Methods of producing amino acid and peptide compounds using enzymes and additives

Abstract

One aspect of the present invention relates to a method for producing an amino acid, the method comprising a step of performing 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 having 90% or more identity 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 amino groups and salts thereof; and a compound B selected from the group consisting of compounds having carbonyl groups 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. [Formula 1] # imgabs0 #

Description

Technical Field

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

[0002] Hitherto, methods for synthesizing amino acids from amines and keto acids using an enzyme that promotes a reductive amination reaction (hereinafter, the enzyme is referred to as a reductive amination enzyme) have been known. For example, Patent Documents 1 to 4 disclose techniques for synthesizing various amino acids using a reductive amination enzyme.

[0003] [Citation list]

[0004] [Patent Document]

[0005] [Patent Document 1] Japanese Patent Publication No. 2003-319788

[0006] [Patent Document 2] Japanese Patent Publication No. 2005-095167

[0007] [Patent Document 3] Japanese Patent Publication No. 2012-080878

[0008] [Patent Document 4] Japanese Patent Publication No. 2012-080879 Summary of the invention

[0009] [Problems to be Solved by the Invention]

[0010] However, the reaction efficiency of the reaction for obtaining an amino acid using a reductive amination enzyme is not always sufficient, and improvement of the efficiency has been desired.

[0011] An object of the present invention is to provide a method for efficiently producing amino acids.

[0012] [Methods of solving the problem]

[0013] The present invention relates to, for example, the following aspects. [1]

[0015] A method for producing an amino acid, the method 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 higher identity with the amino acid sequence represented by SEQ ID NO: 1, a reducing agent, and a compound D represented by the following formula (1):

[0016] (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;

[0017] (ii) 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:

[0018] [Formula 1]

[0019]

[0020] In the formula (1),

[0021] v and w each independently represent 0 or 1,

[0022] At least one of v and w represents 1,

[0023] T represents a carbon atom, a phosphorus atom or a sulfur atom,

[0024] A functional group represented by the following formula (1a):

[0025] [Formula 2]

[0026]

[0027] represents =O, -ORd or a hydroxyl group,

[0028] When each of v and w is 1, the two functional groups represented by the plurality of formula (1a) are the same or different,

[0029] Ra, Rb and Rc each independently represent a hydrogen atom, a C1 to C3 alkyl group, an alkylamino group or -CH2-ORd,

[0030] At least any two of Ra, Rb and Rc are optionally linked to each other to form a ring structure together with T,

[0031] Rd represents a C1 to C3 alkyl group,

[0032] d, e and f each independently represent 0 or 1,

[0033] At least one of d, e and f represents 1,

[0034] When each of v and w is 1, at least one of Ra, Rb and Rc is a methyl group, and none of Ra, Rb and Rc is connected to each other to form a ring structure together with T,

[0035] When at least one of Ra, Rb and Rc is a methylamino group, none of Ra, Rb and Rc are connected to each other to form a ring structure together with T, and

[0036] When the functional group represented by the formula (1a) is a hydroxyl group and T is a carbon atom, v is 1, w is 0, each of d, e and f is 1, and each of Ra, Rb and Rc is a hydrogen atom. [2]

[0038] The production method according to [1], wherein the polypeptide comprises an amino acid sequence having 95% or higher identity with the amino acid sequence represented by SEQ ID NO: 1. [3]

[0040] The production method according to [1] or [2], wherein the polypeptide comprises an amino acid sequence having 98% or higher identity with the amino acid sequence represented by SEQ ID NO: 1. [4]

[0042] The production method according to any one of [1] to [3], wherein in the formula (1), T is a phosphorus atom or a sulfur atom, the functional group represented by the formula (1a) is =O, and each of Ra, Rb and Rc is a methyl group. [5]

[0044] The production method according to any one of [1] to [3], wherein the compound D is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, dimethoxyethane, trimethylphosphine oxide, N,N-dimethylformamide, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol and methylformamide. [6]

[0046] The production method according to any one of [1] to [5], wherein the compound D is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone and trimethyl phosphine oxide. [7]

[0048] The production method according to any one of [1] to [6], wherein the compound D is dimethyl sulfoxide. [8]

[0050] A method for producing an amino acid, the method comprising the steps of performing 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 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 the presence of: a polypeptide having catalytic activity for the intermolecular reductive amination reaction between the compound A and the compound B, or the intramolecular reductive amination reaction of the compound C under at least one reaction condition, a reducing agent, and a compound D',

[0051] wherein the compound D' is at least one compound 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]

[0053] The production method according to [8], wherein the compound D' is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone and trimethylphosphine oxide.

[10]

[0055] The production method according to [8] or [9], wherein the compound D' is dimethyl sulfoxide.

[11]

[0057] The production method according to any one of [1] to

[10] , wherein the reducing agent is at least one compound 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]

[0059] The production method according to any one of [1] to

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

[13]

[0061] The production method according to any one of [1] to

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

[14]

[0063] The production method according to any one of [1] to

[13] , wherein the polypeptide has an amino acid sequence represented by SEQ ID NO: 1, one or more amino acid residues of which are engineered.

[15]

[0065] The production method according to any one of [1] to

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

[16]

[0067] The production method according to any one of [1] to

[15] , wherein the polypeptide comprises an amino acid sequence other than a sequence having 90% or higher sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and one amino acid residue thereof is engineered at either or both of the N-terminus and the C-terminus.

[17]

[0069] The production method 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]

[0071] The production method according to any one of [1] to

[17] , wherein the polypeptide comprises one or more 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.

[19]

[0073] The production method according to any one of [1] to

[18] , wherein the number of amino acid residues of the polypeptide is 300 or more and 400 or less.

[20]

[0075] The production method according to any one of [1] to

[19] , wherein the step is a step of subjecting the compound A and the compound B to an intermolecular reductive amination reaction. [twenty one]

[0077] The production method according to

[20] , wherein the compound A is at least one compound selected from the group consisting of compounds represented by the following formula (2) and salts thereof:

[0078] [Formula 3]

[0079]

[0080] In the formula (2),

[0081] 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, which are optionally substituted, and R 1 and R 2 At least one of them is a hydrogen atom. [twenty two]

[0083] The production method according to

[21] , wherein in the formula (2), R 1 is a hydrogen atom, and R 2 is a hydrogen atom or an alkyl group. [twenty three]

[0085] The production method according to

[21] or

[22] , wherein in the formula (2), R 1 is a hydrogen atom, and R 2 is a hydrogen atom, a methyl group or an ethyl group. [twenty four]

[0087] The production method according to any one of

[20] to

[23] , wherein the compound B is at least one compound selected from the group consisting of compounds represented by the following formula (3) and salts thereof:

[0088] [Formula 4]

[0089]

[0090] [Formula 5]

[0091]

[0092] In the formula (3),

[0093] X represents a carbon atom,

[0094] Y represents a hydrogen atom or a group represented by formula (4),

[0095] n represents an integer of 0 or more and 2 or less, and

[0096] R 6 represents a hydrogen atom, an optionally substituted C1 to C6 alkyl group, an optionally substituted C1 to C6 aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom, or a group containing an oxygen atom,

[0097] In the formula (4),

[0098] [Formula 6]

[0099]

[0100] represents the junction point with X,

[0101] m represents an integer of 0 or more and 6 or less,

[0102] p is 0 or 1,

[0103] q is 0 or 1,

[0104] r is 0 or 1,

[0105] Z 1represents an optionally substituted alkylene group, or an ether bond-containing group having 1 or more and 6 or less carbon atoms, and when m is an integer of 2 or more, a plurality of Z 1 Same or different,

[0106] Z 2 represents a carbon atom,

[0107] R 3 , R 4 and R 5 each independently represents a hydrogen atom, an optionally substituted C1 to C6 alkyl group, an optionally substituted C5 to C 12 an aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom or a group containing an oxygen atom,

[0108] R 3 , R 4 and R 5 Any two or more of are optionally connected to each other to form Z 2 Together they form a ring structure, which is optionally a cycloalkyl group, an aryl group, a heterocyclyl group or a heteroaryl group, and these groups are optionally substituted,

[0109] R 3 , R 4 and R 5 Each optionally with Z 2 To form a double or triple bond, and when R 3 , R 4 and R 5 Any of them is connected to Z through a double bond or a triple bond 2 When bonding, at least one of p, q and r is 0.

[25]

[0111] The production method according to

[24] , wherein in the formula (3), n is 0, R 6 is a hydrogen atom, Y is a C3 to C8 cycloalkyl group or a C6 to C9 aralkyl group, and the aralkyl group is optionally substituted with a C1 to C3 alkyl group or halogen.

[26]

[0113] The production method according to

[24] or

[25] , wherein in the formula (3), n is 0, R 6 is a hydrogen atom, and Y is a (2-chlorophenyl)ethyl group, a phenylmethyl group or a cyclopentyl group.

[27]

[0115] The production method according to any one of

[24] to

[26] , wherein in the formula (2), R1 is a hydrogen atom, and R 2 is a hydrogen atom or a methyl group; and in the formula (3), each of n and m is 0, Y is a group represented by the formula (4), R 3 is a (2-chlorophenyl)methyl group, R 4 , R 5 and R 6 Each of is a hydrogen atom, and each of p, q and r is 1.

[28]

[0117] The production method according to any one of

[24] to

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

[29]

[0119] The production method according to any one of

[24] to

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

[30]

[0121] The production method according to any one of

[24] to

[26] , wherein in the formula (4), R 3 , R 4 and R 5 At least one of them is not methyl.

[31]

[0123] The production method according to any one of [1] to

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

[32]

[0125] The production method according to any one of [1] to

[20] or

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

[33]

[0127] The production method according to any one of [1] to

[20] , or

[31] or

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

[34]

[0129] The production method according to any one of [1] to

[20] , or

[31] or

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

[35]

[0131] The production method according to any one of [1] to

[20] , or

[31] or

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

[36]

[0133] The production method according to any one of [1] to

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

[37]

[0135] The production method according to any one of [1] to

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

[38]

[0137] The production method according to any one of [1] to

[37] , wherein the step is performed under the condition that the ratio of the concentration of the compound A to the concentration of the compound B in the reaction solution is 1 or more.

[39]

[0139] The production method according to any one of [1] to

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

[40]

[0141] The production method according to

[39] , wherein the compound C is a compound represented by the following formula (5):

[0142] [Formula 7]

[0143]

[0144] wherein in the formula (5), n' represents an integer of 0 or more and 2 or less, and 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 are optionally substituted, R 8 represents a hydrogen atom, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom, or a group containing an oxygen atom.

[41]

[0146] The production method according to any one of [1] to

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

[42]

[0148] The production method according to any one of [1] to

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

[43]

[0150] The production method according to any one of [1] to

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

[44]

[0152] The production method according to any one of [1] to

[43] , wherein the step is performed under a temperature condition of 0°C or higher and 50°C or lower.

[45]

[0154] The production method according to any one of [1] to

[44] , wherein the step is performed at a pH of 7 or higher and 11 or lower.

[46]

[0156] The production method according to any one of [1] to

[45] , wherein if compound D or compound D' is a liquid under the conditions of 25°C and 1 atm, the step is performed under the conditions that the concentration of compound D or compound D' in the reaction solution is 1 v / v% or more and 60 v / v% or less, and if compound D or compound D' is a solid under the conditions of 25°C and 1 atm, the step is performed under the conditions that the concentration of compound D or compound D' in the reaction solution is 1 w / v% or more and 60 v / v% or less.

[47]

[0158] The production method according to any one of [1] to

[46] , wherein the step is performed under the condition that the concentration of the polypeptide in the reaction solution is 0.1 μM or more and 10 μM or less.

[48]

[0160] The production method according to any one of [1] to

[47] , wherein the step is performed under the condition that the concentration of the reducing agent in the reaction solution is 0.1 mM or more and 100 mM or less.

[49]

[0162] The production method according to any one of [1] to

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

[50]

[0164] The production method according to any one of [1] to

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

[51]

[0166] The production method according to any one of [1] to

[50] , wherein the yield of the amino acid formed in the step is measured under the following conditions:

[0167] A reaction is carried out in a reaction solution using 50 mM phenylpyruvic acid, 2-oxo-3-(p-tolyl)propionic acid or 2-cyclopentyl-2-oxo-acetic acid, 100 mM D(+)-glucose, 500 mM ammonia, methylamine or ethylamine, 100 mM phosphate buffer solution, 1 mM NADPH, 0.002 unit / μL GDH solution, a polypeptide at a concentration of 2.5 μM, and when the compound D or compound D' is used, 20 v / v% of the compound D or compound D' if the compound D or compound D' is a liquid under the conditions of 25° C. and 1 atm, or 20 w / v% of the compound D or compound D' if the compound D or compound D' is a solid under the conditions of 25° C. and 1 atm, and the yield of the amino acid formed by the reductive amination reaction is determined after 19 hours.

[52]

[0169] The production method according to any one of [1] to

[51] , wherein the catalytic activity of the polypeptide is evaluated under the following conditions:

[0170] A reaction is carried out in a reaction solution using 50 mM sodium 4-(2-chlorophenyl)-2-oxobutyrate, sodium phenylpyruvate or sodium 2-cyclopentyl-2-oxoacetate, 100 mM D(+)-glucose, 500 mM methylamine or ethylamine, 100 mM phosphate buffer solution, 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% of the compound D or compound D' at 25°C or 37°C and pH 8 to 9, and the yield of the amino acid formed by the reductive amination reaction is determined after 3 or 23 hours.

[53]

[0172] A method for producing a peptide compound, the method comprising the following steps:

[0173] (1) producing an amino acid by the production method according to any one of [1] to

[51] ; and

[0174] (2) linking the amino acid with one or more selected from the group consisting of other amino acids and peptides to produce a peptide compound.

[54]

[0176] A reductive amination reaction accelerator represented by the following formula (1), which accelerates the following reaction (i) or (ii):

[0177] (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;

[0178] (ii) 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:

[0179] [Formula 8]

[0180]

[0181] In the formula (1),

[0182] v and w each independently represent 0 or 1,

[0183] At least one of v and w represents 1,

[0184] T represents a carbon atom, a phosphorus atom or a sulfur atom,

[0185] A functional group represented by the following formula (1a):

[0186] [Formula 9]

[0187]

[0188] represents =O, -ORd or a hydroxyl group,

[0189] When each of v and w is 1, the two functional groups represented by the plurality of formula (1a) are the same or different,

[0190] Ra, Rb and Rc each independently represent a hydrogen atom, a C1 to C3 alkyl group, an alkylamino group or -CH2-ORd,

[0191] At least any two of Ra, Rb and Rc are optionally linked to each other to form a ring structure together with T,

[0192] Rd represents a C1 to C3 alkyl group,

[0193] d, e and f each independently represent 0 or 1,

[0194] At least one of d, e and f represents 1,

[0195] When each of v and w is 1, at least one of Ra, Rb and Rc is a methyl group, and none of Ra, Rb and Rc is connected to each other to form a ring structure together with T,

[0196] When at least one of Ra, Rb and Rc is a methylamino group, none of Ra, Rb and Rc are connected to each other to form a ring structure together with T, and

[0197] When the functional group represented by the formula (1a) is a hydroxyl group and T is a carbon atom, each of d, e and f is 1, and each of Ra, Rb and Rc is a hydrogen atom.

[55]

[0199] The reductive amination reaction accelerator according to

[54] , wherein in the formula (1), T represents a phosphorus atom or a sulfur atom, the functional group represented by the formula (1a) is =O, and each of Ra, Rb and Rc is a methyl group.

[56]

[0201] The reductive amination reaction accelerator according to

[54] or

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

[57]

[0203] The reductive amination reaction accelerator according to any one of

[54] to

[56] , which is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone and trimethylphosphine oxide.

[58]

[0205] The reductive amination reaction accelerator according to any one of

[54] to

[57] , which is dimethyl sulfoxide.

[0206] [Beneficial Effects of the Invention]

[0207] According to the present invention, a method for efficiently producing amino acids can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0208] Figure 1 The (2S,3S)-2-amino-3-phenyl-butyric acid (I) obtained in the reaction of the example, the purchased standard compound (2S,3R)-2-amino-3-phenyl-butyric acid hydrochloride (II) and (2S,3S)-2-amino-3-phenyl-butyric acid hydrochloride (III) 1 H-NMR measurement spectrum.

[0209] Figure 2 Chiral HPLC analysis data of (2S,3S)-2-amino-3-phenylbutyric acid (I) obtained by the example reaction and a mixture (V) obtained by mixing purchased standard compounds (III) and (IV) in the following ratio: (III): (IV) = 7:3. DETAILED DESCRIPTION

[0210] Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the embodiments given below.

[0211] In this specification, the term "one or more" means 1 or 2 or more numbers. When the term "one or more" is used in the context of a substituent of a particular group, the term means a number from 1 to the maximum number of substituents that the group accepts. Examples of the term "one or more" specifically include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and / or more numbers.

[0212] In the present specification, the term "to" indicating a range includes values ​​at both ends thereof. For example, "A to B" means a range of A or more and B or less.

[0213] In the present specification, the term "about" when used in combination with a numerical value means a value range of +10% and -10% of the numerical value.

[0214] In this specification, the term "and / or" is intended to include every combination of the terms "and" and "or" in appropriate combinations. Specifically, for example, the term "A, B, and / or C" includes the following seven designs: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B, and C.

[0215] In the present specification, an "alkyl group" is a monovalent group induced by removing an arbitrary hydrogen atom from an aliphatic hydrocarbon, and has a hydrocarbon group or a subset of a hydrocarbon group structure containing hydrogen atoms and carbon atoms but not containing heteroatoms (which refers to atoms other than carbon atoms and hydrogen atoms) or unsaturated carbon-carbon bonds in the main chain. The alkyl group includes not only a straight chain form but also a branched chain form. The alkyl group is preferably a hydrocarbon group having 1 to 20 carbon atoms (C1 to C2). 20 ; In the following, C p To C q It means an alkyl group having p to q carbon atoms, preferably C1 to C 10 The alkyl group is preferably a C1 to C6 alkyl group, and more preferably a C1 to C3 alkyl group. Examples of the alkyl group specifically include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, an isobutyl (2-methylpropyl) group, an n-pentyl group, a sec-pentyl (1-methylbutyl) group, a tert-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-dimethyl The invention also includes but is not limited to: a 1-butyl butyl group, a 2-methylbutyl group, a 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.

[0216] In this specification, an "alkenyl group" is a group having at least one double bond (two adjacent SP 2 The alkenyl group is preferably a monovalent group containing 2 to 4 carbon atoms. Depending on the configuration of the double bond and the substituent (if present), the double bond geometry may present opposite (E) or same (Z) side and cis or trans configuration. Alkenyl groups include not only straight chain forms but also branched chain forms. Alkenyl groups are preferably C2 to C 10Alkenyl groups, more preferably C2 to C6 alkenyl groups. Examples thereof specifically include vinyl groups, allyl groups, 1-propenyl groups, 2-propenyl groups, 1-butenyl groups, 2-butenyl groups (including cis and trans), 3-butenyl groups, pentenyl groups, 3-methyl-2-butenyl groups and hexenyl groups.

[0217] In the present specification, an "alkynyl group" is a monovalent group having at least one triple bond (two adjacent SP carbon atoms). The alkynyl group includes not only a straight chain form but also a branched chain form. The alkynyl group is preferably C2 to C 10 Alkynyl group, more preferably C2 to C6 alkynyl group. Its example specifically includes ethynyl group, 1-propynyl group, propargyl group, 3-butynyl group, pentynyl group, hexynyl group, 3-phenyl-2-propynyl group, 3-(2'-fluorophenyl)-2-propynyl group, 2-hydroxy-2-propynyl group, 3-(3-fluorophenyl)-2-propynyl group and 3-methyl-(5-phenyl)-4-pentynyl group.

[0218] In this specification, "cycloalkyl group" means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group. The cycloalkyl group includes a monocyclic, bicyclic or spirocyclic ring. The preferred example of the cycloalkyl group includes a C3 to C8 cycloalkyl group. Its example specifically includes a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a bicyclo [2.2.1] heptyl group and a spiro [3.3] heptyl group.

[0219] In the present specification, "aryl group" means a monovalent aromatic hydrocarbon ring. Preferred examples of the aryl group include C6 to C 10 Aryl Group. Examples of the aryl group specifically include a phenyl group and a naphthyl (eg, 1-naphthyl and 2-naphthyl) group.

[0220] In this specification, "heterocyclyl group" means a non-aromatic cyclic monovalent group containing carbon atoms and 1 to 5 heteroatoms. The heterocyclyl group may have double bonds and / or triple bonds in the ring. The carbon atoms in the ring may be oxidized to form a carbonyl group, and the ring may be a monocyclic ring or a condensed ring. In the case of a condensed ring, an aromatic ring such as a benzene ring, a pyridine ring or a pyrimidine ring may form a condensed ring with a saturated alicyclic ring such as a cyclopentane ring or a cyclohexane ring, or a saturated heterocyclic ring such as a tetrahydropyran ring, a dioxane ring or a pyrrolidine ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl group), more preferably 4 to 7 (4- to 7-membered heterocyclyl group). Examples of the heterocyclyl group specifically include an azetidinyl group, an oxiranyl group, an oxetanyl group, an azetidinyl group, a dihydrofuranyl group, a tetrahydrofuranyl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydropyridinyl group, a tetrahydropyrimidinyl 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, an 1 , a 2-thiazinyl group, a thiadiazolidinyl group, an azetidinyl group, an oxazolidinone group, a benzodioxanyl group, a benzoxazolyl group, a dioxolanyl group, a dioxanyl group, a tetrahydropyrrolo[1,2-c]imidazolyl group, a thietanyl group, a 3,6-diazabicyclo[3.1.1]heptyl group, a 2,5-diazabicyclo[2.2.1]heptyl group, a 3-oxa-8-azabicyclo[3.2.1]octyl group, a sultamyl group and a 2-oxaspiro[3.3]heptyl group.

[0221] In this specification, "protected heterocyclyl group" means a group in which one or more functional groups, such as an amino group, contained in the "heterocyclyl group" defined above are protected by any protecting group. The protected heterocyclyl group is preferably a protected 4- to 7-membered heterocyclyl group. Specific examples of the protecting group include Boc, Fmoc, Cbz, Troc and Alloc. Specific examples of the protected heterocyclyl include Boc-protected azetidine.

[0222] In this specification, "heterocyclylene group" means a divalent group obtained by removing two hydrogen atoms from a carbon atom present in a non-aromatic ring containing carbon atoms and 1 to 5 heteroatoms. The free valence in the heterocyclylene group may or may not constitute a part of a double bond. The non-aromatic ring containing carbon atoms and 1 to 5 heteroatoms may be the same as the ring described in the "heterocyclylene group". The heterocyclylene group is preferably a 4- to 7-membered heterocyclylene group. Specific examples thereof include tetrahydropyran-4-ylidene groups and azetidine-3-ylidene groups.

[0223] In this specification, "protected heterocyclylene group" means a group in which one or more functional groups, such as an amino group, contained in the "heterocyclylene group" defined above are protected by any protecting group. The protected heterocyclylene group is preferably a protected 4- to 7-membered heterocyclylene group. Specific examples of the protecting group include Boc, Fmoc, Cbz, Troc and Alloc. Specific examples of the protected heterocyclylene group include Boc-protected azetidin-3-ylidene groups.

[0224] In this specification, "heteroaryl group" means an aromatic cyclic monovalent group containing carbon atoms and 1 to 5 heteroatoms. 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 or 7 (6- to 7-membered heteroaryl group). Examples of the heteroaryl group specifically include a furanyl group, a thienyl group, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, an oxadiazolyl group, a thiadiazolyl group, a triazolyl group, a tetrazolyl group, a pyridyl group, a pyrimidinyl group, a pyridazinyl group, a pyrazinyl group, a triazinyl group, a benzofuranyl group, a benzothienyl group, a benzothiadiazolyl group, a benzothiazolyl group, a benzoxazolyl group, a benzoxadiazolyl group, a benzimidazolyl group, an indolyl group, an isoindolyl group, an indazolyl group, a quinolyl group, an isoquinolyl group, an octinyl group, a quinazolinyl group, a quinoxalinyl group, a benzodioxolyl group, an indolizinyl group and an imidazopyridyl group.

[0225] In this specification, "alkoxy group" means an oxy group bonded to the "alkyl" defined above. The alkoxy group is preferably a C1 to C6 alkoxy group. Examples of the alkoxy group specifically include a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, a pentoxy group and a 3-methylbutoxy group.

[0226] In the present specification, "alkylthio group" means a thio group bonded to the "alkyl group" defined above. The alkylthio group is preferably a C1 to C6 alkylthio group. Examples of the alkylthio group may specifically include a methylthio group, an ethylthio group, a 1-propylthio group, a 2-propylthio group, an n-butylthio group, an isobutylthio group, a sec-butylthio group and a tert-butylthio group.

[0227] In this specification, "alkenyloxy group" means an oxy group bonded to the "alkenyl group" defined above. The alkenyloxy group is preferably a C2 to C6 alkenyloxy group. Examples of alkenyloxy groups specifically include vinyloxy groups, allyloxy groups, 1-propyleneoxy groups, 2-propyleneoxy groups, 1-butenyloxy groups, 2-butenyloxy groups (including cis and trans), 3-butenyloxy groups, pentenyloxy groups and hexenyloxy groups.

[0228] In the present specification, a "cycloalkoxy group" means an oxy group bonded to the "cycloalkyl group" defined above. The cycloalkoxy group is preferably a C3 to C8 cycloalkoxy group. Examples of the cycloalkoxy group specifically include a cyclopropyloxy group, a cyclobutyloxy group, and a cyclopentyloxy group.

[0229] In the present specification, an "aryloxy group" means an oxy group bonded to the "aryl group" defined above. The aryloxy group is preferably C6 to C 10 Aryloxy group. Examples of the aryloxy group specifically include a phenoxy group, a 1-naphthoxy group and a 2-naphthoxy group.

[0230] In this specification, "amino group" means -NH2 in a narrow sense, and means -NRR' in a broad sense. Herein, R and R' are each independently selected from a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclyl group, an aryl group and a heteroaryl group, or R and R' form a ring together with the nitrogen atom bonded thereto. Examples of amino groups preferably include -NH2, a mono-C1 to C6 alkylamino group, a di-C1 to C6 alkylamino group and a 4- to 8-membered cyclic amino group.

[0231] In the present specification, "monoalkylamino group" means a group of the above-defined "amino group", wherein R is a hydrogen atom, and R' is the above-defined "alkyl group". The monoalkylamino group is preferably a mono-C1 to C6 alkylamino group, and more preferably a mono-C1 to C3 alkylamino group. Examples of the monoalkylamino group specifically include a methylamino group, an ethylamino group, an n-propylamino group, an isopropylamino group, an n-butylamino group, a sec-butylamino group, and a tert-butylamino group.

[0232] In this specification, "dialkylamino group" means a group of "amino group" defined above, wherein R and R' are each independently "alkyl group" defined above. The dialkylamino group is preferably a di-C1 to C6 alkylamino group, more preferably a di-C1 to C3 alkylamino group. Specific examples of the dialkylamino group include a dimethylamino group and a diethylamino group. In this specification, a monoalkylamino group and a dialkylamino group are also collectively referred to as "alkylamino groups". For example, a "methylamino group" may include a monomethylamino group and a dimethylamino group.

[0233] In this specification, "cyclic amino group" means a group of "amino group" defined above, wherein R and R' form a ring together with the nitrogen atom bonded thereto. The cyclic amino group is preferably a 4- to 8-membered cyclic amino group. Examples of cyclic amino groups specifically include 1-azetidinyl groups, 1-pyrrolidinyl groups, 1-piperidinyl groups, 1-piperazinyl groups, 4-morpholinyl groups, 3-oxazolidinyl groups, 1,1-dioxidothiomorpholinyl-4-yl groups and 3-oxa-8-azabicyclo[3.2.1]oct-8-yl groups.

[0234] In the present specification, "protected amino group" means an amino group protected by any protecting group. Examples of protected amino groups may specifically include amino groups protected by protecting groups such as Boc, Fmoc, Cbz, Troc, Alloc, and the like.

[0235] In this specification, "aminocarbonyl group" means a carbonyl group bonded to the "amino group" defined above. The aminocarbonyl group is preferably -CONH2, a mono-C1 to C6 alkylaminocarbonyl group, a di-C1 to C6 alkylaminocarbonyl group or a 4- to 8-membered cyclic aminocarbonyl group. Examples of aminocarbonyl groups specifically include -CONH2, a dimethylaminocarbonyl group, a 1-azetidinylcarbonyl group, a 1-pyrrolidinylcarbonyl group, a 1-piperidinylcarbonyl group, a 1-piperazinylcarbonyl group, a 4-morpholinylcarbonyl group, a 3-oxazolidinylcarbonyl group, a 1,1-dioxidothiomorpholinyl-4-ylcarbonyl group and a 3-oxa-8-azabicyclo[3.2.1]octane-8-ylcarbonyl group.

[0236] In the present specification, "alkenyloxycarbonyl group" means a carbonyl group bonded to the "alkenyloxy group" defined above. The alkenyloxycarbonyl group is preferably a C2 to C6 alkenyloxycarbonyl group. Examples of the alkenyloxycarbonyl group specifically include a vinyloxycarbonyl group, an allyloxycarbonyl group, a 1-propyleneoxycarbonyl group, a 2-propyleneoxycarbonyl group, a 1-butenyloxycarbonyl group, a 2-butenyloxycarbonyl group (including cis and trans), a 3-butenyloxycarbonyl group, a pentenyloxycarbonyl group and a hexenyloxycarbonyl group.

[0237] In the present specification, an "alkylsulfonyl group" means a sulfonyl group bonded to the above-defined "alkyl group". The alkylsulfonyl group is preferably a C1 to C6 alkylsulfonyl group. Examples of the alkylsulfonyl group specifically include a methylsulfonyl group.

[0238] In this specification, "hydroxyalkyl group" means a group in which one or more hydrogens in the "alkyl group" defined above are replaced by a hydroxyl group. The hydroxyalkyl group is preferably a hydroxy C1 to C6 alkyl group. Examples of the hydroxyalkyl group specifically include a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 2-hydroxy-2-methylpropyl group and a 5-hydroxypentyl group.

[0239] In this specification, "haloalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by halogen. The haloalkyl group is preferably a halogenated C1 to C6 alkyl group, more preferably a C1 to C6 fluoroalkyl group. Examples of haloalkyl groups specifically include difluoromethyl groups, trifluoromethyl groups, 2,2-difluoroethyl groups, 2,2,2-trifluoroethyl groups, 3,3-difluoropropyl groups, 4,4-difluorobutyl groups and 5,5-difluoropentyl groups.

[0240] In the present specification, "cyanoalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by a cyano group. The cyanoalkyl group is preferably a cyano C1 to C6 alkyl group. Examples of the cyanoalkyl group specifically include a cyanomethyl group and a 2-cyanoethyl group.

[0241] In this specification, "aminoalkyl group" means a group in which one or more hydrogens of the "alkyl group" defined above are replaced by the "amino group" defined above. The aminoalkyl group is preferably an amino C1 to C6 alkyl group. Examples of the aminoalkyl group specifically include a 1-pyridylmethyl group, a 2-(1-piperidyl)ethyl group, a 3-(1-piperidyl)propyl group and a 4-aminobutyl group.

[0242] In the present specification, "carboxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by a carboxyl group. The carboxyalkyl group is preferably a C2 to C6 carboxyalkyl group. Examples of the carboxyalkyl group specifically include a carboxymethyl group.

[0243] In the present specification, "alkenyloxycarbonylalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by the "alkenyloxycarbonyl group" defined above. The alkenyloxycarbonylalkyl group is preferably a C2 to C6 alkenyloxycarbonyl-C1 to C6 alkyl group, more preferably a C2 to C6 alkenyloxycarbonyl-C1 or C2 alkyl group. Examples of the alkenyloxycarbonylalkyl group specifically include an allyloxycarbonylmethyl group and a 2-(allyloxycarbonyl)ethyl group.

[0244] In the present specification, "alkoxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by the "alkoxy group" defined above. The alkoxyalkyl group is preferably a C1 to C6 alkoxy-C1 to C6 alkyl group, more preferably a C1 to C6 alkoxy-C1 or C2 alkyl group. Examples of alkoxyalkyl groups specifically include a methoxymethyl group, an ethoxymethyl group, a 1-propoxymethyl group, a 2-propoxymethyl group, a n-butoxymethyl group, an isobutoxymethyl group, a sec-butoxymethyl group, a tert-butoxymethyl group, a pentyloxymethyl group, a 3-methylbutoxymethyl group, a 1-methoxyethyl group, a 2-methoxyethyl group, and a 2-ethoxyethyl group.

[0245] In the present specification, "alkylthioalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by the "alkylthio group" defined above. The alkylthioalkyl group is preferably a C1 to C6 alkylthio-C1 to C6 alkyl group, more preferably a C1 to C6 alkylthio-C1 or C2 alkyl group. Examples of the alkylthioalkyl group specifically include a methylthiomethyl group, an ethylthiomethyl group, a 1-propylthiomethyl group, a 2-propylthiomethyl group, an n-butylthiomethyl group, an isobutylthiomethyl group, a sec-butylthiomethyl group, and a tert-butylthiomethyl group.

[0246] In the present specification, "alkenyloxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by the "alkenyloxy group" defined above. The alkenyloxyalkyl group is preferably a C2 to C6 alkenyloxy-C1 to C6 alkyl group, more preferably a C1 to C6 alkenyloxy-C1 or C2 alkyl group. Examples of the alkenyloxyalkyl group specifically include a vinyloxymethyl group and an allyloxymethyl group.

[0247] In this specification, "cycloalkylalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by the "cycloalkyl group" defined above. The cycloalkylalkyl group is preferably a C3 to C8 cycloalkyl-C1 to C6 alkyl group, more preferably a C3 to C6 cycloalkyl-C1 or C2 alkyl group. Examples of the cycloalkylalkyl group specifically include a cyclopropylmethyl group, a cyclobutylmethyl group, a cyclopentylmethyl group and a cyclohexylmethyl group.

[0248] In the present specification, "cycloalkoxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by the "cycloalkoxy group" defined above. The cycloalkoxyalkyl group is preferably a C3 to C8 cycloalkoxy-C1 to C6 alkyl group, more preferably a C3 to C6 cycloalkoxy-C1 or C2 alkyl group. Examples of the cycloalkoxyalkyl group specifically include a cyclopropyloxymethyl group and a cyclobutyloxymethyl group.

[0249] In this specification, "heterocyclylalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are replaced by the "heterocyclyl group" defined above. The heterocyclylalkyl group is preferably a 4- to 7-membered heterocyclyl-C1 to C6 alkyl group, more preferably a 4- to 7-membered heterocyclyl-C1 or C2 alkyl group. Examples of heterocyclylalkyl groups specifically include 2- (tetrahydro -2H- pyrans -4- bases) ethyl groups and 2- (azetidin -3- bases) ethyl groups.

[0250] In the present specification, "alkylsulfonylalkyl group" means a group in which one or more hydrogen atoms of the above-defined "alkyl group" are substituted by the above-defined "alkylsulfonyl group". The alkylsulfonylalkyl group is preferably a C1 to C6 alkylsulfonyl-C1 to C6 alkyl group, more preferably a C1 to C6 alkylsulfonyl-C1 or C2 alkyl group. Examples of the alkylsulfonylalkyl group specifically include a methylsulfonylmethyl group and a 2-(methylsulfonyl)ethyl group.

[0251] In this specification, "aminocarbonylalkyl group" means a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "aminocarbonyl group" defined above. The aminocarbonylalkyl group is preferably an aminocarbonyl-C1 to C6 alkyl group, more preferably an aminocarbonyl-C1 to C4 alkyl group. Examples of aminocarbonylalkyl groups specifically include methylaminocarbonylmethyl groups, dimethylaminocarbonylmethyl groups, tert-butylaminocarbonylmethyl groups, 1-azetidinylcarbonylmethyl groups, 1-pyrrolidinylcarbonylmethyl groups, 1-piperidinylcarbonylmethyl groups, 4-morpholinylcarbonylmethyl groups, 2-(methylaminocarbonyl)ethyl groups, 2-(dimethylaminocarbonyl)ethyl groups, 2-(1-azetidinylcarbonyl)ethyl groups, 2-(1-pyrrolidinylcarbonyl)ethyl groups, 2-(4-morpholinylcarbonyl)ethyl groups, 3-(dimethylaminocarbonyl)propyl groups and 4-(dimethylaminocarbonyl)butyl groups.

[0252] In the present specification, an "aryloxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by an "aryloxy group" defined above. The aryloxyalkyl group is preferably C6 to C 10 Aryloxy-C1 to C6 alkyl group, more preferably C6 to C 10 Aryloxy-C1 or C2 alkyl group. Examples of the aryloxyalkyl group specifically include a phenoxymethyl group and a 2-phenoxyethyl group.

[0253] In the present specification, an "aralkyl (arylalkyl) group" means a group in which at least one hydrogen atom of the "alkyl group" defined above is substituted by an "aryl group" defined above. The aralkyl group is preferably C7 to C 14 Arylalkyl groups, more preferably C7 to C 10 Aralkyl group. Examples of the aralkyl group specifically include a benzyl group, a phenethyl group and a 3-phenylpropyl group.

[0254] In the present specification, an "aralkyloxy group" means an oxy group bonded to the "aralkyl group" defined above. The aralkyloxy group is preferably C7 to C 14 Aralkyloxy groups, more preferably C7 to C 10 Aralkyloxy group. Examples of the aralkyloxy group specifically include a benzyloxy group, a phenethoxy group and a 3-phenylpropoxy group.

[0255] In the present specification, an "aralkyloxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are substituted by an "aralkyloxy group" defined above. The aralkyloxyalkyl group is preferably C7 to C 14Aralkyloxy-C1 to C6 alkyl group, more preferably C7 to C 14 Aralkyloxy-C1 or C2 alkyl group. Examples of the aralkyloxyalkyl group specifically include a benzyloxymethyl group and a 1-(benzyloxy)ethyl group.

[0256] In this specification, "heteroarylalkyl group" means a group in which at least one hydrogen atom of the "alkyl group" defined above is replaced by the "heteroaryl group" defined above. The heteroarylalkyl group is preferably a 5- to 10-membered heteroaryl-C1 to C6 alkyl group, more preferably a 5- to 10-membered heteroaryl-C1 or C2 alkyl group. Examples of heteroarylalkyl groups specifically include 3-thienylmethyl groups, 4-thiazolylmethyl groups, 2-pyridylmethyl groups, 3-pyridylmethyl groups, 4-pyridylmethyl groups, 2-(2-pyridyl)ethyl groups, 2-(3-pyridyl)ethyl groups, 2-(4-pyridyl)ethyl groups, 2-(6-quinolyl)ethyl groups, 2-(7-quinolyl)ethyl groups, 2-(6-indolyl)ethyl groups, 2-(5-indolyl)ethyl groups, and 2-(5-benzofuranyl)ethyl groups.

[0257] In the present specification, "heteroarylalkoxy group" means an oxy group bonded to the "heteroarylalkyl group" defined above. The heteroarylalkoxy group is preferably a 5- to 10-membered heteroaryl-C1 to C6 alkoxy group, more preferably a 5- to 10-membered heteroaryl-C1 or C2 alkoxy group. Examples of the heteroarylalkoxy group specifically include a 3-thienylmethoxy group and a 3-pyridylmethoxy group.

[0258] In the present specification, "heteroarylalkoxyalkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are replaced by the "heteroarylalkoxy group" defined above. The heteroarylalkoxyalkyl group is preferably a 5- to 10-membered heteroaryl-C1 to C6 alkoxy-C1 to C6 alkyl group, more preferably a 5- to 10-membered heteroaryl-C1 or C2 alkoxy-C1 or C2 alkyl group. Examples of the heteroarylalkoxyalkyl group specifically include a 3-pyridylmethoxymethyl group.

[0259] In the present specification, "heterocyclylene alkyl group" means a group in which one or more hydrogen atoms of the "alkyl group" defined above are replaced by the "heterocyclylene group" defined above. The heterocyclylene alkyl group is preferably a 4- to 7-membered heterocyclylene C1 to C6 alkyl group, more preferably a 4- to 7-membered heterocyclylene C1 to C2 alkyl group. Examples of heterocyclylene alkyl groups specifically include tetrahydro-4H-pyran-4-ylidenemethyl and azetidine-3-ylidenemethyl groups.

[0260] In this specification, "alkoxyalkenyl group" means a group in which one or more hydrogen atoms of the "alkenyl group" defined above are substituted by the "alkoxy group" defined above. The alkoxyalkenyl group is preferably a C1 to C6 alkoxy-C2 to C6 alkenyl group. Examples of alkoxyalkenyl groups specifically include (E)-4-methoxybut-2-ene-1-yl groups.

[0261] In this specification, "aminocarbonylalkenyl group" means a group in which one or more hydrogen atoms of the "alkenyl group" defined above are substituted by the "aminocarbonyl group" defined above. The aminocarbonylalkenyl group is preferably an aminocarbonyl-C2 to C6 alkenyl group. Examples of aminocarbonylalkenyl groups specifically include (E)-3-(dimethylaminocarbonylcarbonyl)-prop-2-en-1-yl groups.

[0262] In this specification, "halogenated alkoxy group" means a group in which one or more hydrogen atoms of the "alkoxy group" defined above are substituted by halogen atoms. The halogenated alkoxy group is preferably a C1 to C6 halogenated alkoxy group. Examples of the halogenated alkoxy group specifically include a difluoromethoxy group, a trifluoromethoxy group, a 2,2-difluoroethoxy group and a 2,2,2-trifluoroethoxy group.

[0263] In this specification, "alkylene group" means a divalent group induced by further removing an arbitrary hydrogen atom from the above-mentioned "alkyl group". The alkylene group is preferably a C4 to C8 alkylene group. Examples of alkylene groups specifically include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)CH2-, -C(CH3)2-, -(CH2)4-, -CH(CH3)CH2CH2-, -C(CH3)2CH2-, -CH2CH(CH3)CH2-, -CH2C(CH3)2-, -CH2CH2CH(CH3)-, -(CH2)5-, -(CH2)6-, -(CH2)7- and -(CH2)8-.

[0264] In the present specification, a "cycloalkylene group" means a divalent group induced by further removing an arbitrary hydrogen atom from the above-mentioned "cycloalkyl group". The cycloalkylene group is preferably a C3 to C8 cycloalkylene group. Examples of the cycloalkylene group specifically 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.

[0265] In the present specification, "alkenylene group" means a divalent group induced by further removing an arbitrary hydrogen atom from the above-mentioned "alkenyl group". Depending on the configuration of the double bond and the substituent (if present), the geometry of the double bond may present opposite side (E) or same side (Z) and cis or trans configuration. The alkenylene group includes straight chain or branched forms, and is preferably C2 to C 10 The alkenylene group is more preferably a C2 to C6 alkenylene group.

[0266] In the present specification, an "alkynylene group" means a divalent group induced by further removing an arbitrary hydrogen atom from the above-mentioned "alkynyl group". The alkynylene group includes a linear or branched form, and is preferably C2 to C 10 The alkynylene group is more preferably a C2 to C6 alkynylene group.

[0267] In this specification, "arylene group" means a divalent group induced by further removing an arbitrary hydrogen atom from the above-mentioned "aryl group". The arylene group may be a monocyclic or condensed ring. The number of atoms constituting the ring is not particularly limited, and is preferably 6 to 10 (C6 to 10 arylene). Examples of arylene groups specifically include 1,2-phenylene groups, 1,3-phenylene groups, 1,4-phenylene groups, 1,2-naphthylene groups, 1,3-naphthylene groups and 1,4-naphthylene groups.

[0268] In the present specification, the "heterocyclylene group" means a divalent group induced by further removing one arbitrary hydrogen atom from the above-mentioned "heterocyclyl group".

[0269] In the present specification, the "heteroarylene group" means a divalent group induced by further removing one arbitrary hydrogen atom from the above-mentioned "heteroaryl group".

[0270] In this specification, "spirocycloalkyl group" means a group formed by sharing a carbon atom in a group bonded thereto with a carbon atom constituting a cycloalkane ring. The spirocycloalkyl group is preferably a C3 to C8 spirocycloalkyl group. Examples thereof specifically include a spirocyclopropyl group, a spirocyclobutyl group, a spirocyclopentyl group, a spirocyclohexyl group, a spirocycloheptyl group and a spirocyclooctyl group.

[0271] In the present specification, a "spiroheterocyclyl group" means a group in which one or more carbon atoms in a "spirocycloalkyl group" are substituted with a heteroatom. Preferred examples of the heterospirocycloalkyl group include a 4-membered to 10-membered spiroheterocyclyl group.

[0272] In this specification, "alicyclic ring" means a non-aromatic hydrocarbon ring. The alicyclic ring may have an unsaturated bond in the ring, and may be a polycyclic ring having two or more rings. The carbon atoms constituting the ring may form a carbonyl group by oxidation. The alicyclic ring is preferably a 3- to 8-membered alicyclic ring. Examples thereof specifically 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.

[0273] In this specification, "heterocycle" means a non-aromatic heterocycle preferably containing 1 to 5, more preferably 1 to 3 heteroatoms in the atoms constituting the ring. The heterocycle may have double bonds and / or triple bonds in the ring. The carbon atoms in the ring may form a carbonyl group by oxidation, and the ring may be a monocyclic, condensed 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). Examples of the heterocyclic ring specifically 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 oxazolidinone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring and an azoline ring, and a ring in which a single bond of one or more of these saturated heterocyclic rings is substituted by a double bond or a triple bond.

[0274] In this specification, "saturated heterocycle" means a non-aromatic heterocycle containing carbon atoms and 1 to 5 heteroatoms in the ring without double bonds and / or triple bonds. Saturated heterocycle can be a monocyclic ring, or can form a condensed ring with another ring (for example, an aromatic ring such as a benzene ring). When a saturated heterocycle forms a condensed ring, the saturated heterocycle is preferably a 4- to 7-membered saturated heterocycle. Examples thereof specifically include azetidine ring, oxetane ring, tetrahydrofuran ring, tetrahydropyran ring, morpholine ring, thiomorpholine ring, pyrrolidine ring, 4-oxopyrrolidine ring, piperidine ring, 4-oxopiperidine ring, piperazine ring, pyrazolidine ring, imidazolidine ring, oxazolidine ring, isoxazolidine ring, thiazolidine ring, isothiazolidine ring, thiadiazolidine ring, oxazolidinone ring, dioxolane, dioxane ring, thietane ring, octahydroindole ring, dihydroindole ring and azepane ring.

[0275] [Peptide compounds]

[0276] In the present specification, "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 of the peptide compound can be, without particular limitation, 5 or more, 7 or more, 8 or more, or 9 or more. The number of amino acid residues of the peptide compound can also be 30 or less, 25 or less, 15 or less, or 13 or less. The number of amino acid residues in the peptide compound can be, for example, 5 or more and 30 or less, 7 or more and 25 or less, 8 or more and 15 or less, and 9 or more and 13 or less, and may be 11. The peptide compound may have a branched structure.

[0277] In this specification, "amino acid" includes natural amino acids and non-natural amino acids. In this specification, "natural amino acid" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg or Pro. Examples of non-natural amino acids include but are not particularly limited to β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α, α-disubstituted amino acids and amino acids with side chains different from natural amino acids. In this specification, non-natural N-substituted amino acids mean N-substituted amino acids other than Pro. In this specification, amino acids accept any conformation. The side chain of the amino acid can be selected without particular restriction, and is freely selected from hydrogen atoms and, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups and cycloalkyl groups. One or two non-adjacent methylene groups of any of these groups may be substituted by oxygen atoms, carbonyl groups (-CO-) or sulfonyl groups (-SO2-). Substituents may be added to each of the groups. This substituent is not limited and may be one or two or more substituents independently selected from any substituents including halogen atoms, O atoms, S atoms, N atoms, B atoms, Si atoms and P atoms. Specifically, examples thereof include optionally substituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups and cycloalkyl groups. In non-limiting aspects, the amino acid in this specification may be a compound having a carboxyl group and an amino group in the same molecule (even in this case, the amino acid also includes imino acids, such as proline and hydroxyproline).

[0278] The main chain amino group of an amino acid may be unsubstituted (NH2 group) or may be substituted (i.e., -NHR group, wherein R represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group or a cycloalkyl group optionally having a substituent, and one or two non-adjacent methylene groups of any of these groups may be substituted by an oxygen atom, a carbonyl group (-CO-) or a sulfonyl group (-SO2-); and the carbon chain bonded to the N atom and the carbon atom at position α may form a ring, such as in proline). The substituents of R are selected in the same manner as the substituents of the amino acid side chains described above. In the case of a substituted main chain amino group, R is included in the "side chain of an amino acid" in the present specification. In the present specification, such an amino acid having a substituted main chain amino group is referred to as an "N-substituted amino acid". In the present specification, examples of "N-substituted amino acids" preferably include, but are not limited to, N-alkyl amino acids, N-C1 to C6 alkyl amino acids, N-C1 to C4 alkyl amino acids, N-methyl amino acids and N-ethyl amino acids.

[0279] In the present specification, "amino acid" includes all isotopes corresponding to each amino acid. An isotope of an "amino acid" is a form in which at least one atom is substituted with an atom having the same atomic number (number of protons) and a different mass number (total number of protons and neutrons) as that of the atom in an abundance ratio different from the natural abundance ratio. In the present specification, examples of isotopes included in "amino acid" include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, which respectively include 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F and 36 Cl.

[0280] In the present 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 and an aralkyl group having a halogen atom as a substituent, and more specifically include a fluoroalkyl group, a difluoroalkyl group and a trifluoroalkyl group.

[0281] In the present specification, examples of the substituent containing an oxygen atom include a hydroxyl group (-OH), an oxy group (-OR), a carbonyl group (-C(=O)-R), a carboxyl group (-CO2H), an oxycarbonyl group (-C(=O)-OR), a carbonyloxy group (-OC(=O)-R), a thiocarbonyl group (-C(=O)-SR), a carbonylthio group (-SC(=O)-R), an aminocarbonyl group (-C(=O)-NHR), a carbonylamino group (-NH-C(=O)-R), an oxycarbonylamino group (-NH-C(=O)-OR), a sulfonylamino group (-NH-SO2-R), an aminosulfonyl group (-SO2-NHR), a sulfamoylamino group (-NH-SO2-NHR), a thiocarboxyl group (-C(=O)-SH) and a carboxycarbonyl group (-C(=O)-CO2H) and the like.

[0282] Examples of oxy (-OR) include alkoxy groups, cycloalkoxy groups, alkenyloxy groups, alkynyloxy groups, aryloxy groups, heteroaryloxy groups and aralkyloxy groups. The alkoxy group is preferably a C1 to C4 alkoxy group or a C1 or C2 alkoxy group, particularly preferably a methoxy group or an ethoxy group.

[0283] 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.

[0284] Examples of the oxycarbonyl group (—C(═O)—OR) include an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group and an aralkyloxycarbonyl group.

[0285] Examples of the carbonyloxy group (—OC(═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.

[0286] 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.

[0287] Examples of the carbonylthio group (—SC(═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.

[0288] Examples of the aminocarbonyl group (-C(=O)-NHR) include an alkylaminocarbonyl group (e.g., a C1 to C6 or C1 to C4 alkylaminocarbonyl group, particularly an ethylaminocarbonyl group and a methylaminocarbonyl group), a cycloalkylaminocarbonyl group, an alkenylaminocarbonyl group, an alkynylaminocarbonyl group, an arylaminocarbonyl group, a heteroarylaminocarbonyl group and an aralkylaminocarbonyl group. Examples thereof also include compounds in which the H atom bonded to the N atom in -C(=O)-NHR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or an aralkyl group.

[0289] Examples of the carbonylamino group (-NH-C(=O)-R) include an alkylcarbonylamino group, a cycloalkylcarbonylamino group, an alkenylcarbonylamino group, an alkynylcarbonylamino group, an arylcarbonylamino group, a heteroarylcarbonylamino group, and an aralkylcarbonylamino group. Examples thereof also include compounds in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0290] Examples of the oxycarbonylamino group (-NH-C(=O)-OR) include an alkoxycarbonylamino group, a cycloalkyloxycarbonylamino group, an alkenyloxycarbonylamino group, an alkynyloxycarbonylamino group, an aryloxycarbonylamino group, a heteroaryloxycarbonylamino group, and an aralkyloxycarbonylamino group. Examples thereof also include compounds in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0291] Examples of sulfonylamino groups (-NH-SO2-R) include alkylsulfonylamino groups, cycloalkylsulfonylamino groups, alkenylsulfonylamino groups, alkynylsulfonylamino groups, arylsulfonylamino groups, heteroarylsulfonylamino groups and aralkylsulfonylamino groups. Examples also include compounds in which the H atom bonded to the N atom in-NH-SO2-R is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or an aralkyl group.

[0292] Examples of aminosulfonyl groups (-SO2-NHR) include alkylaminosulfonyl groups, cycloalkylaminosulfonyl groups, alkenylaminosulfonyl groups, alkynylaminosulfonyl groups, arylaminosulfonyl groups, heteroarylaminosulfonyl groups and aralkylaminosulfonyl groups. Examples thereof also include compounds in which the H atom bonded to the N atom in -SO2-NHR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or an aralkyl group.

[0293] Examples of the sulfamoylamino group (-NH-SO2-NHR) include an alkylsulfamoylamino group, a cycloalkylsulfamoylamino group, an alkenylsulfamoylamino group, an alkynylsulfamoylamino group, an arylsulfamoylamino group, a heteroarylsulfamoylamino group, and an aralkylsulfamoylamino group. The two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with a substituent each 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 the two substituents may form a ring.

[0294] Examples of the substituent containing an S atom include groups such as a thiol group (-SH), a sulfenyl group (-SR), a sulfinyl group (-S(=O)-R), a sulfonyl group (-SO2-R) and a sulfo group (-SO3H).

[0295] Examples of selectable thio groups (—SR) include alkylthio groups, cycloalkylthio groups, alkenylthio groups, alkynylthio groups, arylthio groups, heteroarylthio groups and aralkylthio groups.

[0296] Examples of the sulfonyl group (—SO 2 —R) include an alkylsulfonyl group, a cycloalkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group and an aralkylsulfonyl group.

[0297] In the present specification, examples of substituents containing a nitrogen atom include groups such as an azido group (-N3), a cyano group (-CN), a primary amino group (-NH2), a secondary amino group (-NH-R; also referred to as a monosubstituted amino group), a tertiary amino group (-NR(R'); also referred to as a disubstituted amino group), an amidino group (-C(=NH)-NH2), a substituted amidino group (-C(=NR)-NR'R"), a guanidino group (-NH-C(=NH)-NH2), a substituted guanidino group (-NR-C(=NR"')-NR'R"), an aminocarbonylamino group (-NR-CO-NR'R"), a pyridinyl group, a piperidinyl group, a morpholinyl group and an azetidinyl group.

[0298] 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 a C1 to C6 alkyl group, more preferably a C1 to C4 alkyl group, and even more preferably a methyl group and an ethyl group.

[0299] Examples of tertiary amino groups (-NR(R'): disubstituted amino groups) include amino groups with any two substituents, each of which is 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) amino group. These any two substituents together with the nitrogen atom to which they are attached can form a ring. Examples thereof specifically include dialkylamino groups, particularly C1-C6 dialkylamino groups, C1-C4 dialkylamino groups, dimethylamino groups and diethylamino groups. In this specification, "C p -C q "Dialkylamino group" refers to a group in which the amino group is separated by two C p -C q Alkyl group substituted group. p -C q The alkyl groups may be the same or different.

[0300] 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.

[0301] Examples of the substituted guanidinyl 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, and groups in which these substituents form a ring.

[0302] Examples of the aminocarbonylamino group (—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, and groups in which these substituents form a ring.

[0303] In the present specification, "peptide residue" and "amino acid residue" are also simply referred to as "peptide" and "amino acid", respectively.

[0304] In this specification, the phrase "corresponding to a site" can be used to characterize the amino acid residues in the amino acid sequence of the peptide according to the present embodiment with reference to the amino acid sequence represented by SEQ ID NO: 1. Alignment for determining corresponding sites can be achieved by using various methods within the technical scope of the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (R) (Genetyx). Those skilled in the art can determine appropriate parameters for using sequence alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences to be compared.

[0305] The salt of the compound used herein may preferably be a chemical salt or a pharmaceutical salt. The salt may also be a solvate thereof, preferably a chemical or pharmaceutical solvate thereof. Examples of the salt of the compound include: hydrochloride; hydrobromide; hydroiodide; phosphate; phosphonate; sulfate; sulfonate such as methanesulfonate and p-toluenesulfonate; carboxylates such as acetate, citrate, malate, tartrate, succinate and salicylate; alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt and tetraalkylammonium salt.

[0306] In this specification, solvate refers to those in which the compound and the solvent form a molecular group, and when the solvent is water, it is referred to as a hydrate. The solvate of the salt of the compound disclosed herein is preferably a hydrate, and the specific example of the hydrate includes a monohydrate to a decahydrate, preferably a monohydrate to a pentahydrate, and more preferably a monohydrate to a trihydrate. The solvate of the compound disclosed herein not only includes solvates with a single solvent such as water, alcohol (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), dimethylformamide, but also includes solvates with a variety of solvents.

[0307] [Method of producing amino acids]

[0308] The method for producing an amino acid according to one embodiment comprises the step of performing the following reaction (i) or (ii) in the presence of a polypeptide comprising an amino acid sequence having 90% or higher identity with the amino acid represented by SEQ ID NO: 1, a reducing agent, and a compound D represented by the following formula (1):

[0309] (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;

[0310] (ii) 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.

[0311] Here, the amino acid having a substituted or unsubstituted amino group is formed by an intermolecular reductive amination reaction between the amino group of compound A and the carbonyl group of compound B, or by an intramolecular reductive amination reaction between the amino group and the carbonyl group of compound C.

[0312] According to another embodiment, the method for producing an amino acid comprises the step of carrying out the following reaction (i) or (ii) in the presence of a polypeptide having catalytic activity for 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 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 under at least one reaction condition, a reducing agent, and a compound D' (reaction step):

[0313] (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;

[0314] (ii) 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.

[0315] In the production method, compound D' is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethylphosphine oxide, dimethoxymethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol and methylformamide.

[0316] <Compound D and Compound D'>

[0317] The reaction step of carrying out the reductive amination reaction is carried out in the presence of a compound D represented by the following formula (1):

[0318] [Formula 10]

[0319]

[0320] In formula (1), v and w each independently represent 0 or 1, at least one of v and w represents 1, and T represents a carbon atom, a phosphorus atom or a sulfur atom.

[0321] In formula (1), the functional group represented by the following formula (1a):

[0322] [Formula 11]

[0323]

[0324] Represents =O, -ORd or a hydroxyl group (-OH). In this article, =O as a functional group represented by formula (1a) means that T is bonded to an oxygen atom through a double bond. Specifically, the compound D in which the functional group represented by formula (1a) is =O has a structure represented by T=O. -ORd as a functional group represented by formula (1a) means that T is bonded to ORd through a single bond. Specifically, the compound D in which the functional group represented by formula (1a) is -ORd has a structure represented by T-ORd. The hydroxyl group as a functional group represented by formula (1a) means that T is directly bonded to the hydroxyl group. Specifically, the compound D in which the functional group represented by formula (1a) is -OH has a structure represented by T-OH.

[0325] When each of v and w is 1, the two functional groups represented by multiple formulas (1a) may be the same or different; Ra, Rb and Rc each independently represent a hydrogen atom, a C1 to C3 alkyl group, an alkylamino group or -CH2-ORd; at least any two of Ra, Rb and Rc are optionally connected to each other to form a ring structure together with T; Rd represents a C1 to C3 alkyl group; d, e and f each independently represent 0 or 1; and at least one of d, e and f represents 1.

[0326] When each of v and w is 1, at least one of Ra, Rb and Rc is a methyl group, and none of Ra, Rb and Rc is connected to each other to form a ring structure together with T. For example, when each of v and w is 1, T is a sulfur atom, each functional group represented by formula (1a) is =0, f is 0, and at least one of Ra and Rb can be a methyl group.

[0327] When at least one of Ra, Rb and Rc is a methylamino group, Ra, Rb and Rc are not connected to each other to form a ring structure together with T. When formula (1a) is a hydroxyl group and T is a carbon atom, each of d, e and f is 1, and each of Ra, Rb and Rc is a hydrogen atom.

[0328] The compound D may be a compound represented by the following formula (1-1), wherein T in the formula (1) is a carbon atom:

[0329] [Formula 12]

[0330]

[0331] The compound represented by formula (1-1) may be a compound represented by the following formula (1-1a), wherein v is 1, and w is 0:

[0332] [Formula 13]

[0333]

[0334] In formula (1-1a), when the functional group represented by formula (1a) is =0, two of d, e and f are 1, and the remaining one is 0. When the functional group represented by formula (1a) is =0, the compound represented by formula (1-1a) may be a compound represented by the following formula (1-1b), wherein each of d and e is 1, and f is 0:

[0335] [Formula 14]

[0336]

[0337] The compound represented by formula (1-1b) may be a compound in which one of Ra and Rb is a hydrogen atom or a C1 to C3 alkyl group and the other is an alkylamino group. The alkylamino group may be a monomethylamino group or a dimethylamino group. The compound represented by formula (1-1b) may be, for example, N,N-dimethylformamide, N,N-dimethylacetamide or N-methylformamide.

[0338] In formula (1-1a), when the functional group represented by formula (1a) is a hydroxyl group, each of d, e and f is 1. When the functional group represented by formula (1a) is a hydroxyl group, the compound represented by formula (1-1a) may be a compound represented by the following formula (1-1c), wherein each of d, e and f is 1:

[0339] [Formula 15]

[0340]

[0341] Ra, Rb and Rc may each independently be a hydrogen atom or a C1 to C3 alkyl group. The compound represented by formula (1-1c) may be methanol, in which each of Ra, Rb and Rc is a hydrogen atom.

[0342] In formula (1-1a), when the functional group represented by formula (1a) is -ORd, each of d, e and f can be 1 and each of Rb and Rc can be a hydrogen atom. When the functional group represented by formula (1a) is -ORd, the compound represented by formula (1-1a) can be a compound represented by the following formula (1-1d), wherein each of d, e and f is 1, and each of Rb and Rc is a hydrogen atom. In the compound represented by the following formula (1-1d), Ra can be -CH2-ORd. The compound represented by the following formula (1-1d) can be dimethoxyethane, wherein Ra is -CH2-ORd, and Rd is -CH3:

[0343] [Formula 16]

[0344]

[0345] Compound D may be a compound represented by the following formula (1-2), wherein T in formula (1) is a phosphorus atom:

[0346] [Formula 17]

[0347]

[0348] The compound represented by formula (1-2) may be a compound represented by the following formula (1-2a), wherein v is 1, w is 0, and the functional group represented by formula (1a) is =0:

[0349] [Formula 18]

[0350]

[0351] In formula (1-2a), each of Ra, Rb and Rc may be a C1 to C3 alkyl group. The compound represented by formula (1-2a) may be trimethylphosphine oxide, in which each of Ra, Rb and Rc is a methyl group.

[0352] Compound D may be a compound represented by the following formula (1-3), wherein T in formula (1) is a sulfur atom:

[0353] [Formula 19]

[0354]

[0355] The compound represented by formula (1-3) may be a compound represented by the following formula (1-3a), wherein v is 1, w is 0, the functional group represented by formula (1a) is =0, each of d and e is 1, and f is 0:

[0356] [Formula 20]

[0357]

[0358] In Formula (1-3a), each of Ra and Rb may be a C1 to C3 alkyl group, and Ra and Rb may be connected to each other to form a ring structure together with the sulfur atom (S).

[0359] In the compound represented by formula (1-3a), Ra and Rb may each independently be a methyl group or an ethyl group. The compound represented by formula (1-3a) may be dimethyl sulfoxide in which each of Ra and Rb is a methyl group, or diethyl sulfoxide in which each of Ra and Rb is an ethyl group. The compound represented by formula (1-3a) may be tetramethylene sulfoxide, in which each of Ra and Rb is a C1 to C3 alkyl group, and these alkyl groups are connected to each other to form a ring structure with S.

[0360] The compound represented by formula (1-3) may be a compound represented by the following formula (1-3b), wherein each of v and w is 1, the functional group represented by formula (1a) is =0, each of d and e is 1, and f is 0:

[0361] [Formula 21]

[0362]

[0363] 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 each of Ra and Rb is a methyl group.

[0364] Compound D may be compound D', which is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, trimethyl phosphine oxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetramethylene sulfoxide, diethyl sulfoxide, methanol and methylformamide, because amino acids can be produced in better yields. Compound D or compound D' may be at least one selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone and trimethyl phosphine oxide, and may be dimethyl sulfoxide, because amino acids can be produced in better yields.

[0365] Compound D or compound D' may be liquid or solid under the conditions of 25°C and 1 atm, and may be liquid under the conditions of 25°C and 1 atm because the production efficiency of the amino acid is further improved. Examples of compound D or compound D' that is liquid under the conditions of 25°C and 1 atm include dimethyl sulfoxide, dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, and N-methylformamide.

[0366] <Compound A>

[0367] The 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:

[0368] [Formula 22]

[0369]

[0370] 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, which are optionally substituted, and R 1 and R 2 At least one of them is a hydrogen atom.

[0371] In formula (2), R 1 can be a hydrogen atom, R 2 It may be a hydrogen atom or a C1 to C6 alkyl group, may be a hydrogen atom or a C1 to C3 alkyl group, or may be a hydrogen atom, a methyl group or an ethyl group.

[0372] Compound A may be an alkylamine of formula (2), wherein R 1 and R 2 One of the moieties is an alkyl group, and the other moiety is a hydrogen atom. The alkylamine or its salt may be one or more selected from the group consisting of methylamine, ethylamine and their salts. Compound A may be ammonia, wherein R in formula (2) 1 and R 2 Each of is a hydrogen atom. Compound A may be one or more selected from the group consisting of ammonia, methylamine, ethylamine, and salts thereof.

[0373] <Compound B>

[0374] The 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:

[0375] [Formula 23]

[0376]

[0377] [Equation 24]

[0378]

[0379] In formula (3), X represents a carbon atom, and Y represents a hydrogen atom, or a group represented by formula (4).

[0380] n represents an integer between 0 and 2. n may be 0 or 1, and may be 0.

[0381] R 6 represents a hydrogen atom, an optionally substituted C1 to C6 alkyl group, an optionally substituted C5 to C 12An aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom or a group containing an oxygen atom.

[0382] In formula (4),

[0383] [Formula 25]

[0384]

[0385] Indicates the point of connection with X.

[0386] m represents an integer between 0 and 6. 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.

[0387] p represents 0 or 1, q represents 0 or 1, and r represents 0 or 1.

[0388] Z 1 represents an optionally substituted alkylene group, or an ether bond-containing group having 1 or more and 6 or less carbon atoms. When m is an integer of 2 or more, a plurality of Z 1 They may be the same or different. Examples of the "ether bond-containing group" include alkyl groups substituted by alkoxy groups, aryloxy groups, etc. Specific examples of the ether bond-containing group having 1 to 6 carbon atoms include methoxymethyl groups, ethoxymethyl groups, allyloxymethyl groups, allyloxyethyl groups, and allyloxypropyl groups.

[0389] Z 2 Represents a carbon atom.

[0390] R 3 , R 4 and R 5 each independently represents a hydrogen atom, an optionally substituted C1 to C6 alkyl group, an optionally substituted C5 to C 12 An aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom or a group containing an oxygen atom.

[0391] R 3 , R 4 and R 5 Any two or more of are optionally connected to each other to form Z 2 Together they form a ring structure, which is optionally a cycloalkyl group, an aryl group, a heterocyclyl group or a heteroaryl group, and these groups are optionally substituted.

[0392] R 3 , R4 and R 5 Each optionally with Z 2 To form a double or triple bond, and when R 3 , R 4 and R 5 Any of them is connected to Z through a double bond or a triple bond 2 When bonding, at least one of p, q and r is 0.

[0393] In formula (4), R 3 , R 4 and R 5 At least one of the groups may not be a methyl group.

[0394] Compound B may be a compound represented by the following formula (3'):

[0395] [Equation 26]

[0396]

[0397] In formula (3'), Y' represents a C3 to C8 cycloalkyl group or a C7 to C 10 The aralkyl group is optionally substituted with a C1 to C3 alkyl group or halogen.

[0398] The compound represented by formula (3') or a salt thereof may be at least one 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-oxopropionic acid, 2-oxo-3-phenylbutyric acid, 3,3-dimethyl-2-oxobutyric acid and a salt thereof. Specific examples of the salt include sodium salts, magnesium salts, potassium salts and calcium salts.

[0399] The combination of compound A and compound B can be compound A (ammonia) of formula (2) (wherein R 1 and R 2 Each of which is a hydrogen atom) or a compound of formula (2) A (methylamine) (wherein R 1 is a hydrogen atom and R 2 is a methyl group) and a compound B (4-(2-chlorophenyl)-2-oxobutanoic acid) of the formula (3) (wherein each of n and m is 0, Y is a group represented by the formula (4), R 3 is a (2-chlorophenyl)methyl group, R 4 , R 5 and R 6 Each of n and m is a hydrogen atom, and each of p, q and r is 1) or a compound B (phenylpyruvic acid) of formula (3) (wherein each of n and m is 0, Y is a group represented by formula (4), R 3 is a phenyl group, R 4, R 5 and R 6 Each of which is a hydrogen atom, and each of p, q and r is a combination of 1); or it may be a compound A (ethylamine) of formula (2) (wherein R 1 is a hydrogen atom and R 2 is an ethyl group) and a compound B (phenylpyruvic acid) of the formula (3) (wherein each of n and m is 0, Y is a group represented by the formula (4), R 3 is a phenyl group, R 4 , R 5 and R 6 Each of which is a hydrogen atom, and each of p, q and r is a combination of 1); or it may be a compound A (methylamine) of formula (2) (wherein R 1 is a hydrogen atom and R 2 is a methyl group) and a compound B (2-cyclopentyl-2-oxo-acetic acid) of the formula (3) (wherein each of n and m is 0, Y is a group represented by the formula (4), R 3 , R 4 and Z 2 are connected to each other to form a cyclopentane ring, R 5 and R 6 Each of is a hydrogen atom, and each of p, q and r is a combination of 1).

[0400] The combination of compound A and compound B can be compound A (methylamine) of formula (2) (wherein R 1 is a hydrogen atom and R 2 is a methyl group) and a compound B (3-(4,4-difluorocyclohexyl)-2-oxopropanoic acid) of formula (3) (wherein each of n and m is 0, Y is a group represented by formula (4), R 3 is 4,4-difluorocyclohexyl, R 4 , R 5 and R 6 Each of is a hydrogen atom, and each of p, q and r is a combination of 1).

[0401] The combination of compound A and compound B can be compound A (ammonia) of formula (2) (wherein R 1 and R 2 Each of n and m is a hydrogen atom) and a compound B (2-oxo-3-phenylbutyric acid) of the formula (3) (wherein each of n and m is 0, Y is a group represented by the formula (4), R 3 is a methyl group, R 4 is a phenyl group, R 5 and R 6 Each of is a hydrogen atom, and each of p, q and r is a combination of 1).

[0402] The combination of compound A and compound B can be compound A (methylamine) of formula (2) (wherein R 1 is a hydrogen atom and R 2 is a methyl group) and a compound B (3,3-dimethyl-2-oxobutanoic acid) of formula (3) (wherein each of n and m is 0, Y is a group represented by formula (4), R 3 , R 4 and R 5 Each of R is a methyl group, 6 is a hydrogen atom, and each of p, q and r is a combination of 1).

[0403] <Compound C>

[0404] The 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):

[0405] [Formula 27]

[0406]

[0407] n' represents an integer of 0 or more and 2 or less. n may be 0 or 1, and may be 0.

[0408] 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 are optionally substituted.

[0409] R 8 represents a hydrogen atom, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom, or a group containing an oxygen atom.

[0410] <Peptide>

[0411] The polypeptide comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1. The identity with 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%.

[0412] The identity with the amino acid sequence represented by SEQ ID NO:1 can be determined by the algorithm BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7). Based on this algorithm, programs named BLASTN or BLASTX were developed (Altschul et al., J. Mol. Biol. (1990) 215: 403-10). In the case of analyzing amino acid sequences with BLASTX on the basis of BLAST, the parameters are set to Score=50 and Wordlength=3. In the case of using BLAST and Gapped BLAST programs, the default parameters of each program are used. For the specific methods of these analysis methods, see the information on the BLAST (Basic Local Alignment Search Tool) website of NCBI (National Center for Biotechnology Information) known in the art.

[0413] The polypeptide according to the present embodiment can be an engineered polypeptide having one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1, and 1 to 20, 1 to 15, 1 to 10, 1 to 7 or 1 to 5 amino acid residues can be engineered. The number of engineered amino acid residues can be 3 or less, can be 2 or less, or can be 1.

[0414] Engineering can be one or more selected from the group consisting of substitution, deletion and insertion and can be substitution. Engineering can be conservative engineering. Conservative engineering means that the amino acid residues are engineered so as not to reduce the catalytic activity of interest compared to the polypeptide before engineering.

[0415] Engineering can be the replacement with the natural amino acid different from the amino acid residue before engineering. The natural amino acid for replacement can 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.

[0416] The polypeptide according to the present embodiment may be a polypeptide comprising an engineered sequence having an amino acid residue located at a position corresponding to at least one amino acid residue 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 NO:1 can be a polypeptide comprising an engineered sequence having amino acid residues located at sites corresponding to at least one amino acid residue selected from the group consisting of the following items: a methionine residue at position 141, a histidine residue at position 182, a tryptophan residue at position 253 and a lysine residue at position 260, because the catalytic activity for the reductive amination reaction is further improved, and can be a polypeptide comprising an engineered sequence having amino acid residues located at sites corresponding to at least one amino acid residue selected from the group consisting of the following items: a methionine residue at position 141, a tryptophan residue at position 253 and a lysine residue at position 260.

[0417] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site corresponding to a 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 a histidine residue. The polypeptide may be a polypeptide comprising an 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0418] The polypeptide may include 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, because the catalytic activity for the reductive amination reaction is further improved. Specifically, the polypeptide according to the present embodiment may include 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.

[0419] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site 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 a phenylalanine residue. The polypeptide may be a polypeptide comprising an 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, an aspartic acid residue, a glutamic acid residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0420] The polypeptide may include a sequence in which the amino acid residue at the site corresponding to the phenylalanine residue at position 117 is substituted with a leucine residue, because the catalytic activity for the reductive amination reaction is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence (F117L) in which the amino acid residue at the site 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.

[0421] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site corresponding to a 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 a methionine residue. The polypeptide may be a polypeptide comprising an 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0422] The polypeptide may contain a sequence having the amino acid residue at the site corresponding to the methionine residue at position 141 substituted with at least one amino acid residue selected from the group consisting of a tyrosine residue, a tryptophan residue, a valine residue, a threonine residue, a serine residue, an arginine residue, a leucine residue, a lysine residue, an isoleucine residue, a histidine residue, a phenylalanine residue and an alanine residue, because the catalytic activity for the reductive amination reaction is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence 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 replaced by: a tyrosine residue, a tryptophan residue, a valine residue, a threonine residue, a serine residue, an arginine residue, a leucine residue, a lysine residue, an isoleucine residue, a histidine residue, a phenylalanine residue or an alanine residue (M141Y, M141W, M141V, M141T, M141S, M141R, M141L, M141K, M141I, M141H, M141F or M141A). The polypeptide may include a sequence in which the amino acid residue at the site corresponding to the methionine residue at position 141 is replaced by at least one amino acid residue selected from the group consisting of a tyrosine residue, a tryptophan residue, a valine residue, a lysine residue, an isoleucine residue, a phenylalanine residue and an alanine residue. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence 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 by a tyrosine residue, a tryptophan residue, a valine residue, a lysine residue, an isoleucine residue, a histidine residue, a phenylalanine residue or an alanine residue (M141Y, M141W, M141V, M141K, M141I, M141H, M141F or M141A).

[0423] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site corresponding to a 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 a threonine residue. The polypeptide may be a polypeptide comprising an 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0424] The polypeptide may include a sequence in which the amino acid residue at the site corresponding to the threonine residue at position 156 is substituted with a serine residue, because the catalytic activity for the reductive amination reaction is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence in which the amino acid residue at the site 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 (T156S).

[0425] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site 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 the histidine residue. 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0426] The polypeptide may include a sequence having an amino acid residue at a site corresponding to the histidine residue at position 182 replaced by at least one amino acid residue selected from the group consisting of tyrosine residues, glutamine residues, methionine residues, leucine residues, glycine residues, phenylalanine residues and alanine residues, because the catalytic activity of reductive amination is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence having an amino acid residue at a site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO:1 replaced by tyrosine residues, glutamine residues, methionine residues, leucine residues, glycine residues, phenylalanine residues or alanine residues (H182Y, H182Q, H182M, H182L, H182G, H182F or H182A). The polypeptide may include a sequence having an amino acid residue at a site corresponding to the histidine residue at position 182 replaced by at least one amino acid residue selected from the group consisting of methionine residues, leucine residues and phenylalanine residues. Specifically, the polypeptide according to the present embodiment may comprise an amino acid sequence in which the amino acid residue at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by a methionine residue, a leucine residue or a phenylalanine residue (H182M, H182L or H182F).

[0427] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site corresponding to a 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 a glutamine residue. The polypeptide may be a polypeptide comprising an 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0428] The polypeptide may include 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 at least one amino acid residue selected from the group consisting of a methionine residue and a glutamic acid residue, because the catalytic activity for the reductive amination reaction is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid 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 a methionine residue or a glutamic acid residue (Q186M or Q186E).

[0429] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site corresponding to a 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 an 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue or a tyrosine residue.

[0430] The polypeptide may contain a sequence having the amino acid residue at the site corresponding to the tryptophan residue at position 253 substituted with at least one amino acid residue selected from the group consisting of 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 and alanine, because the catalytic activity for the reductive amination reaction is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is replaced by 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 (W253Y, W253V, W253T, W253S, W253R, W253Q, W253P, W253N, W253M, W253L, W253K, W253I, W253H, W253F or W253A). The polypeptide may include a sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 is replaced by at least one amino acid residue selected from the group consisting of a leucine residue, an isoleucine residue and a histidine residue. Specifically, the polypeptide according to the present embodiment may comprise an amino acid sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by a leucine residue, an isoleucine residue or a histidine residue (W253L, W253I or W253H).

[0431] The polypeptide according to the present embodiment may be a polypeptide comprising a sequence in which an amino acid residue at a site corresponding to a 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 a lysine residue. The polypeptide may be a polypeptide comprising an 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, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0432] The polypeptide may contain a sequence having the amino acid residue at the site corresponding to the lysine residue at position 260 substituted with at least one amino acid residue selected from the group consisting of a tyrosine residue, a tryptophan residue, a threonine residue, a serine residue, an arginine residue, a glutamine residue, an asparagine residue, a methionine residue, a leucine residue, a histidine residue, a glycine residue, a phenylalanine residue, a glutamic acid residue and an alanine residue, because the catalytic activity for the reduction of amino group reaction is further improved. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is replaced by a tyrosine residue, a tryptophan residue, a threonine residue, a serine residue, an arginine residue, a glutamine residue, an asparagine residue, a methionine residue, a leucine residue, a histidine residue, a glycine residue, a phenylalanine residue, a glutamic acid residue or an alanine residue (K260Y, K260W, K260T, K260S, K260R, K260Q, K260N, K260M, K260L, K260H, K260G, K260F, K260E or K260A). The polypeptide may further include a sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 is replaced by at least one amino acid residue selected from the group consisting of a glutamine residue, a methionine residue, a glutamic acid residue and an asparagine residue. Specifically, the polypeptide according to the present embodiment may comprise an amino acid sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by: a glutamine residue, a methionine residue, a glutamic acid residue or an asparagine residue (K260Q, K260M, K260E or K260N).

[0433] The polypeptide according to the present embodiment may comprise the following: an amino acid sequence a1 represented by SEQ ID NO: 4, wherein the amino acid residue represented by X is a valine residue (mutation: M141V); an amino acid sequence a2 represented by SEQ ID NO: 4, wherein the amino acid residue represented by X is a tyrosine residue (mutation: M141Y); an amino acid sequence a3 represented by SEQ ID NO: 6, wherein the amino acid residue represented by X is a leucine residue (mutation: H182L); an amino acid sequence a4 represented by SEQ ID NO: 8, wherein the amino acid residue represented by X is a histidine residue (mutation: W253H); or an amino acid sequence a5 represented by SEQ ID NO: 9, wherein the amino acid residue represented by X is a glutamic acid residue (mutation: K260E) and allowed to stand overnight.

[0434] The polypeptide according to this embodiment may comprise a sequence having 90% or higher sequence identity with amino acid sequence a1, a2, a3, a4 or a5. The sequence identity may be 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher or 99% or higher, or may be 100%.

[0435] The polypeptide according to this embodiment can be a polypeptide comprising the amino acid sequence a1, a2, a3, a4 or a5, one or more of which are engineered amino acid residues, and 1 to 20, 1 to 15, 1 to 10, 1 to 7 or 1 to 5 amino acid residues can be engineered. The number of engineered amino acid residues can be 3 or less, 2 or less, or 1.

[0436] In the polypeptide according to the present embodiment, the amino acid sequence represented by SEQ ID NO: 1 may have two or more engineered sites, or two engineered sites. Hereinafter, preferred embodiments of the polypeptide having two or more modified portions will be described.

[0437] The polypeptide may preferably comprise a sequence comprising engineering of an amino acid residue at a site corresponding to at least one amino acid residue selected from the group consisting of a methionine residue at position 141, a histidine residue at position 182, and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 as a first engineering site, and engineering of an amino acid residue at a site corresponding to at least one amino acid residue selected from the group consisting of a histidine residue at position 182, a tryptophan residue at position 253, and a lysine at position 260 in the amino acid sequence represented by SEQ ID NO: 1 as a second engineering site, wherein the amino acid residue engineered at the second engineering site is located at a site corresponding to an amino acid residue different from the amino acid residue engineered at the first engineering site.

[0438] The polypeptide may preferably comprise a sequence having engineering of an amino acid residue at a site corresponding to a methionine residue at position 141 and a histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1, engineering of an amino acid residue at a site corresponding to a methionine residue at position 141 and a tryptophan residue at position 253, engineering of an amino acid residue at a site corresponding to a methionine residue at position 141 and a lysine residue at position 260, engineering of an amino acid residue at a site corresponding to a histidine residue at position 182 and a tryptophan residue at position 253, engineering of an amino acid residue at a site corresponding to a histidine residue at position 182 and a lysine residue at position 260, or engineering of an amino acid residue at a site corresponding to a tryptophan residue at position 253 and a lysine residue at position 260. The polypeptide may more preferably comprise a sequence having engineering of an amino acid residue at a site corresponding to a methionine residue at position 141 and a lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1, engineering of an amino acid residue at a site corresponding to a methionine residue at position 182 and a lysine residue at position 260, or engineering of an amino acid residue at a site corresponding to a tryptophan residue at position 253 and a lysine residue at position 260.

[0439] The polypeptide may preferably comprise a sequence 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 by an amino acid residue selected from the group consisting of a tyrosine residue, a tryptophan residue, a valine residue, a threonine residue, an arginine residue, a lysine residue, an isoleucine residue and an alanine residue, or more preferably comprise a sequence 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 by an amino acid residue selected from the group consisting of a tyrosine residue, a valine residue and an alanine residue.

[0440] The polypeptide may preferably comprise a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a tyrosine residue, a methionine residue, a leucine residue and a phenylalanine residue, or more preferably comprise a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a methionine residue and a leucine residue.

[0441] The polypeptide may preferably comprise a sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of a tyrosine residue, a threonine residue, a serine residue, an asparagine residue, a methionine residue, a histidine residue and an alanine residue, or more preferably comprise a sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by a histidine residue.

[0442] The polypeptide may preferably comprise a sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of a serine residue, a glutamine residue, an asparagine residue, a leucine residue, an isoleucine residue, a histidine residue, a glycine residue, a phenylalanine residue, a glutamic acid residue and an alanine residue, or more preferably comprise a sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of a glutamine residue and a glutamic acid residue.

[0443] Hereinafter, in the amino acid sequence represented by SEQ ID NO: 1, wherein the amino acid at the position corresponding to position m 1 The amino acid residue X 1 The amino acid residue at the position is X 1 Another amino acid residue other than x 1 Replaced and located at the position corresponding to m 2 The amino acid residue X 2 The amino acid residue at the position is X 2 Another amino acid residue other than x 2 The substituted sequence is called X 1 m 1 x 1 _X 2 m 2 x 2 .

[0444] The amino acid sequence represented by SEQ ID NO:1 in which two amino acid residues are engineered may be, for example, an amino acid sequence selected from group a1 to group a4 consisting of the following amino acid sequences, may preferably be an amino acid sequence selected from group a1 to group a3, may more preferably be an amino acid sequence selected from group a1 and group a2, and may most preferably be an amino acid sequence selected from group a1.

[0445] Group a1:

[0446] M141Y_K260E, H182L_K260Q, M141A_K260E, H182M_K260Q, M141A_K260Q, H182M_K260E, M141V_K260E, W253H_K260E and H182L_K260E.

[0447] Pair a2:

[0448] M141V_K260Q, W253H_K260Q, W253M_K260E, M141Y_H182M, M141Y_K260Q, H182F_K260Q, M141V _K260F, M141V_H182L, M141V_K260L, M141Y_K260G, H182L_W253H, H182F_K260E M141T_K260Q

[0449] Group a3:

[0450] 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.

[0451] Group a4:

[0452] 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.

[0453] The polypeptide may comprise, for example, a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of group a1 to group a4, preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of group a1 to group a3, more preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of group a1 and group a2, and most preferably comprises a sequence having 90% or more sequence identity with an amino acid sequence selected from group a1.

[0454] 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 to a2, and most preferably comprises an amino acid sequence selected from group a1.

[0455] The polypeptide may have three or more engineered sites or three engineered sites in the amino acid sequence shown by SEQ ID NO: 1. In the following, preferred embodiments of the polypeptide having three or more engineered sites are described.

[0456] The polypeptide may preferably comprise a sequence comprising engineering of an amino acid residue at a site corresponding to at least one amino acid residue selected from the group consisting of a methionine residue at position 141 and a histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 as a first engineered site, and engineering of an amino acid residue at a site corresponding to at least one amino acid residue selected from the group consisting of a histidine residue at position 182 and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 as a second engineered site, wherein the engineered amino acid residue at the second engineered site is located at a site that is one amino acid residue different from the amino acid residue engineered at the first engineered site, and engineering of an amino acid residue at a site corresponding to at least one amino acid residue selected from the group consisting of a tryptophan residue at position 253 and a lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 as a third engineered site, wherein the engineered amino acid residue at the third engineered site is located at a site that is one amino acid residue different from the amino acid residues engineered at the first and second engineered sites.

[0457] The polypeptide may preferably comprise a sequence having engineering of amino acid residues at sites corresponding to a methionine residue at position 141, a histidine residue at position 182, and a tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1, engineering of amino acid residues at sites corresponding to a methionine residue at position 141, a histidine residue at position 182, and a lysine residue at position 260, engineering of amino acid residues at sites corresponding to a methionine residue at position 141, a tryptophan residue at position 253, and a lysine residue at position 260, or engineering of amino acid residues at sites corresponding to a histidine residue at position 182, a tryptophan residue at position 253, and a lysine residue at position 260. The polypeptide may more preferably comprise a sequence having engineering of amino acid residues at sites corresponding to a methionine residue at position 141, a histidine residue at position 182, and a lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1, engineering of amino acid residues at sites corresponding to a methionine residue at position 141, a tryptophan residue at position 253, and a lysine residue at position 260, or engineering of amino acid residues at sites corresponding to a histidine residue at position 182, a tryptophan residue at position 253, and a lysine residue at position 260.

[0458] The polypeptide may preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and include a sequence 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 by an amino acid residue selected from the group consisting of: a tyrosine residue, a valine residue, a threonine residue, a serine residue, a leucine residue, a lysine residue, an isoleucine residue, and an alanine residue. The polypeptide may more preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and include a sequence 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 by an amino acid residue selected from the group consisting of: a tyrosine residue, a valine residue, and an isoleucine residue.

[0459] The polypeptide may preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and include a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a methionine residue, a leucine residue, and a phenylalanine residue. The polypeptide may more preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and include a sequence in which the amino acid residue at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a methionine residue and a leucine residue.

[0460] The polypeptide may preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and include a sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a valine residue, a threonine residue, a serine residue, a glutamine residue, a methionine residue, a leucine residue, a histidine residue, a phenylalanine residue, and a glutamic acid residue. The polypeptide may more preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and include a sequence in which the amino acid residue at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a glutamine residue, a methionine residue, a leucine residue, and a histidine residue.

[0461] The polypeptide may preferably have three engineered sites in the amino acid sequence represented by SEQ ID NO: 1, and comprise a sequence in which the amino acid residue at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of: a glutamine residue and a glutamic acid residue.

[0462] Hereinafter, in the amino acid sequence represented by SEQ ID NO: 1, the residue corresponding to position m 1 The amino acid residue X 1 The amino acid sequence at the site is X 1 Another amino acid residue other than x 1 Substitution, corresponding to position m 2 The amino acid residue X 2 The amino acid residue at the position is X 2 Another amino acid residue other than x 2Replace, and corresponding to position m 3 The amino acid residue X 3 The amino acid residue at the position is X 3 Another amino acid residue other than x 3 Replacement is called X 1 m 1 x 1 _X 2 m 2 x 2 _X 3 m 3 x 3 .

[0463] The amino acid sequence represented by SEQ ID NO:1 in which three amino acid residues are engineered may be, for example, an amino acid sequence selected from group b1 to group b4 consisting of the following amino acid sequences, may preferably be an amino acid sequence selected from group b1 to group b3, may more preferably be an amino acid sequence selected from group b1 and group b2, and may most preferably be an amino acid sequence selected from group b1.

[0464] Group b1:

[0465] 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, M141I_W253H_K260E, M141V_H182L_K260E, M141Y_H182L_K260E, H182L_W253M_K260E, M141V_W253Q_K260Q, M141V_W253H_K260Q, M141V_W253L_K260Q, M141I_W253H_K260Q, and M141V_W253M_K260Q.

[0466] Group b2:

[0467] 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, H182M_W253M_K260E, H182L_W253M_K260Q, M141Y_W253H_K260E, M141Y_W253Q_K260E, M141V_H182M_K260Q, H182M_W25 3L_K260E, H182F_W253H_K260E, M141A_H182F_K260Q, M141Y_W253M_K260E, H182M_W253Q_K260Q sum H182M_W253M_K260Q.

[0468] Group b3:

[0469] 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, M141A_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:

[0470] M141A_W253M_K260Q and M141A_H182F_K260E

[0471] The polypeptide may comprise, for example, an amino acid 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 the group consisting of group b1.

[0472] 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 to b2, and most preferably comprises an amino acid sequence selected from group b1.

[0473] The polypeptide according to the present embodiment may be fused to another polypeptide or protein. Specifically, the polypeptide according to the present embodiment may include an amino acid sequence (another amino acid sequence) other than a sequence having 90% or higher sequence identity with the amino acid sequence represented by SEQ ID NO: 1, one of which amino acid residues is engineered at either or both of the N-terminus and the C-terminus. The other amino acid sequence may be, for example, a tag sequence.

[0474] Examples of polypeptides or proteins having additional amino acid sequences include a His tag (6×His, 10×His, etc.) which is a tag consisting of several (e.g., 6 and 10) His (histidine) residues, a streptavidin-binding peptide tag (SBP tag) comprising an amino acid sequence capable of binding to a biotin binding site of streptavidin, GST (glutathione S-transferase), HA (influenza hemagglutinin), an immunoglobulin constant region, B-galactosidase, MBP (maltose binding protein), FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), influenza hemagglutinin (HA), a human c-myc fragment, a VSV-GP fragment, a p18 HIV fragment, a T7-tag, a HSV-tag, an E-tag, a SV40 T antigen fragment, an Ick tag, an α-tubulin fragment, a B-tag, a protein C fragment, Stag, StrepTag, and a HaloTag. 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.

[0475] The polypeptide according to the present embodiment may comprise one or more 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 comprise a streptavidin-binding peptide tag sequence (SBP tag) and a His tag sequence at the C-terminus.

[0476] The polypeptide according to the present embodiment may include the following: an amino acid sequence a1 represented by SEQ ID NO:4, wherein the amino acid residue represented by X is a valine residue (mutation: M141V); an amino acid sequence a2 represented by SEQ ID NO:4, wherein the amino acid residue represented by X is a tyrosine residue (mutation: M141Y); an amino acid sequence a3 represented by SEQ ID NO:6, wherein the amino acid residue represented by X is a leucine residue (mutation: H182L); an amino acid sequence a4 represented by SEQ ID NO:8, wherein the amino acid residue represented by X is a histidine residue (mutation: W253H); or an amino acid sequence a5 represented by SEQ ID NO:9, wherein the amino acid residue represented by X is a glutamic acid residue (mutation: K260E); and a tag sequence. In addition to the amino acid sequence a1, a2, a3, a4 or a5 and the tag sequence, the polypeptide according to the present embodiment may further include a linker sequence that connects the amino acid sequence a1, a2, a3, a4 or a5 to the tag sequence. The linker sequence may comprise, for example, an amino acid sequence represented by GGSS or GGS.

[0477] The polypeptide according to one embodiment may be a polypeptide X1 consisting of an amino acid sequence a1, a2, a3, a4 or a5, a linker sequence bound to the C-terminus of the amino acid sequence a1, a2, a3, a4 or a5 and represented by the amino acid sequence: GGSS, and a His tag bound to the linker sequence and represented by SEQ ID NO: 12. One or more amino acid residues in the polypeptide X1 may be engineered, and 1 to 20, 1 to 15, 1 to 10, 1 to 7 or 1 to 5 amino acid residues may be engineered. The number of engineered amino acid residues in the polypeptide X1 may be 3 or less, may be 2 or less, or may be 1.

[0478] The polypeptide according to one embodiment may be a polypeptide X2 consisting of an amino acid sequence a1, a2, a3, a4 or a5, an SBP tag bound to the C-terminus of the amino acid sequence a1, a2, a3, a4 or a5 and represented by SEQ ID NO: 13, a His tag represented by SEQ ID NO: 12, and a linker sequence connecting the His tag to the SBP tag and represented by the amino acid sequence: GGS. One or more amino acid residues in the polypeptide X2 may be engineered, and 1 to 20, 1 to 15, 1 to 10, 1 to 7 or 1 to 5 amino acid residues may be engineered. The number of engineered amino acid residues in the polypeptide X2 may be 3 or less, may be 2 or less, or may be 1.

[0479] The polypeptide according to the present embodiment can be used as a mixture with another polypeptide, and can be used in an isolated or purified state. In the case of using the polypeptide as a mixture with another polypeptide, the amino acid of interest can be obtained by a step of isolating or purifying the target product.

[0480] The number of amino acid residues in the polypeptide according to the present embodiment in monomeric form may be 300 or more, 310 or more, 320 or more or 325 or more, or may be 330. The number may be 400 or less, 390 or less, 380 or less or 375 or less.

[0481] When an additional amino acid sequence is added neither to the N-terminus nor to the C-terminus, the number of amino acid residues in the polypeptide according to the present embodiment may be 300 or more, 310 or more, 320 or more, or 325 or more, or may be 330. The number may be 360 ​​or less, 350 or less, 340 or less, or 335 or less.

[0482] When an additional amino acid sequence is added to either or both of the N-terminus and the C-terminus, the number of amino acid residues in the polypeptide according to the present embodiment may be 340 or more, 350 or more, 360 or more, or 370 or more, or may be 374. The number may be 400 or less, 390 or less, 380 or less, or 375 or less.

[0483] The polypeptide according to the present embodiment may be used as a monomer, or may be used in the form of an association of two or more monomers.The polypeptide according to the present embodiment may be a homodimer.

[0484] When the polypeptide according to this embodiment is a homodimer, the homodimer has twice the number of amino acid residues as the monomer form.

[0485] In one aspect, the polypeptide has catalytic activity for an intermolecular reductive amination reaction between Compound A and Compound B or an intramolecular reductive amination reaction of Compound C under at least one reaction condition.

[0486] In order to measure the catalytic activity, methylamine as compound A and sodium 4-(2-chlorophenyl)-2-oxobutyrate as compound B can be used in combination. In this case, the catalytic activity of the polypeptide can be evaluated using the MeHph(2-Cl) synthesis activity as an index.

[0487] On the other hand, in order to measure the catalytic activity, ethylamine as the compound A and sodium phenylpyruvate as the compound B may be used in combination. In this case, the catalytic activity of the polypeptide can be evaluated using the EtPhe synthesis activity as an index.

[0488] On the other hand, in order to measure the catalytic activity, methylamine as compound A and sodium 2-cyclopentyl-2-oxoacetate as compound B may be used in combination. In this case, the catalytic activity of the polypeptide may be evaluated using the MeGly(cPent) synthesis activity as an index.

[0489] On the other hand, in order to measure the catalytic activity, ammonia as the compound A and phenylpyruvic acid as the compound B may be used in combination. In this case, the catalytic activity of the polypeptide can be evaluated using the Phe synthesis activity as an index.

[0490] In one aspect, the catalytic activity of the polypeptide can be evaluated using the reaction conditions described in the examples. For example, the concentration of each compound in the reaction solution at the start of the reaction can be: 50 mM of compound B or its salt, 100 mM of D(+)-glucose, 500 mM of compound A or its salt, 100 mM of phosphate buffer solution, 1 mM of NADPH, 0.002 units / μL of GDH solution, 2.5 μM of the polypeptide to be evaluated, and 20 v / v% or 20 w / v% of compound D or compound D'.

[0491] In one embodiment, the catalytic activity can be evaluated by starting the reaction with 50 mM sodium 4-(2-chlorophenyl)-2-oxobutyrate, sodium phenylpyruvate or sodium 2-cyclopentyl-2-oxoacetate, 100 mM D(+)-glucose, 500 mM methylamine or ethylamine, 100 mM phosphate buffer solution, 1 mM NADPH, 0.002 unit / μL GDH solution, 2.5 μM of the polypeptide to be evaluated, 20 v / v% or 20 w / v% of Compound D or Compound D' at 25°C or 37°C and pH 8 to 9 in a reaction solution, and determining the yield of the amino acid formed by the reductive amination reaction after 3 or 23 hours.

[0492] The catalytic activity is calculated based on the amount of amino acid (hereinafter also referred to as "target product") formed by the reductive amination reaction. The amount of the target product formed can be measured using a liquid chromatography mass spectrometer (LCMS). Specifically, the amount can be measured under the conditions described in the examples described later.

[0493] The polypeptide according to the present embodiment can be produced by, for example, a method comprising the following steps: culturing a recombinant cell as a transformant according to a conventional method; and obtaining the polypeptide of interest by isolating or purifying the culture obtained by culturing. The produced polypeptide can also be used without isolating and purifying, or only partially purifying from the culture obtained by culturing.

[0494] A commonly used separation or purification method can be used as a method for obtaining a polypeptide of interest from a culture. Examples of separation or purification methods include: methods utilizing solubility, such as salting out and solvent precipitation; methods utilizing molecular weight differences, such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; methods utilizing charge, such as ion exchange chromatography and hydroxyapatite chromatography; methods utilizing specific affinity, such as affinity chromatography; methods utilizing hydrophobicity differences, such as reversed-phase high performance liquid chromatography; and methods utilizing isoelectric point differences, such as isoelectric focusing electrophoresis.

[0495] When the polypeptide of interest is present in the periplasm or cytoplasm of the recombinant cells (Escherichia coli, etc.) of the culture, the culture is subjected to conventional methods, such as filtration or centrifugation, the bacterial cells or cells are collected, and suspended in an appropriate buffer solution, and the cell wall and / or cell membrane of the cells are broken by, for example, ultrasound, lysozyme, freezing and thawing methods, and then the fraction containing the polypeptide of interest is obtained by methods such as centrifugation or filtration. The fraction is dissolved using a surfactant such as Triton (TM) -X100 to obtain a crude solution. Then, the polypeptide of interest can be separated and purified from the crude solution by using the conventional methods given above.

[0496] The polypeptide according to the present embodiment can be prepared by gene recombination techniques known in the art as described above, and can generally be prepared as follows: using a plasmid containing a wild-type polypeptide gene as a template, using its primers, introducing site-directed mutations into specific positions and specific amino acids by PCR, and digesting the template plasmid with restriction enzymes, followed by transformation of Escherichia coli, etc. to clone a plasmid carrying a mutation of interest.

[0497] In the case of introducing an amino acid mutation into a second amino acid, the primer is then repeatedly used in the same manner as above to carry out site-directed mutagenesis using a plasmid carrying a mutation at a specific site as a template to construct a plasmid DNA encoding a 2-amino acid substitution mutant. Further introduction of amino acid mutations can also be carried out in the same manner as above.

[0498] The prepared DNA and, for example, plasmid pREP4 encoding lac repressor (Laci) are co-transformed into E. coli BL21 strain or the like. The obtained transformed strain is isolated and cultured, and then induced to express with IPTG. The obtained bacterial strain is then disrupted, and the supernatant is applied to an affinity column, for example, using a His tag to purify the polypeptide of interest.

[0499] Alternatively, the polypeptide can also be prepared by synthesizing a gene having a nucleotide sequence encoding the polypeptide of interest and transferring the nucleotide sequence to an expression vector, followed by protein expression, and purifying the polypeptide of interest using an affinity column by using various purification tags.

[0500] The method for producing polypeptide according to the present embodiment is not limited to the above method. Various gene manipulation techniques known in the art can be used, such as point mutation technology, gene synthesis technology and methods for introducing variant fragments using restriction enzymes. Expression is not limited to Escherichia coli, and animal cells or cell-free translation systems can be used. The purification method is not limited to the affinity column using polyhistidine, and various peptide tags or purification columns can be used.

[0501] <Reducing Agent>

[0502] 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.

[0503] In addition, additives 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, which reduce NADP and NAD to NADPH and NADH, respectively, may be further added together. That is, in the reaction step, various additives may be further present, and glucose and glucose dehydrogenase may be further present.

[0504] In the case of using, for example, NADPH as a reducing agent, a reactant that reduces NADP+ (oxidized nicotinamide adenine dinucleotide phosphate) that may be present in the reaction solution to NADPH can be used, because the amount of NADPH used can be reduced. Examples of reactants include a combination of glucose and GDH (glucose dehydrogenase). In the case of using glucose and GDH together with NADPH, the amount of NADPH used can be reduced to a catalytic amount.

[0505] <Reaction Steps>

[0506] The reaction step is carried out in a reaction solution comprising a polypeptide, a reducing agent, compound D, compound A and compound B or compound C. The reaction solution may contain other components, such as a solvent (excluding compounds belonging to compound D). The solvent may be water or a buffer solution. Examples of buffer solutions include phosphate buffer solution, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane) and Bicine (N,N-bis(2-hydroxyethyl)glycine). The reaction step may be carried out under appropriate reaction conditions. "Appropriate reaction conditions" means conditions under which the above-mentioned polypeptide can exhibit catalytic activity.

[0507] The reaction step can be carried out under a temperature condition of 0°C or higher and 50°C or lower. The lower limit of the temperature can be 0°C or higher, 10°C or higher or 20°C or higher. The upper limit of the temperature can be 50°C or lower, 40°C or lower or 30°C or lower. The temperature can be, for example, 0°C or higher and 50°C or lower, 10°C or higher and 40°C or lower or 20°C higher and 30°C or lower. In one aspect, the temperature can be 25°C or can be 16°C. The lower limit of the temperature can be 30°C or higher. The upper limit of the temperature can be 40°C or lower. The temperature can be, for example, 30°C or higher and 40°C or lower. The temperature can also be 37°C. The reaction temperature can be changed stepwise or continuously.

[0508] The reaction step can be carried out under a pH condition of 7 or higher and 11 or lower. The lower limit of pH can be 7 or higher, 7.5 or higher, 8 or higher or 8.5 or higher. The upper limit of pH can be 11 or lower, 10 or lower, 9.5 or lower or 9 or lower. pH can be, for example, between pH 7 and 11, between 8 and 10, or between 8.5 and 9.5. In one aspect, pH can be 8 or can be 9. In this article, pH refers to the pH in the reaction solution at the beginning of the reaction, and accepts pH engineering during the reaction. pH engineering during the reaction can be within 2, can be within 1.5, or can be within 1.

[0509] In the reaction step, the concentration of compound A can be 10mM or higher and 3000mM or lower in terms of the total amount of the reaction solution when the reaction starts. The lower limit of the concentration of compound A can be 10mM or higher, 100mM or higher, 300mM or higher, 500mM or higher, 700mM or higher, 900mM or higher, 1100mM or higher, 1300mM or higher, 1500mM or higher or 1700mM or higher in terms of the total amount of the reaction solution. The upper limit of the concentration of compound A can be 3000mM or lower, 2500mM or lower or 2000mM or lower in terms of the total amount of the reaction solution. The concentration of compound A can be 10mM or higher and 3000mM or lower, or 500mM or higher and 2000mM or lower in terms of the total amount of the reaction solution. In one aspect, the concentration of Compound A may be 500 mM or may be 1750 mM based on the total amount of the reaction solution.

[0510] In the reaction step, the concentration of the compound B or the compound C at the start of the reaction may be 0.001 mM or more and 1000 mM or less based on the total amount of the reaction solution.

[0511] When the reaction starts, the concentration of compound B can be 10mM or higher and 500mM or lower in terms of the total amount of the reaction solution. When the reaction starts, the lower limit of the concentration of compound B can be 10mM or higher, 30mM or higher, 50mM or higher, 100mM or higher, 150mM or higher, 200mM or higher, 250mM or higher, 300mM or higher or 330mM or higher in terms of the total amount of the reaction solution. When the reaction starts, the upper limit of the concentration of compound B can be 500mM or lower, 450mM or lower, 400mM or lower or 380mM or lower in terms of the total amount of the reaction solution. When the reaction starts, the concentration of compound B can be 10mM or higher and 500mM or lower in terms of the total amount of the reaction solution, or 300mM or higher and 400mM or lower. In one aspect, when the reaction starts, the concentration of compound B can be 50mM or can be 350mM in terms of the total amount of the reaction solution.

[0512] When the reaction starts, the concentration of compound C can be 10mM or higher and 500mM or lower in terms of the total amount of the reaction solution. When the reaction starts, the lower limit of the concentration of compound C can be 10mM or higher, 30mM or higher, 50mM or higher, 100mM or higher, 150mM or higher, 200mM or higher, 250mM or higher, 300mM or higher or 330mM or higher in terms of the total amount of the reaction solution. When the reaction starts, the upper limit of the concentration of compound C can be 500mM or lower, 450mM or lower, 400mM or lower or 380mM or lower in terms of the total amount of the reaction solution. When the reaction starts, the concentration of compound C can be 10mM or higher and 500mM or lower in terms of the total amount of the reaction solution, or 300mM or higher and 400mM or lower. In one aspect, when the reaction starts, the concentration of compound C can be 50mM or can be 350mM in terms of the total amount of the reaction solution.

[0513] In the reaction step, the ratio of the number of moles of compound A to the number of moles of compound B (the number of moles of compound A / the number of moles of compound B) at the start of the reaction can be 1 or more based on the total amount of the reaction solution. The lower limit of the ratio of the number of moles of compound A to the number of moles of compound B at the start of the reaction can be 1 or more, 3 or more, 5 or more, 8 or more or 9 or more based on the total amount of the reaction solution. The upper limit of the ratio of the number of moles of compound A to the number of moles of compound B at the start of the reaction can be 100 or less, 50 or less, 30 or less, 20 or less or 15 or less based on the total amount of the reaction solution. The ratio of the number of moles of compound A to the number of moles of compound B at the start of the reaction can be 5 or 10 based on the total amount of the reaction solution.

[0514] If compound D or compound D' is liquid under the conditions of 25 ℃ and 1atm, the reaction step can be carried out under the conditions that the concentration of compound D or compound D' in the reaction solution is 1v / v% or higher and 60v / v% or lower. The lower limit of the concentration of compound D or compound D' in the reaction solution can be 1v / v% or higher, 5v / v% or higher, 10v / v% or higher, 20v / v% or higher, 30v / v% or higher or 40v / v% or higher, because the yield can be further improved. The upper limit of the concentration of compound D or compound D' in the reaction solution can be 60v / v% or lower, 50v / v% or lower or 40v / v% or lower, because the yield can be further improved. In one aspect, the concentration of compound D or compound D' in the reaction solution can be 20v / v% or 10v / 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 beginning of the reaction.

[0515] If compound D or compound D' is solid under the conditions of 25 ℃ and 1atm, the reaction step can be carried out under the conditions that the concentration of compound D or compound D' in the reaction solution is 1w / v% or higher and 60w / v% or lower. The lower limit of the concentration of compound D or compound D' in the reaction solution can be 1w / v% or higher, 5w / v% or higher, 10w / v% or higher, 20w / v% or higher, 30w / v% or higher or 40w / v% or higher, because the yield can be further improved. The upper limit of the concentration of compound D or compound D' in the reaction solution can be 60w / v% or lower, 50w / v% or lower or 40w / v% or lower, because the yield can be further improved. In one aspect, the concentration of compound D or compound D' in the reaction solution can be 20w / v% or 10w / 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 beginning of the reaction.

[0516] The reaction step can be carried out under the condition that the concentration of the polypeptide in the reaction solution is 0.1 μM or higher and 10 μM or lower. The lower limit of the concentration of the polypeptide in the reaction solution can be 0.5 μM or higher, 1.0 μM or higher, 1.5 μM or higher, 2.0 μM or higher, 2.3 μM or higher or 2.4 μM or higher. The upper limit of the concentration of the polypeptide in the reaction solution can be 10 μM or lower, 5 μM or lower, 4 μM or lower, 3 μM or lower, 2.7 μM or lower or 2.6 μM or lower. In one aspect, the concentration of the polypeptide in the reaction solution can be 2.5 μM.

[0517] Reaction step can be carried out under the condition that the concentration of reducing agent in the reaction solution is 0.1mM or higher and 100mM or lower. The lower limit of the concentration of reducing agent in the reaction solution can be 0.1mM or higher, 0.2mM or higher, 0.4mM or higher, 0.6mM or higher, 0.8mM or higher or 1mM or higher. The upper limit of the concentration of reducing agent in the reaction solution can be 100mM or lower, 80mM or lower, 60mM or lower, 40mM or lower, 20mM or lower or 10mM or lower. In one aspect, the concentration of reducing agent in the reaction solution can be 1mM or can be 2mM.

[0518] The yield of the amino acid formed 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 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 formed in the reaction step in the absence of compound D or compound D'. How many times higher the yield of the amino acid formed by the reaction step in the presence of compound D or compound D' is than the yield of the amino acid formed in the reaction step in the absence of compound D or compound D' can be measured at any reaction temperature and any reaction time, but can be determined by calculating the ratio of the yield of the target product under the two conditions, for example, at 25° C. or 37° C., after 1 hour, 3 hours, or 23 hours from the start of the reaction. Specifically, this can be determined by dividing the yield of the amino acid formed by the reaction in the presence of compound D or compound D' by the yield of the amino acid formed in the reaction step in the absence of compound D or compound D'. It should be noted that the yield at 25°C after 3 hours from the start of the reaction is preferably calculated.

[0519] The yield of the amino acid formed 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, and may be 100% or less, 99% or less, 98% or less, 97% or less, or 96% or less.

[0520] The yield of the amino acid formed in the reaction step can be measured under the following conditions.

[0521] A reaction is carried out in a reaction solution using 50 mM phenylpyruvic acid, 2-oxo-3-(p-tolyl)propionic acid or 2-cyclopentyl-2-oxo-acetic acid, 100 mM D(+)-glucose, 500 mM ammonia, methylamine or ethylamine, 100 mM phosphate buffer solution, 1 mM NADPH, 0.002 unit / μL GDH solution, a polypeptide at a concentration of 2.5 μM, and when the compound D or compound D' is used, 20 v / v% of the compound D or compound D' if the compound D or compound D' is a liquid under the conditions of 25° C. and 1 atm, or 20 w / v% of the compound D or compound D' if the compound D or compound D' is a solid under the conditions of 25° C. and 1 atm, and the yield of the amino acid formed by the reductive amination reaction is determined after 19 hours.

[0522] In the present specification, when the reaction step is a step of performing an intermolecular reductive amination reaction between compound A and compound B, the start of the reaction means a later time point among the time points when compound A, compound B, the polypeptide disclosed in the present specification, and the reducing agent are added to the system, and when the reaction step is a step of performing an intramolecular reductive amination reaction of compound C, it means a later time point among the time points when compound C, the polypeptide disclosed in the present specification, and the reducing agent are added to the system.

[0523] The yield of the amino acid formed in the reaction step is calculated by the following method. LCMS analysis is performed on the reaction solution and the calibration curve samples prepared separately (compounds having the same structure or the same UV absorption wavelength as the target product) to obtain their respective UV figures or extracted ion chromatograms. The UV peak area or MS peak area derived from the target product is obtained by the UV figure obtained from the reaction solution or the ion chromatogram extracted. Similarly, the UV peak area or MS peak area of ​​the preparation is obtained from the calibration curve sample. Based on the corresponding relationship between the concentration of the preparation contained in the calibration curve sample and the UV peak area or the MS peak area, the concentration of the target product contained in the reaction solution is calculated by the UV peak area or the MS peak area. When the compound B or compound C in the reaction solution is completely converted into the target product, it is believed that the concentration of the target product in the reaction solution is equal to the concentration of the compound B or compound C in the reaction solution when the reaction starts. In fact, the yield is calculated by dividing the concentration of the target product contained in the reaction solution by the concentration of the compound B or compound C in the reaction solution when the reaction starts.

[0524] The reaction yield can also be calculated by correcting the ratio of the absorbance coefficient of each compound according to the following expression (herein, the reaction yield determined by this method is also referred to as the reaction conversion rate).

[0525] Expression: Reaction conversion rate (%) = S1 / ((S2 / 8.0)+S1)×100

[0526] In the expression, S1 represents the UV peak area of ​​the target product, and S2 represents the UV peak area of ​​compound B or compound C.

[0527] [Method for producing peptide compound]

[0528] The method for producing a peptide compound according to the present embodiment comprises the following steps:

[0529] (1) producing amino acids by the above-mentioned amino acid production method; and

[0530] (2) linking the amino acid with one or more selected from the group consisting of other amino acids and peptides to produce a peptide compound.

[0531] The step of producing the peptide compound can be carried out in an aqueous vehicle or in a mixed solution of an aqueous vehicle and an organic solvent. The aqueous vehicle can 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-bis(2-hydroxyethyl)glycine). Examples of organic solvents include dimethyl sulfoxide.

[0532] [Reductive amination reaction accelerator]

[0533] The reductive amination reaction accelerator according to the present embodiment may be a compound represented by the above-mentioned formula (1), which accelerates the following reaction (i) or (ii):

[0534] (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;

[0535] (ii) 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.

[0536] With regard to specific aspects of the reductive amination reaction accelerator, the aspects described in the method for producing an amino acid can be applied.

[0537] The reductive amination reaction accelerator can be at least one 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, can be at least one selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone and trimethylphosphine oxide, and can be dimethyl sulfoxide.

[0538] The present invention described above can be regarded as the use of the compound represented by formula (1) for promoting the reductive amination reaction of (i) or (ii) described above. The present invention described above can also be regarded as the compound represented by formula (1) for promoting the reductive amination reaction of (i) or (ii) described above. The present invention described above can also be regarded as the use (application) of the compound represented by formula (1) in producing a reductive amination reaction promoter for (i) or (ii) described above. For these specific aspects, the aspects described in the method for producing amino acids can be applied.

[0539] The following abbreviations are used in this specification.

[0540] [Table 1]

[0541]

[0542]

[0543] [Table 2]

[0544]

[0545] [Example]

[0546] The present invention will be further described with reference to examples given below, but the present invention is not limited to these examples.

[0547] The analytical conditions for LCMS are described below.

[0548] LCMS method

[0549] Condition Name: FA05

[0550] Device: Waters Acquity UPLC / SQD

[0551] Column (ID x Length (mm), Particle Size (μm)): Aldrich Ascentis Express C18 (2.1x50,2.7)

[0552] Mobile phase: A) 0.1% FA H2O, B) 0.1% FA MeCN

[0553] Gradient (A / B): 95 / 5 to 0 / 100 (1.0 min) → 0 / 100 (0.4 min)

[0554] Flow rate (ml / min): 1

[0555] Column temperature: 35°C

[0556] Wavelength: Total 210-400nm PDA

[0557] Condition Name: TFA00

[0558] Device: Waters H-class

[0559] Column: (ID x Length (mm), Particle Size (μm)): ACQUITY UPLC HSS T3 (2.1x 50,1.8)

[0560] Mobile phase: A) 0.05% TFA H2O, B) 0.05% TFA MeCN

[0561] 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)

[0562] Flow rate (ml / min): 0.5

[0563] Column temperature: 30°C

[0564] Wavelength: 197nm

[0565] Condition Name: TFA00-QDa

[0566] Device: Waters H-class / QDa

[0567] Column: (ID x Length (mm), Particle Size (μm)): ACQUITY UPLC HSS T3 (2.1x 50,1.8)

[0568] Mobile phase: A) 0.05% TFA H2O, B) 0.05% TFA MeCN

[0569] 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)

[0570] Flow rate (ml / min): 0.5

[0571] Column temperature: 30°C

[0572] Wavelength: 197nm

[0573] Reagents not specifically described were purchased from commercial suppliers.

[0574] Preparation Example 1: Preparation of wild-type NMAADH-C-His

[0575] The gene of SEQ ID NO:1 sequence was synthesized, and its C-terminal was labeled with a joint sequence (GGSS) and a His tag sequence (HHHHHH), and cloned into a vector for expression in Escherichia coli. The expression vector was transformed into a BL21 (DE3) Escherichia coli strain and cultivated. The protein of interest was purified from the supernatant of ultrasonically broken cells using a nickel column. The protein solution was dialyzed (50mM tris hydrochloric acid, 10% glycerol, 150mM sodium chloride, pH 8.0) to obtain a final formulation (SEQ ID NO:10, wild-type NMAADH-His).

[0576] SEQ ID NO: 1:

[0577]

[0578] SEQ ID NO:10:

[0579]

[0580] Preparation Example 2: Preparation of W253H-SBP-His

[0581] A gene of the amino acid sequence represented by SEQ ID NO: 8, wherein X is His, and the C-terminus is tagged with a streptavidin-binding peptide tag sequence (GTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQ), a linker sequence (GGS), and a His tag sequence (HHHHHH), was synthesized and cloned into a vector for expression in E. coli. The expression vector was transferred to a BL21 (DE3) E. coli strain (Novagen), which was then cultured at 18° C. for 2 days using Overnight Express Instant TB Medium (Novagen) to express the protein of interest.

[0582] The bacterial cells obtained were recovered by centrifugation, and the bacterial cells were broken by ultrasound. Lysate was separated by centrifugation, and the supernatant fraction was purified by affinity chromatography using cOmplete His-Tag Purification Resin (Roche). After the fraction containing the protein of interest was recovered, the sample was dialyzed against 50mM Tris-hydrochloric acid (pH 8.0) / 150mM sodium chloride / 10% glycerol and used as the final preparation (SEQ ID NO: 11, W253H).

[0583] The amino acid sequence represented by SEQ ID NO: 8, wherein X is His:

[0584]

[0585] SEQ ID NO:11:

[0586]

[0587] Preparation Example 3: Preparation of variant NMAADH-SBP-His

[0588] The gene of each variant enzyme sequence (SEQ ID No: 14 to 17) was synthesized, and its C-terminus was tagged with a streptavidin-bound peptide tag sequence (GTDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQ), a linker sequence (GGS) and a His tag sequence (HHHHHH), and cloned into a vector for expression in E. coli. The expression vector was transferred to a BL21 (DE3) E. coli strain (Novagen), which was then cultured at 18°C ​​for 2 days using Overnight Express Instant TB Medium (Novagen) to express the protein of interest.

[0589] The bacterial cells obtained by centrifugal recovery, and ultrasonication bacterial cells. By centrifugal separation lysate, and use Ni Sepharose 6Fast Flow (Cytiva) by affinity chromatography purification supernatant fraction. Recover the fraction containing protein of interest and use as 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).

[0590] SEQ ID NO:14:

[0591]

[0592] SEQ ID NO:15:

[0593]

[0594] SEQ ID NO:16:

[0595]

[0596] SEQ ID NO:17:

[0597]

[0598]

[0599] SEQ ID NO:18:

[0600]

[0601] SEQ ID NO: 19:

[0602]

[0603] SEQ ID NO:20:

[0604]

[0605]

[0606] SEQ ID NO:21:

[0607]

[0608] The following reagents were used in this example.

[0609] [Table 3]

[0610]

[0611] The following reagents were prepared in advance and used as follows.

[0612] [Table 4]

[0613]

[0614]

[0615] Synthesis Example 1: Synthesis of the raw material intermediate (E)-4-(2-chlorophenyl)-2-oxobut-3-enoic acid

[0616] [Equation 28]

[0617]

[0618] At 0°C, sodium pyruvate (2.42 g, 0.022 mol) was added to a mixed solution of 1N aqueous sodium hydroxide solution (60 ml) and ethanol (3 ml), and then 2-chlorobenzaldehyde (2.25 ml, 0.02 mol) was added dropwise thereto over 20 minutes or more, and the reaction solution was stirred at 0°C for 2 hours. Then, water (160 ml) was added to the reaction solution, and the mixture was washed with toluene (100 ml). 5N hydrochloric acid was added dropwise to the resulting aqueous layer at 0°C until the pH became 1. The solution was stirred for 30 minutes. The precipitate produced in the solution was recovered by filtration, and the precipitate was washed with cold water. The obtained wet solid was heated and dried under reduced pressure to obtain (E)-4-(2-chlorophenyl)-2-oxobut-2-enoic acid (2.7 g, 0.013 mol, 64% yield).

[0619] LCMS (ESI) m / z = 209.0 (MH) -

[0620] Retention time: 0.52 minutes (analysis conditions FA05)

[0621] Synthesis Example 2: Synthesis of raw material sodium 4-(2-chlorophenyl)-2-oxobutyrate

[0622] [Formula 29]

[0623]

[0624] A suspension of (E)-4-(2-chlorophenyl)-2-oxobut-2-enoic acid (1.00g, 4.75mol) and palladium / carbon (0.20g, 1.88mol) in methanol (20ml) and diphenyl sulfide (7.97μl, 0.047mmol) was stirred at room temperature for 1 hour under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was then concentrated under reduced pressure. 1N aqueous sodium hydroxide solution (15ml) was added to the resulting residue, and the mixture was washed with toluene (15ml) (twice). The water layer was stirred at 0°C for 30 minutes. Then, the precipitate produced in the solution was recovered by filtration, and the precipitate was washed with cold water. The obtained wet solid was heated and dried under reduced pressure to obtain sodium 4-(2-chlorophenyl)-2-oxobutanoate (847mg, 3.61mmol, 75% yield).

[0625] LCMS (ESI) m / z = 211.0 (MH) -

[0626] Retention time: 0.59 minutes (analysis conditions FA05)

[0627] Evaluation Example 1-1: Evaluation of Additives in MeHph(2-Cl) Synthesis 1-1

[0628] A mixed solution of sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration: 50 mM), D(+)-glucose (final concentration: 100 mM), methylamine solution (final concentration: 500 mM), phosphate buffer solution (final concentration: 0.1 M), NADPH solution (final concentration: 1 mM), GDH solution (final concentration: 0.002 unit / μL), any additives described in Table 5 (20 v / v% DMSO, NMP, formamide, 3-methyl-2-oxazolidinone, oxazolidinone, ethanol, dioxane, NMP, 2-propanol, MeCN, THF, TMSO2 based on the reaction solution and 20 w / v% Me3PO2, DMSO2, DESO2 based on the reaction solution), wild-type NMAADH-C-His (final concentration: 2.5 μM) and ultrapure water (pH 8 to 9) was prepared and incubated at 25°C or 37°C for 3 hours. For the additive-free condition (0 v / v%) as a comparative condition, ultrapure water was added instead of the additive.

[0629] The DMSO solution containing hydrochloric acid in an amount of 19 times that of the reaction solution was added to the reaction solution thus incubated so that the final concentration of the hydrogen chloride added was 300 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmospin Filter H, Nacalai Tesque, Inc.) to prepare a sample for LCMS analysis, which was then subjected to LCMS measurement (analysis conditions: FA05) and left to stand overnight. The UV peak area of ​​the title compound MeHph (2-Cl) at 254 nm was calculated to determine the reaction yield. The results are shown in Table 5.

[0630] LCMS (ESI) m / z = 228.1 (M+H) +

[0631] Retention time: 0.38 minutes (analysis conditions FA05)

[0632] [Table 5]

[0633]

[0634]

[0635] Evaluation Example 1-2: Evaluation of Additives in MeHph(2-Cl) Synthesis 1-2

[0636] The same evaluation as in Evaluation Example 1-2 was performed except that any additive described in Table 6 was used (20 v / v%) and the temperature (reaction temperature) at the time of incubation for 3 hours was set to 25° C. The results are shown in Table 6.

[0637] [Table 6]

[0638]

[0639] Evaluation Example 2: Evaluation of the amount of additives added

[0640] Under the same reaction conditions as Evaluation Example 1, at 25°C or 37°C, by only changing the content of DMSO as an additive in the reaction solution from 0v / v% to 40v / v%, the effect of the addition amount on the reaction was evaluated. For the 0v / v% condition, ultrapure water was added instead of the additive. The yield after 3 hours of reaction was evaluated. The sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and was subjected to LCMS measurement (analysis conditions: FA05) and left to stand overnight. The UV peak area of ​​the title compound MeHph (2-Cl) at 254nm was calculated to determine the reaction yield. The results are shown in Table 7.

[0641] LCMS (ESI) m / z = 228.1 (M+H) +

[0642] Retention time: 0.38 minutes (analysis conditions FA05)

[0643] [Table 7]

[0644]

[0645] Evaluation Example 3: Evaluation of the effect of additives in EtPhe synthesis

[0646] The evaluation of the reaction was carried out under the same conditions as the reaction of Evaluation Example 1, using phenylpyruvic acid (final concentration 50mM) instead of sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50mM), using ethylamine solution (final concentration 500mM) instead of methylamine solution (final concentration 500mM), and adding DMSO as an additive to 20v / v% of the total reaction solution volume. The sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and then LCMS measurement (analysis conditions: FA05) was performed and left to stand overnight. The UV peak area of ​​the title compound EtPhe at 254nm was calculated to determine the reaction yield. In this regard, the reaction yield was determined after 3 hours and 23 hours under two conditions of reaction temperature of 25°C and 37°C. The results are shown in Table 8. LCMS (ESI) m / z=194.1 (M+H) +

[0647] Retention time: 0.30 minutes (analysis conditions FA05)

[0648] [Table 8]

[0649]

[0650] Evaluation Example 4: Evaluation of the effect of additives in MeGly (cPent) synthesis

[0651] The evaluation of the reaction was carried out under the same conditions as the reaction of Evaluation Example 1, using sodium 2-cyclopentyl-2-oxoacetate (final concentration 50 mM) instead of sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50 mM), and adding DMSO as an additive to 20 v / v% of the total reaction solution volume. The evaluation of the reaction was determined by the reaction yield after 24 hours under two conditions of reaction temperature of 25°C and 37°C. A hydrochloric acid solution in an amount 7 times that of the reaction solution was added to the reaction solution thus incubated so that the final concentration of the added hydrogen chloride was 200 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmospin Filter H, Nacalai Tesque, Inc.) to prepare a sample for LCMS analysis, which was then left to stand overnight in LCMS measurement (analysis conditions: FA05). The title compound MeGly (cPent) was calculated at 158.1 (M+H) + The mass peak area at 1000 nm was used to determine the reaction yield. The results are shown in Table 9.

[0652] LCMS (ESI) m / z = 158.1 (M+H) +

[0653] Retention time: 0.23 minutes (analysis conditions FA05)

[0654] [Table 9]

[0655]

[0656] Evaluation Example 5: Reaction Example Using Variant Enzyme NMAADH (W253H)

[0657] Using the W253H-SBP-His solution prepared in Preparation Example 2 instead of wild-type NMAADH-C-His, the reaction was evaluated under the same conditions as the reaction of Evaluation Example 1. The evaluation of the reaction was determined after 3 hours under two conditions of reaction temperature of 25°C and 37°C. The sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and then the sample was left stationary overnight in LCMS measurement (analysis conditions: FA05). The UV peak area of ​​the title compound MeHph (2-Cl) at 254nm was calculated to determine the reaction yield. The results are shown in Table 10.

[0658] LCMS (ESI) m / z = 228.1 (M+H) +

[0659] Retention time: 0.38 minutes (analysis conditions FA05)

[0660] [Table 10]

[0661]

[0662] Evaluation Example 6: Evaluation of Additives in MeHph(2-Cl) Synthesis 2

[0663] A mixed solution of sodium 4-(2-chlorophenyl)-2-oxobutyrate (final concentration: 10 mM), D(+)-glucose (final concentration: 20 mM), methylamine solution (final concentration: 100 mM), phosphate buffer solution (final concentration: 0.1 M), NADPH solution (final concentration: 1 mM), GDH solution (final concentration: 0.002 units / 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 the no-additive (0 v / v%) condition as a comparative condition, ultrapure water was added instead of the additive.

[0664] The DMSO solution containing hydrochloric acid in an amount of 7 times that of the reaction solution was added to the reaction solution thus incubated so that the final concentration of the added hydrogen chloride was 300 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmospin Filter H, Nacalai Tesque, Inc.) to prepare a sample for LCMS analysis, which was then subjected to LCMS measurement (analysis conditions: FA05) and left to stand overnight. The UV peak area of ​​the title compound MeHph (2-Cl) at 254 nm was calculated to determine the reaction yield. The results are shown in Table 11.

[0665] LCMS (ESI) m / z = 228.1 (M+H) +

[0666] Retention time: 0.38 minutes (analysis conditions FA05)

[0667] [Table 11]

[0668] additive Yield (%) No Additives 48 DMSO 62

[0669] Evaluation Example 7: Evaluation of Additives in MeGly(cPent) Synthesis Using Variant Enzyme NMAADH(W253H)

[0670] 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-bis(2-hydroxyethyl)glycine (final concentration: 100 mM) were dissolved in distilled water. Additive DMSO (0 to 40 v / v%) was added to the solution, and the pH was then adjusted to 8.8 to 8.9 with a 5 M aqueous sodium hydroxide solution. GDH solution (0.10 unit / μL, 1× TNG solution, final concentration: 0.0040 unit / μL) and variant 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.

[0671] During the incubation period, 50 μL of the reaction solution was sampled, and 750 μL of methanol and 200 μL of 1M hydrochloric acid were added to the sampled solution. The resulting solution was filtered through a 0.5 μm PTFE membrane filter (DISMIC, ADVANTEC) to prepare a sample for LC analysis, which was then subjected to LC measurement (analysis conditions: TFA00) and left to stand overnight. The UV peak area at 197 nm was measured by LC, and the ratio of the absorbance coefficient of each compound was corrected according to the expression X given below to calculate the reaction conversion rate. The change in reaction conversion rate is shown in Table 12.

[0672] Retention time: 1.1 minutes (Analysis conditions: TFA00)

[0673] The resulting solution was measured by LCMS (analysis conditions: TFA00-QDa) to confirm the MS peak of MeGly(cPent).

[0674] Retention time: 1.5 minutes (Analysis conditions: TFA00-QDa)

[0675] LCMS (ESI) m / z = 158.1 (M+H) +

[0676] Expression X: Reaction conversion rate (%) = S 1a / ((S 2a / 8.0)+S 1a )x 100

[0677] In the expression X, S 1a Represents the UV peak area of ​​MeGly (cPent). 2a It represents the UV peak area of ​​sodium 2-cyclopentyl-2-oxoacetate.

[0678] [Table 12]

[0679]

[0680] Evaluation Example 8: Evaluation of Additives in Phe Synthesis

[0681] The evaluation of reaction is carried out under the same conditions as the reaction of evaluation example 1, using phenylpyruvic acid (final concentration 50mM) to replace sodium 4-(2-chlorophenyl)-2-oxobutanoate (final concentration 50mM), using ammonia solution (final concentration 500mM) to replace methylamine solution (final concentration 500mM), and adding DMSO as an additive to 20v / v% of the total reaction solution volume. For the condition of no additive (0v / v%) as a comparative condition, ultrapure water is added to replace the additive. The sample for LCMS analysis is prepared in the same manner as in evaluation example 1, and then LCMS measurement (analysis conditions: FA05) is performed and remains stationary overnight. The UV peak area of ​​the title compound Phe at 254nm is calculated to determine the reaction yield. In this regard, under the conditions of two kinds of reaction temperatures of 25°C and 37°C, the reaction yield is determined after 3 hours and 5 hours. The results are shown in Table 13.

[0682] LCMS (ESI) m / z = 166.1 (M+H) +

[0683] Retention time: 0.27 minutes (analysis conditions FA05)

[0684] [Table 13]

[0685]

[0686] Evaluation Example 9: Evaluation of Additives in Phe Synthesis Using Variant Enzyme NMAADH (W253H)

[0687] Phenylpyruvic acid (final concentration: 50 mM), D (+) -glucose (final concentration: 100 mM), ammonia solution (final concentration: 500 mM), N, N- bicine buffer solution (final concentration: 0.1 M), NADPH solution (final concentration: 1 mM), GDH solution (final concentration: 0.002 units / μ L), additive DMSO (20 v / v % based on the reaction solution), variant NMAADH W253H-SBP-His (final concentration: 2.5 μ M) and a mixed solution of ultrapure water (pH 8 to 9) were prepared and incubated at 25 ° C, and then the reaction was evaluated. For the condition of no additive (0 v / v %) as a comparative condition, ultrapure water was added instead of the additive. The sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and then LCMS measurement (analysis conditions: FA05) was performed and left to stand overnight. The UV peak area of ​​the title compound Phe at 254 nm was calculated to determine the reaction yield. In this regard, the reaction yield was determined after 3 and 5 hours at a reaction temperature of 25°C. The results are shown in Table 14.

[0688] LCMS (ESI) m / z = 166.1 (M+H) +

[0689] Retention time: 0.27 minutes (analysis conditions FA05)

[0690] [Table 14]

[0691]

[0692] Evaluation Example 10: Evaluation of Additives in MeHph(2-Cl) Synthesis 3

[0693] A mixed solution of sodium 4-(2-chlorophenyl)-2-oxobutyrate (final concentration: 50 mM), D(+)-glucose (final concentration: 100 mM), methylamine solution (final concentration: 500 mM), N,N-bicine 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%), variant NMAADH W253H-SBP-His solution (final concentration: 2.5 μM) and ultrapure water was prepared and incubated at 16° C. for 1 hour. For the no additive (0 v / v%) condition as a comparative condition, ultrapure water was added instead of the additive.

[0694] The DMSO solution containing hydrochloric acid in an amount of 19 times that of the reaction solution was added to the reaction solution thus incubated so that the final concentration of the hydrogen chloride added was 300 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmospin Filter H, Nacalai Tesque, Inc.) to prepare a sample for LCMS analysis, which was then subjected to LCMS measurement (analysis conditions: FA05) and left to stand overnight. The UV peak area of ​​the title compound MeHph (2-Cl) at 254 nm was calculated to determine the reaction yield. The results are shown in Table 15.

[0695] LCMS (ESI) m / z = 228.1 (M+H) +

[0696] Retention time: 0.38 minutes (analysis conditions FA05)

[0697] [Table 15]

[0698]

[0699] Evaluation Example 11: Evaluation of Additives in EtPhe Synthesis Using Variant Enzyme NMAADH

[0700] Phenylpyruvate (final concentration: 50 mM), D(+)-glucose (final concentration: 100 mM), ethylamine solution (final concentration: 500 mM), N,N-bicine 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% based on the reaction solution), each variant NMAADH-SBP-His solution (final concentration: 2.5 μM) and a mixed solution of ultrapure water (pH 8 to 9) were prepared and incubated at 37°C, and then the reaction was evaluated. It should be noted that the five variant NMAADH-SBP-His solutions (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) prepared in Preparation Examples 2 and 3 were each used for evaluation. For the condition of no additive (0v / v%) as a comparative condition, ultrapure water was added instead of additive. The sample for LCMS analysis was prepared in the same manner as in Evaluation Example 1, and then LCMS measurement (analysis conditions: FA05) was performed and left to stand overnight. The UV peak area of ​​the title compound EtPhe at 254nm was calculated to determine the reaction yield. In this regard, the reaction yields were determined after 2, 4 and 24 hours at a reaction temperature of 37° C. The results are shown in Tables 16 to 20.

[0701] LCMS (ESI) m / z = 194.1 (M+H) +

[0702] Retention time: 0.30 minutes (analysis conditions FA05)

[0703] [Table 16]

[0704]

[0705] [Table 17]

[0706]

[0707] [Table 18]

[0708]

[0709] [Table 19]

[0710]

[0711] [Table 20]

[0712]

[0713] Evaluation Example 12: Evaluation of additives in the synthesis of (S)-3-(4,4-difluorocyclohexyl)-2-(methylamino)propionic acid using variant enzyme NMAADH

[0714] A mixed solution of 3-(4,4-difluorocyclohexyl)-2-oxopropionic acid (final concentration: 50 mM), D(+)-glucose (final concentration: 100 mM), methylamine solution (final concentration: 500 mM), N,N-bicine 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%), variant NMAADH W253H-SBP-His solution (final concentration: 2.5 μM) and ultrapure water was prepared and incubated at 37° C. for 1 hour. For the no additive (0 v / v%) condition as a comparative condition, ultrapure water was added instead of the additive.

[0715] To the reaction solution thus incubated, a hydrochloric acid solution in an amount three times that of the reaction solution was added so that the final concentration of the added hydrogen chloride was 200 mM. The resulting solution was passed through a 0.45 μm PTFE membrane filter (Cosmospin Filter H, Nacalai Tesque, Inc.) to prepare a sample for LCMS analysis, and then allowed to stand overnight in a LCMS measurement (analysis conditions: FA05). The title compound (S)-3-(4,4-difluorocyclohexyl)-2-(methylamino)propionic acid was calculated at 222.1 (M+H) + The mass peak area at was used to determine the reaction yield. The results are shown in Table 21.

[0716] LCMS (ESI) m / z = 222.1 (M+H) +

[0717] Retention time: 0.36 minutes (analysis conditions FA05)

[0718] [Table 21]

[0719] additive Yield at reaction temperature 37°C (%) No Additives 48 DMSO 64

[0720] Synthesis Example 3: Synthesis of (2S,3S)-2-amino-3-phenyl-butyric acid using variant enzyme W253H-SBP-His and additive (dimethyl sulfoxide)

[0721] In this example, the following reagents were used. Reagents not listed are those in Table 3.

[0722] [Table 22]

[0723] Reagent name CAS Registry Number supplier Sodium 2-oxo-3-phenylbutyrate 125116-72-5 Chemieliva Pharmaceutical 5mol / L hydrochloric acid 7647-01-0 FUJIFILM Wako Pure Chemical Corp. (2S,3R)-2-Amino-3-phenylbutyric acid hydrochloride 143251-58-5 J&W Pharmlab (2S,3S)-2-Amino-3-phenylbutyric acid hydrochloride 53331-55-8 J&W Pharmlab (2R,3R)-2-Amino-3-phenylbutyric acid hydrochloride 143251-57-4 J&W Pharmlab

[0724] The following apparatus and deuterated solvents were used for 1 H-NMR analysis.

[0725] Device: AVANCE III HD 400SMART-BBFO probe (400MHz, Bruker)

[0726] Deuterated solvent: trifluoroacetic acid-d1

[0727] The following abbreviations are used in this example.

[0728] [Table 23]

[0729] abbreviation Compound (I) Compound obtained by the reaction: (2S,3S)-2-amino-3-phenylbutyric acid (II) Standard compound: (2S,3R)-2-amino-3-phenylbutyric acid hydrochloride (III) Standard compound: (2S,3S)-2-amino-3-phenylbutyric acid hydrochloride (IV) Standard compound: (2R,3R)-2-amino-3-phenylbutyric acid hydrochloride

[0730] [Formula 30]

[0731]

[0732] A mixed solution of sodium 2-oxo-3-phenylbutyrate (final concentration 50 mM), D(+)-glucose (final concentration 100 mM), ammonia solution (final concentration 500 mM), N,N-bicine buffer solution (final concentration 0.1 M), NADPH solution (final concentration 1 mM), GDH solution (final concentration 0.002 units / μL), additive DMSO (20 vol%), the variant enzyme W253H-SBP-His prepared in Example 2 (final concentration 20 μM) and ultrapure water was prepared, and incubated at 37°C for 47 hours.

[0733] After incubation, 600 μL of 5 mol / L hydrochloric acid was added to the reaction solution, 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 to 95 / 5) to obtain (2S, 3S)-2-amino-3-phenyl-butyric acid (I) (16.5 mg, 18%).

[0734] LCMS (ESI) m / z = 180.1 (M+H) +

[0735] Retention time: 0.31 minutes (analysis conditions FA05)

[0736] 1H-NMR (400 MHz, trifluoroacetic acid-d1, 298 K) 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)

[0737] The compound (2S,3S)-2-amino-3-phenyl-butyric acid (I) obtained by the reaction 1 The H-NMR and chiral HPLC analysis data were compared with those of purchased standard compounds (2S,3R)-2-amino-3-phenyl-butyric acid hydrochloride (II), (2S,3S)-2-amino-3-phenyl-butyric acid hydrochloride (III) and (2R,3R)-2-amino-3-phenyl-butyric acid hydrochloride (IV) to assign the spatial structures of the obtained compounds.

[0738] according to 1 Comparison of the characteristic proton peaks at positions 2 and 3 in H-NMR analysis ( Figure 1 ) and chiral HPLC analysis results ( Figure 1 and Table 24), the structure of the compound (I) obtained in the reaction was determined to be (2S,3S)-2-amino-3-phenylbutyric acid.

[0739] Chiral HPLC analysis conditions were as follows.

[0740] Device: SHIMADZU Nexera X3

[0741] Column (ID x Length (mm), Particle Size (μm)): DAICEL CORPORATION CHIRALPAK ZWIX (+) (3.0x150, 3.0)

[0742] Mobile phase: MeOH:MeCN:H2O (49:49:2, v / v) containing 50 mM formic acid and 25 mM diethylamine.

[0743] Elution method: Isocratic

[0744] Analysis time: 10 minutes.

[0745] Flow rate (ml / min): 0.5

[0746] Column temperature: 25°C

[0747] Wavelength: 254nm

[0748] Figure 2 and (V) in Table 24 were prepared by mixing purchased standard compounds (III) and (IV) in the following ratio: (III):(IV)=7:3.

[0749] [Table 24]

[0750] PDA Ch1 254nm

[0751]

[0752] PDA Ch1 254nm

[0753]

[0754] Synthesis Example 4: Synthesis of (S)-3,3-dimethyl-2-(methylamino)butyric acid using variant enzyme W253H-SBP-His and additive (dimethyl sulfoxide)

[0755] LC / MS was performed under the following analytical conditions.

[0756] HPLC method

[0757] Device: Waters AQUITY UPLC H-Class / QDa

[0758] Column: ACQUITY UPLC HSS T3 2.1x50mm, 1.8μm

[0759] Solvent: A) 0.05% TFA-H2O, B) 0.05% TFA-CH3CN

[0760] 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)

[0761] Flow rate: 0.5 mL / min Injection volume: 0.5 μl

[0762] Temperature: 30℃

[0763] Wavelength: 197nm

[0764] In this example, the reagents described in the following table were used. The reagents not listed are those in Table 3.

[0765] [Table 25]

[0766]

[0767]

[0768] [Equation 31]

[0769]

[0770] Oxidized nicotinamide adenine dinucleotide phosphate (50 mg) was dissolved in 0.4 mol / L N,N-bicine (2-hydroxyethyl) glycine solution (1.0 mL, pH 9.0) to prepare an oxidized nicotinamide adenine dinucleotide phosphate solution (solution A).

[0771] D-glucose dehydrogenase (50 mg) was dissolved in 0.4 mol / L N,N-bicine solution (1.0 mL, pH 9.0) to prepare a D-glucose dehydrogenase solution (solution B).

[0772] 3,3-Dimethyl-2-oxobutanoic acid (50 mg, 0.38 mmol), N,N-bis(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% sodium hydroxide aqueous solution (88 μL) was added to adjust the pH to 9.0 to prepare a substrate solution (Solution C).

[0773] Solution A (59 μL, 3.8 μmol) and solution B (10 μL, 1.0 wt %) were added to solution C, to which the modified enzyme solution (1.0 to 100 wt %, each added at 73.3 mg / ml solution) was added, and the mixture was stirred at an external temperature of 25° C. for 17 hours, and then shaken at an external temperature of 37° C. for 24 hours.

[0774] Using (S)-2-amino-3,3-dimethylbutyric acid as a standard, the yield of (S)-3,3-dimethyl-2-(methylamino)butyric acid in the reaction solution was calculated based on the UV absorption peak area at 197 nm in LC / MS. The results are shown in Table 26.

[0775] [Table 26]

[0776]

Claims

1. A method for producing an amino acid, the method 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 higher identity with 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 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) 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 the 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, A functional group represented by the following formula (1a): represents =O, -ORd or a hydroxyl group, When each of v and w is 1, the two functional groups represented by the plurality of formula (1a) are the same or different, Ra, Rb and Rc each independently represent a hydrogen atom, a C 1 To C 3 Alkyl group, alkylamino group or -CH 2 -ORd, At least any two of Ra, Rb and Rc are optionally linked to each other to form a ring structure together with T, Rd means C 1 To C 3 Alkyl groups, d, e and f each independently represent 0 or 1, At least one of d, e and f represents 1, When each of v and w is 1, at least one of Ra, Rb and Rc is a methyl group, and none of Ra, Rb and Rc is connected to each other to form a ring structure together with T, When at least one of Ra, Rb and Rc is a methylamino group, none of Ra, Rb and Rc are connected to each other to form a ring structure together with T, and When the functional group represented by the formula (1a) is a hydroxyl group and T is a carbon atom, v is 1, w is 0, each of d, e and f is 1, and each of Ra, Rb and Rc is a hydrogen atom.

2. The method according to claim 1, wherein in the formula (1), T represents a phosphorus atom or a sulfur atom, The functional group represented by the formula (1a) is =0, and Each of Ra, Rb and Rc is a methyl group.

3. The production method according to claim 1 or 2, wherein the compound D is dimethyl sulfoxide.

4. A method for producing an amino acid, comprising the steps of performing 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 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 the presence of: a polypeptide having catalytic activity for the intermolecular reductive amination reaction between the compound A and the compound B, or the intramolecular reductive amination reaction of the compound C under at least one reaction condition, a reducing agent, and a compound D', wherein the compound D' is at least one compound 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 the compound D′ is at least one compound selected from the group consisting of dimethyl sulfoxide, dimethyl sulfone, and trimethylphosphine oxide. The production method according to claim 4 or 5, wherein the compound D' is dimethyl sulfoxide.

7. The production method according to any one of claims 1 to 6, wherein the compound A is at least one compound selected from the group consisting of compounds represented by the following formula (2) and salts thereof: In the 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, which are optionally substituted, and R 1 and R 2 At least one of them is a hydrogen atom.

8. The method according to claim 7, wherein in the formula (2), R 1 is a hydrogen atom, and R 2 An alkyl group.

9. The production method according to claim 7 or 8, wherein in the formula (2), R 1 is a hydrogen atom, and R 2 is a methyl group or an ethyl group.

10. The production method according to any one of claims 1 to 9, wherein the compound B is at least one compound selected from the group consisting of compounds represented by the following formula (3) and salts thereof: In the formula (3), X represents a carbon atom, Y represents a hydrogen atom or a group represented by the formula (4), n represents an integer of 0 or more and 2 or less, and R 6 represents a hydrogen atom, an optionally substituted C 1 To C 6 An alkyl group, an optionally substituted C 1 To C 6 an aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, a group containing a nitrogen atom or a group containing an oxygen atom, In the formula (4), represents the junction point with X, m represents an integer of 0 or more and 6 or less, p is 0 or 1, q is 0 or 1, r is 0 or 1, Z 1 represents an optionally substituted alkylene group, or an ether bond-containing group having 1 or more and 6 or less carbon atoms, and when m is an integer of 2 or more, a plurality of Z 1 Same or different, Z 2 represents a carbon atom, R 3 , R 4 and R 5 Each independently represents a hydrogen atom, an optionally substituted C 1 To C 6 An alkyl group, an optionally substituted C 5 To C 12 an aryl group, an optionally substituted heteroaryl group having 5 or more and 12 or less ring-forming atoms, 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 are optionally connected to each other to form Z 2 Together they form a ring structure, which is optionally a cycloalkyl group, an aryl group, a heterocyclyl group or a heteroaryl group, and these groups are optionally substituted, R 3 , R 4 and R 5 Each optionally with Z 2 To form a double or triple bond, and when R 3 , R 4 and R 5 Any of them is connected to Z through a double bond or a triple bond 2 When bonding, at least one of p, q and r is 0.

11. The method according to claim 10, wherein in the formula (3), n is 0, R 6 is a hydrogen atom, Y is C 3 To C 8 Cycloalkyl groups or C 6 To C 9 An aralkyl group, and the aralkyl group is optionally C 1 To C 3 Alkyl groups or halogen substitution.

12. The production method according to claim 10 or 11, wherein in the formula (3), n is 0, R 6 is a hydrogen atom, and Y is a (2-chlorophenyl)ethyl group, a phenylmethyl group or a cyclopentyl group.

13. The production method according to any one of claims 1 to 12, wherein the steps are performed under appropriate reaction conditions.

14. A method for producing a peptide compound, the method The following steps are involved: (1) producing an amino acid by the production method according to any one of claims 1 to 13; and (2) linking the amino acid with one or more selected from the group consisting of other amino acids and peptides to produce a peptide compound.

15. 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; (ii) 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 the 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, A functional group represented by the following formula (1a): represents =O, -ORd or a hydroxyl group, When each of v and w is 1, the two functional groups represented by the plurality of formula (1a) are the same or different, Ra, Rb and Rc each independently represent a hydrogen atom, a C 1 To C 3 Alkyl group, alkylamino group or -CH 2 -ORd, At least any two of Ra, Rb and Rc are optionally linked to each other to form a ring structure together with T, Rd means C 1 To C 3 Alkyl groups, d, e and f each independently represent 0 or 1, At least one of d, e and f represents 1, When each of v and w is 1, at least one of Ra, Rb and Rc is a methyl group, and none of Ra, Rb and Rc is connected to each other to form a ring structure together with T, When at least one of Ra, Rb and Rc is a methylamino group, none of Ra, Rb and Rc are connected to each other to form a ring structure together with T, and When the functional group represented by the formula (1a) is a hydroxyl group and T is a carbon atom, each of d, e and f is 1, and each of Ra, Rb and Rc is a hydrogen atom.

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