Peptide manufacturing method

Abstract

This technology provides a method for synthesizing peptides while suppressing the use of protective amino acids and inhibiting epimerization. [Solution] A peptide is produced by reacting a compound represented by formula (1) with a compound represented by formula (2) in the presence of a metal salt. TIFF2026067318000019.tif45170 TIFF2026067318000020.tif20170

Description

Technical Field

[0001] This specification relates to a method for producing a peptide and the like.

Background Art

[0002] Peptides are attracting attention as potential pharmaceuticals that combine the advantages of low-molecular-weight and high-molecular-weight pharmaceuticals, and are representative of the next-generation medium-molecular-weight pharmaceuticals.

[0003] Conventionally, when obtaining a peptide by chemical synthesis, a protecting group for protecting an amino group and a condensing agent are required. Further, after the formation of a peptide bond, it is necessary to remove (deprotect) the protecting group. Protected amino acids are expensive, and the generation of waste due to deprotection has been a problem. In addition, in the synthesis of a peptide, it has been a problem to avoid the synthesis of an optically unintended peptide due to epimerization of an amino acid during the formation of a peptide bond.

[0004] As a method for avoiding the use of a protecting group and a condensing agent, a peptide synthesis method using a peptide thiocarboxylate has been proposed (Patent Document 1).

[0005] The present inventors have already reported that a pyridine oxime ester can be used as a selective esterifying agent for hydroxyamide by utilizing the electron-withdrawing property of a cationic organic molecular group generated by coordinating a Lewis acid, which is a metal salt, to the pyridine oxime ester (Patent Document 2).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

[0007] However, the method described in Patent Document 1 may still fail to suppress epimerization even when an epimerization inhibitor and a solubilizing solvent for the protected amino acid are required. On the other hand, even those skilled in the art could not have known that the selective esterifying agent described in Patent Document 2 contributes to the formation of peptide bonds using unprotected amino acids in the main chain.

[0008] This specification provides a technique for synthesizing peptides while suppressing epimerization and minimizing the use of protective amino acids. [Means for solving the problem]

[0009] The present inventors focused on the synthesis of peptides using an esterifying agent already developed by some of the inventors. They found that by using an ester compound obtained by esterifying an amino acid or peptide with this esterifying agent, it is possible to selectively form peptide bonds with the carboxyl group of an amino acid while suppressing epimerization.

[0010] In other words, the use of this esterifying agent enables the extension of amino acids from the N-terminus to the C-terminus and the linking of the N-terminus of the peptide chain to the C-terminus of the peptide. Furthermore, the use of this esterifying agent can be avoided or minimized. In addition, the esterifying agent can be recovered and reused after peptide formation. The following means are provided according to this specification.

[0011] [1] A method for producing a compound represented by formula (3) by reacting a compound represented by the following formula (1) with a compound represented by the following formula (2) in the presence of a metal salt.

[0012] [ka] (In formula (1), A represents a protecting group or an amino acid or peptide which may be protected, V represents a linear alkyl group having 1 to 3 carbon atoms which may have hydrogen atoms substituted, R1 R represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an aryl group, an aralkyl group, an aliphatic heterocyclic group, and an aromatic heterocyclic group. 2 ~R 5 Each of these independently represents a group selected from the group consisting of a hydrogen atom, a halogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, aliphatic heterocyclic group, and aromatic heterocyclic group. [ka] (In formula (2), W represents a linear alkylene group having 1 to 3 carbon atoms, in which hydrogen atoms may be substituted.) [ka] (In formula (3), A, V , W This is equivalent to the expression in equations (1) and (2). [2] In formula (2) above, W represents a linear alkylene group having 1 to 3 carbon atoms, the method according to [1]. [3] The method according to [1] or [2], wherein the metal salt comprises calcium as the metal. [4] The method according to any one of [1] to [3], wherein the metal salt comprises a calcium salt of an amino acid of the compound represented by formula (2). [5] The reaction is carried out in a solvent containing at least dimethyl sulfoxide, according to any one of the methods in [1] to [4]. [6] A method for producing a compound represented by formula (6) by reacting a compound represented by the following formula (4) with a compound represented by the following formula (5) in the presence of a metal salt.

[0013] [ka] (In formula (4), B represents a peptide in which the amino acid residues constituting the peptide may be the same or different, and the active group may be protected, and X represents a protecting group for the N-terminal amino group of B.1 represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, a group having an aliphatic heterocyclic ring, and a group having an aromatic heterocyclic ring, and R 2 ~R 5 each independently represents a group selected from the group consisting of a hydrogen atom, a halogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, a group having an aliphatic heterocyclic ring, and a group having an aromatic heterocyclic ring.) [Chemical formula] (In formula (5), C is a peptide having, at the N-terminus, an α-amino acid, β-amino acid, or γ-amino acid having a carboxy group as a side chain at the β-position or γ-position of the amino group, and the amino acid residues other than the N-terminus may be the same or different, and the active groups in the amino acid residues may be protected, and Y represents an optionally protected amino group, alkylamino group, dialkylamino group, or optionally protected hydroxyl group bonded to the carbon atom of the carbonyl group at the C-terminus of the peptide.) [Chemical formula] (In formula (6), B, C, X, and Y have the same meanings as in formulas (4) and (5).) [7] The method according to [6], wherein in formula (4), B represents a peptide in which some or all of the active groups are not protected, and in formula (5), C represents a peptide in which some or all of the active groups are not protected.) [8] The method according to [6] or [7], wherein the metal salt contains calcium as the metal.) [9] The production method according to any one of [6] to [8], wherein the α-amino acid at the N-terminus of C in formula (5) contains L-aspartic acid or L-glutamic acid.)

[10] A synthetic reagent for peptides, containing a compound represented by formula (7). [Chemical formula] (R 1R represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an aryl group, an aralkyl group, an aliphatic heterocyclic group, and an aromatic heterocyclic group. 2 ~R 5 Each of these independently represents a group selected from the group consisting of a hydrogen atom, a halogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, aliphatic heterocyclic group, and aromatic heterocyclic group. [Brief explanation of the drawing]

[0014] [Figure 1] This diagram shows an overview of this disclosure. [Figure 2] This figure shows the synthesis scheme and results in Example 1. [Figure 3] This figure shows the synthesis scheme and results in Example 2. [Figure 4] This figure shows the synthesis scheme and results in Example 3. [Figure 5] This figure shows the synthesis scheme and results in Example 5. [Modes for carrying out the invention]

[0015] This disclosure relates to the production of peptides, and more particularly to a technique for producing peptides by forming a peptide bond between the carboxyl group of an N-terminal amino acid and the amino group of a C-terminal amino acid.

[0016] Some of the present inventors have already developed an esterifying agent for selectively acyling compounds having a hydroxyamino group, as described in Patent Document 2. Through various studies on this esterifying agent, we have come to focus on the possibility of applying it to the formation of peptide bonds.

[0017] The inventors have found that by using an esterifying agent to activate the carboxyl group of the N-terminal amino acid or peptide and to induce a template effect, and reacting it with the C-terminal amino acid, epimerization of the C-terminal amino acid can be highly suppressed, and amino acids with the intended optical activity can be linked by peptide bonds with high efficiency.

[0018] Although not binding on the disclosures herein, it is inferred that such results are due to the manifestation of the following effects (1) to (3). For reference, these effects are shown in Figure 1.

[0019] (1) By incorporating the carboxyl group of the N-terminal amino acid or peptide into the esterifying agent, the carbonyl carbon atom of the N-terminal carboxyl group is moderately activated. (2) The emergence of a template effect through the coordination of metal ions to the nitrogen atom on the pyridine ring of the esterifying agent portion and the nitrogen atom of the oxime group, as well as to the carbonyl oxygen atom and amino group nitrogen atom of the C-terminal amino acid or peptide to be linked. (3) The emergence of a proximity effect due to the template effect, which selectively brings the nitrogen atom of the amino group of the C-terminal amino acid closer to the carbonyl carbon atom of the activated N-terminal amino acid.

[0020] As shown in Figure 1, the above action allows for the selective proximity of the amino group of the N-terminal amino acid or peptide to the carboxyl group of the C-terminal amino acid or peptide. This is thought to allow for the formation of peptide bonds while avoiding the use of main chain protecting groups and suppressing epimerization. Furthermore, as peptide bonds are formed, the esterifying agent portion is eliminated in an unused structure and can be reused.

[0021] The methods for producing peptides disclosed herein will be described in detail below. There are two main methods for producing peptides: an elongation method, which involves extending a peptide chain by linking peptides one amino acid residue at a time using peptide bonds; and a linking method, which involves linking peptide chains together using peptide bonds. The elongation method will be described first, followed by the linking method.

[0022] (Method for producing peptides by peptide chain elongation reaction: elongation method) The extension method is a method for producing a compound represented by formula (3) by reacting a compound represented by formula (1) with a compound represented by formula (2) in the presence of a metal salt.

[0023] (Compounds represented by formula (1), pyridine oxime / amino acid / peptide compounds) The compound represented by formula (1) disclosed herein is hereinafter referred to as a pyridine oxime / amino acid or peptide compound (hereinafter simply referred to as an oxime / amino acid / peptide compound). An oxime / amino acid / peptide compound has a pyridine oxime portion and an amino acid or peptide portion. The pyridine oxime portion corresponds to the esterifying agent portion described above.

[0024] Regarding the pyridine oxime portion of oxime / amino acid / peptide compounds, R in formula (1) 1 , R 2 , R 3 , R 4 , and R 5 This represents the following base:

[0025] [R 1 ] R 1 This represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, aliphatic heterocyclic group, and aromatic heterocyclic group.

[0026] (Alkyl) R 1The alkyl group represented by may be linear, branched, or cyclic. Preferred alkyl groups include, for example, linear, branched, or cyclic alkyl groups having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms. Specifically, examples include linear or branched alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, cetyl, and stearyl groups; and cycloalkyl groups such as cyclopentyl, methylcyclopentyl, cyclohexyl, methylcyclohexyl, and cyclooctyl groups.

[0027] (alkyl substituents) These alkyl groups may have substituents, and examples of such substituents include hydrocarbon groups, aliphatic heterocyclic groups, aromatic heterocyclic groups, alkoxy groups, alkyl ester groups, alkylenedioxy groups, aryloxy groups, aralkyloxy groups, heteroaryloxy groups, alkylthio groups, arylthio groups, aralkylthio groups, heteroarylthio groups, amino groups, substituted amino groups, cyano groups, hydroxyl groups, oxo groups, nitro groups, mercapto groups, trisubstituted silyl groups, and halogen atoms.

[0028] Examples of hydrocarbon groups that can be substituted for alkyl groups include alkyl groups, alkenyl groups, alkynyl groups, aryl groups, and aralkyl groups.

[0029] The alkyl group to be substituted can be linear, branched, or cyclic. For example, linear, branched, or cyclic alkyl groups having 1 to 20 carbon atoms are preferred. Specifically, examples include linear or branched alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, cetyl, and stearyl groups; and cycloalkyl groups such as cyclopentyl, cyclohexyl, and cyclooctyl groups.

[0030] The alkenyl group to be substituted for the alkyl group may be linear or branched, and examples include alkenyl groups having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms. Specifically, examples include ethenyl group, propenyl group, 1-butenyl group, pentenyl group, hexenyl group, etc.

[0031] The alkynyl group to be substituted for the alkyl group may be linear or branched, and examples include alkynyl groups having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms. Specifically, examples include ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 3-butynyl group, pentynyl group, hexynyl group, etc.

[0032] Examples of aryl groups that can be substituted for alkyl groups include aryl groups having 6 to 20 carbon atoms, specifically phenyl, naphthyl, anthryl, phenanthryl, biphenyl, and terphenyl groups.

[0033] Examples of aralkyl groups to be substituted for alkyl groups include groups in which at least one hydrogen atom of the alkyl group is substituted with the aryl group, for example, aralkyl groups having 7 to 12 carbon atoms are preferred, and specifically include benzyl group, 2-phenylethyl group, 1-phenylpropyl group, 3-naphthylpropyl group, etc.

[0034] Examples of aliphatic heterocyclic groups to be substituted for alkyl groups include monocyclic or polycyclic / fused aliphatic heterocyclic groups having 2 to 14 carbon atoms and containing at least one, preferably 1 to 3, heteroatoms such as nitrogen, oxygen, or sulfur atoms, with 5 to 8 members, preferably 5 or 6 members. Specific examples of aliphatic heterocyclic groups include pyrrolidyl-2-one, piperidino, piperazinyl, morpholino, tetrahydrofuryl, tetrahydropyranyl, and tetrahydrothienyl groups.

[0035] Examples of aromatic heterocyclic groups to be substituted for alkyl groups include monocyclic heteroaryl groups or polycyclic or fused heteroaryl groups having 2 to 15 carbon atoms and containing at least one, preferably 1 to 3, heteroatoms such as nitrogen, oxygen, or sulfur atoms as heteroatoms, with 5 to 8 members, preferably 5 or 6 members. Specifically, examples include furyl, thienyl, pyridyl, pyrimidyl, pyrazyl, pyridazyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, benzofuryl, benzothienyl, quinolyl, isoquinolyl, quinoxalyl, phthalazyl, quinazolyl, naphthyridyl, synnolyl, benzimidazolyl, benzoxazolyl, and benzothiazolyl groups.

[0036] The alkoxy group to be substituted for the alkyl group may be linear, branched, or cyclic, and examples include alkoxy groups having 1 to 6 carbon atoms. Specifically, examples include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, 2-butoxy group, isobutoxy group, tert-butoxy group, n-pentyloxy group, 2-methylbutoxy group, 3-methylbutoxy group, 2,2-dimethylpropyloxy group, n-hexyloxy group, 2-methylpentyloxy group, 3-methylpentyloxy group, 4-methylpentyloxy group, 5-methylpentyloxy group, cyclohexyloxy group, methoxymethoxy group, 2-ethoxyethoxy group, etc.

[0037] The alkyl ester group to be substituted for the alkyl group may be linear, branched, or cyclic. Examples include ester groups having an alkyl group with 1 to 6 carbon atoms. Specifically, examples include methyl ester group, ethyl ester group, n-propyl ester group, isopropyl ester group, n-butyl ester group, 2-butyl ester group, isobutyl ester group, tert-butyl ester group, n-pentyl ester group, 2-methylbutotyl ester group, 3-methylbutyl ester group, 2,2-dimethylpropyl ester group, n-hexyl ester group, 2-methylpentyl ester group, 3-methylpentyl ester group, 4-methylpentyl ester group, 5-methylpentyl ester group, cyclohexyl ester group, methoxymethyl ester group, and 2-ethoxyethyl ester group.

[0038] Examples of alkylenedioxy groups that can be substituted for alkyl groups include alkylenedioxy groups having 1 to 3 carbon atoms, specifically methylenedioxy groups, ethylenedioxy groups, trimethylenedioxy groups, propylenedioxy groups, isopropylidenedioxy groups, and so on.

[0039] Examples of aryloxy groups that can be substituted for alkyl groups include aryloxy groups having 6 to 14 carbon atoms, specifically phenoxy, tolyloxy, xylyloxy, naphthoxy, and anthryloxy groups.

[0040] Examples of aralkyloxy groups that can be substituted for alkyl groups include aralkyloxy groups having 7 to 12 carbon atoms, specifically benzyloxy group, 4-methoxyphenylmethoxy group, 1-phenylethoxy group, 2-phenylethoxy group, 1-phenylpropoxy group, 2-phenylpropoxy group, 3-phenylpropoxy group, 1-phenylbutoxy group, 3-phenylbutoxy group, 4-phenylbutoxy group, 1-phenylpentyloxy group, 2-phenylpentyloxy group, 3-phenylpentyloxy group, 4-phenylpentyloxy group, 5-phenylpentyloxy group, 1-phenylhexyloxy group, 2-phenylhexyloxy group, 3-phenylhexyloxy group, 4-phenylhexyloxy group, 5-phenylhexyloxy group, and 6-phenylhexyloxy group.

[0041] Examples of heteroaryloxy groups to be substituted for alkyl groups include heteroaryloxy groups having 2 to 14 carbon atoms that contain at least one, preferably 1 to 3, heteroatoms such as nitrogen atoms, oxygen atoms, and sulfur atoms as heteroatoms. Specifically, examples include 2-pyridyloxy groups, 2-pyradyloxy groups, 2-pyrimidyloxy groups, and 2-quinolyloxy groups.

[0042] The alkylthio group to be substituted for the alkyl group may be linear, branched, or cyclic. Examples include alkylthio groups having 1 to 6 carbon atoms, specifically methylthio group, ethylthio group, n-propylthio group, isopropylthio group, n-butylthio group, 2-butylthio group, isobutylthio group, tert-butylthio group, pentylthio group, hexylthio group, cyclohexylthio group, etc.

[0043] Examples of arylthio groups that can be substituted for alkyl groups include arylthio groups having 6 to 14 carbon atoms, specifically phenylthio groups, tolylthio groups, xylthio groups, naphthylthio groups, and so on.

[0044] Examples of aralkylthio groups that can be substituted for alkyl groups include aralkylthio groups having 7 to 12 carbon atoms, specifically benzylthio groups and 2-phenethylthio groups.

[0045] Examples of heteroarylthio groups to be substituted for alkyl groups include heteroarylthio groups having 2 to 14 carbon atoms that contain at least one, preferably 1 to 3, heteroatoms such as nitrogen atoms, oxygen atoms, and sulfur atoms as heteroatoms. Specifically, examples include 4-pyridylthio groups, 2-benzimidazolylthio groups, 2-benzoxazolylthio groups, and 2-benzthiazolylthio groups.

[0046] Examples of substituted amino groups that replace alkyl groups include amino groups in which one or two hydrogen atoms of the amino group are replaced by substituents such as alkyl groups, aryl groups, or aralkyl groups. Specific examples of alkyl-substituted amino groups include mono- or dialkylamino groups such as N-methylamino group, N,N-dimethylamino group, N,N-diethylamino group, N,N-diisopropylamino group, and N-cyclohexylamino group. Specific examples of aryl-substituted amino groups include mono- or diarylamino groups such as N-phenylamino group, N,N-diphenylamino group, N,N-ditolylamino group, N-naphthylamino group, and N-naphthyl-N-phenylamino group. Specific examples of aralkyl-substituted amino groups include mono- or dialkylamino groups such as N-benzylamino group and N,N-dibenzylamino group.

[0047] Examples of trisubstituted silyl groups that can be substituted on alkyl groups include trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and triphenylsilyl groups.

[0048] Examples of halogen atoms that can be substituted for alkyl groups include fluorine, chlorine, bromine, and iodine atoms, while examples of halogenated alkyl groups include monofluoromethyl, difluoromethyl, trifluoromethyl, and pentafluoroethyl groups.

[0049] Of these substituents, hydrocarbon groups, aliphatic heterocyclic groups, aromatic heterocyclic groups, alkoxy groups, alkylenedioxy groups, aryloxy groups, aralkyloxy groups, heteroaryloxy groups, alkylthio groups, arylthio groups, aralkylthio groups, heteroarylthio groups, or substituted amino groups may have further substituents selected from the group of substituents listed above.

[0050] (Alkenyl group) R 1 Examples of alkenyl groups represented by include linear, branched, or cyclic alkenyl groups having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms. Specifically, examples include vinyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 3-butenyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1-cyclopentenyl group, 3-cyclopentenyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, 5-hexenyl group, 1-cyclohexenyl group, 3-cyclohexenyl group, and the like.

[0051] Furthermore, these alkenyl groups may have substituents, and examples of such substituents include alkyl groups, aryl groups, heterocyclic groups (aliphatic heterocyclic groups, aromatic heterocyclic groups), halogen atoms, etc. Specific examples of substituents on alkyl groups include those mentioned above.

[0052] (Alkynyl group) R 1Examples of alkynyl groups represented by include linear or branched alkynyl groups having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms. Specifically, examples include ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 2-butynyl group, 3-butynyl group, 1-pentynyl group, 2-pentynyl group, 3-pentynyl group, 4-pentynyl group, 1-hexynyl group, 2-hexynyl group, 3-hexynyl group, 4-hexynyl group, 5-hexynyl group, and the like.

[0053] Furthermore, these alkynyl groups may have substituents, and examples of such substituents include alkyl groups, aryl groups, heterocyclic groups (aliphatic heterocyclic groups, aromatic heterocyclic groups), trisubstituted silyl groups, etc. Specific examples of substituents on alkyl groups include those mentioned above.

[0054] (Aryl group) R 1 Examples of aryl groups represented by include aryl groups having 6 to 20 carbon atoms, specifically phenyl, naphthyl, anthryl, phenanthryl, biphenyl, and terphenyl groups. Phenyl groups are preferred. The aryl group may be substituted. Examples of substituents include various halogen atoms and alkyl groups having approximately 1 to 4 carbon atoms. Specific examples of alkyl group substituents include those mentioned above.

[0055] (Aralkyl group) R 1 Examples of aralkyl groups represented by the above include groups in which at least one hydrogen atom of the alkyl group is substituted with the above aryl group, for example, aralkyl groups having 7 to 12 carbon atoms are preferred, and specifically include benzyl groups, phenylethyl groups such as 2-phenylethyl groups, phenylpropyl groups such as 1-phenylpropyl groups, naphthylpropyl groups such as 3-naphthylpropyl groups, etc. Specific examples include those described above as substituents on the alkyl group.

[0056] (Groups having an aliphatic heterocycle) R 1 The aliphatic heterocycle in the group having an aliphatic heterocycle represented by is not particularly limited, but examples include monocyclic aliphatic heterocycle groups having 2 to 14 carbon atoms and containing at least one, preferably 1 to 3, heteroatoms such as nitrogen, oxygen, and sulfur atoms as heteroatoms, with 5 to 8 members, preferably 5 or 6 members, or polycyclic or fused aliphatic heterocycle groups. Specific examples of aliphatic heterocycle groups include, for example, pyrrolidyl-2-one group, piperidino group, piperazinyl group, morpholino group, tetrahydrofuryl group, tetrahydropyranyl group, and tetrahydrothienyl group.

[0057] Such aliphatic heterocycle groups are not particularly limited, but examples include groups in which an aliphatic heterocycle is bonded to a carbon atom of the carbonyl group of an oxime / amino acid / peptide compound via an alkylene group having approximately 1 to 4 carbon atoms.

[0058] (Group having an aromatic heterocycle) R 1 The aromatic heterocyclic groups represented by are not particularly limited, but examples include monocyclic heteroaryl groups or polycyclic or fused heteroaryl groups having 2 to 15 carbon atoms and containing at least one, preferably 1 to 3, heteroatoms such as nitrogen, oxygen, or sulfur atoms as heteroatoms, with 5 to 8 members, preferably 5 or 6 members. Specifically, examples include furyl group, thienyl group, pyridyl group, indolyl group, pyrimidyl group, pyrazyl group, imidazolyl group, pyridazyl group, pyrazolyl group, imidazolyl group, oxazolyl group, thiazolyl group, benzofuryl group, benzothienyl group, quinolyl group, isoquinolyl group, quinoxalyl group, phthalazyl group, quinazolyl group, naphthylyl group, sinnolyl group, benzimidazolyl group, benzoxazolyl group, benzothiazolyl group, and the like. Furthermore, the aromatic heterocycle may be substituted, and examples of substituents include those described above as substituents on alkyl groups.

[0059] Such aromatic heterocyclic groups are not particularly limited, but examples include groups in which an aromatic heterocyclic group is bonded to a carbon atom of the carbonyl group of an oxime / amino acid / peptide compound via an alkylene group having approximately 1 to 4 carbon atoms. An example of such an aromatic heterocyclic group is the indolylmethyl group.

[0060] R 1 While not particularly limited, examples include linear alkyl groups having 1 to 4 carbon atoms, such as hydrogen atoms, methyl groups, and ethyl groups. Furthermore, from the viewpoint of electron-donating properties, the above-mentioned substituted aryl groups, aralkyl groups, aliphatic heterocycles, and aromatic heterocycles may be preferred. [R 2 ]~[R 5 ] R 2 ~R 5 Each of these independently represents a group selected from the group consisting of hydrogen atoms, halogen atoms, alkyl groups, aryl groups, aralkyl groups, aliphatic heterocyclic groups, and aromatic heterocyclic groups. Examples of halogen atoms include fluorine, chlorine, bromine, and iodine. Alkyl groups, aryl groups, aralkyl groups, aliphatic heterocyclic groups, and aromatic heterocyclic groups have already been explained. 2 ~R 5 While not particularly limited, these could all be, for example, hydrogen atoms.

[0061] [A] In oxime / amino acid / peptide compounds, A in the amino acid or peptide portion represents an amino group protecting group or a peptide whose N-terminus is protected by such a protecting group.

[0062] [Protecting group for amino group] As the protecting group for the amino group, known protecting groups used as protecting groups for the N-terminus of amino groups or peptides can be used. Examples include tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), 9-fluorenylmethoxycarbonyl group (Fmoc), acetyl group (Ac), and benzoyl group (Bz). When X is a protecting group for the amino group, the amino acid or peptide portion is an amino acid.

[0063] The amino acids used are not particularly limited. Examples include α-amino acids, β-amino acids, and γ-amino acids (all of which include N-alkyl amino acids). In N-alkyl acids, any alkyl group having 1 to 20 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, heptyl, and hexyl groups, can be used, but examples include alkyl groups having 1 to 6 carbon atoms and alkyl groups having 1 to 4 carbon atoms.

[0064] Examples of α-amino acids include, but are not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, hydroxyproline, ornithine, citrulline, N-methylglycine (sarcosine), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, and α-aminoisobutyric acid. Examples of β-amino acids include, but are not limited to, β-alanine. Examples of γ-amino acids include, but are not limited to, γ-aminobutyric acid. Furthermore, amino acids may be either L-type or D-type optical isomers. When obtaining naturally derived peptides, L-amino acids can be used as the amino acids contained in the oxime / amino acid / peptide compound.

[0065] [V] In the amino acid / peptide portion, V represents a linear alkylene group with 1 to 3 carbon atoms, which may have a hydrogen atom substituted. When V represents a methylene group, which may have a hydrogen atom substituted, NH-V(CO)- is an α-amino acid residue. When V represents an ethylene group, which may have a hydrogen atom substituted, NH-V(CO)- is a β-amino acid residue. When V represents an n-propylene group, which may have a hydrogen atom substituted, NH-V(CO)- is a γ-amino acid residue. Examples of α-amino acid residues, β-amino acid residues, and γ-amino acid residues are the amino acid residues that correspond to the α-amino acid, β-amino acid, and γ-amino acid residues already explained.

[0066] In V, the group substituting the hydrogen atom of the alkylene group corresponds to the side chains of various amino acids that can constitute the α-amino acid residue, β-amino acid residue, and γ-amino acid residue corresponding to NH-V(CO)-. Furthermore, the group substituting the hydrogen atom of the alkylene group in V includes groups having various substituents or parts thereof that can constitute the side chains of known amino acids. Examples include alkyl groups, amino groups, carboxyl groups having linear alkyl groups with 1 to 2 carbon atoms, carboxyl groups, hydroxyl groups, aliphatic aromatic rings, heterocycles, as well as alkylamino groups and dialkylamino groups. The alkyl group in alkylamino groups and dialkylamino groups can be any alkyl group with 1 to 20 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, heptyl, and hexyl groups, but examples include alkyl groups with 1 to 6 carbon atoms and alkyl groups with 1 to 4 carbon atoms.

[0067] [Protecting group] If the substituent has an active group (such as a hydroxyl group, amino group, or carboxyl group), the active group may or may not be protected. In this manufacturing method, since the carboxyl group at the C-terminus of the amino acid or peptide portion binds the peptide with high selectivity, even if V has a carboxyl group or an amino group, it may not need to be protected.

[0068] The type of protecting group is determined appropriately depending on the type of side chain. For example, if the amino acid is lysine and has an amino group in V, known amino group protecting groups such as tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), and 9-fluorenylmethoxycarbonyl group (Fmoc) can be used as protecting groups. If the amino acid is glutamic acid or aspartic acid and has a carboxyl group in V, benzyl ester (Bzl), tert-butyl ester (t-Bu), alkyl group, etc. can be used as protecting groups. Furthermore, if the amino acid is serine or threonine and has a hydroxyl group in V, benzyl group or tert-butyl group can be used as protecting groups. If the amino acid is tyrosine and has a phenolic hydroxyl group in V, 2-bromobenzyloxycarbonyl (Z(2Br)) or tert-butyl group can be used as protecting groups. If V contains a cysteine ​​side chain (having a sulfhydryl group), the protecting groups that can be used are 4-methylbenzyl group (Bzl(4Me)), trityl group (Trt), tert-butyl group, and N-(acetyl)aminomethyl group (Acm). If the amino acid has a guanidino group in V, such as arginine, the protecting group that can be used is p-toluenesulfonyl group (p-Ts). If the amino acid has an imidazole ring in V, such as histidine, the π-nitrogen can be protected with a benzyloxymethyl group (Bom) or a tert-butoxymethyl group (Bum), and the τ-nitrogen can be protected with a 2,4-dinitrophenyl group (Dnp), a trityl group, etc.

[0069] [Peptide with N-terminus protected by protecting group] When A is a peptide whose N-terminus is protected by a protecting group, the amino acid or peptide portion becomes a peptide. A peptide is a polymer in which the aforementioned amino acids are linked by peptide bonds in any order. The usable amino acids have already been described. When obtaining naturally derived peptides, L-amino acid residues can be used as the amino acid residues constituting the peptide. Peptides are not particularly limited, but examples include structures in which 2 to 100, 2 to 50, 2 to 30, 2 to 20, 2 to 10, 2 to 6, or 2 to 4 amino acids are linked together. Also, the R at the C-terminal amino acid residue of the peptide. 6 This has already been explained.

[0070] (Manufacturing of oxime / amino acid / peptide compounds) Oxime / amino acid / peptide compounds can be synthesized by those skilled in the art based on known techniques. For example, an amino acid or peptide with a protected N-terminus corresponding to the amino acid / peptide portion in formula (1), and the aforementioned R 1 ~R 5 A pyridine oxime compound represented by formula (7) and ethyl cyano(hydroxyimino) (Oxyma Pure (trade name)) are dissolved in a solvent such as dichloromethane, and then stirred in an ice bath for 1 hour. After that, a carbodiimide activator such as diisopropylcarbodiimide is added, and the mixture is stirred at room temperature for about 1 hour. The mixture is then concentrated using an evaporator and vacuum dried to obtain the compound represented by formula (1), thereby producing an oxime / amino acid / peptide compound. Such oxime / amino acid / peptide compounds are also synthetic substrates (synthetic intermediates) in peptide production methods. The pyridine oxime compound, Oxima, and DIC can each be used in amounts ranging from 1 to 1.2 moles per mole of amino acid or peptide.

[0071] [ka]

[0072] (Compound represented by formula (2): amino acid) The compound represented by formula (2) is an amino acid. In formula (2), W is synonymous with V in formula (2). That is, W represents a linear alkylene group having 1 to 3 carbon atoms, which may be substituted. When W represents a methylene group, which may be substituted, the compound represented by formula (2) is an α-amino acid. When W represents an ethylene group, which may have a hydrogen atom substituted, the compound represented by formula (2) is a β-amino acid. When W represents an n-propylene group, which may have a hydrogen atom substituted, the compound represented by formula (2) is a γ-amino acid. The embodiments of α-amino acids, β-amino acids, and γ-amino acids have already been described.

[0073] Furthermore, similar to V, the groups in W that substitute a hydrogen atom of the alkylene group have structures corresponding to α-amino acids, β-amino acids, and γ-amino acids as compounds represented by formula (2). In addition, the groups that substitute a hydrogen atom of the alkylene group in W include groups having each substituent or a part thereof that can constitute the side chain of known amino acids. Examples include alkyl groups, amino groups, carboxyl groups having linear alkyl groups with 1 to 2 carbon atoms, carboxyl groups, hydroxyl groups, etc., as well as alkylamino groups and dialkylamino groups. The alkyl group in alkylamino groups and dialkylamino groups can be any alkyl group with 1 to 20 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, heptyl, and hexyl groups, but examples include alkyl groups with 1 to 6 carbon atoms and alkyl groups with 1 to 4 carbon atoms.

[0074] Furthermore, when the substituent in W is an active group, the active group may be protected as appropriate. The protective group configuration described for V can be applied.

[0075] The peptide production method uses a metal salt. The metal M in the metal salt can be any monovalent or polyvalent metal with two or more valencies, including those conventionally known to constitute salts. Examples of metal M include Ca, Mg, K, Na, Al, B, Ti, Zr, Sn, Zn, Ga, Bi, Sb, Si, Cd, V, Mo, W, Mn, Fe, Cu, Co, Pb, Ni, Ag, and rare earth metals. Among these, Ca is sometimes preferred. Using Ca can sometimes significantly reduce peptide yield and epimerization.

[0076] Furthermore, any known anionic group can be used as the anionic group without particular limitations. Examples of such anionic groups include propanoic acid, acetic acid, and trimethyl acetate.

[0077] The metal salt may be supplied, for example, as the calcium salt of the compound (amino acid) represented by formula (2). This suppresses the epimerization rate and simultaneously improves the peptide yield. The calcium salt of the acid in the amino acid sequence represented by formula (2) can be produced, for example, by the method described in the following paper (Hashimoto, C.; Takeguchi, K.; Kodomari, M. Synlett, 2011, 10, 1427-1430).

[0078] In the extension method, a peptide represented by formula (3) can be obtained by reacting appropriate amounts of oxime / amino acid / peptide compounds, amino acids, and metal salts, etc., with the reaction solvent, reaction temperature, and reaction time appropriately set. Theoretically, oxime / amino acid / peptide compounds and amino acids react in equimolar (equivalent) amounts, but this can be adjusted as needed. For example, 1 mole of oxime / amino acid / peptide compound can be used in ratios of approximately 1-4 moles, 1-3 moles, or 1-2 moles of amino acids.

[0079] The amount of metal salt used is not particularly limited, but for example, it can be used in ratios of approximately 1-4 moles, 1-3 moles, or 1-2 moles per mole of amino acid.

[0080] The reaction solvent is not particularly limited, as long as it has sufficient solubility for oxime / amino acid / peptide compounds, amino acids, and metal salts. For example, one or more solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethoxyethane (DME), and acetonitrile (CH3CN) can be used in appropriate combinations. For example, using a solvent containing DMSO may be preferable from the viewpoint of yield and epimerization rate.

[0081] The concentrations of the oxime / amino acid / peptide compound and amino acid in the reaction solvent are not particularly limited and can be set as appropriate.

[0082] The reaction temperature and time are not particularly limited, but for example, they can be set at temperatures of approximately 0°C to 50°C, 10°C to 45°C, or 15°C to 40°C for approximately 3 to 20 hours. Those skilled in the art can appropriately set the reaction temperature and time considering the yield of the peptide produced and epimerization, etc.

[0083] These peptide chain elongation reactions can be carried out in an oxidizing atmosphere such as air or an inert gas atmosphere, although this is not particularly limited. Examples of inert gases include nitrogen gas, argon gas, and one or more other gases. The reaction can also be carried out at atmospheric pressure, or under pressurized or depressurized conditions as appropriate.

[0084] According to the extension method, a peptide represented by formula (3) can be obtained. Simultaneously, a pyridine oxime compound represented by formula (7), which corresponds to the pyridine oxime portion in the oxime / amino acid / peptide compound, can be obtained as a detached product. This pyridine oxime compound can be reused by recovering it from the solvent.

[0085] In the extension method, a new oxime / amino acid / peptide compound can be produced from the peptide represented by formula (3) produced by the extension method and the compound represented by formula (7), in accordance with the previously described method for producing oxime / amino acid / peptide compounds. An extended peptide can then be produced by linking a new amino acid to the C-terminus of this obtained peptide by a peptide bond. The compound represented by formula (7) is a peptide synthesis reagent disclosed herein.

[0086] (Manufacturing method by linking reaction between peptide chains: linking method) In the linking method, the compound represented by formula (6) can be produced by reacting the compound represented by formula (4) with the compound represented by formula (5) in the presence of a metal salt.

[0087] For the metal salt in the linking method, the embodiments described in the extension method can be applied.

[0088] (Compounds represented by formula (4), oxime / peptide compounds) The compounds represented by formula (4) disclosed herein are hereinafter referred to as pyridine oxime / peptide compounds (hereinafter also simply as oxime / peptide compounds). An oxime / peptide compound has a pyridine oxime moiety and a peptide moiety.

[0089] Regarding the pyridine oxime portion of the oxime / peptide compound, R in formula (4) 1 , R 2 , R 3 , R 4 , and R 5 The same embodiments described in equation (1) can be applied to this.

[0090] In oxime / peptide compounds, B represents a peptide. The amino acid residues constituting this peptide may be the same or different. The amino acid residues constituting B include various forms of amino acids already described for V and W in formulas (1) and (2).

[0091] In addition to the side chains of amino acid residues in the peptide, substituents on the alkylene groups of β-amino acids and γ-amino acids may also be protected. That is, B represents a peptide in which some or all of the side chains are not protected. In this method, since selective coupling reactions are possible, the active group does not need to be protected. Regarding protecting groups, various forms of protecting groups have already been described with respect to formulas (1) and (2), etc.

[0092] The number of amino acid residues in a peptide chain is not particularly limited, but examples include structures with 2 to 30, 2 to 20, 2 to 10, 2 to 6, and 2 to 4 amino acid residues linked together. The amino acid residues in the peptide may be either L-forms or D-forms, but L-forms can be used when referring to naturally derived peptides.

[0093] X represents a protecting group for the N-terminal amino group of B. The previously described embodiment of the protecting group for the N-terminal amino group of A can be applied to this protecting group.

[0094] Oxime / peptide compounds can be obtained, for example, by using the peptide synthesis reagent represented by formula (7) with the extension reaction described above or a separately obtained peptide, in accordance with the method for producing oxime / amino acid / peptide compounds. Such oxime / peptide compounds also serve as synthetic substrates (synthetic intermediates) in peptide production methods.

[0095] (Compounds and peptides represented by formula (5)) In formula (5), C represents a peptide. C has an α-amino acid, β-amino acid, or γ-amino acid at its N-terminus, with a carboxyl group as a side chain at the β- or γ-position of the amino group. By having these amino acids at the N-terminus, a template effect can be expressed through coordination between the oxygen atom of the β-carboxyl group or γ-carboxyl group, the nitrogen atom of the amino group of the N-terminal amino acid residue, and the N atom in the oxime / peptide compound. As a result, the formation of a peptide bond between the activated carbonyl group in the oxime / peptide compound and the N-terminal amino group is promoted.

[0096] The amino acid residues of a peptide other than the N-terminus may be the same or different, and the active group at the amino acid residue may be protected. The active group does not need to be protected, as selective ligation reactions are possible. That is, C represents a peptide in which some or all of the side chains are not protected. The amino acid residues in the peptide can be any of the various forms of amino acids described in formula (1), etc.

[0097] The number of amino acid residues in a C peptide is not particularly limited, but examples include structures with 2 to 30, 2 to 20, 2 to 10, 2 to 6, and 2 to 4 amino acid residues linked together. The amino acid residues in the peptide may be either the L-form or the D-form, but the L-form can be used when referring to naturally derived peptides.

[0098] Y represents an optionally protected amino group, alkylamino group, dialkylamino group, or optionally protected hydroxyl group bonded to the carbon atom of the carbonyl group at the C-terminus of the peptide. Various embodiments of the protecting group described above can be applied as appropriate. In this method, since selective coupling reactions are possible, these C-terminuses do not need to be protected.

[0099] The compound represented by formula (5) can be obtained by removing a protecting group as appropriate using the extension and linking reactions disclosed herein, as well as a separately obtained peptide.

[0100] In the coupling reaction, the amounts of solvent, oxime / peptide compound, and peptide used can be appropriately applied to the embodiments of the solvent, oxime / amino acid / peptide compound, and amino acid used in the extension reaction. Similarly, the reaction temperature, time, and atmosphere in the coupling reaction can be appropriately applied to the embodiments of the extension reaction.

[0101] According to the linking method, the amino group at the N-terminus of peptide C is selectively linked to the carboxyl group at the C-terminus of peptide B via a peptide bond through the template effect and proximity effect, thereby obtaining the peptide represented by formula (6). Simultaneously, the pyridine oxime compound represented by formula (7) can be obtained as a detached product. This pyridine oxime compound can be reused by recovering it from the solvent. Furthermore, the peptide thus obtained can be further extended by the extension method or linking method as needed.

[0102] (Peptide synthesis reagents) The peptide synthesis reagents disclosed herein include a compound represented by formula (7). 1 ~R 5 The embodiments described for the compound already represented by formula (1) can be applied. This type of compound can be obtained by those skilled in the art, for example, through synthesis. This peptide synthesis reagent can be used in both the extension and linking methods.

[0103] (Other aspects) As described above, all peptide production methods disclosed herein can adopt the method of using the peptide synthesis reagent represented by formula (7). Furthermore, the oxime / amino acid / peptide compounds represented by formulas (1) and (4) are peptide synthesis substrates (synthetic intermediates) obtained by linking a peptide synthesis reagent with an amino acid or peptide by an ester reaction, and according to this specification, embodiments of such synthetic substrates (synthetic intermediates) and methods for producing them are also disclosed. [Examples]

[0104] The following describes specific examples that embody the disclosures of this specification. These examples are for illustrative purposes only and are not limiting. Note that all amino acids used as synthetic substrates below are in their L-form. [Examples]

[0105] In this example, reaction conditions were investigated for peptide synthesis by peptide elongation. For explanation purposes, the synthesis scheme using one reaction condition is described below, and the various conditions investigated, along with the peptide isolation yield and epimerization rate, are shown in the table below.

[0106] [ka]

[0107] As shown in the synthesis scheme above, first, valine (4 molar equivalents), calcium propanoate (2 molar equivalents), and potassium acetate (4 molar equivalents) were heated in DMSO at 80°C for 65 minutes. Next, this reaction mixture (suspension) was added to a DMSO solution containing a mixture of 1 molar equivalent each of an oxime / peptide compound having a glycine-phenylalanine dipeptide, and the mixture was reacted at 30°C for 4 hours. After the reaction, peptide 3a and dipeptide 3b were obtained. The yields and epimerization rates of peptide 3a and dipeptide 3b are shown in Table 1.

[0108] The epimerization rate (ee) was determined by the peak area (PA) ratio in HPLC. Epimerization rate (%) = PA of the LD form of peptide 3a / (PA of the LD form of peptide 3a + PA of the LL form of peptide 3a) × 100

[0109] Based on the results shown in Figure 2, the previously described reaction conditions were selected as one of the suitable reaction conditions from the viewpoint of reaction yield, epimerization rate, and reagent cost. [Examples]

[0110] (Synthesis of peptide-active esters and peptide elongation reactions) In this example, N-terminally protected dipeptide 1 was activated to synthesize pyridine oxime ester 2, which was then reacted with unprotected amino acids in the presence of a calcium salt to synthesize tripeptide 3. The epimerization rate (dr) was determined by HPLC analysis of the reaction mixture. The yield is the isolation yield. Figure 3 shows the synthesis scheme and results in this example. Note that Z represents Cbz.

[0111] The scheme in this example was as follows: Cbz-Gly-AA2-OH (1,0.1 mmol), ketoxime (S1,0.12 mmol), and Oxyma pure (0.12 mmol) were weighed into a well-dried round-bottom flask and purged with nitrogen. Dichloromethane (DCM,1 mL) was added and dissolved, then the mixture was stirred at -10°C for 5 minutes. Diisopropylcarbodiimide (DIC,0.12 mmol) was added and the mixture was stirred for 2 hours, then the temperature was raised to room temperature and the mixture was stirred for 2 hours. Subsequently, the reaction mixture was concentrated in an evaporator and dried under vacuum to obtain oxime ester 2. Meanwhile, in a separate flask, an arbitrary amino acid H-AA3-OH (0.4 mmol), calcium propanoate (0.2 mmol), and potassium acetate (0.4 mmol) were suspended in DMSO (1.0 mL) and stirred at 60°C for 1 hour. After this solution was cooled, it was added to the flask containing the previously prepared oxime ester 2 and stirred at 30°C for 17 hours. The reaction mixture was purified by direct reverse-phase preparative HPLC to obtain tripeptide 3.

[0112] As shown in Figure 3, tripeptides were obtained for various dipeptides and amino acids.

[0113] Since the amounts of substrates and reagents used differed slightly for each extended amino acid, individual information for representative examples is provided below. Yield, properties, and MS data are also shown. All compounds other than the representative examples, as shown in the synthesis scheme in Figure 3, were synthesized and identified using the same method as the representative examples.

[0114] 3a: Z-Gly-Phe-Val-OH: Z-Gly-Phe-OH (2a, 37.0 mg), ketoxime (S1, 17.2 mg), Oxyma pure (17.7 mg), DCM (1 mL), DIC (19.5 μL), H-Val-OH (48.9 mg), Calcium propionate (38.9 mg), KOAc (40.9 mg), DMSO (1 mL) Yield: 37.6 mg (80%), Appearance: colorless oil HRMS (ESI-orbitrap) m / z: calculated for C 24 H 28 N3O6 - [M-H] - : 454.1984, found 454.1986.

[0115] 3b: Z-Gly-Phe-Leu-OH: Z-Gly-Phe-OH (2a, 35.5 mg), ketoxime (S1, 16.4 mg), Oxyma pure (17.1 mg), DCM (1 mL), DIC (19.0 μL), H-Leu-OH (52.8 mg), Calcium propionate (37.4 mg), KOAc (39.8 mg), DMSO (1 mL) Yield: 29.5 mg (63%), Appearance: colorless oil HRMS (ESI-orbitrap) m / z: calculated for C 25 H 30 N3O6 - [M-H] - : 468.2140, found 468.2143.

[0116] 3c: Z-Gly-Phe-Ile-OH: Z-Gly-Phe-OH (2a, 35.5 mg), ketoxime (S1, 16.3 mg), Oxyma pure (17.1 mg), DCM (1 mL), DIC (19.0 μL), H-Ile-OH (52.3 mg), Calcium propionate (37.3 mg), KOAc (39.6 mg), DMSO (1 mL) Yield: 39.8 mg (85%), Appearance: colorless oil HRMS (ESI-orbitrap) m / z: calculated for C 25 H 30 N3O6 - [M-H] - : 468.2140, found 468.2146.

[0117] 3f: Z-Gly-Phe-Trp-OH: Z-Gly-Phe-OH (2a, 35.5 mg), ketoxime (S1, 16.3 mg), Oxyma pure (17.0 mg), DCM (1 mL), DIC (19.0 μL), H-Trp-OH (81.4 mg), Calcium propionate (37.2 mg), KOAc (39.6 mg), DMSO (1 mL) Yield: 46.9 mg (87%), Appearance: pale brown solid HRMS (ESI-orbitrap) m / z: calculated for C 30 H 29 N4O6 - [M-H] - : 541.2093, found 541.2099.

[0118] 3g: Z-Gly-Phe-Cys(Mob)-OH: Z-Gly-Phe-OH (2a, 37.0 mg), ketoxime (S1, 17.3 mg), Oxyma pure (17.7 mg), DCM (1 mL), DIC (19.5 μL), H-Cys(Mob)-OH (100.1 mg), Calcium propionate (38.9 mg), KOAc (41.0 mg), DMSO (1 mL) Yield: 55.8 mg (93%), Appearance: pale yellow solid HRMS (ESI-orbitrap) m / z: calculated for C 30 H 32 N3O7S - [M-H] - : 578.1966, found 578.1975.

[0119] 3h: Z-Gly-Leu-Ala-OH: Z-Gly-Leu-OH (2h, 32.6 mg), ketoxime (S1, 16.5 mg), Oxyma pure (17.3 mg), DCM (1 mL), DIC (18.5 μL), H-Ala-OH (36.1 mg), Calcium propionate (37.4 mg), KOAc (39.4 mg), DMSO (1 mL) Yield: 31.5 mg (80%), Appearance: colorless oil HRMS (ESI-orbitrap) m / z: calculated for C 19 H 26 N3O6<00> - [M-H] - : 392.1827, found 392.1830.

[0120] 3o: Z-Gly-Tyr-Ala-OH: Z-Gly-Tyr-OH (2o, 38.0 mg), ketoxime (S1, 16.9 mg), Oxyma pure (17.5 mg), DCM (1 mL), DIC (18.5 μL), H-Ala-OH (35.9 mg), Calcium propionate (37.7 mg), KOAc (39.5 mg), DMSO (1 mL) Yield: 33.4 mg (75%), Appearance: white amorphous HRMS (ESI-orbitrap) m / z: calcd for C 22 H 24 N3O7 - [MH] - : 442.1620, found 442.1621.

[0121] 3p: Z-Gly-Lys(Z)-Ala-OH: Z-Gly- Lys(Z)-OH (2p, 46.8 mg), ketoxime (S1, 16.4 mg), Oxyma pure (17.1 mg), DCM (1 mL), DIC (18.5 μL), H-Ala-OH (35.8 mg), Calcium propionate (37.6 mg), KOAc (39.8 mg), DMSO (1 mL) Yield: 44.0 mg (81%), Appearance: white amorphous HRMS (ESI-orbitrap) m / z: calcd for C 27 H 33 N4O8 - [MH] - : 541.2304, found 541.2305.

Example

[0122] (The activity of the ペプチド active synthetic とペプチド elongation reaction 2) This example shows how the yield and epimerization rate (dr) were improved by using an improved method for substrates that showed insufficient reaction yield and epimerization rate (dr) in Example 2. The synthesis scheme and results for this example are shown below and in Figure 4. All compounds described in this synthesis scheme, other than the representative example, were synthesized and identified in the same way as the representative example.

[0123] The synthesis scheme is as follows: Cbz-Gly-AA2-OH (1,0.1 mmol), ketoxime (S1,0.12 mmol), and oxyma pure (0.12 mmol) are weighed into a well-dried round-bottom flask and purged with nitrogen. DCM (1 mL) is added and dissolved, then the mixture is stirred in an ice bath for 5 minutes. DIC (0.12 mmol) is added and the mixture is stirred for 1 hour, then the temperature is raised to room temperature and the mixture is stirred for 2 hours. Subsequently, the reaction mixture is concentrated in an evaporator and dried under vacuum to obtain oxime ester 2. Meanwhile, (H-Ala-O)2Ca (0.2 mmol) and DMSO (1.0 mL), prepared according to the method described in the paper (Hashimoto, C.; Takeguchi, K.; Kodomari, M. Synlett, 2011, 10, 1427-1430), are added to the flask containing ester 2 and stirred at 30°C for 17 hours. After adding an aqueous KH2PO4 solution, the resulting white solid was filtered, and the filtrate was concentrated to obtain a crude product. This was then purified by reverse-phase medium-pressure flash chromatography to obtain tripeptide 3.

[0124] In the aforementioned paper, amino acids (20 mmol) dissolved in distilled water (100 ml) were added to calcium hydroxide (0.815 g, 11.0 mmol) dissolved in 50 ml of distilled water. After stirring at room temperature for 30 minutes, the water was evaporated, and the solid was dried on silica gel under reduced pressure for 1 day. The solid was ground using a mortar and pestle, and (H-AA-OH)2Ca was washed with THF and diethyl ether on filter paper in a funnel, and dried on silica gel under reduced pressure to obtain the desired result. This amino acid calcium exhibits excellent solubility in DMF, as described in paper 1.

[0125] As shown in Figure 4, tripeptides were obtained from various dipeptides and amino acids. By pre-treating the amino acids as calcium salts, the epimerization rate was improved for all amino acids.

[0126] Since the amount of substrate and reagents used differed slightly for each extended amino acid, individual information for representative examples is provided below. Yield, characteristics, and MS data are also shown.

[0127] 3i: Z-Gly-Val-Ala-OH: Z-Gly-Val-OH (2i, 33.6 mg), ketoxime (S1, 18.1 mg), Oxyma pure (18.1 mg), DCM (1.1 mL), DIC (20.0 μL), (H-Ala-O)2Ca (47.3 mg), DMSO (2.2 mL) Yield: 37.8 mg (92%), Appearance: white solid HRMS (ESI-orbitrap) m / z: calcd for C 18 H 24 N3O6 - [MH] - : 378.1671, found 378.1673.

[0128] 3j: Z-Gly-Ile-Ala-OH: Z-Gly-Ile-OH (2j, 32.4 mg), ketoxime (S1, 16.6 mg), Oxyma pure (17.3 mg), DCM (1.0 mL), DIC (18.5 μL), (H-Ala-O)2Ca (43.7 mg), DMSO (2.0 mL) Yield: 28.5 mg (72%), Appearance: white solid HRMS (ESI-orbitrap) m / z: calcd for C 19 H 26 N3O6 - [MH] - : 392.1827, found 392.1827.

[0129] 3k: Z-Gly-Ser(tBu)-Ala-OH: Z-Gly- Ser(tBu)-OH (2k, 36.1 mg), ketoxime (S1, 16.8 mg), Oxyma pure (18.5 mg), DCM (1.0 mL), DIC (19.0 μL), (H-Ala-O)2Ca (44.3 mg), DMSO (2.0 mL) Yield: 41.8 mg (96%), Appearance: white solid HRMS (ESI-orbitrap) m / z: calcd for C 20 H 28 N3O7 - [MH] - : 422.1933, found 422.1933. [Examples]

[0130] (Continuous peptide elongation reaction) In this example, tetrapeptides were synthesized from Boc-protected amino acids via a series of reactions, following the procedure in Example 3. The synthesis scheme and results are shown below.

[0131] [ka]

[0132] The details of the synthesis scheme were as follows: Boc-Trp-OH (608.7 mg, 2.00 mmol), ketoxime (S1, 327 mg, 2.40 mmol), and Oxyma pure (341 mg, 2.40 mmol) were weighed into a well-dried round-bottom flask and purged with nitrogen. DCM (20 mL) was added and dissolved, then the mixture was stirred in an ice bath for 5 minutes. DIC (371 μL, 2.40 mmol) was added and the mixture was stirred for 1 hour, then the temperature was raised to room temperature and stirred for 1 hour. Subsequently, the reaction mixture was concentrated in an evaporator and dried under vacuum to obtain the corresponding oxime ester. For the next reaction, 1 / 10 of this oxime ester, 0.2 mmol, was dispensed and used.

[0133] To the oxime ester dispensed into a test tube, (H-Ala-O)2Ca (86.5 mg, 0.4 mmol) and DMSO (4.0 mL) were added, and the mixture was stirred at 30°C for 17 hours. After adding an aqueous KH2PO4 solution, the resulting white solid was filtered, and the filtrate was concentrated to obtain a crude product. This was treated with a Biotage solid-phase cartridge Isolute C18 (EC) to obtain Boc-Trp-Ala-OH. This peptide was used in the next reaction without further purification.

[0134] Following the procedure described above, H-Gly-OH was extended to obtain a reaction solution containing Boc-Trp-Ala-Gly-Gly-OH. After adding an aqueous KH2PO4 solution to this solution, the resulting white solid was filtered, and the filtrate was concentrated to obtain a crude product. Finally, the product was purified by reverse-phase medium-pressure chromatography to obtain Boc-Trp-Ala-Gly-Gly-OH (91.1 mg, 93%) as a pale yellow amorphous material.

[0135] From these results, it was found that tetrapeptides can be obtained by repeatedly performing oxyesterification of crude products such as Boc-Trp-Ala-OH and extension with unprotected amino acid salts. [Examples]

[0136] (Condensation of peptide chains) In this example, a larger oligopeptide was synthesized by condensing a peptide-derived pyridine oxime ester with an unprotected peptide. The epimerization rate (dr) was determined by HPLC analysis of the reaction mixture. The yield is the isolation yield. Figure 5 shows the synthesis scheme and results in this example.

[0137] The details of the synthetic scheme will be described using Entry3 as a representative. For other Entries, the same operations are performed. A well-dried glass microtube (1 mL) was charged with a peptide Dan-(Pro-Pro-Gly)5-OH (4.76 mg, 31.9 μmol) with its N-terminus protected by a Dansyl group for fluorescence detection, a ketoxime (S1, 1.30 mg, 95.7 μmol), and Oxyma pure (1.36 mg, 95.7 μmol), and then purged with nitrogen. After adding DCM (22 μL) to dissolve, it was stirred in an ice bath for 5 minutes. A solution of DIC (1.48 μL, 95.7 μmol) dissolved in DCM (10 μL) was added and stirred for 1 hour, then the temperature was raised to room temperature and stirred for 17 hours. The reaction solution was blown with Ar gas to concentrate and dried under vacuum to obtain an oxime ester. On the other hand, angiotensin II acetate (5.00 mg, 47.9 μmol) was weighed into another 2 mL microtube made of PP, and a stock solution (50 μL, each salt containing 47.9 μmol) of calcium propionate and potassium acetate dissolved in DMSO was added, and stirred at 40 °C for 30 minutes (Solution 1). Subsequently, the previously synthesized oxime ester was dissolved in DMSO (13.8 μL), and Solution 1 was added to this solution, and stirred at 40 °C for 17 hours. The reaction solution was directly purified by reversed-phase preparative HPLC to obtain Dan-(Pro-Pro-Gly)5-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH (5.4 mg, 68%). The mass spectrometry results were as follows. HRMS(ESI-orbitrap) m / z:calcd for C 122 H 169 N 29 O 29 S 2+ [M+2H] 2+ : 1268.6192, found 1268.6175.

[0138] From the above results, it was found that an oxime ester of a peptide chain with a free C-terminus (unprotected side chain) and a peptide chain with aspartic acid and glutamic acid at the N-terminus (unprotected side chain) can be linked by a peptide bond. According to Entry 1 and 2, it was found that even with a total of 10 residues, the epimerization rate could be kept low. Furthermore, according to Entry 2 to 5, it was found that if the number of amino acid residues is around 25 or less, the synthesis yield will easily exceed 50%. In addition, from the results of Entry 3 and 4, it was found that selective linking by peptide bonds is possible not only by using an Asp residue with a β-carboxyl group at the N-terminus, but also by using a Glu residue with a γ-carboxyl group. [Examples]

[0139] (Control experiment using succinimide ester) A comparative experiment was conducted on the condensation reaction (coupling reaction) of aspartic acid using pyridine oxime ester 2a, a peptide synthesis reagent used herein, and a common active ester (succinimide ester) 2b. The method was carried out in accordance with Example 5. The synthesis scheme and results are shown below. When 2b was used, epimerization proceeded (86:14 dr), while when 2a was used, epimerization was found to be suppressed (>99:1 dr).

[0140] [ka]

Claims

1. A method for producing a compound represented by formula (3) by reacting a compound represented by the following formula (1) with a compound represented by the following formula (2) in the presence of a metal salt. 【Chemistry 12】 (In formula (1), A represents a protecting group or an amino acid or peptide which may be protected, V represents a linear alkylene group having 1 to 3 carbon atoms which may have hydrogen atoms substituted, R １ R represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an aryl group, an aralkyl group, an aliphatic heterocyclic group, and an aromatic heterocyclic group. ２ ~R ５ Each of these independently represents a group selected from the group consisting of a hydrogen atom, a halogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, aliphatic heterocyclic group, and aromatic heterocyclic group. 【Chemistry 13】 (In formula (2), W represents a linear alkyl group having 1 to 3 carbon atoms, which may have hydrogen atoms substituted.) 【Chemistry 14】 (In formula (3), A, V , W This is equivalent to the expression in equations (1) and (2).

2. The method according to claim 1, wherein in formula (2), W represents a linear alkylene group having 1 to 3 carbon atoms, the active group of which may be protected.

3. The method according to claim 1, wherein the metal salt contains calcium as the metal.

4. The method according to claim 3, wherein the metal salt comprises a calcium salt of an amino acid of a compound represented by formula (2).

5. The method according to claim 1, wherein the reaction is carried out in a solvent containing at least dimethyl sulfoxide.

6. A method for producing a compound represented by formula (6) by reacting a compound represented by the following formula (4) with a compound represented by the following formula (5) in the presence of a metal salt. 【Chemistry 15】 (In formula (4), B represents a peptide in which the amino acid residues constituting the peptide may be the same or different, and the active group at the amino acid residue may be protected, and X represents the protecting group at the N-terminus of B. １ R represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an aryl group, an aralkyl group, an aliphatic heterocyclic group, and an aromatic heterocyclic group. ２ ~R ５ Each of these independently represents a group selected from the group consisting of a hydrogen atom, a halogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, aliphatic heterocyclic group, and aromatic heterocyclic group. 【Chemistry 16】 (In formula (5), C represents a peptide having an α-amino acid, β-amino acid, or γ-amino acid at the N-terminus, with a carboxyl group as a side chain at the β- or γ-position of the amino group, and the amino acid residues other than the N-terminus may be the same or different, and the active group at the amino acid residue may be protected; and Y represents an optionally protected amino group, alkylamino group, dialkylamino group, or optionally protected hydroxyl group bonded to the carbon atom of the carbonyl group at the C-terminus of the peptide.) 【Chemistry 17】 (In equation (6), B, C, X, and Y are the same as those in equations (4) and (5).)

7. The method according to claim 6, wherein the metal salt contains calcium as the metal.

8. The manufacturing method according to claim 6, wherein the α-amino acid at the N-terminus of C in formula (5) comprises L-aspartic acid or L-glutamic acid.

9. A peptide synthesis reagent comprising a compound represented by formula (7). [Chemistry 18] (R １ represents a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, a group having an aliphatic heterocyclic ring, and a group having an aromatic heterocyclic ring, and R ２ to R ５ each independently represent a group selected from the group consisting of a hydrogen atom, a halogen atom, an optionally substituted alkyl group, aryl group, aralkyl group, a group having an aliphatic heterocyclic ring, and a group having an aromatic heterocyclic ring.)

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