Method for producing n-substituted peptide

Abstract

To provide a method for rapidly synthesizing a variety of C-terminal-free N-substituted peptides in a high yield while suppressing epimerization.SOLUTION: The present invention provides a method for producing an N-substituted peptide represented by the following formula (6), comprising a step of reacting a first amino acid represented by the following formula (1), a second amino acid represented by the following formula (2), a silylating agent, a condensing agent, a tertiary amine, and an imidazole derivative.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing N-substituted peptides. [Background technology]

[0002] N-substituted peptides, which have a substituent (e.g., a methyl group) on the nitrogen atom that constitutes the amide bond, have attracted much attention as drug candidate molecules because they may have high biocompatibility, cell membrane permeability, and resistance to degradative enzymes. One method for obtaining N-substituted peptides is to elongate the peptide toward the C-terminus using N-substituted amino acids with both C- and N-terminus free. It is well known that N-substituted amino acids with both C- and N-terminus free react preferentially at the N-terminus side under basic conditions, so this method allows the elongated C-terminus free peptide to be obtained directly without a deprotection step. This makes it possible to reduce the number of steps and production costs, and improve atom economy. In general, C- and N-free amino acids often have poor solubility in solvents other than water. On the other hand, using water as a solvent increases the risk of causing undesired hydrolysis of intermediates generated in the reaction. As a method to solve this problem, Non-Patent Document 1 uses a combination of a silylating agent that enables temporary protection and isobutyl chloroformate to increase the solubility of the free amino acids at both the C and N termini in an organic solvent, and also simultaneously removes the temporary silyl protection during post-treatment, thereby preventing an increase in the number of steps. It has been reported that this allows peptides to be efficiently elongated using N-unsubstituted amino acids, but since the amino group of N-substituted amino acids has poor reactivity, it is impossible to obtain N-substituted peptides in high yields using the method of Non-Patent Document 1. In addition, the yield of N-substituted peptides can be improved by reacting for a long period of time, but in this case, racemization (epimeriization) in the N-substituted peptides is likely to occur due to the long reaction time. In addition, when using a flow method that is advantageous for continuous production, if the reaction time is long, a significantly long reaction tube is required, which is also a problem in that convenience during reaction is lost.

[0003] Thus, there has yet to be provided a method for obtaining N-substituted peptides in high yields in a short period of time using a wide variety of C-terminally unprotected N-substituted amino acids.

[0004] The only example that reported synthesis of C-terminal unprotected N-methylated peptides via temporary silyl protection is Non-Patent Document 2. Non-Patent Document 2 reports that the desired reaction hardly proceeded when isobutyl chloroformate was used as an activating agent, whereas the N-methylated peptide was obtained in a constant yield while avoiding side reactions by using an activating agent having a bulky special structure. However, there is still a demand for a method for producing N-substituted peptides optimized for a flow method that is advantageous for industrial continuous production. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Indian Journal of Chemistry -Section B 2004, 43, 1282-1287. [Non-Patent Document 2] Organic Letters 2020, 22, 8039-8043. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for synthesizing a variety of C-terminal free N-substituted peptides in high yield and quickly while suppressing epimerization. [Means for solving the problem]

[0007] The present inventors have conducted intensive research to achieve the above-mentioned object. As a result, they have found that various N-substituted peptides can be obtained quickly and in high yield while suppressing epimerization by reacting an N-terminal protected amino acid with a C-terminal unprotected N-substituted amino acid in the presence of a condensing agent, a tertiary amine, a silylating agent, and an imidazole derivative. In this case, since many silylating agents are present in the reaction system, it is thought that if an imidazole derivative as a nucleophilic agent is added thereto, the two will easily react with each other, and the imidazole derivative will no longer function. However, contrary to this concern, it has actually been found that the imidazole derivative fully exerts its function even in the presence of a silylating agent, and accelerates the progress of the target reaction. In addition, it has been found that when an imidazole derivative is optionally used in combination with an acid (particularly a strong acid), the yield of the target reaction can be further improved and epimerization can be further suppressed. Based on such findings, the present inventors have conducted further research and completed the present invention. That is, the present invention includes the following configurations.

[0008] Term 1. General formula (6):

[0009] [ka]

[0010] [In the formula, P 1 R represents a protecting group for an amino group. 1 represents a hydrocarbon group having 1 to 10 carbon atoms. 1 and A.A. 2 are the same or different and represent amino acid residues. A method for producing an N-substituted peptide represented by the following formula: General formula (1):

[0011] [ka]

[0012] [In the formula, each symbol is the same as defined above.] and a first amino acid represented by General formula (2):

[0013] [ka]

[0014] [In the formula, each symbol is the same as defined above.] and a second amino acid represented by A silylating agent; General formula (3):

[0015] [ka]

[0016] [In the formula, X represents a halogen atom, and Y represents a hydrocarbon group having 1 to 10 carbon atoms.] A condensing agent represented by the formula: General formula (4):

[0017] [ka]

[0018] [In the formula, R 2 , R 3 and R 4 are the same or different and each represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. 2 , R 3 and R 4 may be linked to form a substituted or unsubstituted ring (excluding an imidazole ring), and the ring may have one or more heteroatoms. and a tertiary amine represented by the formula: General formula (5):

[0019] [ka]

[0020] [In the formula, R5 represents a hydrocarbon group having 1 to 10 carbon atoms. and an imidazole derivative represented by the formula:

[0021] Item 2. The method according to Item 1, further comprising using an acid in the above step.

[0022] Item 3. The method according to Item 2, wherein the acid is a Bronsted acid having an acid dissociation constant (pKa) in water of 3 or less.

[0023] Section 4. Said R 2 , R 3 and R 4 and n is not a methyl group.

[0024] Item 5. In the above process, further comprising reacting a compound represented by general formula (7):

[0025] [ka]

[0026] [In the formula, R 6 , R 7 and R 8 are the same or different and each represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. 6 , R 7 and R 8 may be linked to form a substituted or unsubstituted ring, and the ring may contain one or more heteroatoms. 6 , R 7 and R 8 At least one of the groups is a methyl group. Item 5. The method according to item 4, wherein a tertiary amine represented by the following formula is used:

[0027] Item 6. The silylating agent is represented by the formula: H 4-a-b SiZ a R b [In the formula, Z may be the same or different and represents a halogen atom; R may be the same or different and represents a hydrocarbon group having 1 to 10 carbon atoms; a is an integer of 1 to 3, b is an integer of 1 to 3, and 2≦a+b≦4.] 5. The method according to any one of items 1 to 4, wherein the organohalosilane is represented by the formula: Manufacturing method.

[0028] Item 7. The method according to any one of Items 1 to 5, wherein Y is an isopropyl group.

[0029] Section 8. Said R 5 8. The method according to any one of items 1 to 7, wherein is an alkyl group having 1 to 3 carbon atoms.

[0030] Item 9. The method according to any one of Items 1 to 8, wherein the reaction temperature in the above step is 10 to 100° C.

[0031] Item 10. The method according to any one of Items 1 to 9, wherein the reaction time of the above step is less than 120 minutes.

[0032] Term 11. Formula (II):

[0033] [ka] TIFF2025037736000009.tif177170

[0034] [In the formula, P 2 are the same or different and represent a protecting group for a hydrogen atom or an amino group. An N-substituted peptide represented by the formula: Effect of the Invention

[0035] According to the present invention, it is possible to synthesize a variety of N-substituted peptides with free C-terminus rapidly and in high yield while suppressing epimerization. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a schematic diagram showing a schematic configuration of a flow reactor (microflow reactor). [Diagram 2] Chromatograms of peptides represented by Fmoc-L-Phe-L-MePhe-OH (retention time: 12.1 minutes), Fmoc-L-Phe-D-MePhe-OH (retention time: 13.3 minutes), and Fmoc-D-Phe-L-MePhe-OH (retention time: 14.9 minutes). [Diagram 3] 1 shows the results of evaluating the epimerization rate of the N-substituted peptide (Fmoc-L-Phe-L-MePhe-OH) obtained by the production method of the present invention immediately after production, one day later, and one week later. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

[0038] In this specification, the expression of a numerical range "A to B" means "greater than or equal to A and less than or equal to B."

[0039] 1. First Amino Acid In the present invention, the first amino acid used as a substrate is not particularly limited as long as it is one that is used as a substrate in a normal peptide elongation reaction toward the C-terminus. Examples of the first amino acid include those represented by the general formula (1):

[0040] [ka]

[0041] [In the formula, P 1 represents a protecting group for an amino group. 1 indicates an amino acid residue.] Examples of the compound include compounds represented by the following formula:

[0042] In the general formula (1), P 1 As the protecting group for the amino group represented by the formula (I), any protecting group known as a protecting group for an amino group can be used. For example, alkyl-type protecting groups such as tert-butyl (t-Bu) group, benzyl group, allyl group, methyl group, and triphenylmethyl group (Trt); aryl-type protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide-type protecting groups such as formyl group and acetyl group (Ac); phthalimide-type protecting groups such as phthaloyl group (Phth); benzyloxycarbonyl group (Cbz) and the like. ), carbamate-type protecting groups such as a tert-amyloxycarbonyl group (Aoc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a tert-butyloxycarbonyl group (Boc), an allyloxycarbonyl group (Alloc), or a 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), or a (2-trimethylsilyl)-ethanesulfonyl group (SES).

[0043] In the present invention, the protecting group of the amino group includes a tag used to improve the problem of peptide solubility. For example, the tag may be a hydrophobic tag consisting of an aromatic ring substituted with multiple long-chain alkoxy groups. This allows the reaction established in the solid-phase peptide synthesis method to be applied as is. Here, "solubility" means the ease with which a molecule dissolves in a solvent. In addition, in the present invention, the protecting group of the amino group also includes a solid-phase support. As the solid-phase support, a wide variety of solid-phase supports used in the conventional solid-phase peptide synthesis method can be adopted.

[0044] In the general formula (1), AA 1Any of natural amino acid residues and synthetic amino acid residues can be used as the amino acid residue represented by the formula (I): glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), threonine (Thr), lysine (Lys), 5-hydroxylysine (Hyl), arginine (Arg), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), cysteine ​​(CySH), cystine (CySH), sucrose (Ss ... Examples of amino acids that may be used include, but are not limited to, cysteine ​​(Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine (β-Ala), N-methyl-β-alanine, N-methyl-phenylalanine, N-phenyl-glycine, N-indolyl-glycine, sarcosine (Sar), 2-aminoisobutyric acid (Aib), γ-aminobutyric acid, kainic acid, and derivatives thereof.

[0045] In the general formula (1), AA 1 The amino acid residue represented by is not limited to an amino acid residue derived from an α-amino acid, but may be any amino acid residue derived from a β-amino acid or a γ-amino acid, etc.

[0046] In addition, in the general formula (1), the amino acid residue may have a protecting group depending on the type of the side chain. When the amino acid residue has, for example, a hydroxyl group, a thiol group, an amino group, and / or a carboxyl group in the side chain, it is preferable that the amino acid residue has a protecting group from the viewpoint of avoiding side reactions. On the other hand, when the amino acid residue does not have a hydroxyl group, a thiol group, an amino group, and / or a carboxyl group in the side chain, it is preferable that the amino acid residue does not have a protecting group from the viewpoint of reducing the deprotection step, improving atom economy, and reducing waste. According to the production method of the present invention, even if the amino acid residue does not have a protecting group, side reactions derived from the side chain are unlikely to occur.

[0047] The protecting group that the amino acid residue may have may be any protecting group known to be a protecting group for the side chain of an amino acid residue. Examples of such protecting groups include alkyl protecting groups such as tert-butyl (t-Bu) group, benzyl group, allyl group, methyl group, and triphenylmethyl group (Trt); silyl protecting groups such as tert-butyldimethylsilyl group (TBS); aryl protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide protecting groups such as formyl group and acetyl group (Ac); phthalimide protecting groups such as phthaloyl group (Phth); Examples of the protecting group include carbamate-type protecting groups such as benzyloxycarbonyl group (Cbz), tert-amyloxycarbonyl group (Aoc), 9-fluorenylmethoxycarbonyl group (Fmoc), tert-butyloxycarbonyl group (Boc), allyloxycarbonyl group (Alloc), and 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfenyl group (Npys), 2-nitrobenzenesulfonyl group (Ns), and (2-trimethylsilyl)-ethanesulfonyl group (SES). The number of protecting groups in the case where the protecting group is present is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

[0048] In addition, in the general formula (1), the amino acid residue may further have a substituent at the main carbon chain thereof. The substituent is not particularly limited, and examples thereof include hydrocarbon groups such as alkyl groups, aryl groups, and heteroaryl groups.

[0049] In general formula (1), the alkyl group which the amino acid residue may have as a substituent is not particularly limited, and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, etc., as well as branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, etc., and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8), such as a cyclopentyl group, a cyclohexyl group, etc.

[0050] In general formula (1), the aryl group which the amino acid residue may have as a substituent is not particularly limited, and any of a monocyclic aryl group, a fused ring aryl group, and a polycyclic aryl group can be used. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a hydroxyphenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, an indolyl group, an imidazolyl group, a pyrenyl group, and a triphenylenyl group.

[0051] In general formula (1), the heteroaryl group which the amino acid residue may have as a substituent is not particularly limited, and both a monocyclic heteroaryl group and a polycyclic heteroaryl group can be adopted. Examples thereof include a pyrrolidyl group, a pyrrolyl group, a tetrahydrothienyl group, a thienyl group, an oxolanyl group, a furanyl group, an imidazolyl group, an N-methylimidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, an N,N-dimethyl-4-aminopyridyl group, a pyrazyl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

[0052] From the above, specific examples of the first amino acid used in the present invention include, for example,

[0053] [ka]

[0054] etc.

[0055] The first amino acid represented by the general formula (1) may be either an L-type or a D-type optical isomer. In general, epimerization of amino acid residues during amidation reaction in peptide synthesis is one of the most serious problems. In this regard, the production method of the present invention makes it possible to synthesize a variety of N-substituted peptides in high yields while suppressing epimerization of amino acid residues by rapidly progressing the target reaction.

[0056] 2. The second amino acid The second amino acid that can be used in the present invention is not particularly limited as long as it is a free N-substituted amino acid at both the C- and N-terminus, and can be, for example, a second amino acid represented by the general formula (2):

[0057] [ka]

[0058] [In the formula, R 1 represents a hydrocarbon group having 1 to 10 carbon atoms. 2 indicates an amino acid residue.] Examples of the compound include compounds represented by the following formula:

[0059] In the general formula (2), AA 2 Any of natural amino acid residues and synthetic amino acid residues can be used as the amino acid residue represented by the formula (I): glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), threonine (Thr), lysine (Lys), 5-hydroxylysine (Hyl), arginine (Arg), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), cysteine ​​(CySH), cystine (CySH), sucrose (Ss ... Examples of amino acids that may be used include, but are not limited to, cysteine ​​(Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine (β-Ala), N-methyl-β-alanine, N-methyl-phenylalanine, N-phenyl-glycine, N-indolyl-glycine, sarcosine (Sar), 2-aminoisobutyric acid (Aib), γ-aminobutyric acid, kainic acid, and derivatives thereof.

[0060] It is common general knowledge that sarcosine (Sar; N-methylated glycine) is a rare example of an N-substituted amino acid that exhibits higher reactivity than the corresponding unsubstituted amino acid (unsubstituted glycine). According to the production method of the present invention, an N-substituted peptide can be synthesized using an N-substituted amino acid that exhibits relatively low reactivity.

[0061] In the general formula (2), AA 2 The amino acid residue represented by is not limited to an amino acid residue derived from an α-amino acid, but may be any amino acid residue derived from a β-amino acid or a γ-amino acid, etc.

[0062] In the general formula (2), R 1 The hydrocarbon group represented by is not particularly limited, and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as n-propyl, n-butyl, and n-pentyl groups; branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as isopropyl, sec-butyl, and tert-butyl groups; cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8), such as cyclopentyl and cyclohexyl groups; aryl groups having 6 to 10 carbon atoms (particularly 6 to 8), such as phenyl and naphthyl groups; and heteroaryl groups having 6 to 10 carbon atoms (particularly 6 to 8), such as imidazolyl, pyridyl, and indolyl groups. 1 From the viewpoint of improving the yield, the hydrocarbon group represented by the formula (1) is preferably a hydrocarbon group having 1 to 3 carbon atoms, more preferably a methyl group or an ethyl group, and further preferably a methyl group. 1 The hydrocarbon group represented by the formula (I) may have a substituent. The substituent is not particularly limited, and examples thereof include the above-mentioned alkyl groups, aryl groups, and heteroaryl groups. According to the production method of the present invention, the amino group at the N-terminus of the amino acid is represented by the formula (I). 1 Even when the aryl group is substituted with , the peptide elongation reaction can proceed rapidly.

[0063] In addition, in the general formula (2), the amino acid residue may have a protecting group depending on the type of the side chain. In addition, in the general formula (2), the amino acid residue may further have a substituent at the main chain carbon. In the general formula (2), the protecting group and / or the substituent that the amino acid residue may have include the above-mentioned AA 1 It is possible to employ a protecting group and / or a substituent which the amino acid residue represented by the formula (I) may have.

[0064] From the above, specific examples of the second amino acid used in the present invention include, for example,

[0065] [ka]

[0066] etc.

[0067] The second amino acid represented by the general formula (2) may be either an L-type or a D-type optical isomer. In general, epimerization of amino acid residues during amidation reaction in peptide synthesis is one of the most serious problems. In this regard, the production method of the present invention allows the target reaction to proceed rapidly, thereby making it possible to synthesize a variety of N-substituted peptides in high yield while suppressing epimerization of amino acid residues.

[0068] The amount of the second amino acid used is not particularly limited and can be appropriately selected from a wide range. From the viewpoints of suppressing the production of impurities, improving the yield, etc., the amount of the second amino acid used is preferably about 0.1 to 10 moles, more preferably about 0.5 to 5 moles, and even more preferably about 1 to 3 moles, per mole of the first amino acid.

[0069] 3.Silylation Agent The silylating agent that can be used in the present invention is not particularly limited, and examples thereof include silylating agents represented by the formula (I): H 4-a-b SiZ a R b (I) [In the formula, Z may be the same or different and represents a halogen atom; R may be the same or different and represents a hydrocarbon group having 1 to 10 carbon atoms; a is an integer of 1 to 3, b is an integer of 1 to 3, and 2≦a+b≦4.] Examples of organohalosilanes include those represented by the following formula:

[0070] In formula (I), examples of the halogen atom represented by Z include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0071] In formula (I), the hydrocarbon group represented by R is preferably a hydrocarbon group having 1 to 6 carbon atoms (for example, an alkyl group, an aryl group), more preferably an alkyl group having 1 to 3 carbon atoms, and even more preferably an alkyl group having 1 or 2 carbon atoms.

[0072] In formula (I), a is preferably 1 or 2, and more preferably 1.

[0073] In formula (I), b is preferably 2 or 3, and more preferably 3.

[0074] Specific examples of the organohalosilanes include dimethylchlorosilane, dimethyldichlorosilane, trimethylchlorosilane, trimethylbromosilane, trimethyliodosilane, triethylchlorosilane, triethylbromosilane, triethyliodosilane, triisopropylchlorosilane, dimethylethylchlorosilane, dimethylisopropylchlorosilane, and tert-butyldiphenylchlorosilane.

[0075] From the above, examples of suitable silylating agents include, for example,

[0076] [ka]

[0077] etc.

[0078] The silylating agents can be used alone or in combination of two or more kinds.

[0079] The amount of the silylating agent used is not particularly limited and can be appropriately selected from a wide range. From the viewpoint of suppressing the generation of impurities, improving the yield, etc., the amount is preferably about 1 to 10 moles, more preferably about 2 to 5 moles, and even more preferably about 2 to 3 moles, per mole of the second amino acid. When two or more kinds of silylating agents are used in combination, it is preferable to adjust the total amount to be within the above range.

[0080] 4. Condensing Agent The condensing agent that can be used in the present invention is represented by the general formula (3):

[0081] [ka]

[0082] [In the formula, X represents a halogen atom, and Y represents a hydrocarbon group having 1 to 10 carbon atoms.] It is a compound represented by the formula:

[0083] In the general formula (3), examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. From the viewpoints of yield, economy, safety, and the like, a chlorine atom or a bromine atom is preferred, and a chlorine atom is more preferred.

[0084] In general formula (3), the hydrocarbon group represented by Y is not particularly limited and includes linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as n-propyl, n-butyl, and n-pentyl, as well as branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as isopropyl and sec-butyl. Of these, the hydrocarbon group represented by Y is preferably a branched alkyl group having 3 to 10 carbon atoms, more preferably a branched alkyl group having 3 to 6 carbon atoms, and even more preferably an isopropyl group, from the viewpoint of reactivity with the first amino acid and the tertiary amine.

[0085] From the above, the condensing agent used in the present invention is, for example,

[0086] [ka]

[0087] etc.

[0088] These condensing agents can be used alone or in combination of two or more kinds.

[0089] The amount of the condensation agent used is not particularly limited and can be appropriately selected from a wide range. From the viewpoint of suppressing the generation of impurities, improving the yield, etc., the amount is preferably about 0.1 to 10 moles, more preferably about 0.2 to 5 moles, and even more preferably about 0.5 to 3 moles, per mole of the first amino acid. When two or more kinds of condensation agents are used in combination, it is preferable to adjust the total amount to be within the above range.

[0090] 5. Tertiary amines The tertiary amine that can be used in the present invention has the general formula (4):

[0091] [ka]

[0092] [In the formula, R 2 , R 3 and R 4 are the same or different and each represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. 2 , R 3 and R 4 may be linked to form a substituted or unsubstituted ring (excluding an imidazole ring), and the ring may have one or more heteroatoms. The compound is represented by the formula (hereinafter, sometimes referred to as "first tertiary amine").

[0093] In the general formula (4), R 2 , R 3 and R 4 The alkyl group represented by the formula (I) is not particularly limited, and examples thereof include linear alkyl groups having 1 to 10 (particularly 1 to 6) carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, and the like, as well as branched alkyl groups having 3 to 10 (particularly 3 to 6) carbon atoms, such as an isopropyl group, an isobutyl group, a sec-butyl group, an tert-butyl group, and the like.

[0094] In the general formula (4), R 2 , R 3 and R 4 The aryl group represented by the formula (I) is not particularly limited, and any of a monocyclic aryl group, a fused ring aryl group, and a polycyclic aryl group can be used. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, a pyrenyl group, and a triphenylenyl group.

[0095] In the general formula (4), R 2 , R 3 and R 4 The heteroaryl group represented by the formula (I) is not particularly limited, and both a monocyclic heteroaryl group and a polycyclic heteroaryl group can be adopted. Examples thereof include a pyrrolidyl group, a pyrrolyl group, a tetrahydrothienyl group, a thienyl group, an oxolanyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazyl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

[0096] In the general formula (4), R 2 , R 3 and R 4may be bonded to form a substituted or unsubstituted ring (excluding an imidazole ring), and the ring may have one or more heteroatoms. The ring is not particularly limited, and examples thereof include an aziridine ring, an azirine ring, a diaziridine ring, a diazirine ring, an azetidine ring, an azeto ring, a diazeto ring, a pyrrolidine ring, a pyrrole ring, an imidazolidine ring, a piperidine ring, a piperazine ring, and a morpholine ring.

[0097] In the general formula (4), R 2 , R 3 and R 4 The substituents which may be possessed by the alkyl group, aryl group, heteroaryl group, and the ring formed by linking these groups are not particularly limited, and examples thereof include a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the above alkyl group, the above aryl group, an alkoxy group, an amino group, etc. The number of the substituents, when possessed, is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

[0098] In the general formula (4), although there is no particular limitation, from the viewpoint of yield improvement, epimerization suppression, etc., R 2 , R 3 and R 4 It is preferable that at least one of R is not a methyl group. 2 , R 3 and R 4 More preferably, at least two of R 2 , R 3 and R 4 It is more preferable that none of the first tertiary amines is a methyl group. Specifically, the first tertiary amine is preferably triethylamine (Et3N), diisopropylethylamine (i-Pr2NEt), or the like.

[0099] The first tertiary amine may be used alone or in combination of two or more kinds.

[0100] The amount of the first tertiary amine used is not particularly limited, but is preferably about 0.5 to 10 moles, more preferably about 2 to 8 moles, and even more preferably about 3 to 5 moles, per mole of the first amino acid, from the viewpoints of improving the yield, suppressing epimerization, etc. When two or more kinds of first tertiary amines are used in combination, it is preferable to adjust the total amount to be within the above range.

[0101] In the production method of the present invention, from the viewpoints of improving the yield and suppressing epimerization, it is also preferable that the compound represented by the general formula (7):

[0102] [ka]

[0103] [In the formula, R 6 , R 7 and R 8 are the same or different and each represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. 6 , R 7 and R 8 may be linked to form a substituted or unsubstituted ring, and the ring may contain one or more heteroatoms. 6 , R 7 and R 8 At least one of the groups is a methyl group. In the production method of the present invention, when an imidazole derivative and two kinds of tertiary amines (a first tertiary amine and a second tertiary amine) are used in combination, the target amidation reaction can be more rapidly carried out, and therefore, various N-substituted peptides can be easily obtained rapidly and in high yield while suppressing epimerization.

[0104] In the general formula (7), R 6 , R 7 and R 8The alkyl group, aryl group, and heteroaryl group represented by the formula (1), the ring formed by linking these groups, and the substituents that these groups may have can be the same as those in the above general formula (4). 6 , R 7 and R 8 A ring formed by combining two or more of these may be an imidazole ring. 6 , R 7 and R 8 At least one of the groups is a methyl group.

[0105] In the general formula (7), although there is no particular limitation, from the viewpoint of yield improvement, epimerization suppression, etc., R 6 , R 7 and R 8 At least two of the above are preferably methyl groups. Specifically, the second tertiary amine is preferably trimethylamine (Me3N), dimethylethylamine (Me2NEt), dimethyl n-hexylamine (Me2Nn-Hex), 1-methylpyrrolidine, dimethylbenzylamine (Me2NBn), 4-dimethylaminopyridine (DMAP), or the like.

[0106] The second tertiary amine may be used alone or in combination of two or more kinds.

[0107] The amount of the second tertiary amine used is not particularly limited, but is preferably about 0.01 to 5 moles, more preferably about 0.02 to 2 moles, and even more preferably about 0.05 to 1 mole, per mole of the first amino acid, from the viewpoints of improving the yield, suppressing epimerization, etc. When two or more types of second tertiary amines are used in combination, it is preferable to adjust the total amount to be within the above range.

[0108] 6. Imidazole derivatives The imidazole derivative that can be used in the present invention is represented by the general formula (5):

[0109] [ka]

[0110] [In the formula, R 5 represents a hydrocarbon group having 1 to 10 carbon atoms. It is a compound represented by the formula:

[0111] In the general formula (5), R 5 The hydrocarbon group represented by is not particularly limited, and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as n-propyl, n-butyl, and n-pentyl groups; branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as isopropyl, sec-butyl, and tert-butyl groups; cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8), such as cyclopentyl and cyclohexyl groups; aryl groups having 6 to 10 carbon atoms (particularly 6 to 8), such as phenyl and naphthyl groups; and heteroaryl groups having 6 to 10 carbon atoms (particularly 6 to 8), such as imidazolyl, pyridyl, and indolyl groups. 5 From the viewpoint of improving the yield, the hydrocarbon group represented by the formula: is preferably a hydrocarbon group having 1 to 3 carbon atoms, more preferably a methyl group or an ethyl group, and even more preferably a methyl group.

[0112] Specifically, the imidazole derivative is preferably N-methylimidazole (NMM), N-ethylimidazole, N-isopropylimidazole, or N-phenylimidazole, and more preferably N-methylimidazole.

[0113] The imidazole derivatives may be used alone or in combination of two or more kinds.

[0114] The amount of the imidazole derivative used is not particularly limited, but is preferably about 0.1 to 10 moles, more preferably about 0.5 to 5 moles, and even more preferably about 1 to 3 moles, per mole of the first amino acid, from the viewpoints of improving the yield, suppressing epimerization, etc. When two or more imidazole derivatives are used in combination, it is preferable to adjust the total amount to be within the above range.

[0115] 7. Acid The acid that can be used in the present invention is not particularly limited, and a wide variety of known organic or inorganic acids can be used, and a wide variety of known Lewis acids or Bronsted acids can be used.

[0116] Examples of acids that can be used in the present invention include Lewis acids such as aluminum chloride, iron chloride, lithium chloride, aluminum nitrate, zinc nitrate, cobalt nitrate, copper nitrate, nickel nitrate, nickel sulfate, and bismuth chloride, and Bronsted acids such as trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, paratoluenesulfonic acid, acetic acid, hydrofluoric acid, hydrochloric acid (hydrochloric acid), hydrobromic acid, perchloric acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0117] Among these, from the viewpoints of improving the yield and suppressing epimerization, the acid is preferably a Brønsted acid, more preferably a Brønsted acid having an acid dissociation constant (pKa) in water of 3 or less, still more preferably a Brønsted acid having an acid dissociation constant (pKa) in water of 2 or less, and particularly preferably a Brønsted acid having an acid dissociation constant (pKa) in water of 1 or less.

[0118] The acids can be used alone or in combination of two or more kinds.

[0119] The amount of the acid used is not particularly limited, but is preferably about 0.01 to 5 moles, more preferably about 0.1 to 1 mole, and even more preferably about 0.1 to 0.8 moles, relative to 1 mole of the first amino acid, from the viewpoints of improving the yield, suppressing epimerization, etc. When two or more acids are used in combination, it is preferable to adjust the total amount to be within the above range.

[0120] 8. N-Substituted Peptides The N-substituted peptide of the present invention has the general formula (6):

[0121] [ka]

[0122] [In the formula, P 1 R represents a protecting group for an amino group. 1 represents a hydrocarbon group having 1 to 10 carbon atoms. 1 indicates an amino acid residue. 2 and A.A. 2 are the same or different and represent amino acid residues. It is a compound represented by the formula:

[0123] In formula (6), each symbol is the same as defined above.

[0124] Specific examples of the N-substituted peptides that are the subject of the present invention include, for example,

[0125] [ka]

[0126] etc.

[0127] The N-substituted peptide represented by the general formula (6) may be any of the optical isomers L,L, L,D, D,L, and D,D, depending on the structure of the starting material.

[0128] The N-substituted peptides obtained by the production method of the present invention can be obtained either alone or in combination of two or more kinds.

[0129] The N-substituted peptides obtained by the production method of the present invention are useful as peptide drugs, and as raw materials or intermediates thereof.

[0130] Conventionally, flow synthesis of N-substituted peptides, which can be applied to a wide variety of substrates, has been impossible to realize because of the long reaction time required. Therefore, the production method of the present invention has the advantage that it can provide a variety of N-substituted peptides quickly and in high yield while suppressing epimerization. In addition, the method of the present invention is also useful in opening the way to continuous multi-step synthesis of N-substituted peptides.

[0131] 9.New compounds Among the N-substituted peptides obtained by the production method of the present invention, those having the formula (II):

[0132] [ka] TIFF2025037736000023.tif178170

[0133] [In the formula, P 2 are the same or different and represent a protecting group for a hydrogen atom or an amino group. The N-substituted peptide represented by the formula (I) is a novel compound not yet described in the literature.

[0134] In the above formula (II), P 2 As the protecting group for the amino group represented by the formula (I), any protecting group known as a protecting group for an amino group can be used. For example, alkyl-type protecting groups such as tert-butyl (t-Bu) group, benzyl group, allyl group, methyl group, and triphenylmethyl group (Trt); aryl-type protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide-type protecting groups such as formyl group and acetyl group (Ac); phthalimide-type protecting groups such as phthaloyl group (Phth); benzyloxycarbonyl group (Cbz) and the like. ), carbamate-type protecting groups such as tert-amyloxycarbonyl group (Aoc), 9-fluorenylmethoxycarbonyl group (Fmoc), tert-butyloxycarbonyl group (Boc), allyloxycarbonyl group (Alloc), and 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfenyl group (Npys), 2-nitrobenzenesulfonyl group (Ns), and (2-trimethylsilyl)-ethanesulfonyl group (SES). 2 Among these, the protecting group for the amino group represented by the formula (I) is preferably a benzyloxycarbonyl group (Cbz), a 9-fluorenylmethoxycarbonyl group (Fmoc), or a tert-butyloxycarbonyl group (Boc).

[0135] P2 When is a protecting group for an amino group, P can be obtained by a known deprotection step depending on the type of the protecting group. 2 can be eliminated and converted into a hydrogen atom.

[0136] 10. Method for producing N-substituted peptides The method for producing the N-substituted peptide, which is the object of the present invention, comprises a step of reacting the first amino acid (N-terminal protected amino acid) with the second amino acid (C-terminal unprotected N-substituted amino acid) in the presence of the condensing agent, the first tertiary amine, the silylating agent, and the imidazole derivative. There is no particular limitation on the reaction step, and all components can be added simultaneously or sequentially.

[0137] The production method of the present invention preferably includes a step (A1) of contacting the first amino acid (N-terminal protected amino acid), the condensing agent, the first tertiary amine, and optionally the second tertiary amine, and a step (A2) of contacting the second amino acid (C,N-terminally N-substituted amino acid), the silylating agent, the imidazole derivative, optionally the acid, and optionally the first tertiary amine, and more preferably further includes a step (B) of contacting the mixture obtained by the step (A1), the mixture obtained by the step (A2), optionally the imidazole derivative, and optionally the acid. In addition, the step (A2) preferably includes a step (A2a) of contacting the second amino acid, the silylating agent, the imidazole derivative, and the first tertiary amine, and a step (A2b) of contacting the mixture obtained by the step (A2a), the imidazole derivative, and the acid. The first tertiary amine used in each reaction step may be the same or different between the reaction steps, and this also applies to the acid and the imidazole derivative.

[0138] Each reaction step of the present invention can be carried out by either a batch method or a flow method. Among them, from the viewpoints of high safety and ease of scale-up, it is more preferable to carry out the reaction by a flow method, and from the viewpoints of precise control of reaction temperature and reaction time, it is even more preferable to carry out the reaction by a microflow method.

[0139] In this specification, the batch method means a batch operation, specifically, a series of operations in which the substances to be reacted are charged into a reaction vessel or reaction apparatus at once, reacted, and then removed after equilibrium or a certain reaction rate is reached.

[0140] In this specification, the flow method generally means a continuous operation carried out in a flow reactor. Specifically, the flow method means that the reaction proceeds by continuously feeding and discharging the raw materials and the substances required for the treatment into and from the apparatus (flow reactor) at a constant rate (flow rate).

[0141] In this specification, the microflow method means a flow method (continuous operation) using a microflow reactor.

[0142] In particular, the reaction step of the present invention can be carried out using a microflow reactor (hereinafter, may be understood as a "flow reactor" in a broad sense). The microflow reactor includes, for example, a flow path for transporting a fluid containing a raw material or an intermediate used in the reaction, a pump for feeding the fluid into the flow path, and a mixer for mixing the fluid.

[0143] Regarding the use of a microflow reactor, from the viewpoints of improving the yield and avoiding side reactions, in the production method of the present invention, it is preferable to carry out the above steps (A1) and (A2b) in a microflow reactor (flow reactor), and it is more preferable to carry out the above step (B) in a microflow reactor (flow reactor).

[0144] Hereinafter, the configuration of the microflow reactor and the method for producing an N-substituted peptide of the present invention using the same will be described with reference to Fig. 1. However, it goes without saying that the production method of the present invention is not limited to the embodiment of Fig. 1.

[0145] 1 is a schematic diagram showing a general configuration of a microflow reactor 1. The microflow reactor 1 can include a syringe pump 11 that contains a first liquid, a syringe pump 12 that contains a second liquid, and a syringe pump 13 that contains a third liquid.

[0146] As an example, the first liquid may contain a first amino acid and a first tertiary amine, the second liquid may contain a condensing agent, and the third liquid may contain a second amino acid, a silylating agent, an imidazole derivative, optionally an acid, and optionally a first tertiary amine. As another example, the third liquid may be obtained by mixing a mixture of a second amino acid, a silylating agent, an imidazole derivative, and optionally a first tertiary amine, optionally an acid, and optionally an imidazole derivative. Alternatively, the third liquid may be obtained by mixing a silylated second amino acid obtained by reacting a second amino acid, a silylating agent, an imidazole derivative, and optionally a first tertiary amine in a flask, optionally an acid, and optionally an imidazole derivative.

[0147] The microflow reactor 1 can include flow paths F1, F2, F3, F4, and F5 for transporting fluids. The inner diameter of the flow path may be, for example, 0.1 to 10 mm, or 0.3 to 1 mm. The microflow reactor 1 can include mixers M1 and M2 for mixing fluids. The mixer is not particularly limited, but for example, a V-shaped mixer, a T-shaped mixer, or the like, in which the fluids are mixed by being introduced therein, can be used. In such a mixer, the interfacial area between two liquids is increased by the entrainment flow generated in the flow path, and two or more reaction liquids can be easily mixed in several milliseconds. The inner diameter of the flow path inside the mixer may be, for example, 0.1 to 10 mm, or 0.2 to 1 mm.

[0148] The inner diameter of the flow path may be the diameter of the inner part of the flow path (the part through which the fluid passes) in the cross section of the flow path in a direction perpendicular to the longitudinal direction of the flow path. If the shape of the inner part of the flow path is not a perfect circle, the inner diameter of the flow path may be the diameter of the shape of the inner part of the flow path converted into a perfect circle based on the area standard.

[0149] Syringe pumps 11, 12, 13, and 14, mixers M1 and M2, and flow paths F1, F2, F3, F4, and F5 may be formed of, for example, a resin such as plastic or elastomer, glass material, metal, ceramic, or the like.

[0150] By operating the syringe pump 11, the first liquid can move through the flow path F1 and flow into the mixer M1. By operating the syringe pump 12, the second liquid can move through the flow path F2 and flow into the mixer M1. The first liquid and the second liquid are mixed by the mixer M1 to become a first mixed liquid, which can be sent to the flow path F4. On the other hand, by operating the syringe pump 13, the third liquid moves through the flow path F3 and flows into the mixer M2, where it is mixed with the first mixed liquid to become a second mixed liquid, which can be sent to the flow path F5. The second mixed liquid can move through the flow path F5 and be used in a subsequent reaction, or can be stored in any test tube, or can be used for isolation, purification, and in-line analysis.

[0151] According to the microflow reactor 1, it is easy to increase the area for heat exchange per volume of the reaction solution. In addition, it is easy to control the reaction time by adjusting the flow rate and the length of the flow path. This makes it easy to strictly control the reaction solution, and as a result, it is easy to minimize the progress of undesired side reactions and to improve the yield of the target product.

[0152] After the reaction using the microflow reactor is completed, the target compound can be obtained in high purity and high yield through normal isolation and purification steps as necessary. In addition, when the target compound is obtained in high yield through the reaction using the microflow reactor, the isolation and purification steps can be omitted or simplified, and the target compound can be directly subjected to crystallization or analysis.

[0153] Furthermore, while conventional peptide synthesis requires complicated isolation and purification steps, the present invention uses a condensing agent that produces fewer impurities after the reaction and is easy to remove, making it easy to obtain cyclic peptides in high yields. This makes it possible to omit or simplify these steps, and the resulting peptides can be directly subjected to crystallization or analysis.

[0154] The method for producing an N-substituted peptide of the present invention is not limited to one carried out using a microflow reactor, and may be, for example, a batch vessel having a small volume and capable of achieving a high stirring speed. The volume of the mixing section of the batch vessel may be 1 to 100 mL, or may be 5 to 50 mL.

[0155] As described above, the method for producing the N-substituted peptide of the present invention can be carried out by a liquid phase method, and therefore can be easily scaled up.

[0156] The reaction temperature in the reaction step of the present invention is preferably 10 to 100°C, more preferably 20 to 70°C, and even more preferably 40 to 60°C, from the viewpoints of improving the yield and suppressing epimerization.

[0157] The reaction time in the reaction step of the present invention is preferably from 1 second to less than 120 minutes, more preferably from 10 seconds to less than 60 minutes, and even more preferably from 60 seconds to 5 minutes, from the viewpoint of suppressing epimerization.

[0158] The solvent used in the reaction step of the present invention is not particularly limited, and for example, any organic solvent in which the substrate can be dissolved can be used. From the viewpoint of the solubility of the compound, however, it is preferable to use an aprotic polar solvent.

[0159] The aprotic polar solvent is not particularly limited, but includes halogen-based solvents such as dichloromethane and chloroform; ketone-based solvents such as acetone and methyl ethyl ketone; ether-based solvents such as 1,4-dioxane, tetrahydrofuran, and diethyl ether; amide-based solvents such as N,N-dimethylformamide and N,N-diethylformamide, sulfoxide-based solvents such as dimethyl sulfoxide, nitrile-based solvents such as acetonitrile, and mixtures thereof. Among them, as the reaction solvent, ether-based solvents, nitrile-based solvents, and the like are preferred from the viewpoints of substrate solubility, low reactivity, ease of removal, and the like, and 1,4-dioxane, acetonitrile, and the like are more preferred. These solvents can be used alone or in combination of two or more kinds.

[0160] The solvent used in the reaction step of the present invention may contain components other than the organic solvent, such as water, as long as the effects of the present invention are not significantly impaired. However, when the obtained N-substituted peptide is used as is in a subsequent reaction and the subsequent reaction does not favor water, it is preferable that the solvent used in the reaction step does not contain water. In this case, the N-substituted peptide obtained by the production method of the present invention can be used in the subsequent reaction without isolation and / or purification, which is advantageous in large-scale synthesis and / or continuous multi-step synthesis.

[0161] In the method of the present invention, in addition to the above-mentioned components, additives can be used as appropriate within the range that does not impair the effects of the present invention. EXAMPLES

[0162] The following Reference Examples and Examples will be given to more clearly illustrate the features of the present invention, but the present invention is not limited to the following Examples. In order to simplify the description in the specification, the following abbreviations may be used in the Reference Examples, Examples, and Tables in the Examples. Abbreviations: Meanings are: Fmoc: 9-fluorenylmethyloxycarbonyl group, Bn: benzyl group, Boc: tert-butoxycarbonyl group, NMI: N-methylimidazole, HCl: hydrogen chloride, Me2NBn: dimethylbenzylamine, DIEA: N,N-diisopropylethylamine, Me: methyl group, i-Pr: isopropyl group, Ph: phenyl group, TMS: trimethylsilyl group, THF: tetrahydrofuran, MeCN: acetonitrile, TMSCl: trimethylsilyl group. chlorosilane, BSA: N,O-bistrimethylsilylacetamide, TIPSCl: triisopropylchlorosilane, TBDPSCl: tert-butyldiphenylchlorosilane, TESCl: triethylchlorosilane, DMSCl: dimethylchlorosilane, Phe: phenylalanine, Gly: glycine, Val: valine, MeVal: α-methylvaline, Asp: aspartic acid, Ser: serine, Lys: lysine, Sar: sarcosine, Pro: proline.

[0163] [Example 1] Synthesis of N-substituted peptides N-substituted peptides were synthesized by the method shown below (Table 1).

[0164] [Table 1]

[0165] A 1,4-dioxane solution (flow rate: 1.20 mL / min) of an N-terminal protected amino acid represented by Fmoc-L-Phe-OH (0.333 M, 1.0 molar equivalent), dimethylbenzylamine (0.0167 or 0.0333 M, 0.05 or 0.10 molar equivalent), and N,N-diisopropylethylamine (0.366 or 0.466 M, 1.1 or 1.4 molar equivalent), and a 1,4-dioxane solution (flow rate: 2.0 mL / min) of isopropyl chloroformate (0.230 or 0.300 M, 1.15 or 1.50 molar equivalent) were introduced into a T-shaped mixer at 40° C. using a syringe pump. The resulting mixture was introduced into reaction tube 1 (inner diameter: 0.800 mm, length: 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. Also, a mixture was obtained by mixing an unprotected N-substituted amino acid represented by HL-MePhe-OH, a silylating agent (0.48-1.20M, 2.4-6.0 molar equivalents) listed in Table 1, N,N-diisopropylethylamine (0.24-0.60M, 1.2-3.0 molar equivalents), N-methylimidazole (0-0.60M, 0-3.0 molar equivalents), and acetonitrile at room temperature. An acetonitrile solution (flow rate: 1.2mL / min) obtained by adding N-methylimidazole and hydrochloric acid (hydrogen chloride) (0 or 0.060M, 0 or 0.3 molar equivalents, respectively) to the mixture and the mixture in reaction tube 1 were introduced into a T-shaped mixer at a temperature (20-70°C) listed in Table 1 using a syringe pump. The resulting mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 20.7 m, volume: 10.4 mL, reaction time: 2 min) at the same temperature. After reaching a steady state over about 60 s, the reacted mixture was poured into ethyl acetate (3 mL) and 0.33 M hydrochloric acid (3 mL) at room temperature for 30-60 s. The aqueous layer was extracted with ethyl acetate three times. The organic layer was washed with saturated saline, dried over magnesium sulfate, filtered, and concentrated in vacuum at room temperature. The yields of the obtained N-substituted peptide represented by Fmoc-L-Phe-L-MePhe-OH (compound 6a) and its epimer (compound epi-6a; Fmoc-D-Phe-L-MePhe-OH) were determined by HPLC-UV analysis using a calibration curve (column: COSMOSIL 5C). 18-AR-II 4.6 mm I.D. × 150 mm, solvent: acetonitrile + 0.1% formic acid / H2O + 0.1% formic acid (0-20 min: 30-100%, 20-25 min: 100%, 25-27 min: 30%, 27-30 min: 30%), flow rate: 1.0 mL / min, detection wavelength: 254 nm, temperature: 40 °C, retention time: 17.3 min). The results are shown in Table 1.

[0166] When no imidazole derivative was used, the yield of N-substituted peptide (compound 6a) was low, and the same amount of epimer (compound epi-6a) was produced (Comparative Example 1-1 and Comparative Example 1-2). On the other hand, when an imidazole derivative was used, epimerization was suppressed, and N-substituted peptide (compound 6a) was obtained in high yield in a short time of about 2 minutes (Example 1-1). Furthermore, when hydrochloric acid (hydrogen chloride) was further used, the yield was further improved, and epimerization did not occur substantially (Example 1-5).

[0167] [ka]

[0168] Fmoc-L-Phe-L-MePhe-OH (Example 3-1): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 98.7 mg, 0.180 mmol, 90% White solid; mp 85-87℃; 1H NMR (400 MHz, CDCl3, mixture of 3 rotamers, major rotamer): δ 7.76 (d, J = 7.6 Hz, 2H), 7.54 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 7.2 Hz, 1H), 7.43-7.14 (m, 12H), 7.04 (d, J= 6.8 Hz, 2H), 5.76 (d, J = 9.2 Hz, 1H), 5.13 (dd, J = 5.2, 10.4 Hz, 1H), 4.81 (dt, J = 7.2, 8.4 Hz, 1H), 4.31 (dd, J = 7.6, 10.4Hz, 1H), 4.03-4.28 (m, 2H), 2.80-3.42 (m, 4H), 2.75 (s, 3H) ppm; 13 C NMR (100 MHz, CDCl3, major rotamer): δ 174.05, 172.79, 155.79, 143.98, 141.39, 136.64, 136.14, 129.69, 129.40, 128.89, 128.64, HRMS (ESI): calcd for [C 34 H 32 N2O5+H] + 549.2384, found 549.2384 [Example 2] Study of imidazole derivatives and solvents Next, the hydrocarbon group R 5 The effects of the hydrocarbon group R shown in Table 2 and the reaction solvent were evaluated. 5 N-substituted peptides were synthesized in the same manner as in Examples 1-5, except that solvent A and / or solvent B were used.

[0169] [Table 2]

[0170] From the results in Table 2, R 5 It can be seen that the yield of the desired N-substituted peptide is improved when is a methyl group (Examples 1-5, 2-1 and 2-2). In addition, the N-substituted peptide was obtained in high yield regardless of the solvent used (Examples 1-5, 2-1 and 2-2). In particular, it is considered that the yield is improved when an ether solvent is used in combination with acetonitrile.

[0171] [Example 3] Substrate range study Next, in order to evaluate the range of substrates applicable as the N-terminal amino acid and the C-terminal amino acid, N-substituted peptides were synthesized using amino acids having various amino acid residues. Specifically, N-substituted peptides were synthesized using the first amino acid and the second amino acid shown in Table 3. As shown in Table 3, the amounts of various reactants used were increased compared to those in Examples 1 and 2.

[0172] [Table 3]

[0173] The results in Table 1 show that the desired reaction could be carried out regardless of which amino acid residue was used (Examples 3-1 to 3-13). These results show that the production method of the present invention can be applied to a wide variety of N-substituted amino acids and N-substituted peptides.

[0174] [ka]

[0175] Boc-L-Phe-L-MePhe-OH (Example 3-2): Collection time: 60 s Purification method: The residue was purified by silica gel column chromatography (Hex / EtOAc / AcOH=200 / 50 / 1 to 0 / 250 / 1) 147.0 mg, 0.345 mmol, 86% White solid; mp 174-177 ℃; 1 H NMR (400 MHz, CDCl3, approximately 3 : 7 mixture of 2 rotamers): δ 7.36-7.18 (m, 8H), 7.08 (d, J = 6.8 Hz, 1.4H), 6.94 (d, J = 6.8 Hz, 0.6H), 5.27-5.21 (m, 1H), 5.07 (dd, J = 5.6, 10.4 Hz, 0.7H), 5.02 (dd, J = 4.4, 9.6 Hz, 0.3H), 4.74 (dd, J = 6.8, 15.2 Hz, 0.7H), 4.45-4.37 (m, 0.3 H), 3.37 (dd, J = 5.2, 14.4 Hz, 0.7H), 3.30 (dd, J = 5.2, 14.8 Hz, 0.3H), 3.10-2.95 (m, 1.6H), 2.90-2.70 (m, 3.8H), 2.52 (dd, J = 9.6, 14.0 Hz, 0.3H), 1.94 (dd, J = 4.8, 14.0 Hz, 0.3H), 1.37 (s, 6.3H), 1.32 (s, 2.7H) ppm; 13 C NMR (100 MHz, CDCl3, major rotamer): δ 172.47, 172.22, 154.97, 137.32, 136.54, 129.67, 129.39, 128.91, 128.47, 128.38, 126.69, 79.48, 58.56, 51.63, 39.05, 34.58, 32.60, 28.36 ppm; HRMS (ESI): calcd for [C 24 H 30 N2O5+H] +427.2227, found 427.2226

[0176]

Chem.

[0177] Cbz-L-Phe-L-MePhe-OH (Example 3-3): Collection time: 30 s Purification method: The residue was purified by silica gel column chromatography (Hex / EtOAc / AcOH = 200 / 50 / 1 to 200 / 100 / 1) 73.5 mg, 0.160 mmol, 80% White solid; mp 55 - 59 °C; 1 H NMR (400 MHz, CDCl3, approximately 3 : 7 mixture of 2 rotamers): δ 7.38 - 7.13 (m, 13H), 7.06 (d, J = 7.2 Hz, 1.4H), 6.91 - 6.89 (m, 0.6H), 5.51 (d, J = 9.2 Hz, 0.7H), 5.44 (d, J = 6.8 Hz, 0.3H), 5.10 - 4.93 (m, 3H), 4.79 (dd, J = 7.2, 15.6 Hz, 0.7H), 4.47 - 4.42 (m, 0.3H), 3.38 - 3.29 (m, 1H), 3.05 - 2.82 (m, 2.4H), 2.99 (s, 0.9H), 2.73 (s, 2.1 H), 2.73 (s, 2H), 2.51 (dd, J = 9.6, 14.0 Hz, 0.3H), 1.88 (dd, J = 4.8, 14.4 Hz, 0.3H) ppm; 1313C NMR (100 MHz, CDCl3, major rotamer): δ 174.00, 172.77, 155.78, 136.68, 136.49, 136.08, 129.64, 129.34, 128.90, 128.63, 128.60, 128.29, 127.94, 127.05, 126.90, 66.87, 59.52, 52.27, 38.98, 34.44, 33.43 ppm; HRMS (ESI): calcd for [C 27 H 28 N2O5+H] + 461.2071, found 461.2074

[0178]

Chem.

[0179] Fmoc-L-Phe-Pro-OH (Example 3-4): Collection time: 30 s Purification method: The residue was purified by silica gel column chromatography (Hex / EA / MeOH = 100 / 100 / 1) 80.1 mg, 0.165 mmol, 83% White solid; mp 98 - 103 °C; 11H NMR (400 MHz, CDCl3, approximately 1:10 mixture of 2 rotamers, major rotamer): δ 7.78 - 7.73 (m, 2H), 7.60 - 7.54 (m, 2H), 7.41 (t, J = 8.0 Hz, 2H), 7.33 - 7.29 (m, 4H), 7.25 - 7.22 (m, 3H), 5.66 (d, J = 8.4 Hz, 1H), 4.71 (dd, J = 8.4, 14.8 Hz, 1H), 4.59 (dd, J = 3.2, 8.0 Hz, 1H), 4.39 (dd, J = 7.6, 10.8 Hz, 1H), 4.31 (dd, J = 7.2, 10.4 Hz, 1H), 4.19 (t, J = 7.2 Hz, 1H), 3.60 - 3.46 (m, 1H), 3.11 - 3.01 (m, 2H), 2.90 - 2.85 (m, 1H), 2.32 - 2.27 (m, 1H), 2.05 - 1.94 (m, 1H), 1.90 - 1.74 (m, 2H) ppm; 13 13C NMR (100 MHz, CDCl3, major rotamer): δ 174.12, 172.15, 156.05, 143.90, 141.37, 135.84, 129.72, 128.69, 127.82, 127.17, 125.35, 125.27, 120.08, 67.28, 59.55, 54.11, 47.44, 47.16, 38.99, 28.39, 24.88 ppm; HRMS (ESI): calcd for [C 29 H 28 N2O5+H] + 485.2071, found 485.2073

[0180]

Chem.

[0181] Fmoc-L-Phe-Sar-OH (Example 3 - 5): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 87.1 mg, 0.190 mmol, 95% White solid; mp 94 - 96 ℃; 1 1H NMR (400 MHz, CDCl3, approximately 1 : 4 mixture of 2 rotamers): δ 7.76 - 7.74 (m, 2H), 7.57 - 7.50 (m, 2H), 7.38 (t, J = 7.2 Hz, 2H), 7.30 - 7.17 (m, 7H), 6.05 (d, J = 8.8 Hz, 0.2H), 5.94 (d, J = 8.8 Hz, 0.8H), 4.96 (dd, J = 11.2, 15.2 Hz, 0.8H), 4.80 (dd, J = 6.8, 15.2 Hz, 0.2H), 4.40 - 3.87 (m, 5H), 3.07 (dd, J = 7.2, 13.2 Hz, 1H), 2.98 (dd, J = 6.4, 13.2 Hz, 1H), 2.95 (s, 0.6H), 2.91 (s, 2.4H) ppm; 13 13C NMR (100 MHz, CDCl3, major rotamer): δ 172.97, 172.41, 156.01, 143.87, 141.35, 135.90, 129.65, 128.62, 127.80, 127.17, 125.33, 125.29, 120.05, 67.27, 52.15, 49.83, 47.15, 39.14, 36.59 ppm; HRMS (ESI): calcd for [C 27 H 26 N2O5 + H] + 459.1914, found 459.1914

[0182]

Chem.

[0183] Fmoc-L-Phe-L-MeAla-OH (Example 3-6): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 87.2 mg, 0.185 mmol, 92% White solid; mp 82-86 ℃; 1 H NMR (400 MHz, CDCl3, mixture of 3 rotamers, major rotamer): δ 7.76 (d, J = 7.2 Hz, 2H), 7.58-7.53 (m, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 8.0 Hz, 3H), 7.24-7.19 (m, 4H), 5.95-5.85 (m, 1H), 5.09 (dd, J = 7.2, 14.8 Hz, 1H) 4.96-4.86 (m, 1H), 4.38-4.32 (m, 1H), 4.27 (t, J = 7.6 Hz, 1H), 4.17 (t, J = 6.8 Hz, 1H), 3.13-2.95 (m, 2H), 2.86 (s, 3H), 1.41 (d, J = 7.2 Hz, 3H) ppm; 13 C NMR (100 MHz, CDCl3, major rotamer): δ 175.21, 172.67, 156.02, 143.94, 141.38, 135.90, 129.72, 128.62, 127.82, 127.16, 125.35, 125.29, 120.08, 67.24, 53.15, 52.43, 47.19, 38.91, 31.90, 14.16 ppm; HRMS (ESI): calcd for [C 28 H 28 N2O5+H] + 473.2071, found 473.2066

[0184]

Chem.

[0185] Fmoc-L-Phe-L-MeVal-OH (Example 3-7): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 81.0 mg, 0.162 mmol, 81% White solid; mp 84-90 ℃; 1 1H NMR (400 MHz, CDCl3, mixture of 3 rotamers, major rotamer): δ 7.77-7.73 (m, 2H), 7.58-7.47 (m, 2H), 7.42-7.37 (m, 2H), 7.33-7.19 (m 7H), 5.78 (d, J = 8.8 Hz, 1H), 4.99-4.91 (m, 1H), 4.78-4.71 (m, 1H), 4.38-4.28 (m, 2H) 4.20-4.13 (m, 1H), 3.14-2.95 (m, 2H), 2.81 (s, 3H), 2.23-2.17 (m, 1H), 1.03 (d, J = 6.4 Hz, 3H), 0.80 (d, J = 6.4 Hz, 3H) ppm; 13 13C NMR (100 MHz, CDCl3, major rotamer): δ 174.13, 173.63, 156.06, 143.93, 143.88, 141.37, 135.85, 129.56, 128.68, 127.83, 127.18, 125.31, 120.08, 67.28, 62.48, 52.55, 47.16, 39.21, 31.70, 27.26, 19.85, 18.91 ppm; HRMS (ESI): calcd for [C 30 H 32 N2O5+H] + 501.2384, found 501.2383

[0186]

Chem.

[0187] Fmoc-Gly-L-MePhe-OH (Example 3-8): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 76.5 mg, 0.167 mmol, 83% White solid; mp 86 - 89 ℃; 1 1H NMR (400 MHz, CDCl3, approximately 1 : 4 mixture of two rotamers): δ 7.76 (d, J = 7.6 Hz, 2H), 7.61 - 7.52 (m, 2H), 7.39 (t, J = 7.6 Hz, 2H), 7.32 - 7.15 (m, 7H), 5.84 (brs, 1H), 5.11 (dd, J = 5.2, 11.2 Hz, 0.8H), 4.56 (dd, J = 4.0, 10.0 Hz, 0.2H), 4.50 - 4.15 (m, 3H), 4.03 (dd, J = 4.4, 17.2 Hz, 1H), 3.88 (dd, J = 4.4, 17.2 Hz, 1H), 3.44 - 3.32 (m, 1H), 3.20 - 3.00 (m, 1H), 2.98 (s, 0.5H), 2.78 (s, 2.5H) ppm; 13 13C NMR (100 MHz, CDCl3, major rotamer): δ 174.06, 169.69, 156.51, 143.94, 141.40, 136.70, 129.18, 128.89, 128.77, 127.85, 127.20, 125.31, 120.10, 67.40, 60.00, 47.18, 42.93, 34.43, 32.77 ppm; HRMS (ESI): calcd for [C 29 H 28 N2O5 + H] + 459.1914, found 459.1911

[0188] [Chemistry]

[0189] Fmoc-β-Ala-L-MePhe-OH (Example 3-9): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 93.6 mg, 0.198 mmol, 99% White solid; mp 86 - 89 °C; 1 H NMR (400 MHz, CDCl3, mixture of at least 3 rotamers, major rotamer): δ 7.76 (d, J = 7.2 Hz, 2H), 7.60 - 7.62 (m, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.23 - 7.20 (m, 2H), 7.17 - 7.13 (m, 3H), 5.45 (brt, J = 6.0 Hz, 1H), 5.11 (dd, J = 5.2, 11.2 Hz, 1H), 4.37 (d, J = 6.8 Hz, 2H), 4.20 (t, J = 6.8 Hz, 1H), 3.41 - 3.36 (m, 3H), 3.10 (dd, J = 10.8, 14.0 Hz, 1H), 2.73 (s, 3H), 2.48 - 2.36 (m, 2H) ppm; 13 C NMR (100 MHz, CDCl3, major rotamer): δ 174.03, 173.13, 156.74, 144.05, 141.40, 136.91, 128.82, 128.70, 127.82, 127.18, 126.84, 125.24, 120.09, 66.80, 59.47, 47.31, 36.76, 34.53, 33.82, 33.64 ppm; HRMS (ESI): calcd for [C28 H 28 N2O5 + H + 473.2071, found 473.2072

[0190]

Chem.

[0191] Fmoc-L-Lys(NBoc)-L-MePhe-OH (Example 3-10): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 111.3 mg, 0.177 mmol, 88% White solid; mp 86 - 89 °C; 1 1H NMR (400 MHz, CDCl3, mixture of at least 3 rotamers, major rotamer): δ 7.76 (d, J = 7.2 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.23 - 7.09 (m, 5H), 5.83 - 5.78 (m, 1H), 5.28 (brs, 1H), 4.68 (brs, 1H), 4.58 (brs, 1H), 4.32 (d, J = 6.8 Hz, 2H), 4.20 - 4.13 (m, 1H), 3.50 - 3.38 (m, 1H), 3.15 - 2.90 (m, 5H), 2.92 (s, 3H), 1.60 - 1.20 (m, 4H), 1.42 (s, 9H) ppm; 13 13C NMR (100 MHz, CDCl3, assignment of 13 13C NMR signals were very difficult due to the existence of rotamers, see copy of 1313C NMR spectrum in Page XX); HRMS (ESI): calcd for [C 36 H 34 N3O7+H] + 630.3174, found 630.3175

[0192]

Chem.

[0193] Fmoc-L-Val-L-MePhe-OH (Example 3-11): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 80.0 mg, 0.160 mmol, 80% White solid; mp 91 - 94 °C; 1 1H NMR (400 MHz, CDCl3, mixture of 3 rotamers, major rotamers): δ 7.77 (d, J = 7.6 Hz, 2H), 7.60 (dd, J = 1.6, 7.2 Hz, 2H), 7.43 - 7.36 (m, 2H), 7.34 - 7.27 (m, 2H), 7.24 - 7.12 (m, 4H), 7.08 - 7.02 (m, 1H), 5.53 (d, J = 9.6 Hz, 1H), 5.28 (dd, J = 5.2, 10.8 Hz, 1H), 4.62 - 4.32 (m, 2H), 4.29 - 4.23 (m, 1H), 4.21 - 4.13 (m, 1H), 3.41 (dd, J = 5.6, 12.2 Hz, 1H), 3.06 - 2.99 (m, 1H), 2.95 (s, 3H), 1.96 (sext, J = 6.8 Hz, 1H), 0.93 (dd, J = 7.2, 6H) ppm; 1313C NMR (100 MHz, CDCl3, major rotamer): δ 174.44, 173.45, 156.42, 144.01, 141.41, 136.53, 128.85, 128.58, 127.87, 127.21, 126.94, 125.38, 120.11, 67.18, 58.75, 55.90, 47.28, 34.45, 33.38, 31.25, 19.50, 17.67 ppm; HRMS (ESI): calcd for [C 30 H 32 N2O5+H] + 501.2384, found 501.2381

[0194]

Chem.

[0195] Fmoc-Aib-L-MePhe-OH (Example 3-12): Collection time: 30 s flow & batch: 20 min (flow 2 min + batch 18 min) Purification method: The residue was purified by GPC (CHCl3) 69.7 mg, 0.143 mmol, 72% White solid; mp 92-95 °C; 11H NMR (400 MHz, CDCl3): δ 7.73 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz, 2H), 7.30 (td, J = 1.6, 7.2 Hz, 2H), 7.23 (t, J = 6.8 Hz, 2H), 7.17 (d, J = 6.8 Hz, 3H), 5.24 (brs, 1H), 4.74 (brs, 1H), 4.54 (brs, 2H), 4.14 (t, J = 6.0 Hz, 1H), 3.46 (d, J = 10.4 Hz, 1H), 3.16 (d, J = 11.6 Hz, 1H), 2.61 (s, 3H), 1.37 (s, 3H), 1.31 (s, 3H) ppm; 13 13C NMR (100 MHz, CDCl3, major rotamer): δ 173.05, 173.26, 155.33, 143.68, 141.50, 137.41, 129.20, 128.58, 127.88, 127.23, 126.85, 125.02, 120.12, 66.25, 63.06, 57.20, 47.50, 35.33, 34.32, 25.78, 25.46 ppm; HRMS (ESI): calcd for [C 29 H 30 N2O5+H] + 487.2227, found 487.2228

[0196]

Chem.

[0197] Fmoc-L-Phe-BnGly-OH (Example 3-13): Collection time: 30 s Purification method: The residue was purified by GPC (CHCl3) 77.6 mg, 0.145 mmol, 73% White solid; mp 73-78℃; 1 H NMR (400 MHz, CDCl3, mixture of at least 3 rotamers, 2 : 3 mixture of two rotamers): δ 7.80-7.00 (m, 18H), 6.22-6.12 (m, 0.4H), 6.10-6.00 (m, 0.6H), 5.10-5.00 (m, 0.6H), 4.97-4.80 (m, 0.4H), 4.58 (d, J = 16.0 Hz, 1H), 4.46 (d, J = 16.0 Hz, 1H), 4.37-4.18 (m, 2H), 4.20-3.78 (m, 3H), 3.20-2.90 (m, 2H) ppm; 13 C NMR (100 MHz, CDCl3, assignment of 13 C NMR signals were very difficult due to the existence of rotamers, see copy of 13 C NMR spectrum in Page XX; HRMS (ESI): calcd for [C 33 H 30 N2O5+H] + 535.2227, found 535.2228 [Example 4] Consideration of epimerization In order to demonstrate the usefulness of the present invention, it is important to verify whether epimerization occurs during peptide chain elongation. In normal peptide chain elongation, the amino acid used as the nucleophile does not cause epimerization, so it is sufficient to pay attention to epimerization during the reaction when the amino acid used as the electrophile or the peptide is activated. On the other hand, when an N-substituted amino acid is used as the nucleophile as in the present invention, the following study revealed that epimerization of the amino acid residue on the nucleophile side occurs during storage of the solution of the obtained N-substituted peptide (Figure 2).

[0198] In this study, we investigated the possibility that Fmoc-Phe-MePhe-OH obtained by the present invention may undergo epimerization during storage in solution. The following three peptides were prepared and subjected to HPLC analysis under the conditions shown in Figure 2, which showed good separation. Fmoc-L-Phe-L-MePhe-OH(Exp 3250-5) Fmoc-L-Phe-D-MePhe-OH(Exp 3336-2) Fmoc-D-Phe-L-MePhe-OH(Exp 3335-2) Therefore, Fmoc-L-Phe-L-MePhe-OH was dissolved in acetonitrile, and the progress of epimerization over time was followed by HPLC analysis (Figure 3). As a result, the amount of Fmoc-D-Phe-L-MePhe-OH in which the electrophile Phe was epimerized was hardly increased or decreased, while the amount of Fmoc-L-Phe-D-MePhe-OH in which the nucleophile Phe was epimerized was observed to increase over time. Therefore, it was found that when the N-substituted peptide obtained by synthesis is stored in a solution, the C-terminus side (the amino acid residue used as the nucleophile) undergoes epimerization. It was also found that none of the amino acid residues of the obtained peptide are epimerized if it is stored in a solid state at a low temperature. In other words, it was found that in verifying epimerization in the present invention, it is necessary to analyze the obtained sample immediately after the reaction.

[0199] [ka]

[0200] Fmoc-L-Phe-D-MePhe-OH(Exp 3336-2): Conditions were different from the general procedure. The solution of S1: Me2NBn (0.05 equiv), i-Pr2NEt (1.10 equiv); The solution of S2: IPCF (1.15 equiv); The solution of S3: H-D-MePhe-OH (1.50 equiv), TMSCl (3.00 eq.), DIEA (1.50 equiv.), NMI (1.80 equiv.) Collection time: 45 s Purification method: The residue was purified by GPC (CHCl3) 129.8mg, 0.237 mmol, 79% White solid; mp 88-92 ℃; 1 H NMR (400 MHz, CDCl3, mixture of 3 rotamers, major rotamer): δ 7.72 (d, J = 7.6 Hz, 2H), 7.54 (d, J= 7.6 Hz, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.12-7.42 (m, 12H), 7.02-6.93 (m, 2H), 5.98 (d, J = 8.8 Hz, 1H), 5.22 (dd, J = 5.2, 10.8 Hz, 1H), 4.83 (dd, J = 7.2, 10.8 Hz, 1H), 4.30 (dd, J = 7.6, 10.0 Hz, 1H), 4.18 (t, J = 7.2 Hz, 1H), 4.12 (t, J = 7.2 Hz, 1H), 3.39 (dd, 5.2, 14.8 Hz, 1H), 3.01 (dd, J = 10.8, 14.4 Hz, 1H), 2.81 (s, 3H), 2.54-2.70 (m, 2H) ppm; 1313C NMR (100 MHz, CDCl3, major rotamer): δ 173.69, 173.24, 156.01, 143.93, 141.36, 136.78, 136.02, 129.47, 129.00, 128.85, 128.57, 127.80, 127.18, 127.05, 125.36, 125.31, 120.05, 67.28, 59.55, 52.41, 47.16, 38.64, 34.65, 33.39 ppm; HRMS (ESI): calcd for [C 34 H 32 N2O5+H] + 549.2384, found 549.2384

[0201]

Chem.

[0202] Fmoc-D-Phe-L-MePhe-OH (Exp 3335-2): Conditions were different from the general procedure. The solution of S1: Me2NBn (0.05 equiv), i-Pr2NEt (1.10 equiv); The solution of S2: IPCF (1.15 equiv); The solution of S3: H-D-MePhe-OH (1.50 equiv), TMSCl (3.00 eq.), DIEA (1.50 equiv.), NMI (1.80 equiv.) Collection time: 60 s Purification method: The residue was purified by GPC (CHCl3) 171.6 mg, 0.313 mmol, 78% White solid; mp 87 - 93 °C; 1H NMR (400 MHz, CDCl3, mixture of 3 rotamers, major rotamers): δ 7.72 (d, J = 7.6 Hz, 2H), 7.54 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.10-7.43 (m, 12H), 7.05-6.90 (m, 2H), 6.01 (d, J = 8.4 Hz, 1H), 5.22 (d, J = 4.4, 10.4 Hz, 1H), 4.83 (dt, J = 7.6, 7.6 Hz, 1H), 4.30 (dd, J = 4.0, 9.6 Hz, 1H), 4.18 (dd, J = 7.2, 10.0 Hz, 1H), 4.11 (t, J = 6.4 Hz, 1H) 3.39 (dd, J = 5.2, 14.8 Hz, 1H), 3.01 (dd, J = 10.8, 14.0 Hz, 1H), 2.81 (s, 3H), 2.70-2.50 (m, 2H) ppm; 13 C NMR (100 MHz, CDCl3, major rotamer): δ 173.71, 173.25, 156.02, 143.93, 141.35, 136.78, 136.02, 129.47, 128.99, 128.84, 128.56, 127.79, 127.17, 127.04, 125.36, 125.30, 120.04, 67.27, 59.52, 52.41, 47.15, 38.62, 34.65, 33.38 ppm; HRMS (ESI): calcd for [C 34 H 32 N2O5+H] + 549.2384, found 549.2384

Claims

1. General formula (6): 【Chemistry 1】 [In the formula, P １ R represents a protecting group for an amino group. １ represents a hydrocarbon group having 1 to 10 carbon atoms. １ and A.A. ２ are the same or different and represent amino acid residues. A method for producing an N-substituted peptide represented by the following formula: General formula (1): 【Chemistry 2】 [In the formula, each symbol is the same as above.] and a first amino acid represented by General formula (2): 【Chemistry 3】 [In the formula, each symbol is the same as above.] and a second amino acid represented by A silylating agent; General formula (3): 【Chemistry 4】 [In the formula, X represents a halogen atom, and Y represents a hydrocarbon group having 1 to 10 carbon atoms.] and a condensing agent represented by the formula: General formula (4): 【Chemistry 5】 [In the formula, R ２ , R ３ and R ４ are the same or different and each represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. ２ , R ３ and R ４ may be linked to form a substituted or unsubstituted ring (excluding an imidazole ring), and the ring may have one or more heteroatoms. and a tertiary amine represented by the formula: General formula (5): 【Chemistry 6】 [In the formula, R ５ represents a hydrocarbon group having 1 to 10 carbon atoms. and an imidazole derivative represented by the formula:

2. The method according to claim 1 , further comprising using an acid in the step.

3. The method according to claim 2, wherein the acid is a Bronsted acid having an acid dissociation constant (pKa) in water of 3 or less.

4. The R ２ , R ３ and R ４ The method according to any one of claims 1 to 3, wherein none of

5. In the above process, a compound represented by the general formula (7): 【Chemistry 7】 [In the formula, R ６ , R ７ and R ８ are the same or different and each represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. ６ , R ７ and R ８ may be linked to form a substituted or unsubstituted ring, and the ring may contain one or more heteroatoms. ６ , R ７ and R ８ At least one of these is a methyl group. The method according to claim 4, wherein a tertiary amine represented by the formula:

6. The silylating agent has the formula: H ４－ａ－ｂ SiZ ａ R ｂ [In the formula, Z may be the same or different and each represents a halogen atom; R may be the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms; a is an integer of 1 to 3, b is an integer of 1 to 3, and 2≦a+b≦4.] The method according to any one of claims 1 to 3, wherein the organohalosilane is represented by the formula: Manufacturing method.

7. The method according to any one of claims 1 to 3, wherein Y is an isopropyl group.

8. The R ５ The method according to any one of claims 1 to 3, wherein is an alkyl group having 1 to 3 carbon atoms.

9. The method according to any one of claims 1 to 3, wherein the reaction temperature in the above step is 10 to 100°C.

10. The method according to any one of claims 1 to 3, wherein the reaction time of the step is less than 120 minutes.

11. Formula (II): 【Chemistry 8】 【change】 [In the formula, P ２ are the same or different and represent a hydrogen atom or an amino-protecting group. The N-substituted peptide represented by the formula:

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