mM D(+)-glucose, 500 mM ammonia, methylamine, or ethylamine, 100 mM phosphate buffer, 1 mM NADPH, 0.002 unit / μL GDH solution, and 2.5 μM polypeptide at a concentration; when Compound D or Compound D' is used, Compound D or Compound D' is 20 v / v % if Compound D or Compound D' is liquid under conditions of 25°C and 1 atmosphere, or 20 w / v % if Compound D or Compound D' is solid under conditions of 25°C and 1 atmosphere; and the reaction is initiated under conditions of 37°C, pH 8 to 9, and the yield of the amino acid produced by the reductive amination reaction is determined after 19 hours have elapsed.

[0250] In this specification, the term "start time of the reaction" refers to the latest of the times when compound A, compound B, the polypeptide disclosed herein, and a reducing agent are added to the system when the reaction step is a step of carrying out an intermolecular reductive amination reaction between compound A and compound B, and refers to the latest of the times when compound C, the polypeptide disclosed herein, and a reducing agent are added to the system when the reaction step is a step of carrying out an intramolecular reductive amination reaction of compound C.

[0251] The yield of the amino acid produced in the reaction step is calculated by the following method. First, the reaction solution and a separately prepared calibration curve sample (a compound having the same structure as the target product or a structure showing the same UV absorption wavelength) are subjected to LCMS analysis, and a UV chart or extracted ion chromatogram is obtained for each. The UV peak area or MS peak area derived from the target product is obtained from the UV chart or extracted ion chromatogram obtained from the reaction solution. Similarly, the UV peak area or MS peak area of ​​the standard is obtained from the calibration curve sample. Based on the correspondence between the concentration of the standard contained in the calibration curve sample and the UV peak area or MS peak area, the concentration of the target product contained in the reaction solution is calculated from the UV peak area or MS peak area. When all of compound B or compound C in the reaction solution is converted to the target product, the concentration of the target product in the reaction solution is considered to be equal to the concentration of compound B or compound C in the reaction solution at the start of the reaction. The yield is calculated by dividing the concentration of the target product actually contained in the reaction solution by the concentration of compound B or compound C in the reaction solution at the start of the reaction. The reaction yield can also be calculated using the following formula by correcting the ratio of the extinction coefficients of each compound (in this specification, the reaction yield determined by this method may be referred to as the reaction conversion rate). Formula: Reaction conversion rate (%) = S １ / ((S ２ / 8.0) + S １ ) × 100 In the formula, S １ indicates the UV peak area of ​​the target product. ２ indicates the UV peak area of ​​Compound B or Compound C.

[0252] [Method for Producing Peptide Compound] The method for producing a peptide compound according to this embodiment comprises the following steps: (1) producing an amino acid by the above-described method for producing an amino acid; and (2) linking the amino acid to one or more selected from the group consisting of other amino acids and other peptides to produce a peptide compound.

[0253] The step of producing a peptide compound may be carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent. The aqueous medium may be water or a buffer solution. Examples of the buffer solution include phosphate buffer, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane), and bicine (N,N-di(2-hydroxyethyl)glycine). Examples of the organic solvent include dimethyl sulfoxide.

[0254] [Reductive Amination Reaction Accelerator] The reductive amination reaction accelerator according to this embodiment may be a compound represented by the above formula (1) that accelerates the following reaction (i) or (ii): (i) an intermolecular reductive amination reaction between compound A selected from the group consisting of compounds having an amino group and salts thereof, and compound B selected from the group consisting of compounds having a carbonyl group and salts thereof, or (ii) an intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof.

[0255] Specific embodiments of the reductive amination reaction promoter are the same as those described in the method for producing amino acids.

[0256] The reductive amination reaction accelerator may be one or more selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, dimethoxyethane, trimethylphosphine oxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide, or may be one or more selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide, or may be dimethyl sulfoxide.

[0257] The present invention as described above can also be understood as the use of a compound represented by formula (1) above for promoting the reductive amination reaction of (i) or (ii) above. The present invention as described above can also be understood as a compound represented by formula (1) above for use in promoting the reductive amination reaction of (i) or (ii) above. The present invention as described above can also be understood as the use (application) of a compound represented by formula (1) above for producing a reductive amination reaction promoter of (i) or (ii) above. These specific embodiments can be applied to the embodiments described in the method for producing an amino acid.

[0258] The following abbreviations are used in this specification:

[0259] The present invention is further illustrated by, but not limited to, the following examples.

[0260] The LCMS analysis conditions are as follows. LCMS method condition name: FA05 Apparatus: Waters Acquity UPLC / SQD Column (ID x length (mm), particle size (µm)): Aldrich Ascentis Express C18 (2.1 x 50, 2.7) Mobile phase: A) 0.1% FA H ２ O, B) 0.1% FA MeCN Gradient (A / B): 95 / 5 - 0 / 100 (1.0 min) ⇒ 0 / 100 (0.4 min) Flow rate (ml / min): 1 Column temperature: 35 ° C Wavelength: 210-400 nm PDA total

[0261] Condition name: TFA00 Apparatus: Waters H class 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 H ２ O, 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 Wavelength: 197 nm

[0262] Condition name: TFA00-QDa Apparatus: Waters H class / QDa Column: (ID x length (mm), particle size (μm)): ACQUITY UPLC HSS T3 (2.1 x 50, 1.8) Mobile phase: A) 0.05% TFA H ２ O, 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 Wavelength: 197 nm

[0263] Reagents not otherwise specified were purchased from commercial suppliers.

[0264] Preparation Example 1: Preparation of wild-type NMAADH-C-His A gene was synthesized by adding a linker sequence (GGSS) and a His tag sequence (HHHHHH) to the C-terminus of SEQ ID NO: 1, and cloned into an E. coli expression vector. This expression vector was transformed into the BL21(DE3) E. coli strain and cultured. The target protein was purified from the supernatant of ultrasonically disrupted cells using a nickel column. The protein solution was dialyzed (50 mM Tris-HCl, 10% glycerol, 150 mM sodium chloride, pH 8.0) to obtain the final preparation (SEQ ID NO: 10, wild-type NMAADH-His).

[0265] SEQ ID NO: 1: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSMTCVVPHGARKPLFGTNPIAFAAPCA EHDPIVFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSW EFDWSGHPGAKTPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRRYREREVAEEEEGVAVTEQELKGLKELLG

[0266] SEQ ID NO: 10: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSMTCVVPHGARKPLFGTNPIAFAAPCAEHDPI VFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDDPKAILEGGALLPFGGHKGSALSMMMVELLAAAALTGGHFSWEFDWSGH PGAKTPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRYREREVAEEEEGVAVTEQELKGLKELLGGGSSHHHHHHH

[0267] Preparation Example 2: 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 target protein was expressed by culturing it at 18°C ​​for 2 days using Overnight Express Instant TB Medium (Novagen).

[0268] The resulting bacterial cells were collected by centrifugation and disrupted by sonication. The lysate was fractionated by centrifugation, and the supernatant fraction was purified by affinity chromatography using cComplete His-Tag Purification Resin (Roche). After collecting the fraction containing the target protein, the sample was dialyzed against 50 mM Tris-HCl (pH 8.0) / 150 mM sodium chloride / 10% glycerol to obtain the final preparation (SEQ ID NO: 11, W253H).

[0269] The amino acid sequence represented by SEQ ID NO: 8, wherein X is His: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSMTCVVPHGARKPLFGTNPIAFAAPCA EHDPIVFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSW EFDHSGHPGAKTPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRRYREREVAEEEEGVAVTEQELKGLKELLG

[0270] SEQ ID NO: 11: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSMTCVVPHGARKPLFGTNPIAFAAPCAEHDPIVFDMATSAMAHGDVQI AARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSWEFDHSGHPGAKTPWTGQLIIVIDPGKAEGQRF AQRSRELVEHMQAVGLTRMPGERRYREREVAEEEEGVAVTEQELKGLKELLGGTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGGSHHHHH

[0271] Preparation Example 3: Preparation of modified NMAADH-SBP-His Genes were synthesized by adding a streptavidin-binding peptide tag sequence (GTDEKTTGWRGGHVVEGLAGEELEQLRARLEHHPQ), a linker sequence (GGS), and a His tag sequence (HHHHHH) to the C-terminus of each of the modified enzyme sequences (SEQ ID NOS: 14 to 17), and the genes were cloned into an E. coli expression vector. This expression vector was introduced into the BL21(DE3) E. coli strain (Novagen), and the target protein was expressed by culturing it at 18°C ​​for 2 days using Overnight Express Instant TB Medium (Novagen).

[0272] The resulting cells were collected by centrifugation and disrupted by sonication. The lysate was fractionated by centrifugation, and the supernatant was purified by affinity chromatography using Ni Sepharose 6 Fast Flow (Cytiva). Fractions containing the target protein were collected and used as the final preparation (SEQ ID NO: 18, M141V_W253Q_K260E-SBP-His; SEQ ID NO: 19, M141I_W253H_K260E-SBP-His; SEQ ID NO: 20, M141V_W253L_K260Q-SBP-His; SEQ ID NO: 21, M141V_W253L_K260E-SBP-His).

[0273] SEQ ID NO: 14: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSVTCVVPHGARKPLFGTNPIAFAAPCA EHDPIVFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSW EFDQSGHPGAETPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRRYREREVAEEEEGVAVTEQELKGLKELLG

[0274] SEQ ID NO: 15: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSITCVVPHGARKPLFGTNPIAFAAPCA EHDPIVFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSW EFDHSGHPGAETPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRRYREREVAEEEEGVAVTEQELKGLKELLG

[0275] SEQ ID NO: 16: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSVTCVVPHGARKPLFGTNPIAFAAPCA EHDPIVFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSW EFDLSGHPGAQTPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRRYREREVAEEEEGVAVTEQELKGLKELLG

[0276] SEQ ID NO: 17: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSVTCVVPHGARKPLFGTNPIAFAAPCA EHDPIVFDMATSAMAHGDVQIAARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSW EFDLSGHPGAETPWTGQLIIVIDPGKAEGQRFAQRSRELVEHMQAVGLTRMPGERRRYREREVAEEEEGVAVTEQELKGLKELLG

[0277] SEQ ID NO: 18: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSVTCVVPHGARKPLFGTNPIAFAAPCAEHDPIVFDMATSAMAHGDVQI AARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAAALTGGHFSWEFDQSGHPGAETPWTGQLIIVIDPGKAEGQRF AQRSRELVEHMQAVGLTRMPGERRYREREVAEEEEGVAVTEQELKGLKELLGGTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGGSHHHHH

[0278] SEQ ID NO: 19: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSITCVVPHGARKPLFGTNPIAFAAPCAEHDPIVFDMATSAMAHGDVQI AARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSWEFDHSGHPGAETPWTGQLIIVIDPGKAEGQRF AQRSRELVEHMQAVGLTRMPGERRYREREVAEEEEGVAVTEQELKGLKELLGGTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGGSHHHHH

[0279] SEQ ID NO: 20: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSVTCVVPHGARKPLFGTNPIAFAAPCAEHDPIVFDMATSAMAHGDVQI AARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAALTGGHFSWEFDLSGHPGAQTPWTGQLIIVIDPGKAEGQRF AQRSRELVEHMQAVGLTRMPGERRYREREVAEEEEGVAVTEQELKGLKELLGGTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGGSHHHHH

[0280] SEQ ID NO: 21: MRVPFTELQSLLQAIFQRHGCSEAVARVLAHNCASAQRDGAHSHGVFRMPGYVSTLASGWVDGQATPQVSDVAAGYVRVDAAGGFAQPALAAARELLVAKARSAGIAVLAIHNSHHFAALWPDVEPFAEEGLVALSVVNSVTCVVPHGARKPLFGTNPIAFAAPCAEHDPIVFDMATSAMAHGDVQI AARAGQQLPEGMGVDADGQPTTDPKAILEGGALLPFGGHKGSALSMMVELLAAALTGGHFSWEFDLSGHPGAETPWTGQLIIVIDPGKAEGQRF AQRSRELVEHMQAVGLTRMPGERRYREREVAEEEEGVAVTEQELKGLKELLGGTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGGSHHHHH

[0281] The following reagents were used in this example:

[0282] The following reagents were prepared in advance and used as follows:

[0283]

[0284] Synthesis Example 1: Synthesis of raw material intermediate (E)-4-(2-chlorophenyl)-2-oxobut-3-enoic acid Sodium pyruvate (2.42 g, 0.022 mol) was added to a mixed solution of 1N aqueous sodium hydroxide (60 ml) and ethanol (3 ml) at 0°C, followed by dropwise addition of 2-chlorobenzaldehyde (2.25 ml, 0.02 mol) over 20 minutes. The reaction mixture was stirred at 0°C for 2 hours. Water (160 ml) was then added to the reaction mixture, which was then washed with toluene (100 ml). 5N hydrochloric acid was added dropwise to the resulting aqueous layer at 0°C until the pH reached 1, and the resulting solution was stirred for 30 minutes. The precipitate formed in the solution was collected by filtration and washed with cold water. The resulting wet solid was dried under reduced pressure with heating to obtain (E)-4-(2-chlorophenyl)-2-oxobut-3-enoic acid (2.7 g, 0.013 mol, 64% yield). LCMS (ESI) m / z = 209.0 (M-H). － Retention time: 0.52 minutes (Analysis conditions FA05)

[0285] Synthesis Example 2: Synthesis of starting material sodium 4-(2-chlorophenyl)-2-oxobutanoate A suspension of (E)-4-(2-chlorophenyl)-2-oxobut-3-enoic acid (1.00 g, 4.75 mol) and palladium / carbon (0.20 g, 1.88 mol) in methanol (20 ml) and diphenyl sulfide (7.97 μl, 0.047 mmol) was stirred at room temperature for 1 hour under a hydrogen atmosphere. The reaction solution was filtered, and the resulting filtrate was concentrated under reduced pressure. A 1N aqueous solution of sodium hydroxide (15 ml) was added to the resulting residue, and the mixture was washed twice with toluene (15 ml). The aqueous layer was stirred at 0°C for 30 minutes, and the precipitate that formed in the solution was collected by filtration and washed with cold water. The resulting wet solid was dried under reduced pressure with heating to give sodium 4-(2-chlorophenyl)-2-oxobutanoate (847 mg, 3.61 mmol, 75% yield). LCMS (ESI) m / z=211.0 (MH) － Retention time: 0.59 minutes (Analysis conditions FA05)

[0286] Evaluation Example 1-1: Evaluation of additives in synthesis of MeHph(2-Cl) 1-1 Sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), methylamine solution (final concentration 500 mM), phosphate buffer (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), any of the additives listed in Table 5 (DMSO, NMP, formamide, 3-methyl-2-oxazolidinone, oxanolidinone, ethanol, dioxane, NMP, 2-propanol, MeCN, THF, TMSO ２ In the case of ３ PO, DMSO ２ , DESO ２ In the case of (1), a mixed solution (pH 8-9) of wild-type NMAADH-C-His (final concentration 2.5 μM) and ultrapure water was prepared and incubated for 3 hours at 25°C or 37°C. For comparison, ultrapure water was added instead of the additive in the absence of additive (0 v / v%) condition.

[0287] After incubation, a 19-fold volume of hydrochloric acid-containing DMSO solution was added to the reaction solution so that the final concentration of hydrogen chloride added was 300 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmos Spin Filter H, Nacalai Tesque) to prepare a sample for LCMS analysis, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area at 254 nm of the target product, MeHph(2-Cl), was calculated to determine the reaction yield. The results are shown in Table 5. LCMS (ESI) m / z = 228.1 (M+H) ＋ Retention time: 0.38 minutes (Analysis conditions FA05)

[0288]

[0289] Evaluation Example 1-2: Evaluation of additives in synthesis of MeHph(2-Cl) 1-2 Evaluation was carried out in the same manner as in Evaluation Example 1-2, except that one of the additives (20 v / v %) listed in Table 6 was used and the temperature (reaction temperature) during the 3-hour incubation was set to 25° C. The results are shown in Table 6.

[0290] Evaluation Example 2: Evaluation of the Amount of Additive Added Under the same reaction conditions as in Evaluation Example 1, at 25°C or 37°C, only the content of DMSO as an additive relative to the reaction solution was varied from 0 v / v% to 40 v / v%, and the effect on the reaction depending on the amount added was evaluated. Under the 0 v / v% condition, ultrapure water was added instead of the additive. The yield after 3 hours of reaction was evaluated. Samples for LCMS analysis were prepared in the same manner as in Evaluation Example 1, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area at 254 nm of the target product MeHph(2-Cl) was calculated to determine the reaction yield. The results are shown in Table 7. LCMS (ESI) m / z = 228.1 (M+H) ＋ Retention time: 0.38 minutes (Analysis conditions FA05)

[0291]

[0292] Evaluation Example 3: Evaluation of the Effect of Additives on EtPhe Synthesis The reaction was evaluated under the same conditions as in Evaluation Example 1, except that phenylpyruvic acid (final concentration 50 mM) was used instead of sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50 mM), and ethylamine solution (final concentration 500 mM) was used instead of methylamine solution (final concentration 500 mM). DMSO was added as an additive to a concentration of 20 v / v% relative to the total reaction solution. A sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area at 254 nm of the target product, EtPhe, was calculated to determine the reaction yield. The reaction yields were determined after 3 hours and 23 hours at two reaction temperatures of 25°C and 37°C. The results are shown in Table 8. LCMS (ESI) m / z = 194.1 (M+H) ＋ Retention time: 0.30 minutes (Analysis conditions FA05)

[0293] Evaluation Example 4: Evaluation of the Effect of Additives on MeGly(cPent) Synthesis The reaction was evaluated under the same conditions as in Evaluation Example 1, except that 2-cyclopentyl-2-oxoacetate sodium (final concentration 50 mM) was used instead of 4-(2-chlorophenyl)-2-oxobutanoate sodium (final concentration 50 mM), and DMSO was added as an additive at 20 v / v% relative to the total reaction solution volume. The reaction was evaluated by determining the reaction yield after 24 hours at two reaction temperatures of 25°C and 37°C. After incubation, a 7-fold volume of aqueous hydrochloric acid solution was added to the reaction solution so that the final concentration of hydrogen chloride added was 200 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmos Spin Filter H, Nacalai Tesque) to prepare a sample for LCMS analysis, and LCMS measurement (analysis conditions: FA05) was performed. Target compound MeGly(cPent) 158.1 (M+H) ＋ The mass peak area of ​​the compound was calculated to determine the reaction yield. The results are shown in Table 9. LCMS (ESI) m / z = 158.1 (M+H) ＋ Retention time: 0.23 minutes (Analysis conditions FA05)

[0294] Evaluation Example 5: Reaction example using modified enzyme NMAADH (W253H) Under the same conditions as the reaction in Evaluation Example 1, the reaction was evaluated using the W253H-SBP-His solution prepared in Preparation Example 2 instead of wild-type NMAADH-C-His. The reaction was evaluated after 3 hours at two reaction temperatures of 25°C and 37°C. A sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and LCMS measurement (analysis conditions: FA05) was carried out. The UV peak area at 254 nm of the target product MeHph(2-Cl) was calculated to determine the reaction yield. The results are shown in Table 10. LCMS (ESI) m / z = 228.1 (M+H) ＋ Retention time: 0.38 minutes (Analysis conditions FA05)

[0295] Evaluation Example 6: Evaluation of additives in MeHph(2-Cl) synthesis 2 A mixed solution of sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 10 mM), D(+)-glucose (final concentration 20 mM), methylamine solution (final concentration 100 mM), phosphate buffer (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), additive DMSO (20 v / v%), wild-type NMAADH-C-His (final concentration 2.5 μM), and ultrapure water was prepared and incubated at 25° C. for 1 hour. For comparison, ultrapure water was added instead of the additive under additive-free (0 v / v%) conditions.

[0296] After incubation, a 7-fold volume of hydrochloric acid-containing DMSO solution was added to the reaction solution so that the final concentration of hydrogen chloride added was 300 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmos Spin Filter H, Nacalai Tesque) to prepare a sample for LCMS analysis, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area at 254 nm of the target product MeHph(2-Cl) was calculated to determine the reaction yield. The results are shown in Table 11. LCMS (ESI) m / z = 228.1 (M+H) ＋ Retention time: 0.38 minutes (Analysis conditions FA05)

[0297] Evaluation Example 7 Evaluation of Additives in MeGly(cPent) Synthesis Using Modified Enzyme NMAADH(W253H) Sodium 2-cyclopentyl-2-oxoacetate (final concentration 100 mM), D(+)-glucose (final concentration 200 mM), methylamine hydrochloride (final concentration 500 mM), NADPH (final concentration 2.0 mM), and N,N-di(2-hydroxyethyl)glycine (final concentration 100 mM) were dissolved in distilled water, and the additive DMSO (0 to 40 v / v%) was added, followed by adjusting the pH to 8.8 to 8.9 with 5 M aqueous sodium hydroxide solution. A GDH solution (0.10 units / μL, 1x TNG solution, final concentration 0.0040 units / μL) and a modified NMAADH W253H-SBP-His solution (0.67 mM, final concentration 5.0 μM) were added, and the mixture was incubated at 25° C. for 24 hours.

[0298] 50 μL of the reaction solution during incubation was sampled, and 750 μL of methanol and 200 μL of 1 M hydrochloric acid were added. The resulting solution was filtered through a 0.5 μm PTFE membrane filter (DISMIC, ADVANTEC) and used as a sample for LC analysis, whereupon LC measurement (analysis conditions: TFA00) was carried out. The UV peak area at 197 nm of LC was measured, and the reaction conversion rate was calculated by correcting the ratio of the extinction coefficients of each compound based on the following formula X. The progress of the reaction conversion rate is shown in Table 12. Retention time: 1.1 minutes (analysis conditions: TFA00). The resulting solution was also subjected to LCMS measurement (analysis conditions: TFA00-QDa), and the MS peak of MeGly(cPent) was confirmed. Retention time: 1.5 minutes (analysis conditions: TFA00-QDa). LCMS (ESI) m / z = 158.1 (M+H) ＋ Formula X: Reaction conversion rate (%) = S １ａ / ((S ２ａ / 8.0) + S １ａ ) × 100 In formula X, S １ａ indicates the UV peak area of ​​MeGly(cPent). ２ａ indicates the UV peak area of ​​sodium 2-cyclopentyl-2-oxoacetate.

[0299] Evaluation Example 8: Evaluation of Additives in Phe Synthesis The reaction was evaluated under the same conditions as in Evaluation Example 1, except that phenylpyruvic acid (final concentration 50 mM) was used instead of sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50 mM), and ammonia solution (final concentration 500 mM) was used instead of methylamine solution (final concentration 500 mM). DMSO was added as an additive at 20 v / v% of the total reaction solution. For comparison, ultrapure water was added instead of the additive in the absence of additive (0 v / v%). A sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area at 254 nm of the target product, Phe, was calculated to determine the reaction yield. The reaction yield was determined after 3 hours and 5 hours at two reaction temperatures of 25°C and 37°C. The results are shown in Table 13. LCMS (ESI) m / z = 166.1 (M+H) ＋ Retention time: 0.27 minutes (Analysis conditions FA05)

[0300] Evaluation Example 9: Evaluation of additives in Phe synthesis using modified enzyme NMAADH (W253H)

[0043] A mixed solution (pH 8-9) of phenylpyruvic acid (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), ammonia solution (final concentration 500 mM), N,N-di(2-hydroxyethyl)glycine buffer solution (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), additive DMSO (20% v / v relative to the reaction solution), modified NMAADH W253H-SBP-His (final concentration 2.5 μM), and ultrapure water was prepared and incubated at 25°C, and the reaction was evaluated. For comparison, ultrapure water was added instead of the additive under conditions without additives (0% v / v). Samples for LCMS analysis were prepared in the same manner as in Evaluation Example 1, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area of ​​the target compound Phe at 254 nm was calculated to determine the reaction yield. The reaction yield was determined after 3 hours and 5 hours at a reaction temperature of 25°C. The results are shown in Table 14. LCMS (ESI) m / z = 166.1 (M+H) ＋ Retention time: 0.27 minutes (Analysis conditions FA05)

[0301] Evaluation Example 10: Evaluation of additives in MeHph(2-Cl) synthesis 3 A mixed solution containing sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), methylamine solution (final concentration 500 mM), N,N-di(2-hydroxyethyl)glycine buffer solution (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), additive DMSO (20 v / v%), modified NMAADH W253H-SBP-His solution (final concentration 2.5 μM), and ultrapure water was prepared and incubated at 16° C. for 1 hour. For comparison, ultrapure water was added instead of the additive under conditions without additive (0 v / v%).

[0302] After incubation, a 19-fold volume of hydrochloric acid-containing DMSO solution was added to the reaction solution so that the final concentration of hydrogen chloride added was 300 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmos Spin Filter H, Nacalai Tesque) to prepare a sample for LCMS analysis, and LCMS measurement (analysis conditions: FA05) was performed. The UV peak area at 254 nm of the target product MeHph(2-Cl) was calculated to determine the reaction yield. The results are shown in Table 15. LCMS (ESI) m / z = 228.1 (M+H) ＋ Retention time: 0.38 minutes (Analysis conditions FA05)

[0303] Evaluation Example 11: Evaluation of additives in EtPhe synthesis using modified NMAADH enzyme

[0113] A mixed solution (pH 8-9) of phenylpyruvic acid (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), ethylamine solution (final concentration 500 mM), N,N-di(2-hydroxyethyl)glycine buffer solution (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), additive DMSO (20 v / v% relative to the reaction solution), various modified NMAADH-SBP-His solutions (final concentration 2.5 μM), and ultrapure water was prepared and incubated at 37°C to evaluate the reaction. The five types of modified NMAADH-SBP-His solutions prepared in Preparation Examples 2 and 3 (M141V_W253Q_K260E-SBP-His solution, M141I_W253H_K260E-SBP-His solution, M141V_W253L_K260Q-SBP-His solution, M141V_W253L_K260E-SBP-His solution, and W253H-SBP-His solution) were used for evaluation. For comparison, ultrapure water was added instead of the additive in the absence of additives (0 v / v%). Samples for LCMS analysis were prepared in the same manner as in Evaluation Example 1, and LCMS measurements (analysis conditions: FA05) were performed. The UV peak area at 254 nm of the target compound, EtPhe, was calculated, and the reaction yield was determined. The reaction yield was determined after 2 hours, 4 hours, and 24 hours at a reaction temperature of 37° C. The results are shown in Tables 16 to 20. LCMS (ESI) m / z=194.1 (M+H) ＋ Retention time: 0.30 minutes (Analysis conditions FA05)

[0304] Evaluation Example 12 Evaluation of Additives in the Synthesis of (S)-3-(4,4-Difluorocyclohexyl)-2-(methylamino)propanoic Acid Using Modified NMAADH Enzyme A mixed solution containing 3-(4,4-difluorocyclohexyl)-2-oxopropanoic acid (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), methylamine solution (final concentration 500 mM), N,N-di(2-hydroxyethyl)glycine buffer solution (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), additive DMSO (20 v / v%), modified NMAADH W253H-SBP-His solution (final concentration 2.5 μM), and ultrapure water was prepared and incubated at 37° C. for 1 hour. For comparison, ultrapure water was added instead of the additive in the absence of additive (0 v / v %) condition.

[0305] After incubation, a hydrochloric acid solution in an amount three times the volume of the reaction solution was added to the reaction solution so that the final concentration of hydrogen chloride added was 200 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmos Spin Filter H, Nacalai Tesque) to prepare a sample for LCMS analysis, and LCMS measurement (analysis conditions: FA05) was carried out. The target compound (S)-3-(4,4-difluorocyclohexyl)-2-(methylamino)propanoic acid had an affinity of 222.1 (M+H). ＋ The mass peak area of ​​the compound was calculated to determine the reaction yield. The results are shown in Table 21. LCMS (ESI) m / z = 222.1 (M+H) ＋ Retention time: 0.36 minutes (Analysis conditions FA05)

[0306] Synthesis Example 3: Synthesis of (2S,3S)-2-amino-3-phenyl-butanoic acid using modified enzyme W253H-SBP-His and additive (dimethyl sulfoxide) The following reagents were used in this example. Reagents not listed in Table 3 were used.

[0307] １ ​H-NMR analysis was performed using the following equipment and deuterated solvent: Equipment: AVANCE III HD 400 SMART-BBFO probe (400 MHz, Bruker) Deuterated solvent: Trifluoroacid-d １

[0308] The following abbreviations are used in this example: A mixed solution of sodium 2-oxo-3-phenylbutanoate (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), ammonia solution (final concentration 500 mM), N,N-di(2-hydroxyethyl)glycine buffer solution (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 unit / μL), additive DMSO (20 vol%), the modified enzyme W253H-SBP-His prepared in Preparation Example 2 (final concentration 20 μM), and ultrapure water was prepared, and the mixture was incubated at 37°C for 47 hours. To the reaction solution after incubation, 600 μL of 5 mol / L hydrochloric acid was added, and the resulting solution was purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution = 100 / 0 → 95 / 5) to obtain (2S,3S)-2-amino-3-phenyl-butanoic acid (I) (16.5 mg, 18%). LCMS (ESI) m / z = 180.1 (M+H) ＋ Retention time: 0.31 minutes (Analysis conditions FA05) １ H-NMR (400MHz, Trifluoroacid-d １ , 298K) δ 7.32-7.21 (3H, m), 7.18-7.16 (2H, m), 4.94 (1H, d, J = 5.6 Hz), 3.55-3.48 (1H, m), 1.45 (1H, d, J = 6.8 Hz). The compound (2S, 3S)-2-amino-3-phenyl-butanoic acid (I) obtained in this reaction and the purchased standard compounds (2S, 3R)-2-amino-3-phenyl-butanoic acid hydrochloride (II), (2S, 3S)-2-amino-3-phenyl-butanoic acid hydrochloride (III), and (2R, 3R)-2-amino-3-phenyl-butanoic acid hydrochloride (IV) １ The stereostructure of the obtained compound was identified by comparing the H-NMR and chiral HPLC analytical data. １From the comparison of the characteristic 2- and 3-position proton peaks in H-NMR analysis (FIG. 1) and the comparison of the chiral HPLC analysis results (FIG. 1 and Table 24), the structure of compound (I) obtained in this reaction was identified as (2S,3S)-2-amino-3-phenyl-butanoic acid.

[0309] The chiral HPLC analysis conditions are as follows: Apparatus: SHIMADZU Nexera X3 Column (ID x length (mm), particle size (μm)): DAICEL CHIRALPAK ZWIX (+) (3.0 x 150, 3.0) Mobile phase: MeOH:MeCN:H containing 50 mM formic acid and 25 mM diethylamine ２ 0 (49:49:2, v / v) solution Elution method: isocratic Analysis time: 10 minutes Flow rate (ml / min): 0.5 Column temperature: 25°C Wavelength: 254 nm

[0310] (V) in FIG. 2 and Table 24 was prepared by mixing purchased standard compounds (III) and (IV) in a ratio of (III):(IV)=7:3.

[0311] Synthesis Example 4: Synthesis of (S)-3,3-dimethyl-2-(methylamino)butyric acid using modified enzyme W253H-SBP-His and additive (dimethyl sulfoxide) LC / MS was performed under the following analytical conditions. HPLC method: Apparatus: Waters AQUITY UPLC H-Class / QDa Column: ACQUITY UPLC HSS T3 2.1 x 50 mm, 1.8 μm Solvent: A) 0.05% TFA-H ２ O, B) 0.05% TFA-CH ３ CN Gradient: 0% B (0 min) → 98% B (5.0 min) → 98% B (6.0 min) → 0% B (6.01 min) → 0% B (8.0 min) Flow rate: 0.5 mL / min Injection volume: 0.5 μl Temperature: 30° C. Wavelength: 197 nm

[0312] In this example, the reagents listed in the table below were used. Reagents not listed were those listed in Table 3.

[0313] Oxidized nicotinamide adenine dinucleotide phosphate (50 mg) was dissolved in 0.4 mol / L N,N-di(2-hydroxyethyl)glycine solution (1.0 mL, pH 9.0) to prepare an oxidized nicotinamide adenine dinucleotide phosphate solution (Solution A). D-glucose dehydrogenase (50 mg) was dissolved in 0.4 mol / L N,N-di(2-hydroxyethyl)glycine solution (1.0 mL, pH 9.0) to prepare a D-glucose dehydrogenase solution (Solution B). 3,3-Dimethyl-2-oxobutyric acid (50 mg, 0.38 mmol), N,N-di(2-hydroxyethyl)glycine (130 mg, 0.77 mmol), methylamine hydrochloride (130 mg, 1.9 mmol), and D-glucose (140 mg, 0.77 mmol) were dissolved in distilled water (0.80 mL) and dimethyl sulfoxide (0.20 mL). A 50 w / v% aqueous sodium hydroxide solution (88 μL) was added, and the pH was adjusted to 9.0 to prepare a substrate solution (Solution C). Solution A (59 μL, 3.8 μmol) and Solution B (10 μL, 1.0 wt%) were added to Solution C, and modified enzyme solution (1.0 to 100 wt%, added as a 73.3 mg / mL solution, respectively) was added, and the mixture was stirred at an external temperature of 25 ° C for 17 hours, followed by shaking at an external temperature of 37 ° C for 24 hours. The yield of (S)-3,3-dimethyl-2-(methylamino)butyric acid in the reaction solution was calculated from the UV absorption peak area at 197 nm in LC / MS using (S)-2-amino-3,3-dimethylbutyric acid as a standard. The results are shown in Table 26.

Claims

1. A method for producing amino acids, comprising the steps of carrying out the following reaction (i) or (ii) in the presence of a polypeptide containing an amino acid sequence having 90% or more identity with the amino acid sequence represented by Sequence ID No. 1, a reducing agent, and a compound D represented by the following formula (1): (i) Intermolecular reductive amination reaction between compound A, selected from the group consisting of compounds having an amino group and salts thereof, and compound B, selected from the group consisting of compounds having a carbonyl group and salts thereof. (ii) Intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof. 【Chemistry 1】 [In equation (1), v and w each independently represent either 0 or 1. One or more of v and w represent 1. T represents a carbon atom, a phosphorus atom, or a sulfur atom. The following formula (1a) 【Chemistry 2】 The functional group represented by =O, -ORd, or hydroxyl group, When both v and w are 1, the two functional groups represented by the multiple formulas (1a) may be the same or different. Ra, Rb, and Rc are each independently hydrogen atoms, C １ ~C ３ Alkyl group, alkylamino group, or -CH ２ - Represents ORd, Two or more of Ra, Rb, and Rc may be linked together with T to form a ring structure. Rd is C １ ~C ３ Represents an alkyl group, d, e, and f each independently represent either 0 or 1. One or more of d, e, and f represent 1. When both v and w are 1, one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc are not linked to each other to form a ring structure with T. If one or more of Ra, Rb, and Rc are methylamino groups, then Ra, Rb, and Rc do not link to each other to form a ring structure with T. When the functional group represented by formula (1a) is a hydroxyl group and T is a carbon atom, then v is 1, w is 0, d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.

2. In formula (1) above T is a phosphorus atom or a sulfur atom, The functional group represented by formula (1a) is =O, Ra, Rb, and Rc are all methyl groups. The manufacturing method according to claim 1.

3. The manufacturing method according to claim 1, wherein the compound D is dimethyl sulfoxide.

4. The process includes carrying out an intermolecular reductive amination reaction between compound A, selected from the group consisting of compounds having an amino group and salts thereof, and compound B, selected from the group consisting of compounds having a carbonyl group and salts thereof, or an intramolecular reductive amination reaction of compound C, selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof, under at least one reaction condition, in the presence of a polypeptide, a reducing agent, and compound D', wherein the intermolecular reductive amination reaction between compound A and compound B, or the intramolecular reductive amination reaction of compound C, is performed. A method for producing amino acids, wherein compound D' is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethylphosphine oxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol, and methylformamide.

5. The production method according to claim 4, wherein compound D' is one or more compounds selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide.

6. The manufacturing method according to claim 4, wherein the compound D' is dimethyl sulfoxide.

7. The manufacturing method according to any one of claims 1 to 6, wherein compound A is one or more compounds selected from the group consisting of compounds represented by the following formula (2) and salts thereof. 【Transformation 3】 [In equation (2), R １ and R ２ each independently 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, and these groups may be substituted, and at least one of R １ or R ２ is a hydrogen atom.]

8. In the above formula (2), R １ is a hydrogen atom, R ２ The manufacturing method according to claim 7, wherein is an alkyl group.

9. In the above formula (2), R １ is a hydrogen atom, R ２ The manufacturing method according to claim 7, wherein is a methyl group or an ethyl group.

10. The manufacturing method according to any one of claims 1 to 6, wherein compound B is one or more compounds selected from the group consisting of compounds represented by the following formula (3) and salts thereof. 【Chemistry 4】 【Transformation 5】 [In equation (3), X represents a carbon atom, Y represents a hydrogen atom or a group represented by the above formula (4). n represents an integer between 0 and 2, R ６ C is a hydrogen atom, and may be substituted. １ ~C ６ Alkyl alkyl groups, or substituted C １ ~C ６ This refers to an aryl group, a heteroaryl group having 5 to 12 atoms forming a ring that may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom. In equation (4), 【Transformation 6】 This indicates the connection point with X, m represents an integer between 0 and 6, p is either 0 or 1, q is either 0 or 1, r is either 0 or 1, Z １ This represents an alkylene group that may be substituted, or an ether bond-containing group having 1 to 6 carbon atoms, and if m is an integer of 2 or more, there are multiple Z groups. １ They may be the same or different. Z ２ This represents a carbon atom. R ３ , R ４ and R ５ Each of these is independently a hydrogen atom, or a substituted C. １ ~C ６ Alkyl alkyl groups, or substituted C ５ ~C １２ This refers to an aryl group, a heteroaryl group having 5 to 12 atoms forming a ring that may be substituted, a group containing a nitrogen atom, or a group containing an oxygen atom. R ３ , R ４ and R ５ Any two or more of these are connected to form Z ２ They may form a ring structure together, and these ring structures may be cycloalkyl groups, aryl groups, heterocyclyl groups, or heteroaryl groups, and these groups may be substituted. R ３ , R ４ , and R ５ is, Z ２ It may also form a double bond or triple bond with R ３ , R ４ , and R ５ One of the following is a double or triple bond Z ２ When combined with , one or more of p, q, or r are 0.

11. In equation (3) above, n is 0, R ６ Y is a hydrogen atom, and Y is C ３ ~C ８ Cycloalkyl groups, or C ６ ~C ９ This shows an aralkyl group, and the aralkyl group is C １ ~C ３ The manufacturing method according to claim 10, which may be substituted with an alkyl group or halogen.

12. In equation (3) above, n is 0, R ６ The manufacturing method according to claim 10, wherein is a hydrogen atom and Y is a (2-chlorophenyl)ethyl group, a phenylmethyl group, or a cyclopentyl group.

13. The manufacturing method according to any one of claims 1 to 6, wherein the above step is carried out under appropriate reaction conditions.

14. A method for producing a peptide compound, comprising the following steps. (1) A step of producing an amino acid by the manufacturing method described in any one of claims 1 to 6; and (2) A step of producing a peptide compound by linking the amino acid with one or more selected from the group consisting of other amino acids and other peptides.

15. Reductive amination accelerators represented by the following formula (1) that promote the reaction (i) or (ii) below: (i) Intermolecular reductive amination reaction between compound A, selected from the group consisting of compounds having an amino group and salts thereof, and compound B, selected from the group consisting of compounds having a carbonyl group and salts thereof. (ii) Intramolecular reductive amination reaction of compound C selected from the group consisting of compounds having an amino group and a carbonyl group and salts thereof. 【Transformation 7】 [In equation (1), v and w each independently represent either 0 or 1. One or more of v and w represent 1. T represents a carbon atom, a phosphorus atom, or a sulfur atom. The following formula (1a) 【Transformation 8】 The functional group represented by =O, -ORd, or hydroxyl group, When both v and w are 1, the two functional groups represented by the multiple formulas (1a) may be the same or different. Ra, Rb, and Rc are each independently hydrogen atoms, C １ ~C ３ Alkyl group, alkylamino group, or -CH ２ - Represents ORd, Two or more of Ra, Rb, and Rc may be linked together with T to form a ring structure. Rd is C １ ~C ３ Represents an alkyl group, d, e, and f each independently represent either 0 or 1. One or more of d, e, and f represent 1. When both v and w are 1, one or more of Ra, Rb, and Rc are methyl groups, and Ra, Rb, and Rc are not linked to each other to form a ring structure with T. If one or more of Ra, Rb, and Rc are methylamino groups, then Ra, Rb, and Rc do not link to each other to form a ring structure with T. When the functional group represented by formula (1a) is a hydroxyl group and T is a carbon atom, then d, e, and f are all 1, and Ra, Rb, and Rc are all hydrogen atoms.

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