Double-residue extension peptide and preparation method thereof

Abstract

Various peptides having extended double residues can be prepared by reacting a first amino acid or peptide represented by formula (1), a halogenating agent, a tertiary amine represented by formula (3), an amino acid N-carboxylic acid anhydride (NCA) represented by formula (4), and a second amino acid or peptide represented by formula (5). # imgabs0 # # imgabs1 # imgabs2 # imgabs3 #

Description

Technical Field

[0001] The present invention relates to a double-residue extended peptide and a preparation method thereof. Background Art

[0002] The demand for peptides is increasing in the applications of medicines, foods, cosmetics, etc. However, in existing peptide synthesis methods, excessive high-priced reactants are required, which will produce a large amount of waste from the reactants, so the increase in environmental burden or preparation cost becomes a problem. In addition, when each peptide is extended by 1 residue, a larger protective group equal to or larger than that of an amino acid is used, and a deprotection process of the protective group is further required, so the decrease in atom economy and the increase in the number of processes also become a major problem. Therefore, a rapid and efficient peptide synthesis method is sought.

[0003] As a method for reducing the number of steps for peptide synthesis, it is known that there is a method for synthesizing tripeptides or oligopeptides by a one-pot synthesis method. Among them, Shin et al. reported a one-pot tripeptide synthesis using dehydrogenated amino acid N-carboxylic anhydride (dehydrogenated NCA) (for example, with reference to non-patent literature 1 and 2). However, the method of non-patent literature 1 and 2 is only to improve the yield by using dehydrogenated NCA that is more stable than common amino acid N-carboxylic anhydride (NCA), and it is impossible to extend a wide range of amino acid residues using common NCA.

[0004] In addition, it is also shown that carbamate-protected NCA is useful in one-pot tripeptide synthesis (for example, see non-patent document 3). However, the method of non-patent document 3 requires the additional preparation of carbamate-protected NCA, and the need to remove the protecting group remains unchanged. In addition, there is no mention of the epimerization of amino acid residues that is a problem in conventional peptide synthesis, particularly in peptide synthesis under alkaline conditions.

[0005] On the other hand, there are also reports of synthesizing oligopeptides by one-pot method without using unstable NCA (for example, refer to non-patent document 4). However, the method of non-patent document 4 uses a protecting group and an expensive reagent, and it is necessary to remove the protecting group and the impurities from the reagent, and the reaction time is long. Therefore, it cannot be called an excellent peptide synthesis method that can be applied to a wide range of amino acids.

[0006] Therefore, it has been necessary to stabilize all the extended amino acids by protecting or dehydrogenating them. That is, there has been no method for rapidly obtaining various peptides using unprotected amino acids. Therefore, such a method is sought.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent literature 1: Chem. Lett. 1994, 1909-1910.

[0010] Non-patent document 2: Bull. Chem. Soc. Jpn. 1994, 67, 2151-2161.

[0011] Non-patent document 3: Tetrahedran Lett. 1995, 36, 807-810.

[0012] Non-patent document 4: Org. Biomol. Chem. 2017, 15, 7533-7542. Summary of the invention

[0013] 1. Technical issues to be resolved

[0014] The present invention is accomplished in view of the above-mentioned current state of the prior art, and its main purpose is to provide a method for preparing various di-residue extended peptides using amino acid N-carboxylic anhydride (NCA).

[0015] (II) Technical solution

[0016] In order to achieve the above-mentioned purpose, the inventors of the present application have conducted careful research. As a result, it was found that by reacting a first amino acid or peptide, a halogenating agent, a tertiary amine, an amino acid N-carboxylic anhydride (NCA) and a second amino acid or peptide, the N-terminus of the amino acid or peptide is extended with a di-residue, thereby various peptides with di-residue extension can be obtained in a short time. The inventors of the present application have conducted further research based on the above-mentioned insights and completed the present invention. That is, the present invention includes the following structure.

[0017] Item 1. A method for preparing a di-residue extended peptide represented by the following general formula (6):

[0018] [Chemical formula 1]

[0019]

[0020] In formula (6), R 1 represents a hydrogen atom or an alkyl group; R 4 , R 8 and R 10 The same or different, represents a hydrogen atom or a methyl group; R 2 , R 5 , R 6 , R 9 and R 11 Same or different, represents the side chain of the amino acid residue; R 2 , R 5 , R 6 , R 9and R 11 They may form a ring together with the adjacent nitrogen atom through the carbon atom to which they are bonded or not; R 3 , R 7 and R 12 are the same or different and represent a hydrogen atom or a substituted or unsubstituted monovalent organic group; 1 represents a hydrogen atom, an amino protecting group, a label or a solid phase carrier; P 2 represents a hydrogen atom, a protecting group of a carboxyl group, a label or a solid phase carrier; n1 represents an integer greater than 0, and when n1 is an integer greater than 2, n1 R 1 , R 2 and R 3 Optional the same or different; n2 represents an integer greater than 0, when n2 is an integer greater than 2, n2 R 10 , R 11 and R 12 Optional same or different,

[0021] The preparation method comprises: a reaction step of reacting a first amino acid or peptide represented by the following general formula (1), a halogenating agent, a tertiary amine represented by the following general formula (3), an amino acid N-carboxylic anhydride (NCA) represented by the following general formula (4), and a second amino acid or peptide represented by the following general formula (5),

[0022] [Chemical formula 2]

[0023]

[0024] In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , P 1 and n1 and R 1 , R 2 , R 3 , R 4 , R 5 , P 1 and n1 are the same,

[0025] [Chemical formula 3]

[0026]

[0027] In formula (3), R 13 , R 14 , R 15 are the same or different and represent substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or, R 13 , R 14 and R15 Two or more of the groups are connected to form a ring with or without one or more heteroatoms or substituents,

[0028] [Chemical formula 4]

[0029]

[0030] In formula (4), R 6 and R 7 With the R 6 and R 7 same,

[0031] [Chemical formula 5]

[0032]

[0033] In formula (5), R 8 , R 9 , R 10 , R 11 , R 12 , P 2 and n2 and the R 8 , R 9 , R 10 , R 11 , R 12 , P 2 The same as n2.

[0034] Item 2. The preparation method according to Item 1, wherein the reaction step comprises:

[0035] (A) a reaction step of reacting a first amino acid or peptide represented by the general formula (1), a halogenating agent, a tertiary amine represented by the general formula (3), and an amino acid N-carboxylic anhydride (NCA) represented by the general formula (4); and

[0036] (B) A reaction step of reacting the reaction mixture obtained in the step (A) with the second amino acid or peptide represented by the general formula (5).

[0037] Item 3. The preparation method according to Item 1 or 2, wherein the step (A) comprises:

[0038] (A1) a reaction step of reacting a first amino acid or peptide represented by the general formula (1), a halogenating agent, and a tertiary amine represented by the general formula (3); and

[0039] (A2) A reaction step of reacting the reaction mixture obtained in the step (A1) with the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4).

[0040] Item 3-1. The preparation method according to any one of Items 1 to 3, wherein the R 2 , R 5 , R 6 , R 9 and R 11 are the side chains of natural amino acid residues.

[0041] Item 3-2. The production method according to any one of Items 1 to 3-1, wherein n1 is an integer of 0 to 100.

[0042] Item 3-3. The preparation method according to any one of Items 1 to 3-2, wherein n2 is an integer of 0 to 100.

[0043] Item 4. The preparation method according to any one of Items 1 to 3-3, wherein the halogenating agent is thionyl represented by the following general formula (2),

[0044] [Chemical formula 6]

[0045]

[0046] In formula (2), X's are the same or different and represent a halogen atom.

[0047] Item 5. The production method according to any one of Items 1 to 4, wherein the tertiary amine comprises an aliphatic amine and / or a heterocyclic aromatic amine.

[0048] Item 6. The production method according to any one of Items 1 to 5, wherein the reaction temperature of the reaction step is -80 to 100°C.

[0049] Item 7. The production method according to any one of Items 1 to 6, wherein the reaction time of the reaction step is less than 60 minutes.

[0050] Item 8. The production method according to any one of Items 1 to 7, wherein the reaction step is performed by a flow method.

[0051] Item 9. The production method according to any one of Items 1 to 8, wherein the reaction step is performed by a microflow method.

[0052] Item 10. An acylated NCA represented by the following general formula (8):

[0053] [Chemical formula 7]

[0054]

[0055] In formula (8), R 1represents a hydrogen atom, or a substituted or unsubstituted monovalent organic group; R 4 Represents a hydrogen atom or a methyl group; R 2 , R 5 and R 6 Same or different, represents the side chain of the amino acid residue; R 2 , R 5 and R 6 They may form a ring together with the adjacent nitrogen atom through the carbon atom to which they are bonded or not; R 3 and R 7 represents a hydrogen atom, or a substituted or unsubstituted monovalent organic group; P 1 represents a hydrogen atom, an amino protecting group, a label or a solid phase carrier; n1 represents an integer greater than 0, and when n1 is an integer greater than 2, n1 R 1 ~R 3 Optional same or different.

[0056] Item 11. A peptide represented by the following general formula (9):

[0057] [Chemical formula 8]

[0058]

[0059] In formula (9), R 16 , R 17 and R 18 Same or different, represents the side chain of the amino acid residue; R 16 , R 17 and R 18 Through the carbon atom to which they are bonded, they may form a ring together with the adjacent nitrogen atom or not; 3 represents a protecting group for a hydrogen atom or an amino group; P 4 It represents a protecting group for a hydrogen atom or a carboxyl group.

[0060] Item 12. A peptide consisting of the amino acid sequence represented by SEQ ID NO:1.

[0061] (III) Beneficial effects

[0062] According to the present invention, a first amino acid or peptide, amino acid N-carboxylic anhydride (NCA), a specific halogenating agent and a specific tertiary amine are combined and reacted with a second amino acid or peptide, thereby causing the N-terminus of the first amino acid or peptide to undergo di-residue extension, thereby enabling various di-residue extended peptides to be obtained in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a schematic diagram showing the schematic structure of a microfluidic reactor.

[0064] Figure 2 The chromatograms are of the tripeptide (compound 6i) represented by Fmoc-L-Cys(t-Bu)-L-Phe-L-Ala-Ot-Bu obtained in Example 6 and of the tripeptide (compounds epi-6i-1 and epi-6i-2) obtained in Example 9 as its diastereomers (chromatographic column: DAICEL CHIRALPAK IB 4.6 mm×25 cm, solvent: 10% hexane solution of isopropanol, flow rate: 1.0 mL / min, detection wavelength: 254 nm).

[0065] Figure 3 Chromatograms of the tripeptide (compound 6o) represented by Fmoc-L-Ala-L-Cys(t-Bu)-L-Ala-Ot-Bu obtained in Example 6 and chromatograms of the tripeptide (compounds epi-6o-1 and epi-6o-2) obtained in Example 9 as its diastereomers (chromatographic column: DAICEL CHIRALPAK IB 4.6 mm×25 cm, solvent: 10% hexane solution of isopropanol, flow rate: 1.0 mL / min, detection wavelength: 254 nm).

[0066] Figure 4 Chromatograms of the tripeptide (compound 6a) represented by Fmoc-L-Phe-L-Phe-L-Ala-Ot-Bu obtained in Example 4 and chromatograms of the tripeptides (compounds epi-6a-1 and epi-6a-2) obtained in Comparison Examples 1 and 2 as its diastereomers (chromatographic column: DAICEL CHIRALPAK IH 4.6 mm×25 cm, solvent: 10% hexane solution of isopropanol, flow rate: 1.0 mL / min, detection wavelength: 254 nm). DETAILED DESCRIPTION

[0067] In the present specification, “comprising” is a concept that includes “including”, “consist essentially of” and “consist of”.

[0068] In addition, in this specification, the expression of the numerical range of "A to B" means "A or more and B or less".

[0069] The method for preparing a di-residue extended peptide of the present invention comprises the step of reacting a first amino acid or peptide, a halogenating agent, a tertiary amine, an amino acid N-carboxylic anhydride (NCA), and a second amino acid or peptide.

[0070] Furthermore, in the method of the present invention, by using an amino acid N-carboxyanhydride (NCA) whose N-terminus is not protected, it is possible to omit a deprotection step required every time an amino acid is extended in a conventional peptide synthesis.

[0071] Furthermore, in the method of the present invention, even when an amino acid that is easily diastereoisomerized is contained, diastereoisomerization of the amino acid is less likely to occur.

[0072] Furthermore, when the microfluidic method is used in the method of the present invention, by appropriately selecting a halogenating agent and a tertiary amine, a high-purity target peptide can be easily obtained if the peptide is a crystallizable peptide.

[0073] (1) First amino acid or peptide

[0074] The first amino acid or peptide that can be used in the present invention is a compound represented by the following general formula (1).

[0075] [Chemical formula 9]

[0076]

[0077] In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , P 1 and n1 and R 1 , R 2 , R 3 , R 4 , R 5 , P 1 The same as n1.

[0078] In the general formula (1), R 1 The group represented is a hydrogen atom or an alkyl group.

[0079] In the general formula (1), as long as R 1 The alkyl group represented is not particularly limited to an alkyl group having 1 to 10 carbon atoms, and examples thereof include chain alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6) such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl, branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6) such as isopropyl, isobutyl, sec-butyl, and tert-butyl (t-Bu), and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8) such as cyclopentyl and cyclohexyl.

[0080] In the general formula (1), R 2 and R 5The groups represented are the same or different and are side chains of amino acid residues. In the present specification, the side chain of an amino acid residue refers to a monovalent group obtained by removing a group represented by the following formula from an amino acid having an amino group and a carboxyl group in one molecule.

[0081] [Chemical formula 10]

[0082]

[0083] In the general formula (1), R 2 and R 5 The side chains of the amino acid residues represented are not limited to those derived from α-amino acids, and side chains derived from β-amino acids, γ-amino acids, and the like can also be employed.

[0084] In the general formula (1), R 2 and R 5 The side chain of the amino acid residue represented can also adopt any of the side chains of natural amino acid residues and synthetic amino acid residues, for example, 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 (Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine, N-methyl-β-alanine, sarcosine, γ-aminobutyric acid, kainic acid or side chains of natural amino acid residues such as derivatives thereof.

[0085] In the general formula (1), R 2 and R 5The side chain of the amino acid residue represented can adopt the side chain of the amino acid residue with a protecting group according to the type of the protected functional group. When the above-mentioned amino acid residue is an amino acid residue with an amino group or a carboxyl group in the side chain, from the perspective of avoiding side reactions, the side chain of the amino acid residue with a protecting group is preferably used. On the other hand, when the above-mentioned amino acid residue is composed only of an amino acid residue with a functional group other than an amino group and a carboxyl group in the side chain, from the perspective of reducing the deprotection process, improving atom economy (Atom Economy), and reducing waste, the side chain of the amino acid residue without a protecting group is preferably used. According to the preparation method of the present invention, even if the side chain of the above-mentioned amino acid residue does not have a protecting group, the side reaction caused by the side chain is not easy to occur.

[0086] As the protecting group possessed by the side chain of the above-mentioned amino acid residue, any known protecting group as a protecting group possessed by the side chain of the amino acid residue can be adopted, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB), p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl, acetyl (Ac) and the like; phthaloyl (Ph th) and other phthalimide-type protecting groups; carbamate-type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethanesulfonyl (SES), etc.

[0087] In the general formula (1), R 3 The group represented is a hydrogen atom or a substituted or unsubstituted organic group.

[0088] In the general formula (1), R 3 The organic group represented is preferably a hydrocarbon group, and examples of the hydrocarbon group include a substituted or unsubstituted alkyl group and a substituted or unsubstituted aryl group.

[0089] In the general formula (1), R 3The alkyl group represented is not particularly limited, and examples thereof include chain alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6) such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl, branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6) such as isopropyl, isobutyl, sec-butyl, and tert-butyl, and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8) such as cyclopentyl and cyclohexyl.

[0090] In the general formula (1), R 3 The aryl group represented is not particularly limited, and any of a monocyclic aryl group, a condensed ring aryl group and a polycyclic aryl group can be used. For example, phenyl, hydroxyphenyl, naphthyl, anthracenyl, phenanthrenyl, biphenyl, terphenyl, fluorenyl, indolyl, imidazolyl, pyrenyl, triphenylene and the like aryl groups having 6 to 18 carbon atoms (especially 6 to 14) can be listed.

[0091] In the general formula (1), R 3 The represented hydrocarbon group may have a substituent. As the substituent, for example, a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the above-mentioned alkyl group, the above-mentioned aryl group, an alkoxy group (methoxy group, ethoxy group, propoxy group, etc.), a carboxyl group, an amino group, etc. can be listed, without particular limitation. When having a substituent, the number of the substituent is not particularly limited, preferably 1 to 6, more preferably 1 to 3.

[0092] In the general formula (1), R 3 The substituent of the represented hydrocarbon group may have a protecting group. As the above-mentioned protecting group, any known protecting group as a protecting group of the above-mentioned substituent can be used, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB), p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl, acetyl (Ac) and the like; phthaloyl (Phth) and the like; Phthalimide type protecting groups; carbamate type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethylsulfonyl (SES), etc. The number of protecting groups when there is a protecting group is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

[0093] In the general formula (1), R 4 The group represented is a hydrogen atom or a methyl group.

[0094] In addition, in the general formula (1), P 1 The group represented is a protective group of a hydrogen atom or an amino group, a label or a solid phase carrier. From the perspective of avoiding side reactions, the protective group of the amino group is preferred. On the other hand, from the perspective of reducing the deprotection process, improving atom economy and reducing waste, P 1 On the other hand, when n1 in the general formula (1) is relatively large, that is, when the peptide represented by the general formula (1) is a long-chain peptide, from the perspective of being applicable to the preparation method of the present invention, P 1 The represented group is preferably a tag or a solid support.

[0095] As the above P 1 The protecting group of the amino group represented by the above-mentioned can be any known protecting group as a protecting group of the amino group, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB) and p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl and acetyl (Ac) and the like; phthaloyl (Phth) and the like; Phthalimide type protecting groups; carbamate type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethanesulfonyl (SES), etc.

[0096] As the above P 1 The represented tags can be widely used for improving the solubility of peptides, for example, hydrophobic tags composed of aromatic rings substituted with multiple long-chain alkoxy groups can be listed. Thus, the reaction established by the peptide solid phase synthesis method can be directly applied. Here, "solubility" refers to the ease with which a molecule dissolves in a solvent.

[0097] As the above P 1 As the solid phase support shown, a solid phase support generally used in a solid phase synthesis method for a peptide can be used.

[0098] In the general formula (1), n1 is an integer greater than 0, and is usually an integer from 0 to 100. From the perspective of improving the yield and suppressing side reactions, it is preferably an integer from 0 to 50, more preferably an integer from 0 to 10, and even more preferably an integer from 0 to 2. When n1 is an integer greater than 2, n1 R1 , R 2 and R 3 Same or different.

[0099] The amino acids constituting the first amino acid or the peptide represented by the general formula (1) may be either an L-type or a D-type optical isomer. In addition, the diastereoisomerization of the amino acid residues is usually the biggest problem during the amidation reaction in peptide synthesis, but in the production method of the present invention, the diastereoisomerization of the amino acid residues can be suppressed.

[0100] In summary, specific examples of the first amino acid or peptide represented by the general formula (1) include the following.

[0101] [Chemical formula 11]

[0102]

[0103] (2) Halogenating agent

[0104] The halogenating agent that can be used in the present invention is an electrophilic halogenating agent.

[0105] As the electrophilic halogenating agent, for example, there can be mentioned phosphorus halides represented by PX3 [wherein X is the same or different and represents a halogen atom; in this specification, the same shall apply hereinafter] or PX5, thionyl represented by SOX2, sulfonyl represented by SO2X2, oxalyl halide represented by (COX)2, etc. Among them, thionyl is preferred from the viewpoint of reactivity or price.

[0106] The above-mentioned thionyl group can be represented by the following general formula (2).

[0107] [Chemical formula 12]

[0108]

[0109] In formula (2), X's are the same or different and represent a halogen atom.

[0110] The groups represented by the above X are the same or different and are halogen atoms.

[0111] 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 economy, safety and the like, a chlorine atom or a bromine atom is preferred, and a chlorine atom is more preferred.

[0112] As described above, specific examples of the thionyl represented by the general formula (2) used in the present invention include the following thionyl and the like.

[0113] [Chemical formula 13]

[0114]

[0115] The halogenating agent that can be used in the present invention may be used alone or in combination of two or more.

[0116] The amount of the halogenating agent that can be used in the present invention is not particularly limited. From the perspective of suppressing the generation of impurities and improving the yield, for example, relative to 1 mole of the first amino acid or peptide represented by the general formula (1), it is usually preferably about 0.1 to 10 moles, more preferably about 0.2 to 5 moles, and further preferably about 0.5 to 3 moles. When two or more halogenating agents that can be used in the present invention are used in combination, it is preferably adjusted in a manner that the total amount is within the above range.

[0117] (3) Tertiary amine

[0118] The tertiary amine that can be used in the present invention is a compound represented by the following general formula (3).

[0119] [Chemical formula 14]

[0120]

[0121] In formula (3), R 13 , R 14 and R 15 are the same or different and represent a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; or, R 13 , R 14 and R 15 Two or more of the groups are connected to form a ring which may or may not have one or more heteroatoms or substituents.

[0122] In the general formula (3), R 13 , R 14 and R 15 The alkyl group represented is not particularly limited, and examples thereof include chain alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6) such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl, and branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6) such as isopropyl, isobutyl, sec-butyl, and tert-butyl.

[0123] In the general formula (3), R 13 , R 14 and R 15 The aryl group represented is not particularly limited, and any one of a monocyclic aryl group, a condensed ring aryl group and a polycyclic aryl group can be used. For example, aryl groups having 6 to 18 carbon atoms (especially 6 to 14) such as phenyl, naphthyl, anthracenyl, phenanthrenyl, biphenyl, terphenyl, fluorenyl, pyrenyl and triphenylene can be listed.

[0124] In the general formula (3), R13 , R 14 and R 15 The heteroaryl group represented is not particularly limited, and any of a monocyclic heteroaryl group and a polycyclic heteroaryl group can be used, for example, pyrrolidinyl, pyrrolyl, tetrahydrothienyl, thienyl, tetrahydrofuranyl (oxolanyl group), furanyl, imidazolyl, N-methylimidazolyl, pyrazolyl, thiazolyl, oxazolyl, piperidinyl, pyridyl, N,N-dimethyl-4-aminopyridinyl, pyrazinyl, indolyl, isoindolyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl and the like can be cited.

[0125] In the general formula (3), R 13 , R 14 and R 15 The alkyl, aryl and heteroaryl groups represented may have a substituent. As the substituent, for example, a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the above-mentioned alkyl group, the above-mentioned aryl group, an alkoxy group, an amino group, etc. can be listed without particular limitation. The number of substituents when having a substituent is not particularly limited, preferably 1 to 6, more preferably 1 to 3.

[0126] R 13 , R 14 and R 15 In the above, two or more of them are connected to form a ring with or without one or more heteroatoms or substituents. As the above ring, for example, an aziridine ring, an aziridine ring, a diaziridine ring, a diaziridine ring, an azetidine ring, an azetadiene ring, a diazetadiene ring, a pyrrolidine ring, a pyrrole ring, an imidazolidine ring, an imidazole ring, a pyridine ring, a piperidine ring, a piperazine ring, a morpholine ring, etc. can be listed without particular limitation.

[0127] In summary, specific examples of the tertiary amine used in the present invention include the following tertiary amines.

[0128] [Chemical formula 15]

[0129]

[0130] The tertiary amine represented by the general formula (3) can be used alone or in combination of two or more. The tertiary amine can include, for example, aliphatic amines such as triethylamine (Et3N), diisopropylethylamine (i-Pr2NEt), dimethylbenzylamine (Me2NBn), N-methylmorpholine (NMM); and / or heterocyclic aromatic amines such as pyridine, 4-dimethylaminopyridine (DMAP), and N-methylimidazole (NMI).

[0131] Among them, from the perspective of improving the yield, etc., the tertiary amine represented by the general formula (3) preferably includes an aliphatic amine and a heterocyclic aromatic amine, more preferably includes at least one aliphatic amine selected from the group consisting of triethylamine (Et3N), diisopropylethylamine (i-Pr2NEt) and dimethylbenzylamine (Me2NBn), and at least one heterocyclic aromatic amine selected from the group consisting of pyridine, 4-dimethylaminopyridine (DMAP) and N-methylimidazole (NMI), and particularly preferably includes at least one aliphatic amine selected from the group consisting of triethylamine (Et3N), diisopropylethylamine (i-Pr2NEt) and dimethylbenzylamine (Me2NBn), and a heterocyclic aromatic amine that is N-methylimidazole (NMI).

[0132] The amount of the tertiary amine used is not particularly limited. From the perspectives of improving yield, inhibiting diastereomerization, and economic efficiency, for example, relative to 1 mole of the first amino acid, it is usually preferably about 0.1 to 10 moles, more preferably about 0.2 to 5 moles, and further preferably about 0.5 to 3 moles. When two or more tertiary amines represented by the general formula (3) are used in combination, it is preferably adjusted in a manner such that the total amount is within the above range. In addition, as the amount of the tertiary amine used increases, the yield of the di-residue extended peptide as the target product will also increase, but even if the tertiary amine is used in excess, the yield of the di-residue extended peptide as the target product will hardly increase. Therefore, it is preferred to appropriately adjust the amount of the tertiary amine used from the perspective of economic efficiency.

[0133] (4) Amino acid N-carboxylic anhydride (NCA)

[0134] The amino acid N-carboxylic anhydride (NCA) that can be used in the present invention is a compound represented by the following general formula (4).

[0135] [Chemical formula 16]

[0136]

[0137] In formula (4), R 6 and R 7 With the R 6 and R 7 same.

[0138] In the general formula (4), R 6 The group represented is the side chain of the amino acid residue. 6 The side chains of the amino acid residues represented are not limited to those derived from α-amino acids, and side chains derived from β-amino acids, γ-amino acids, and the like can also be employed.

[0139] In the general formula (4), as R 6The side chain of the amino acid residue represented can be any of the side chains of natural amino acid residues and synthetic amino acid residues, for example, 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 (Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine, N-methyl-β-alanine, sarcosine, γ-aminobutyric acid, kainic acid or side chains of natural amino acid residues such as derivatives thereof.

[0140] In the general formula (4), R 6 The side chain of the amino acid residue represented can adopt the side chain of the amino acid residue with a protecting group according to the type of the protected functional group. When the above-mentioned amino acid residue is an amino acid residue with an amino group or a carboxyl group in the side chain, from the perspective of avoiding side reactions, the side chain of the amino acid residue with a protecting group is preferably used. On the other hand, when the above-mentioned amino acid residue is composed only of an amino acid residue with a functional group other than an amino group and a carboxyl group in the side chain, from the perspective of reducing the deprotection process, improving atom economy, and reducing waste, the side chain of the amino acid residue without a protecting group is preferably used. According to the preparation method of the present invention, even if the above-mentioned amino acid residue does not have a protecting group, the side reaction caused by the side chain is not easy to occur.

[0141] As the protecting group possessed by the side chain of the above-mentioned amino acid residue, any known protecting group as a protecting group possessed by the side chain of the amino acid residue can be adopted, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB), p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl, acetyl (Ac) and the like; phthaloyl (Ph th) and other phthalimide-type protecting groups; carbamate-type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethanesulfonyl (SES), etc.

[0142] The amino acid N-carboxylic anhydride (NCA) represented by the general formula (4) may be any optical isomer of the L-type and the D-type. In addition, during the amidation reaction in peptide synthesis, the diastereoisomerization of the amino acid residues is generally problematic, but in the production method of the present invention, the diastereoisomerization of the amino acid residues can be suppressed.

[0143] In the general formula (4), R 7 The group represented is a hydrogen atom or a substituted or unsubstituted organic group.

[0144] In the general formula (4), as R 7 The organic group represented is preferably a hydrocarbon group, and examples of the hydrocarbon group include a substituted or unsubstituted alkyl group and a substituted or unsubstituted aryl group.

[0145] In the general formula (4), R 7 The alkyl group represented is not particularly limited, and examples thereof include chain alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6) such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl, branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6) such as isopropyl, isobutyl, sec-butyl, and tert-butyl, and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8) such as cyclopentyl and cyclohexyl.

[0146] In the general formula (4), R 7The aryl group represented is not particularly limited, and any of a monocyclic aryl group, a condensed ring aryl group and a polycyclic aryl group can be used. For example, phenyl, hydroxyphenyl, indolyl, imidazolyl, naphthyl, anthracenyl, phenanthrenyl, biphenyl, terphenyl, fluorenyl, pyrenyl, triphenylene and other aryl groups having 6 to 18 carbon atoms (especially 6 to 14).

[0147] In the general formula (4), R 7 The represented hydrocarbon group may have a substituent. As the substituent, for example, a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the above-mentioned alkyl group, the above-mentioned aryl group, an alkoxy group (methoxy group, ethoxy group, propoxy group, etc.), a carboxyl group, an amino group, etc. can be listed, without particular limitation. When having a substituent, the number of the substituent is not particularly limited, preferably 1 to 6, more preferably 1 to 3.

[0148] In the general formula (4), R 7 The substituent of the represented hydrocarbon group may have a protecting group. As the above-mentioned protecting group, any known protecting group as a protecting group of the above-mentioned substituent can be used, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB), p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl, acetyl (Ac) and the like; phthaloyl (Phth) and the like; Phthalimide type protecting groups; carbamate type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethylsulfonyl (SES), etc. The number of protecting groups when there is a protecting group is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

[0149] In summary, specific examples of the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4) include the following.

[0150] [Chemical formula 17]

[0151]

[0152] The amount of NCA used is not particularly limited. From the perspectives of improving yield, inhibiting epimerization, and economic efficiency, for example, the amount is usually 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 relative to 1 mole of the first amino acid. When two or more NCAs represented by the general formula (4) are used in combination, they are preferably adjusted so that the total amount is within the above range.

[0153] (5) Second amino acid or peptide

[0154] The second amino acid or peptide that can be used in the present invention is a compound represented by the following general formula (5).

[0155] [Chemical formula 18]

[0156]

[0157] In formula (5), R 8 , R 9 , R 10 , R 11 and R 12 , P 2 and n2 and the R 8 , R 9 , R 10 , R 11 and R 12 , P 2 The same as n2.

[0158] In the general formula (5), R 8 and R 10 The groups represented are the same or different and are hydrogen atoms or methyl groups.

[0159] In the general formula (5), R 9 and R 11 The groups represented are the same or different and are side chains of amino acid residues. 9 and R 11 The side chains of the amino acid residues represented are not limited to those derived from α-amino acids, and side chains derived from β-amino acids, γ-amino acids, and the like can also be employed.

[0160] In the general formula (5), R 9 and R 11The side chain of the amino acid residue represented can also adopt any of the side chains of natural amino acid residues and synthetic amino acid residues, for example, 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 (Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine, N-methyl-β-alanine, sarcosine, γ-aminobutyric acid, kainic acid or side chains of natural amino acid residues such as derivatives thereof.

[0161] In the general formula (5), R 9 and R 11 The side chain of the amino acid residue represented can adopt the side chain of the amino acid residue with a protecting group according to the type of the protected functional group. When the above-mentioned amino acid residue is an amino acid residue with an amino group or a carboxyl group in the side chain, from the perspective of avoiding side reactions, the side chain of the amino acid residue with a protecting group is preferably used. On the other hand, when the above-mentioned amino acid residue is composed only of an amino acid residue with a functional group other than an amino group and a carboxyl group in the side chain, from the perspective of reducing the deprotection process, improving atom economy, and reducing waste, the side chain of the amino acid residue without a protecting group is preferably used. According to the method of the present invention, even if the side chain of the above-mentioned amino acid residue does not have a protecting group, the side reaction caused by the side chain is not easy to occur.

[0162] As the protecting group possessed by the above-mentioned amino acid residue, any known protecting group as a protecting group possessed by the side chain of the amino acid residue can be adopted, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB) and p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl and acetyl (Ac) and the like; phthaloyl (Phthyl) and the like; ) and other phthalimide-type protecting groups; carbamate-type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethanesulfonyl (SES), and the like.

[0163] In the general formula (5), R 12 The group represented is a hydrogen atom or a substituted or unsubstituted organic group.

[0164] In the general formula (5), R 12 The organic group represented is preferably a hydrocarbon group, and examples of the hydrocarbon group include a substituted or unsubstituted alkyl group and a substituted or unsubstituted aryl group.

[0165] In the general formula (5), R 12 The alkyl group represented is not particularly limited, and examples thereof include chain alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6) such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl, branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6) such as isopropyl, isobutyl, sec-butyl, and tert-butyl, and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8) such as cyclopentyl and cyclohexyl.

[0166] In the general formula (5), R 12 The aryl group represented is not particularly limited, and any of a monocyclic aryl group, a condensed ring aryl group and a polycyclic aryl group can be used. For example, phenyl, hydroxyphenyl, indolyl, imidazolyl, naphthyl, anthracenyl, phenanthrenyl, biphenyl, terphenyl, fluorenyl, pyrenyl, triphenylene and other aryl groups having 6 to 18 carbon atoms (especially 6 to 14).

[0167] In the general formula (5), R 12The represented hydrocarbon group may have a substituent. As the substituent, for example, a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the above-mentioned alkyl group, the above-mentioned aryl group, an alkoxy group, a carboxyl group, an amino group, etc. can be listed, without particular limitation. When having a substituent, the number of the substituent is not particularly limited, preferably 1 to 6, more preferably 1 to 3.

[0168] In the general formula (5), R 12 The substituent of the represented hydrocarbon group may have a protecting group. As the above-mentioned protecting group, any known protecting group as a protecting group of the above-mentioned substituent can be used, for example, alkyl type protecting groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protecting groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protecting groups such as p-methoxybenzyl (PMB), p-methoxyphenyl (PMP) and the like; amide type protecting groups such as formyl, acetyl (Ac) and the like; phthaloyl (Phth) and the like; Phthalimide type protecting groups; carbamate type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethylsulfonyl (SES), etc. The number of protecting groups when there is a protecting group is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

[0169] In addition, in the general formula (5), P 2 The group represented is a hydrogen atom, a carboxyl protecting group, a label or a solid phase carrier. From the perspective of avoiding side reactions, a carboxyl protecting group is preferred. On the other hand, from the perspective of reducing the deprotection process, improving atom economy and reducing waste, P 2 On the other hand, when n2 in the general formula (5) is relatively large, that is, when the peptide represented by the general formula (5) is a long-chain peptide, from the perspective of being applicable to the preparation method of the present invention, P 2 The represented group is preferably a tag or a solid support.

[0170] As the above P 2The protective group of the carboxyl group represented by the above-mentioned can be any protective group known as a protective group of the carboxyl group, for example, alkyl type protective groups such as tert-butyl (t-Bu), benzyl, allyl, methyl, trityl (Trt) and the like; silyl type protective groups such as tert-butyldimethylsilyl (TBS) and the like; aryl type protective groups such as p-methoxybenzyl (PMB) and p-methoxyphenyl (PMP) and the like; amide type protective groups such as formyl and acetyl (Ac) and the like; phthaloyl (Phth) and the like; Phthalimide type protecting groups; carbamate type protecting groups such as benzyloxycarbonyl (Cbz), tert-amyloxycarbonyl (Aoc), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), 2,2,2-triethoxycarbonyl (Troc); sulfonamide type protecting groups such as 3-nitro-2-pyridinesulfinyl (Npys), 2-nitrobenzenesulfonyl (Ns), (2-trimethylsilyl)-ethanesulfonyl (SES), etc.

[0171] As the above P 2 The represented tags can be tags for improving the solubility of peptides, for example, hydrophobic tags composed of aromatic rings substituted with multiple long-chain alkoxy groups, etc. Thus, the reaction established by the peptide solid phase synthesis method can be directly applied. Among them, "solubility" refers to the ease with which a molecule dissolves in a solvent.

[0172] As the above P 2 As the solid phase support shown, a solid phase support generally used in a solid phase synthesis method for a peptide can be used.

[0173] In the general formula (5), n2 is an integer greater than 0, and is usually an integer from 0 to 100. From the perspective of improving the yield and suppressing side reactions, it is preferably an integer from 0 to 50, more preferably an integer from 0 to 10, and even more preferably an integer from 0 to 2. When n2 is an integer greater than 2, the number of R 10 ~R 12 Same or different.

[0174] The amino acids constituting the second amino acid or the peptide represented by the general formula (5) may be either the L-type or the D-type optical isomers.

[0175] As the peptide represented by general formula (5), a two-residue extended peptide obtained by the preparation method of the present invention or a similar preparation method can also be used. By the above manner, i.e., by repeating the preparation method of the present invention or a similar preparation method, an oligopeptide or polypeptide of a desired length can be effectively obtained in a short time.

[0176] In summary, specific examples of the second amino acid or peptide represented by the general formula (5) include the following.

[0177] [Chemical formula 19]

[0178]

[0179] The amount of the second amino acid or peptide used is not particularly limited. From the perspectives of improving yield, inhibiting epimerization, and economic efficiency, for example, relative to 1 mol of the first amino acid, it is usually preferably about 0.1 to 10 mol, more preferably about 0.2 to 5 mol, and further preferably about 0.5 to 3 mol. When two or more second amino acids or peptides represented by the general formula (5) are used in combination, it is preferably adjusted so that the total amount is within the above range.

[0180] 6. Two-residue extended peptides

[0181] The di-residue extended peptide as the target product of the present invention is preferably a compound represented by the following general formula (6).

[0182] [Chemical formula 20]

[0183]

[0184] In formula (6), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , P 1 , P 2 , n1 and n2 and the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , P 1 , P 2 , n1 and n2 are the same.

[0185] Among the compounds obtained by the production method of the present invention or a similar production method, the compound represented by the following formula is a novel compound not described in the literature.

[0186] [Chemical formula 21]

[0187]

[0188]

[0189] 7. Other conditions

[0190] The solvent used in the reaction step of the present invention is not particularly limited. For example, any organic solvent that dissolves the substrate, water, or a mixed solvent thereof can be used. However, from the viewpoint of the solubility of the substrate, an aprotic polar solvent is preferably included.

[0191] In addition, the aprotic polar solvent used in the reaction process of the present invention is not particularly limited, and examples thereof include halogen solvents such as dichloromethane and chloroform; ketone solvents such as acetone and methyl ethyl ketone; ether solvents such as 1,4-dioxane, tetrahydrofuran, and diethyl ether; amide solvents such as N,N-dimethylformamide and N,N-diethylformamide, sulfoxide solvents such as dimethyl sulfoxide, nitrile solvents such as acetonitrile, and mixtures of these solvents. Among them, from the perspectives of substrate solubility, low reactivity, and ease of removal, halogen solvents and nitrile solvents are preferred, and dichloromethane and acetonitrile are more preferred. These solvents can be used alone or in combination of two or more.

[0192] In particular, as a solvent for dissolving a peptide that is difficult to dissolve only in an organic solvent, a mixed solvent of water and an aprotic polar solvent is preferably used, and a mixed solvent of water and acetonitrile, water and 1,4-dioxane, or water and tetrahydrofuran is more preferably used. In terms of volume ratio, the mixing ratio of the above mixed solvents is preferably about 10:90 to 90:10, more preferably about 75:25 to 25:75, and even more preferably 50:50.

[0193] In addition to the above-mentioned components, additives can be appropriately used in the present invention within a range that does not impair the effects of the present invention. Examples of the above-mentioned additives include hydroxybenzotriazole or its analogs, metal salts, and the like.

[0194] From the perspective of suppressing epimerization and shortening the reaction time, the reaction temperature of the reaction process of the present invention is preferably -80~100°C, more preferably 0~80°C, and further preferably 0~60°C. The reaction process of the present invention includes reaction processes (A), (A1), (A2) and (B) described later. The reaction time can be set to the time for performing the amide bond formation reaction twice. From the perspective of suppressing epimerization, the reaction time is preferably 0.01 seconds to 12 hours, more preferably 1 second to 6 hours, and further preferably 10 seconds to 1 hour. When the reaction process of the present invention is carried out by a microfluidic method, it is preferred that the reaction time can be set to less than 10 minutes, more preferably less than 5 minutes, and further preferably less than 1 minute.

[0195] 8. Preparation of double residue extended peptides

[0196] The di-residue extended peptide as the target product of the present invention can be prepared by reacting an amino acid or peptide represented by the general formula (1), a halogenating agent, a tertiary amine represented by the general formula (3), an amino acid N-carboxylic anhydride (NCA) represented by the general formula (4), and an amino acid or peptide represented by the general formula (5). The reaction is not particularly limited, and all components can be added simultaneously or sequentially.

[0197] In the production method of the present invention, the first amino acid or peptide represented by the general formula (1), the halogenating agent and the tertiary amine represented by the general formula (3) are reacted to produce an acid halide represented by the following general formula (7).

[0198] [Chemical formula 22]

[0199]

[0200] In formula (7), R 1 , R 2 , R 3 , R 4 , R 5 , P 1 , n1 and X and the R 1 , R 2 , R 3 , R 4 , R 5 , P 1 , n1 and X are the same.

[0201] Since the acid halide is unstable, it is preferred to precisely control the reaction temperature and reaction time of the reaction from the viewpoint of suppressing side reactions such as epimerization and autopolymerization.

[0202] In the production method of the present invention, acylated NCA represented by the following general formula (8) can be produced by reacting with the acid halide represented by the general formula (7) and the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4).

[0203] [Chemical formula 23]

[0204]

[0205] In formula (8), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , P 1 and n1 and R1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , P 1 The same as n1.

[0206] Since the acylated NCA is unstable, it is preferred to precisely control the reaction temperature and reaction time of the reaction from the viewpoint of suppressing side reactions such as epimerization and autopolymerization.

[0207] In the past, it was believed that acylated NCA from conventional amino acid N-carboxylic anhydride (NCA) rather than from dehydrogenated amino acid N-carboxylic anhydride (dehydro-NCA) was unstable and therefore could not be synthesized (Organic Synthetic Chemistry, 1989, Vol. 47, No. 9, 782-794). In contrast, the preparation method of the present invention allows the synthesis of acylated NCA from conventional NCA and its use in peptide synthesis. In addition, the preparation method of the present invention allows the separation of the acylated NCA that may be generated as an intermediate. In the preparation method of the present invention, the above-mentioned double-residue extended peptide can be generated by reacting the acylated NCA represented by the above-mentioned general formula (8) and the second amino acid or peptide represented by the above-mentioned general formula (5).

[0208] Therefore, in the production method of the present invention, since highly reactive reaction intermediates can be generated sequentially in a short time, a di-residue extended peptide can be easily and efficiently obtained, and epimerization of each amino acid residue can be easily suppressed.

[0209] Therefore, since a highly reactive reaction intermediate can be generated more efficiently, the preparation method of the present invention preferably adopts:

[0210] (A) a reaction step of reacting the first amino acid or peptide represented by the general formula (1), the halogenating agent, the tertiary amine represented by the general formula (3), and the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4); and

[0211] (B) a reaction step of reacting the reaction mixture obtained in the step (A) with the second amino acid or peptide represented by the general formula (5),

[0212] Furthermore, the step (A) preferably comprises:

[0213] (A1) a reaction step of reacting a first amino acid or peptide represented by the general formula (1), a halogenating agent, and a tertiary amine represented by the general formula (3); and

[0214] (A2) A reaction step of reacting the reaction mixture obtained in the step (A1) with the second amino acid N-carboxylic anhydride (NCA) represented by the general formula (4).

[0215] The reaction process of the present invention can be carried out by any of a batch method and a flow method. From the perspective of high safety or ease of scale-up, the flow method is more preferred, and from the perspective of being able to precisely control the reaction temperature or reaction time, the microfluidic method is further preferred.

[0216] In addition, in this specification, the batch method refers to a batch operation, specifically, a series of operations of adding the substances involved in the reaction into a reaction container or a reaction device at one time to react, and taking them out after reaching equilibrium or a certain reaction rate.

[0217] In this specification, the flow method refers to a continuous operation, specifically, an operation in which a raw material and a substance required for processing the raw material are continuously fed into a device at a certain ratio (flow rate) and discharged.

[0218] In this specification, the microfluidic method refers to a flow method (continuous operation) using a microfluidic reactor.

[0219] The reaction process of the present invention can be implemented using a microfluidic reactor. The microfluidic reactor includes, for example: a pipeline for conveying a fluid containing raw materials or intermediates for reaction; a pump for conveying the fluid into the pipeline; and a mixer for mixing the fluid.

[0220] Below, refer to Figure 1 The morphology of the above-mentioned microfluidic reactor and the method for preparing the di-residue extended peptide of the present invention using the microfluidic reactor are described. Figure 1 The implementation limitations are obvious.

[0221] Figure 1 Schematic diagram showing a schematic configuration of the microfluidic reactor 1. The microfluidic reactor 1 may include a syringe pump 11 containing a first liquid, a syringe pump 12 containing a second liquid, a syringe pump 13 containing a third liquid, a syringe pump 14 containing a fourth liquid, and a syringe pump 15 containing a fifth liquid.

[0222] As an example, the first liquid can contain a first amino acid or peptide and a tertiary amine, the second liquid can contain a halogenating agent, the third liquid can contain amino acid N-carboxylic anhydride (NCA), the fourth liquid can contain a tertiary amine, and the fifth liquid can contain a second amino acid or peptide. As another example, in one of the above examples, the first liquid can contain a first amino acid or peptide, and the second liquid can contain a halogenating agent and a tertiary amine, but the present invention is not limited to any one embodiment.

[0223] The microfluidic reactor 1 can be equipped with pipes F1~F9 for conveying fluids. The inner diameter of the pipe can be, for example, 0.1~50mm, or 0.3~1mm. The microfluidic reactor 1 can be equipped with mixers M1~M4 for mixing fluids. There is no particular limitation on the mixer, and for example, a V-type mixer, a T-type mixer, etc. that achieve fluid mixing by allowing the fluid to flow in can also be used. In such a mixer, the interfacial area (interfacial area) of the two liquids is increased by the entrained flow (entrainment flow) generated in the pipe, and it is easy to mix the reaction liquid of more than two liquids within a few milliseconds. The inner diameter of the pipe inside the mixer can be, for example, 0.1~50mm, or 0.2~1mm.

[0224] The inner diameter of the pipe can be set to the diameter of the inner portion of the pipe (the portion through which the fluid passes) in the cross section of the pipe in a direction that intersects the longitudinal direction of the pipe at right angles. When the shape of the inner portion of the pipe is not a perfect circle, the inner diameter of the pipe can be set to the diameter when the shape of the inner portion of the pipe is converted into a perfect circle based on the area standard.

[0225] As an example, the syringe pumps 11 to 15 and the pipes F1 to F9 can be formed using resin such as plastic or elastomer, glass material, metal, ceramic, or the like.

[0226] By starting the syringe pump 11, the first liquid can move in the pipeline F1 and flow into the mixer M1. By starting the syringe pump 12, the second liquid can move in the pipeline F2 and flow into the mixer M1. In addition, the first liquid and the second liquid are mixed by the mixer M1 to form a first mixed liquid, which can be transported to the pipeline F6. On the other hand, by starting the syringe pump 13, the third liquid moves in the pipeline F3, flows into the mixer M2, mixes with the first mixed liquid, forms a second mixed liquid, and can be transported to the pipeline F7. Similarly, by starting the syringe pump 14, the fourth liquid moves in the pipeline F4, flows into the mixer M3, mixes with the second mixed liquid, forms a third mixed liquid, and can be transported to the pipeline F8. By starting the syringe pump 15, the fifth liquid moves in the pipeline F5, flows into the mixer M4, mixes with the third mixed liquid, forms a fourth mixed liquid, and can be transported to the pipeline F9. In addition to being stored in the test tube 21, the fourth mixed liquid can also be used for separation, purification processes and online analysis.

[0227] By using the above-mentioned microfluidic reactor 1, it is easy to increase the area of ​​heat exchange of the reaction solution per unit volume. In addition, it is easy to control the reaction time by the flow rate or the length of the pipeline. Therefore, it is easy to strictly control the reaction solution, and as a result, it is easy to minimize the conduction of unwanted side reactions and easily improve the yield of the target product.

[0228] Therefore, the method for producing a two-residue extended peptide of the present invention can be carried out by a liquid phase method and can therefore be easily scaled up.

[0229] For the use of a microfluidic reactor, when synthesizing an acyl halide represented by the general formula (7), for example, the first liquid may contain a first amino acid or peptide represented by the general formula (1) and a tertiary amine represented by the general formula (3), and the second liquid may contain a halogenating agent, or the first liquid may contain a first amino acid or peptide represented by the general formula (1), and the second liquid may contain a halogenating agent and a tertiary amine represented by the general formula (3). From the perspective of suppressing side reactions, it is preferred that the first liquid contain a first amino acid or peptide represented by the general formula (1) and a tertiary amine represented by the general formula (3), and the second liquid contain a halogenating agent.

[0230] In the use of the microfluidic reactor, when synthesizing the acylated NCA represented by the general formula (8), the first mixed liquid contains the acyl halide represented by the general formula (7), the third liquid contains the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4), and, for example, in addition to the first liquid or the second liquid, the fourth liquid may contain the same tertiary amine represented by the general formula (3) as the first liquid or the second liquid, or the fourth liquid may contain a tertiary amine represented by the general formula (3) different from the first liquid or the second liquid. From the perspective of effectively activating the acyl halide represented by the general formula (7) and the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4), it is preferred that the fourth liquid contains a second tertiary amine represented by the general formula (3) different from the first tertiary amine contained in the first liquid or the second liquid.

[0231] Among them, the acid halide represented by the general formula (7) may be one synthesized by a method other than the microfluidic method, but from the viewpoint of improving the reaction efficiency and suppressing the side reaction, the acid halide synthesized by the microfluidic method is preferred.

[0232] Among them, the amino acid N-carboxylic anhydride (NCA) represented by general formula (4) can also use amino acid N-carboxylic anhydride (NCA) synthesized by methods other than microfluidic method, but from the perspective of improving reaction efficiency and inhibiting side reactions, amino acid N-carboxylic anhydride (NCA) synthesized by microfluidic method is preferred. The inventors of this application also reported the microfluidic synthesis of α-amino acid N-carboxylic anhydride and β-amino acid N-carboxylic anhydride (α-NCA and β-NCA) (for example, referring to Angew. Chem. Int. Ed., 2018, 57(35), 11389-11393 and Chem. Asian J., 2020, 15(1), 79-84).

[0233] In addition, the method for preparing the di-residue extended peptide of the present invention is not limited to the use of a microfluidic reactor, and for example, a batch processing container with a small volume and a high stirring speed can also be used. The volume of the mixing portion of the batch processing container can be 1 to 100 mL, or 5 to 50 mL.

[0234] After the reaction using the microfluidic reactor is completed, the di-residue extended peptide as the target compound can be obtained through a common separation and purification process as necessary.

[0235] Furthermore, in conventional peptide synthesis, complicated operations are required in the separation and purification steps. However, according to the present invention, the impurities generated after the reaction are small, and an easily removable reactant is used, so that a di-residue extended peptide can be easily obtained at a high yield. Therefore, these steps can be omitted or simplified, and crystallization or analysis can be directly performed.

[0236] Example

[0237] Hereinafter, examples and comparative examples will be described to further clarify the features of the present invention, but the present invention is not limited to the following examples.

[0238] In order to simplify the description of the specification, the following abbreviations are sometimes used in the reference examples, examples and tables in the examples. In the substituents, the abbreviations have the following meanings:

[0239] Cbz: benzyloxycarbonyl, Boc: tert-butoxycarbonyl, Fmoc: 9-fluorenylmethyloxycarbonyl, NMI: N-methylimidazole, NMM: N-methylmorpholine, Me2NBn: dimethylbenzylamine, Et3N: triethylamine, i-Pr2NEt: N,N-diisopropylethylamine, DMAP: 4-dimethylaminopyridine, Bn: benzyl, Me: methyl, t-Bu: tert-butyl, Phe: phenylalanine, Ala: alanine, β-Ala: β-alanine, Val: valine, Tyr: tyrosine, Asp: aspartic acid, Ser: serine, Cys: cysteine, Trp: tryptophan, Lys: lysine, Thr: threonine.

[0240] [Example 1]

[0241] Study on the scope of tertiary amines

[0242] Using the various tertiary amines shown in Table 1, acylated NCA was synthesized by the following method (Table 1). In addition, since acylated NCA is unstable, it is derived into a stable amide, and the yield of the acylated NCA is indirectly evaluated based on the yield of the amide. In addition, in order to simplify the study of tertiary amines, stable pivaloyl chloride was used instead of unstable amino acid chloride, but from the perspective of side reactions such as self-polymerization or dimerization, it is obvious that it is not easy to convert pivaloyl chloride into amino acid chloride.

[0243] Using a syringe pump, a dichloromethane solution (CH2Cl2; flow rate: 1.20 mL / min) of an amino acid N-carboxylic anhydride represented by L-Phe-NCA (compound 3a; 0.300 M, 1.00 molar equivalent) and pivaloyl chloride (0.300 M, 1.00 molar equivalent) and a dichloromethane solution (flow rate: 2.00 mL / min) of a tertiary amine (0.180+0.180 M, 1.00+1.00 molar equivalent) were introduced into a T-type mixer at 20° C. The resulting mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, length: 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. Next, using a syringe pump, the obtained mixture and a dichloromethane solution of isopropylamine (3.00 M, 10.0 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C. At the same temperature, the obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 1459 mm, volume: 733 μL, reaction time: 10 seconds). After reaching a steady state after 60 seconds, the reacted mixture was poured into 1 M hydrochloric acid (1.5 mL) and dichloromethane (6 mL) at room temperature for 25 seconds. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The results were analyzed by HPLC-UV using a calibration curve (chromatographic column: COSMOSIL 5C 18 -AR-II 4.6mmI.D.×150mm, solvent: acetonitrile+0.1% formic acid / H2O+0.1% formic acid (0-20 minutes: 30~100%, 20-25 minutes: 100%, 25-27 minutes: 30%, 27-30 minutes: 30%), flow rate: 1.0mL / min, detection wavelength: 254nm, temperature: 40°C, retention time: 8.7 minutes), determine the yield of (S)-N-isopropyl-3-phenyl-2-pivalamidopropionamide (compound S1) obtained.

[0244] Since the desired compound S1 can be obtained by using a variety of tertiary amines, it can be understood that the desired acylated NCA is generated (Examples 1-1 to 1-7). Among them, when two tertiary amines are combined, compound S1 is obtained in high yield (Examples 1-4 to 1-7). In particular, when N,N-diisopropylethylamine and N-methylimidazole are used as tertiary amines, compound S1 can be obtained quantitatively (>99%), and purification by silica gel column chromatography is not required, and the reaction results can be quickly analyzed (Example 1-7).

[0245] [Table 1]

[0246]

[0247] [Chemical formula 24]

[0248]

[0249] (S)-N-isopropyl-3-phenyl-2-pivaloylaminopropionamide (Compound S1):

[0250] White solid, melting point 168-171℃, IR (neat): 3285, 2969, 1633, 1556, 1216 cm -1 , [α] 23 D = -17.3 (c 2.05, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.30-7.20(m, 5H), 6.45(d, J=6.4Hz, 1H), 5.82(d, J=7.2Hz, 1H), 4.57(ddd, J=6.4, 6.4, 6.4Hz, 1H), 4.00-3.91(m, 1H), 3.10(dd, J=6.4, 13.2Hz, 1H), 2.97(dd, J=6.4, 13.2Hz, 1H), 1.15(s,9H), 1.04(d, J=6.4Hz, 3H), 0.98(d, J=6.4Hz, 3H) ppm; 13 C NMR (100 MHz, CDCl3): δ 178.4, 170.1, 137.0, 129.5, 128.6, 127.0, 54.5, 41.5, 38.9, 38.8, 27.5, 22.6, 22.5 ppm; HRMS (ESI): calcd for (theoretical value) [C 17 H 26 N2O2+H] + 291.2067, found (experimental value) 291.2066 and [C 17 H 26 N2O2+Na] + 313.1886, found 313.1885.

[0251] [Example 2]

[0252] Study on the dosage of tertiary amine and halogenating agent and reaction time

[0253] Next, the amino acid chlorides were synthesized by the following method (Table 2). In addition, since the amino acid chlorides were unstable, they were derived into stable amides, and the yield of the amino acid chlorides was indirectly evaluated based on the yield of the amides. The effects of the amount of tertiary amine and halogenating agent used and the reaction time on the yield of the target product were evaluated.

[0254] Using a syringe pump, a solution of an N-terminal protected amino acid represented by Fmoc-L-Phe-OH (compound 1a; 0.300 M, 1.00 molar equivalent) and a tertiary amine (X molar equivalent) (flow rate: 1.20 mL / min), and a solution of thionyl chloride (SOCl2; X molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C. At the same temperature, the obtained mixture was introduced into reaction tube 1 (inner diameter: 0.800 mm, reaction time: Y seconds). Next, using a syringe pump, the obtained mixture and a solution of isopropylamine (3.00 M, 10.0 molar equivalent) were introduced into a T-type mixer at 20°C (flow rate: 1.20 mL / min). At the same temperature, the obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 1459 mm, volume: 733 μL, reaction time: 10 seconds). After reaching a steady state after 60 seconds, the reaction mixture was poured into 1M hydrochloric acid (1.5 mL) and dichloromethane (6 mL) at room temperature for 25 seconds, the aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The HPLC-UV analysis using a calibration curve (chromatographic column: COSMOSIL 5C 18 -AR-II 4.6mmI.D.×150mm, solvent: acetonitrile+0.1% formic acid / H2O+0.1% formic acid (0-20 minutes: 30~100%, 20-25 minutes: 100%, 25-27 minutes: 30%, 27-30 minutes: 30%), flow rate: 1.0mL / min, detection wavelength: 254nm, temperature: 40℃, retention time: 14.3 minutes), determine the yield of (9H-fluoren-9-yl)methyl (S)-(1-(isopropylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (compound S2).

[0255] The desired compound S2 can be obtained by the conditions of Examples 2-1 to 2-8, and it can be understood that the desired amino acid chloride is generated. Among them, when 1.2 molar equivalents of thionyl chloride are used and the reaction time is set to 10 seconds, compound S2 (Example 2-5) is obtained with an extremely high separation yield. Therefore, the conditions of Example 2-5 are used in the subsequent examples.

[0256] [Table 2]

[0257]

[0258] [Chemical formula 25]

[0259]

[0260] (9H-fluoren-9-yl)methyl (S)-(1-(isopropylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (Compound S2):

[0261] Chromatographic conditions: CH2Cl2:MeOH=98:2~80:20, 63.3mg, 0.15mmol, 98%.

[0262] White solid, melting point 188-191℃, IR (neat): 3295, 1697, 1651, 1541, 1261, 757,740 cm -1 ; [α] 24 D = -0.95 (c 2.05, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.6Hz, 2H), 7.56(dd, J=7.6Hz, 2H), 7.40(dd, J=7.6, 7.6Hz, 2H), 7.33-7.21(m, 7H), 5.05(d, J=6.0Hz, 1H), 5.25(brs, 1H), 4.45-4.28(m, 3H), 4.20(t, J=6.8Hz, 1H), 4.00-3.91(m, 1H), 3.16-3.14(m, 1H), 2.97-2.92(m, 1H), 1.03(d, J=6.4Hz, 3H),0.92(d, J=6.4Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 169.7, 156.0, 143.9, 141.4,136.8, 129.5, 128.8, 127.9, 127.21, 127.17, 125.1, 120.1, 67.1, 56.7, 47.3,41.6, 39.4, 22.6, 22.5 ppm; HRMS (ESI): calcd for [C 27 H 28 N2O3+H] + 429.2173, found429.2171 and [C 27 H 28N2O3+Na] + 451.1992, found 451.1992.

[0263] [Example 3]

[0264] Synthesis and separation of intermediates

[0265] Next, based on the results of Examples 1 and 2, acylated NCA was synthesized and isolated starting from the first amino acid by the following method.

[0266] Using a syringe pump, N-terminal protected amino acid represented by Fmoc-L-Phe-OH (compound 1a; 0.300M, 1.00 molar equivalent) and dichloromethane solution of N,N-diisopropylethylamine (0.360M, 1.2 molar equivalent) (flow rate: 1.20 mL / min), and dichloromethane solution of thionyl chloride (0.216M, 1.20 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20° C. The obtained mixture was introduced into reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. Next, the obtained mixture and a dichloromethane solution of an amino acid N-carboxylic anhydride represented by L-Phe-NCA (compound 3a; 0.300 M, 1.00 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.216 M, 1.2 molar equivalent) and N-methylimidazole (0.180 M, 1.00 molar equivalent) (flow rate: 2.00 mL / min) were injected into the T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into a reaction tube 3 (inner diameter: 0.800 mm, length: 2122 mm, volume: 1067 μL, reaction time: 10 seconds). After reaching a steady state after 90 seconds, the reacted mixture was poured into 1M hydrochloric acid (1.5 mL) and dichloromethane (8 mL) at room temperature for 60 seconds. The aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The residue was purified by recrystallization using dichloromethane and hexane.

[0267] The yield of acylated NCA (compound 4a) represented by Fmoc-L-Phe-L-Phe-NCA was 189.6 mg (0.34 mmol), with a yield of 94%. Acylated NCA, which was previously thought to be impossible to synthesize, was successfully obtained with high yield and high purity.

[0268] [Chemical formula 26]

[0269]

[0270] Fmoc-L-Phe-L-Phe-NCA (Compound 4a)

[0271] Colorless amorphous solid, 1 H NMR (400MHz, CDCl3): δ 7.77-7.75(m, 2H), 7.54(d, J=7.2Hz, 2H), 7.42-7.38(m, 2H), 7.31-7.25(m, 8H), 7.21(d, J=7.2Hz, 2H), 7.09-7.08(m, 2H), 5.52(ddd, J=5.2, 8.0Hz, 1H), 5.30(d, J=8.0Hz, 1H), 4.97(d, J=2.8Hz, 1H), 4.40(d, J=6.8Hz, 2H), 4.21(t, J=6.8Hz, 1H), 3.54(dd, J=5.2,14.0Hz, 1H), 3.27(d, J=2.8, 14.0Hz, 1H), 3.20(dd, J=5.2, 14.0Hz, 1H), 2.86(dd, J=8.0, 14.0Hz, 1H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.8, 165.1, 155.7,147.5, 143.8, 141.4, 135.1, 131.9, 129.8, 129.4, 129.3, 128.9, 128.4, 127.9,127.6, 127.2, 125.1, 120.1, 67.3, 60.9, 55.9, 47.2, 37.6, 34.9 ppm; HRMS(ESI): calcd for [C 34 H 28 N2O6+Na] + 583.1840, found 583.1874.

[0272] [Example 4]

[0273] Study on the dosage of amino acids and reaction time

[0274] Next, a tripeptide (Compound 6a) (Table 3) was synthesized by the following method. Specifically, the following method refers to a method of reacting the acylated NCA (Compound 4a) of Example 3 with a second amino acid. The effects of the amount of the second amino acid used and the reaction time on the yield of the target product were evaluated.

[0275] Using a syringe pump, N-terminal protected amino acid represented by Fmoc-L-Phe-OH (compound 1a; 0.300M, 1.00 molar equivalent) and dichloromethane solution of N,N-diisopropylethylamine (0.360M, 1.2 molar equivalent) (flow rate: 1.20 mL / min), and dichloromethane solution of thionyl chloride (0.216M, 1.20 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20° C. The obtained mixture was introduced into reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. Next, the obtained mixture and a dichloromethane solution of an amino acid N-carboxylic anhydride represented by L-Phe-NCA (compound 3a; 0.300 M, 1.00 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.216 M, 1.2 molar equivalent) and N-methylimidazole (0.180 M, 1.00 molar equivalent) (flow rate: 2.00 mL / min) were injected into the T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into reaction tube 3 (inner diameter: 0.800 mm, length: 2122 mm, volume: 1067 μL, reaction time: 10 seconds). Using a syringe pump, the obtained mixture and a dichloromethane solution of a C-terminal protected amino acid represented by HL-Ala-Ot-Bu (compound 5a; X molar equivalent) (flow rate: 1.20 mL / min) were injected into a T-type mixer at 20°C. At the same temperature, the obtained mixture was introduced into reaction tube 4 (inner diameter: 0.800 mm, reaction time: Y seconds). After reaching a steady state after about 60 seconds, the reacted mixture was poured into 1 M hydrochloric acid (1.5 mL) and dichloromethane (8 mL) at room temperature for 25 seconds. The aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated brine, dried with magnesium sulfate, filtered, and vacuum concentrated at room temperature. The results were analyzed by HPLC-UV using a calibration curve (chromatographic column: COSMOSIL5C18 -AR-II 4.6mmI.D.×150mm, solvent: acetonitrile + 0.1% formic acid / H2O + 0.1% formic acid (0-20 minutes: 30~100%, 20-25 minutes: 100%, 25-27 minutes: 30%, 27-30 minutes: 30%), flow rate: 1.0mL / min, detection wavelength: 254nm, temperature: 40°C, retention time: 17.3 minutes), determine the yield of the tripeptide represented by Fmoc-L-Phe-L-Phe-L-Ala-Ot-Bu (compound 6a).

[0276] All conditions tried can obtain the desired compound 6a (Examples 4-1 to 3-4). Among them, when 1.2 molar equivalents of C-terminal protected amino acid was used and the reaction time was set to 10 seconds, since compound 6a can be obtained in a sufficiently high yield, from the perspective of suppressing epimerization and shortening the reaction time, the conditions of Example 4-2 were used in the subsequent examples.

[0277] [Table 3]

[0278]

[0279] [Chemical formula 27]

[0280]

[0281] Fmoc-L-Phe-L-Phe-L-Ala-Ot-Bu (Compound 6a):

[0282] Purification method: CH2Cl2 / hexane recrystallization

[0283] White solid, melting point 162-164℃, IR(neat): 3286, 1732, 1704, 1644, 1539, 1451,1254, 1146, 741 cm -1 ; [α] 25 D = -21.7 (c 0.88, CHCl3); 1H NMR (400MHz, CDCl3): δ 7.75(d,J=7.6Hz, 2H), 7.50(dd, J=7.6, 7.6Hz, 2H), 7.39(dd, J=7.6, 7.6Hz, 2H), 7.31-7.07(m, 12H), 6.67(brs, 1H), 6.54(brs, 1H), 5.44(d, J=6.0Hz, 1H), 4.68(d, J=6.4Hz, 1H), 4.46(brs, 1H), 4.42-4.38(m, 1H), 4.34-4.31(m, 1H), 4.24(brs, 1H),4.14(t, J=6.8Hz, 1H), 3.01-2.99(m, 4H), 1.43(s, 9H), 1.28(d, J=6.0Hz, 3H)ppm; 13 C NMR (100MHz, CDCl3): δ 171.6, 170.8, 169.8, 156.1, 143.8, 141.4, 136.3,129.4, 128.8, 128.6, 127.9, 127.2, 127.1, 125.2, 125.1, 120.1, 82.1, 67.2,56.2, 54.4, 48.9, 47.2, 38.5, 38.4, 28.0, 18.6 ppm; HRMS(ESI): calcd for[C 40 H 43 N3O6+Na] + 684.3044, found 684.3041.

[0284] [Example 5]

[0285] Synthesis of tripeptide 6a by batch method

[0286] Next, in order to evaluate the influence of the synthesis method, tripeptide 6a (Table 4) was synthesized by a batch method. In the batch method, the reaction was carried out under the same conditions as the flow method with respect to the amount of compound and solvent used and the temperature.

[0287] A dichloromethane solution (0.50 mL) of an N-terminal protected amino acid represented by Fmoc-L-Phe-OH (compound 1a; 0.300 M, 1.00 molar equivalent) and N,N-diisopropylethylamine (0.360 M, 1.2 molar equivalent) was vigorously stirred (magnetic stirrer, 1000 rpm). A dichloromethane solution (0.83 mL) of thionyl chloride (0.216 M, 1.20 molar equivalent) was added thereto in one portion at 20°C under an argon atmosphere. After stirring at the same temperature for 10 seconds, a dichloromethane solution (0.50 mL) of an amino acid N-carboxylic anhydride represented by L-Phe-NCA (compound 3a; 0.300 M, 1.0 molar equivalent) was added in one portion at 20°C under an argon atmosphere. After stirring at the same temperature for 10 seconds, a dichloromethane solution (0.83 mL) of N,N-diisopropylethylamine (0.216 M, 1.2 molar equivalents) and N-methylimidazole (0.180 M, 1.0 molar equivalents) was added all at once at 20°C under an argon atmosphere. After stirring at the same temperature for 10 seconds, a dichloromethane solution (0.50 mL) of a C-terminal protected amino acid represented by HL-Ala-Ot-Bu (compound 5a; 0.360 M, 1.20 molar equivalents) was added all at once at 20°C under an argon atmosphere. The reaction mixture was washed with 1 M hydrochloric acid and saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The reaction mixture was analyzed by HPLC-UV using a calibration curve (chromatographic column: COSMOSIL 5C 18 -AR-II 4.6mmI.D.×150mm, solvent: acetonitrile + 0.1% formic acid / H2O + 0.1% formic acid (0-20 minutes: 30~100%, 20-25 minutes: 100%, 25-27 minutes: 30%, 27-30 minutes: 30%), flow rate: 1.0mL / min, detection wavelength: 254nm, temperature: 40°C, retention time: 17.3 minutes), the yield of the tripeptide (compound 6a) represented by the obtained Fmoc-L-Phe-L-Phe-L-Ala-Ot-Bu was determined, and the reaction results can be quickly analyzed without silica gel column chromatography. Although the yield is lower than that of the microflow method, the desired tripeptide (compound 6a) can be obtained with high reproducibility by the batch method (Table 4).

[0288] [Table 4]

[0289]

[0290] [Example 6]

[0291] Study on the substrate range of tripeptide 6

[0292] Next, in order to evaluate the substrate range of the tripeptide, tripeptides were synthesized using amino acids having various amino acid residues (Table 5).

[0293] Using a syringe pump, a dichloromethane solution of an N-terminal protected amino acid (compound 1; 0.300 M, 1.00 molar equivalent) and N,N-diisopropylethylamine (0.360 M, 1.2 molar equivalent) (flow rate: 1.20 mL / min) and a dichloromethane solution of thionyl chloride (0.216 M, 1.20 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C. The resulting mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. Using a syringe pump, the resulting mixture and a dichloromethane solution of an amino acid N-carboxylic anhydride (compound 3; 0.300 M, 1.00 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C. At the same temperature, the obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds). Using a syringe pump, the obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.216 M, 1.2 molar equivalents) and N-methylimidazole (0.180 M, 1.00 molar equivalents) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C. At the same temperature, the obtained mixture was introduced into reaction tube 3 (inner diameter: 0.800 mm, reaction time: 10 seconds or 15 seconds). Using a syringe pump, the obtained mixture and a dichloromethane solution of a C-terminal protected amino acid (compound 5; 0.360 M, 1.20 molar equivalents) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C. At the same temperature, the obtained mixture is introduced into a reaction tube 4 (inner diameter: 0.800mm, length: 2519mm, volume: 1267μL, reaction time: 10 seconds). Then, as needed, batch stirring is performed at room temperature for 1 minute (compounds 6h, 6j, 6n and 6p). After reaching a steady state after 90 seconds, the reacted mixture is poured into 1M hydrochloric acid (1.5mL) and dichloromethane (8mL) at room temperature for 25 seconds. The water layer is extracted with dichloromethane, the organic layer is washed with saturated sodium bicarbonate water and saturated saline, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The residue is purified by recrystallization. In addition, when using compound 5 which requires a longer reaction time, the obtained mixture is poured into a dichloromethane solution of MePhe-OMe (1.50 molar equivalents) at room temperature for 25 seconds. Then, it is stirred for 3 hours at the same temperature (compound 6q). 1M hydrochloric acid was added thereto, the aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The residue was purified by silica gel column chromatography. Purification conditions, yields, and yields are shown in Table 5.

[0294] [Chemical formula 28]

[0295]

[0296] [Table 5]

[0297]

[0298] [Chemical formula 29]

[0299]

[0300] Cbz-L-Phe-L-Phe-L-Ala-Ot-Bu (Compound 6b):

[0301] White solid, melting point 112-115℃, IR(neat): 3290, 1734, 1703, 1644, 1538, 1148cm -1 ; [α] 26 D = -17.7 (c 1.41, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.36-7.07(m, 15H), 6.55(d, J=7.2Hz, 1H), 6.50(d, J=5.2Hz, 1H), 5.35(d, J=6.4Hz, 1H), 5.06(d, J=12.4Hz, 1H), 5.01(d, J=12.4Hz, 1H), 4.66(ddd, J=7.2, 7.2, 7.2Hz, 1H), 4.44-4.42(m, 1H), 4.35-4.28(m, 1H), 3.06-2.94(m, 4H), 1.44(s, 9H), 1.28(d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.6, 170.9, 170.0, 156.1, 136.4,136.3, 129.4, 128.7, 128.6, 128.2, 128.1, 127.1, 127.0, 82.1, 67.1, 56.2,54.3, 48.9, 38.6, 38.5, 28.0, 18.5 ppm; HRMS(ESI): calcd for [C 33 H 39 N3O6+Na] + 596.2731, found 596.2732.

[0302] [Chemical formula 30]

[0303]

[0304] Cbz-β-Ala-L-Phe-L-Ala-Ot-Bu (compound 6c):

[0305] White solid, melting point 97-99℃, IR(neat): 3279, 1726, 1641, 1550, 1455, 1369,1251, 1149 cm -1 ; [α] 26 D = -0.77 (c 1.25, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.32-7.09(m,10H), 6.71(d, J=6.8Hz, 1H), 6.57(d, J=7.2Hz, 1H), 5.56(brs, 1H), 5.10-5.03(m,2H), 4.72(ddd, , 2.40-2.35(m, 2H),1.43(s, 9H), 1.29 (d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.8, 171.4,170.4, 156.6, 136.6, 136.4, 129.4, 128.7, 128.6, 128.1, 127.1, 82.2, 66.7,54.4, 48.9, 38.6, 37.2, 36.1, 28.0, 18.5 ppm; HRMS(ESI): calcd for [C 27 H 35 N3O6+Na] + 520.2418, found 520.2419.

[0306] [Chemical formula 31]

[0307]

[0308] Cbz-L-Ala-L-Phe-L-Ala-Ot-Bu (Compound 6d: Angew. Chem. Int. Ed. 2006, 45, 1248-1252.):

[0309] White solid, melting point 129-133℃, IR (neat): 3310, 3272, 1733, 1698, 1645, 1539,1239, 1150 cm -1 ; [α] 26 D = -31.0 (c 1.02, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.38-7.30(m,5H), 7.29-7.09(m, 5H), 6.82(d, J=6.8Hz, 1H), 6.67(d, J=5.6Hz, 1H), 5.46(d, J=6.8Hz, 1H), 5.11(d, J=12.0Hz, 1H), 5.05(d, J=12.0Hz, 1H), 4.73(ddd, J=6.8,6.8, 6.8Hz, 1H), 4.38-4.31(m, 1H), 4.27-4.24(m, 1H), 3.06(d, J=6.8Hz, 2H),1.44(s, 9H), 1.30(d, J=6.8Hz, 3H), 1.30(d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 172.2, 171.7, 170.0, 156.1, 136.4, 136.3, 129.5, 128.7, 128.4, 128.2, 127.1, 82.1, 67.2, 54.3, 50.8, 48.9, 38.5, 28.0, 18.7, 18.5 ppm; HRMS(ESI): calcd for [C 27 H 35 N3O6+Na] + 520.2418, found 520.2419.

[0310] [Chemical formula 32]

[0311]

[0312] Fmoc-L-Val-L-Phe-L-Ala-Ot-Bu (Compound 6e):

[0313] White solid, melting point 192-196℃, IR (neat): 3289, 1734, 1696, 1644, 1541, 1247, 1147 cm -1 ; [α] 24 D = -26.2 (c 1.41, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.6Hz,2H), 7.59(d, J=7.6Hz, 2H), 7.41-7.38(m, 2H), 7.33-7.26(m, 2H), 7.23-7.05(m,5H), 6.64(d, J=7.2Hz, 1H), 6.55(d, J=6.8Hz, 1H), 5.43(d, J=8.0Hz, 1H), 4.74-4.70(m, 1H), 4.45(dd, J=6.8Hz, 10.0Hz, 1H), 4.35-4.31(m, 2H), 4.20(t, J=6.8Hz, 1H), 4.04-4.01(m, 1H), 3.07-3.01(m, 2H), 2.10-2.07(m, 1H), 1.43(s,9H), 1.29(d, J=6.4Hz, 3H), 0.91(d, J=6.4Hz, 3H), 0.84(d, J=6.4Hz, 3H) ppm; 13 CNMR(100MHz, CDCl3): δ 171.6, 171.2, 170.0, 156.5, 144.0, 141.5, 136.4, 129.4,128.7, 127.9, 127.2, 125.2, 120.1, 82.1, 67.2, 60.5, 54.3, 48.9, 47.3, 38.5,31.1, 28.1, 19.3, 18.6, 17.8 ppm; HRMS(ESI): calcd for [C 36 H 43 N3O6+Na] + 636.3044, found 636.3044.

[0314] [Chemical formula 33]

[0315]

[0316] Fmoc-L-Tyr(t-Bu)-L-Phe-L-Ala-Ot-Bu (Compound 6f):

[0317] White solid, melting point 122-125℃, IR (neat): 3290, 1733, 1703, 1644, 1540, 1507,1236, 1160 cm -1 ; [α] 24 D = -11.7 (c 1.70, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.6Hz, 2H), 7.53-7.51(m, 2H), 7.40(dd, J=7.6, 7.6Hz, 2H), 7.33-7.29(m, 2H), 7.22-7.00(m, 7H), 6.88(d, J=8.0Hz, 2H), 6.49(brs, 1H), 6.37(brs, 1H), 5.23(brs, 1H), 4.62(ddd, J=6.8, 6.8, 6.8Hz, 1H), 4.41-4.30(m, 4H), 4.15(t, J=6.8Hz, 1H), 3.01-2.94(m, 4H), 1.44(s, 9H), 1.30(s, 9H), 1.29(d, J=10.8Hz, 3H)ppm; 13 C NMR (100MHz, CDCl3): δ 171.6, 170.7, 169.7, 154.7, 143.8, 141.4, 136.3,130.9, 129.9, 129.4, 128.7, 127.9, 127.24, 127.18, 125.2, 125.1, 124.4,120.1, 82.1, 78.5, 67.3, 56.3, 54.4, 48.9, 47.2, 38.4, 37.6, 29.0, 28.1, 18.5ppm; HRMS(ESI): calcd for [C 44 H 51 N3O7+Na] + 756.3619, found 756.3622.

[0318] [Chemical formula 34]

[0319]

[0320] Fmoc-L-Asp(OBn)-L-Phe-L-Ala-Ot-Bu (Compound 6g):

[0321] White solid, melting point 112-115℃, IR(neat): 3294, 1736, 1697, 1646, 1541, 1227,1148, 739 cm -1 ; [α] 24 D = -7.4 (c 1.82, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.6Hz, 2H), 7.56(d, J=7.6Hz, 2H), 7.42-7.10(m, 14H), 6.92(d, J=7.6Hz, 1H), 6.41(d, J=6.0Hz, 1H), 5.78(d, J=8.4Hz, 1H), 5.12(s, 2H), 4.63-4.54(m, 2H),4.43-4.32(m, 3H), 4.19(t, J=6.8Hz, 1H), 3.08-2.98(m, 3H), 2.76(dd, J=6.0,16.4Hz, 1H), 1.43(s, 9H), 1.29 (d, J=6.8Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ171.7, 170.2, 169.7, 156.1, 143.8, 141.4, 136.4, 135.4, 129.4, 128.8, 128.6,128.4, 127.9, 127.3, 127.2, 125.2, 120.2, 82.1, 67.5, 67.1, 54.7, 51.3, 48.9,47.2, 38.0, 36.2, 28.0, 18.5 ppm; HRMS(ESI): calcd for [C 42 H 45 N3O8+Na] + 742.3099, found 742.3098.

[0322] [Chemical formula 35]

[0323]

[0324] Fmoc-L-Ser(t-Bu)-L-Phe-L-Phe-OMe (Compound 6h):

[0325] White solid, melting point 131-135℃, IR(neat): 3294, 2974, 1745, 1698, 1647, 1541,1241, 1195, 738 cm -1 ; [α] 25 D = +11.4 (c 1.75, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d,J=7.2Hz, 2H), 7.58(d, J=7.2Hz, 2H), 7.39(dd, J=7.2, 7.2Hz, 2H), 7.31(dd, J=7.2, 7.2Hz, 2H), 7.29-7.15(m, 10H), 7.01(d, J=6.0Hz, 2H), 6.40(d, J=5.2Hz,1H), 5.63(d, J=5.2Hz, 1H), 4.75-4.71(m, 1H), 4.63(ddd, J=6.8, 6.8, 6.8Hz,1H), 4.39-4.33(m, 2H), 4.21(t, J=7.6Hz, 1H), 4.14(brs, 1H), 3.74-3.62(m, 4H), 3.26(dd, J=8.0Hz, 1H), 3.09-2.95(m, 4H), 1.12(s, 9H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.5, 170.3, 156.2, 143.9, 143.8, 141.4, 136.4, 135.9, 129.4, 129.3, 128.8, 128.7, 127.9, 127.2, 125.2, 120.1, 74.6, 67.3, 61.6, 54.5,53.6, 52.4, 47.2, 37.9, 27.4 ppm; HRMS(ESI): calcd for [C 41 H 45 N3O7+Na] + 714.3150, found 714.3150.

[0326] [Chemical formula 36]

[0327]

[0328] Fmoc-L-Cys(t-Bu)-L-Phe-L-Ala-Ot-Bu (Compound 6i):

[0329] White solid, melting point 126-128℃, IR(neat): 3290, 1732, 1708, 1645, 1538, 1452,1245, 1150, 737 cm -1 ; [α] 24 D = -17.0 (c 1.81, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.77(d,J=7.6Hz, 2H), 7.58 (d, J=7.6Hz, 2H), 7.40(dd, J=7.6, 7.6Hz, 2H), 7.31(dd, J=7.6, 7.6Hz, 2H), 7.26-7.15(m, 5H), 6.80(d, J=7.2Hz, 1H), 6.53(d, J=5.2Hz,1H), 5.67(brs, 1H), 4.69(ddd, J=7.2, 7.2, 7.2Hz, 1H), 4.43-4.28(m, 4H), 4.21(t, J=7.2Hz, 1H), 3.15-3.06(m, 2H), 2.93(dd, J=5.6, 13.2Hz, 1H), 2.80(dd, J=6.8, 13.2Hz, 1H), 1.44(s, 9H), 1.30(s, 12H) ppm; 13 C NMR (100MHz, CDCl3): δ171.6, 170.0, 169.7, 156.1, 143.8, 143.8, 141.4, 136.3, 129.5, 128.8, 127.9,127.2, 125.2, 120.1, 82.0, 67.4, 55.0, 54.5, 48.9, 47.2, 43.4, 38.0, 31.0,30.8, 28.0, 18.5 ppm; HRMS(ESI): calcd for [C 38 H 47 N3O6S1+Na] + 696.3078, found696.3078.

[0330] [Chemical formula 37]

[0331]

[0332] Fmoc-L-Trp(Boc)-L-Phe-L-Phe-OMe (Compound 6j):

[0333] White solid, melting point 184-188℃, IR(neat): 3304, 1732, 1706, 1672, 1643, 1539,1453, 1369, 1270, 1160, 739 cm -1 ; [α] 24 D = -10.0 (c 1.30, CHCl3); 1 H NMR (400MHz, CDCl3): δ 8.11(d, J=8.0Hz, 1H), 7.73(d, J=7.2Hz, 2H), 7.57(d, J=7.6Hz, 1H), 7.51-7.46(m, 3H), 7.39-7.35(m, 2H), 7.32-7.15(m, 7H), 7.13-7.07(m, 3H), 6.99-6.96(m, 4H), 6.68(d, J=4.8Hz, 1H), 6.37(brs, 1H), 5.52(brs, 1H), 4.73-4.67(m,1H), 4.60-4.51(m, 2H), 4.34(dd, J=7.6, 10.4Hz, 1H), 4.25-4.23(m, 1H), 4.14(t,J=7.2Hz, 1H), 3.61(s, 3H), 3.13-2.89(m, 6H), 1.61(s, 9H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.4, 171.0, 170.0, 156.2, 149.6, 143.8, 141.4, 136.2, 135.8, 135.6, 130.4, 129.3, 129.2, 128.7, 128.6, 127.8, 127.2, 127.1, 125.2, 124.8,124.6, 122.9, 120.1, 119.1, 115.5, 115.3, 83.9, 67.4, 55.0, 54.4, 53.6, 52.3,47.1, 38.2, 38.0, 28.3, 27.9 ppm; HRMS(ESI): calcd for [C 50 H 50 N4O8+Na] + 857.3521, found 857.3523.

[0334] [Chemical formula 38]

[0335]

[0336] Fmoc-L-Lys(Cbz)-L-Ala-L-Ala-Ot-Bu (Compound 6k):

[0337] White solid, melting point 156-159℃, IR(neat): 3287, 1732, 1716, 1684, 1638, 1541,1261, 1151, 739 cm -1 ; [α] 24 D = -8.1 (c 1.73, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.75(d, J=7.2Hz, 2H), 7.57(d, J=7.2Hz, 2H), 7.40-7.26(m, 9H), 6.71(d, J=7.2Hz, 1H), 6.66(d, J=6.8Hz, 1H), 5.68(d, J=6.0Hz, 1H), 5.10-5.07(m, 3H), 4.51-4.37(m,4H), 4.19(t, J=6.8Hz, 1H), 3.20-3.15(m, 1H), 1.84(brs, 1H), 1.68-1.67(m, 1H),1.51-1.31(m, 19H) ppm; 13 C NMR (100MHz, DMSO-d6): δ 172.0, 171.63, 171.56, 156.1, 156.0, 143.9, 143.8, 140.7, 137.3, 128.3, 127.7, 127.6, 127.1, 125.3, 120.1,80.3, 65.6, 65.1, 54.5, 48.3, 47.6, 46.7, 31.6, 29.1, 27.6, 22.8, 18.4, 16.8ppm; HRMS(ESI): calcd for [C 39 H 48 N4O8+H] + 701.3544, found 701.3545, [C 39 H 48 N4O8+Na] + 723.3364, found 723.3363.

[0338] [Chemical formula 39]

[0339]

[0340] Fmoc-L-Thr(Bn)-L-Ala-L-Ala-Ot-Bu (Compound 61):

[0341] White solid, melting point 182-185℃, IR (neat): 3294, 1732, 1705, 1641, 1537, 1451,1245, 1154, 739 cm -1 ; [α] 24 D = -9.6 (c 1.28, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.6Hz, 2H), 7.60(d, J=7.2Hz, 2H), 7.41-7.29(m, 9H), 7.02(d, J=7.2Hz, 1H), 6.69(d, J=6.8Hz, 1H), 5.82(d, J=6.4Hz, 1H), 4.68(d, J=11.6Hz, 1H), 4.57-4.36(m, 6H), 4.22(t, J=6.8Hz, 1H), 4.16-4.15(m, 1H), 1.46(s, 9H), 1.34(d, J=7.6Hz, 3H), 1.32(d, J=6.8Hz, 3H), 1.17(d, J=6.0Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.9, 171.1, 169.4, 156.4, 144.0, 143.8, 141.4, 137.9, 128.6, 128.1, 127.9, 127.2, 125.2, 120.1, 82.1, 74.8, 71.7, 67.3, 58.0, 49.1, 48.9,47.3, 28.1, 18.5, 18.2, 15.2 ppm; HRMS(ESI): calcd for [C 36 H 43 N3O7+Na] + 652.2993, found 652.2990.

[0342] [Chemical formula 40]

[0343]

[0344] Fmoc-L-Phe-L-Ala-L-Ala-Ot-Bu (Compound 6m):

[0345] White solid, melting point 153-156℃, IR(neat): 3296, 1731, 1707, 1643, 1539, 1451,1254, 1154, 739 cm -1 ; [α] 25 D = -16.5 (c 1.53, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.74(d,J=7.6Hz, 2H), 7.51 (dd, J=7.6, 7.6Hz, 2H), 7.38(dd, J=7.6, 7.6Hz, 2H), 7.29-7.18(m, 7H), 6.92(brs, 2H), 5.73(d, J=7.2Hz, 1H), 4.59-4.52(m, 2H), 4.43-4.37(m, 2H), 4.27-4.23(m, 1H), 4.15(t, J=6.8Hz, 1H), 3.07(d, J=5.6Hz, 2H), 1.44(s, 9H), 1.34(d, J = 6.8 Hz, 6H) ppm; 13 C NMR (100MHz, CDCl3): δ 172.0, 171.4,170.9, 156.1, 143.9, 141.4, 136.4, 129.5, 128.7, 127.8, 127.2, 125.3, 125.2,120.1, 82.2, 67.2, 56.1, 49.0, 48.9, 47.2, 38.8, 28.1, 18.9, 18.6 ppm; HRMS(ESI): calcd for [C 34 H 39 N3O6+Na] + 608.2731, found 608.2729.

[0346] [Chemical formula 41]

[0347]

[0348] Cbz-L-Ala-L-Asp(Ot-Bu)-L-Phe-OMe (Compound 6n):

[0349] White solid, melting point 139-142℃, IR(neat): 3299, 1731, 1653, 1523, 1217, 1155,cm -1; [α] 23 D = +19.1 (c 1.1, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.38-7.13(m, 11H), 7.04(d, J=7.2Hz, 1H), 5.26(brs, 1H), 5.11(d, J=12.4Hz, 1H), 5.07(d, J=12.4Hz, 1H), 4.79-4.74(m, 2H), 4.21-4.18(m, 1H), 3.69(s, 3H), 3.13(dd, J=6.0, 13.6Hz,1H), 3.05(dd, J=6.0, 13.6Hz, 1H), 2.91-2.88(m, 1H), 2.53(dd, J=6.4, 16.4Hz,1H), 1.43(s, 9H), 1.31(d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 172.2,171.5, 170.1, 156.1, 136.3, 135.9, 129.4, 128.71, 128.68, 128.3, 128.2,127.2, 82.1, 67.2, 53.8, 52.4, 50.8, 49.2, 37.7, 36.8, 28.1, 18.7 ppm; HRMS(ESI): calcd for [C 29 H 37 N3O8+Na] + 578.2473, found 578.2473.

[0350] [Chemical formula 42]

[0351]

[0352] Cbz-L-Ala-L-Cys(Bn)-L-Ala-Ot-Bu (compound 6o):

[0353] Yellow amorphous solid, IR(neat): 3301, 1731, 1692, 1641, 1538, 1239, 1146, 698cm -1 ; [α] 23 D = -23.4 (c 1.08, CHCl3); 1H NMR (400MHz, CDCl3): δ 7.34-7.21(m, 10H), 6.97(d, J=5.6Hz, 1H), 6.90(d, J=6.4Hz, 1H), 5.42(d, J=6.4Hz, 1H), 5.11(s, 2H),4.56-4.54(m, 1H), 4.43-4.38(m, 1H), 4.28-4.26(m, 1H), 3.74(s, 2H), 2.91(dd, J=5.2, 13.6Hz, 1H), 2.73(dd, J=6.4, 13.6Hz, 1H), 1.45(s, 9H), 1.37(d, J=7.6Hz,3H), 1.35 (d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 172.3, 171.6, 169.4,156.1, 138.2, 136.2, 129.2, 128.8, 128.7, 128.4, 128.2, 127.4, 82.2, 67.2,52.3, 50.9, 49.2, 36.6, 33.7, 28.1, 18.8, 18.4 ppm; HRMS(ESI): calcd for[C 28 H 37 N3O6S1+Na] + 566.2295, found 556.2297.

[0354] [Chemical formula 43]

[0355]

[0356] Cbz-L-Ala-L-Tyr(t-Bu)-L-Phe-OMe (Compound 6p):

[0357] White solid, melting point 163-166℃, IR(neat): 3296, 1742, 1698, 1646, 1538, 1507,1236, 1163 cm -1 ; [α] 24 D = -3.3 (c 1.85, CHCl3); 1H NMR (400MHz, CDCl3): δ 7.36-7.18(m,8H), 7.06-7.00(m, 4H), 6.86(d, J=8.4Hz, 2H), 6.68(d, J=6.8Hz, 1H), 6.41(d, J=5.6Hz, 1H), 5.30(d, J=6.8Hz, 1H), 5.11(d, J=12.4Hz, 1H), 5.03(d, J=12.4Hz,1H), 4.75(ddd, J=5.6Hz, 1H), 4.60(ddd, J=6.8, 6.8, 6.8Hz, 1H), 4.21-4.17(m, J=6.4Hz, 1H), 3.66(s, 3H), 3.07(dd, J=5.6, 13.2Hz, 1H), 3.00-2.95(m, 3H), 1.30(s, 9H), 1.26(d, J=6.8Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 172.2, 171.4,170.3, 154.6, 136.2, 135.8, 131.1, 129.9, 129.3, 128.7, 128.4, 128.3, 127.2,124.4, 78.5, 67.3, 54.4, 53.5, 52.4, 50.7, 37.9, 37.5, 28.9, 18.5 ppm; HRMS(ESI): calcd for [C 34 H 41 N3O7+H] + 604.3017, found 604.3016, [C 34 H 41 N3O7+Na] + 626.2837, found 626.2837.

[0358] [Chemical formula 44]

[0359]

[0360] Fmoc-L-Phe-L-Phe-L-MePhe-OMe (Compound 6q):

[0361] White solid, melting point 80-84℃, IR(neat): 3296, 1739, 1724, 1637, 1538, 1496,1452, 1256, 741 cm -1 ; [α]21 D = -55.9 (c 0.77, CHCl3); 1 H NMR (400MHz, CDCl3): ( 1 H NMR spectrum shows conformational isomers, only the NMR signal of the major isomer is shown) δ 7.76 (d, J=7.6Hz, 2H), 7.55-7.50(m, 2H), 7.40(dd, J=7.6, 7.6Hz, 2H), 7.31 (dd, J=7.6, 7.6Hz, 2H), 7.25-7.10(m, 15H), 6.51(d, J=7.6Hz, 1H), 5.26-5.24(m, 1H), 5.13(d, J=7.6Hz, 1H),5.01-4.95(m, 1H), 4.46-4.42(m, 1H), 4.40-4.35(m, 1H), 4.30-4.26(m, 1H), 4.19-4.12(m, 1H), 3.68(s, 3H), 3.32(dd, J=4.8, 14.0Hz, 1H), 3.02-2.80(m, 5H), 2.68(s, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.3, 170.8, 169.9, 155.8, 143.9, 141.4, 136.7, 136.3, 135.9, 129.7, 129.5, 129.0, 128.8, 128.6, 128.5, 127.9, 127.2,127.1, 127.0, 125.3, 125.2, 120.1, 67.1, 58.7, 55.8, 52.4, 50.5, 47.2, 38.9,38.3, 34.8, 32.7 ppm; HRMS (ESI): calcd for [C 44 H 43 N3O6+Na] + 732.3044, found732.3046.

[0362] [Example 7]

[0363] Synthesis of protected Stellarin G precursor (compound 7a)

[0364] Next, a protected Stellarin G precursor (Compound 7a; SEQ ID NO: 1) as a pentapeptide was synthesized by the following method. Specifically, by the method of the present invention, a tripeptide was first synthesized, and then the tripeptide was di-residue extended to obtain a pentapeptide.

[0365] (Step 1: Synthesis of tripeptide (Compound 6r))

[0366] Using a syringe pump, a dichloromethane solution (flow rate: 1.20 mL / min) of an N-terminal protected amino acid represented by Cbz-L-Leu-OH (compound 1b; 0.300 M, 1.0 molar equivalent) and N,N-diisopropylethylamine (0.360 M, 1.20 molar equivalent) and a dichloromethane solution (flow rate: 2.00 mL / min) of thionyl chloride (0.216 M, 1.2 molar equivalent) were introduced into a T-type mixer at 20° C. The resulting mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of amino acid N-carboxylic anhydride represented by L-Ala-NCA (compound 3b; 0.300M, 1.0 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.216M, 1.2 molar equivalent) and N-methylimidazole (0.180M, 1.00 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into reaction tube 3 (inner diameter: 0.800 mm, length: 3133 mm, volume: 1600 μL, reaction time: 15 seconds). Using a syringe pump, the obtained mixture and a dichloromethane solution of a C-terminal protected amino acid represented by H-Gly-Ot-Bu (compound 5b; 0.360 M, 1.2 molar equivalents) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C. At the same temperature, the obtained mixture was introduced into reaction tube 4 (inner diameter: 0.800 mm, length: 2519 mm, volume: 1267 μL, reaction time: 10 seconds). After reaching a steady state after 90 seconds, the reacted mixture was poured into 1 M hydrochloric acid (1.5 mL) and dichloromethane (8 mL) at room temperature for 60 seconds. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with a saturated NaHCO3 aqueous solution and a saturated saline solution, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. Palladium carbon (10 wt %) was added to a methanol solution containing a semi-pure tripeptide (compound 6q; 1.0 molar equivalent) represented by the obtained Cbz-L-Leu-L-Ala-Gly-Ot-Bu at room temperature. The obtained mixture was stirred at room temperature under a hydrogen atmosphere. After stirring for 4 hours, the obtained mixture was filtered through cerium silica and concentrated in vacuo at room temperature to obtain a semi-pure tripeptide (compound 6r).

[0367] (Step 2: Synthesis of Pentapeptide (Compound 7a))

[0368] Using a syringe pump, a dichloromethane solution (flow rate: 1.20 mL / min) of an N-terminal protected amino acid represented by Cbz-L-Ala-OH (compound 1c; 0.300 M, 1.0 molar equivalent) and N,N-diisopropylethylamine (0.360 M, 1.2 molar equivalent) and a dichloromethane solution (flow rate: 2.00 mL / min) of thionyl chloride (0.216 M, 1.2 molar equivalent) were introduced into a T-type mixer at 20° C. The resulting mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of amino acid N-carboxylic anhydride represented by L-Tyr(t-Bu)-NCA (compound 3c; 0.300M, 1.0 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.216M, 1.2 molar equivalent) and N-methylimidazole (0.180M, 1.0 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into a reaction tube 3 (inner diameter: 0.800 mm, length: 2122 mm, volume: 1067 μL, reaction time: 10 seconds). After reaching a steady state after 70 seconds, the obtained mixture was poured into a test tube containing a dichloromethane solution (1.2 molar equivalents) of a semi-pure tripeptide (compound 6r) represented by HL-Leu-L-Ala-Gly-Ot-Bu at room temperature for 50 seconds. After stirring at room temperature for 10 minutes, 1M hydrochloric acid (1.5 mL) and dichloromethane (8 mL) were added. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with a saturated NaHCO3 aqueous solution and saturated saline, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The crude product was recrystallized from dichloromethane / hexane. The protected StellarinG precursor (compound 7a) consisting of the amino acid sequence represented by SEQ ID NO: 1 can be obtained with a high yield of 80% without the need for silica gel column chromatography.

[0369] [Chemical formula 45]

[0370]

[0371] [Chemical formula 46]

[0372]

[0373] Protection of Stellarin G precursor (Compound 7a):

[0374] Colorless amorphous solid, IR(neat): 3282, 2977, 1734, 1715, 1705, 1682, 1630,1540, 1407, 1455, 1366, 1237, 1161 cm -1 ; [α] 23 D = -26.1 (c 2.09, CHCl3); 1 H NMR (400MHz, CD3OD): δ 7.36-7.30(m, 5H), 7.13(d, J=8.4Hz, 2H), 6.87(d, J=8.4Hz, 2H), 5.11(d, J=12.4Hz, 1H), 5.00(d, J=12.4Hz, 1H), 4.65(q, J=4.8Hz, 1H),4.45-4.40(m, 2H), 4.11(q, J=7.2Hz, 1H), 3.89-3.77(m, 2H), 3.12(dd, J=4.8,13.6Hz, 1H), 2.95(dd, J=8.4, 13.6Hz, 1H), 1.63-1.62(m, 3H), 1.45(s, 9H), 1.39(d, J=7.6Hz, 3H), 1.28(s, 9H), 1.24(d, J=7.2Hz, 3H), 0.92-0.87(m, 6H) ppm; 13 CNMR (100MHz, CD3OD): δ 175.6, 175.1, 174.2, 173.6, 170.1, 158.4, 155.4, 137.9,133.1, 130.9, 129.5, 129.1, 128.8, 125.1, 82.7, 79.4, 67.8, 56.0, 53.4, 52.4,50.2, 42.8, 41.6, 37.5, 29.2, 28.3, 25.7, 23.6, 22.0, 18.2, 18.0 ppm; HRMS(ESI): calcd for [C 39 H 57 N5O9+Na] + 762.4048, found 762.4049.

[0375] [Example 8]

[0376] Synthesis of beef flavor enhancing peptide (compound 9b)

[0377] Next, a beefy meaty peptide (compound 9b; SEQ ID NO: 7) as an octapeptide was synthesized by the following method. Specifically, a tripeptide was first synthesized by the method of the present invention, and then the tripeptide was di-residue extended to obtain a pentapeptide, and then the pentapeptide was di-residue extended to obtain a heptapeptide, and the heptapeptide was mono-residue extended to obtain an octapeptide.

[0378] (Step 1: Synthesis of tripeptide (Compound 6t))

[0379] Using a syringe pump, a dichloromethane solution (flow rate: 1.20 mL / min) of an N-terminal protected amino acid represented by Cbz-L-Ser(t-Bu)-OH (compound 1d; 0.324 M, 1.1 molar equivalent) and N,N-diisopropylethylamine (0.389 M, 1.3 molar equivalent) and a dichloromethane solution (flow rate: 2.00 mL / min) of thionyl chloride (0.233 M, 1.3 molar equivalent) were introduced into a T-type mixer at 20° C. The resulting mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of amino acid N-carboxylic anhydride represented by L-Leu-NCA (compound 3d; 0.324M, 1.1 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.233M, 1.3 molar equivalent) and N-methylimidazole (0.194M, 1.1 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into a reaction tube 3 (inner diameter: 0.800 mm, length: 2122 mm, volume: 1067 μL, reaction time: 10 seconds). After reaching a steady state after about 90 seconds, the obtained mixture was poured into a dichloromethane solution of a C-terminal protected amino acid represented by H-Ala-Ot-Bu・HCl (compound 5a; 1.0 molar equivalent) and N,N-diisopropylethylamine (1.0 molar equivalent) at room temperature for 265 seconds. After stirring at room temperature for 1 minute, 1M hydrochloric acid (5 mL) was added for quenching. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with a saturated NaHCO3 aqueous solution, water, and saturated saline, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The crude product was recrystallized using hexane. At room temperature, 10 wt % palladium on carbon (25 wt %) was added to a methanol solution containing the obtained tripeptide represented by Cbz-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (compound 6s; 1.0 molar equivalent). The obtained mixture was stirred at room temperature under a hydrogen atmosphere. After stirring for 3 hours, the obtained mixture was filtered through cerium silicate and concentrated in vacuo at room temperature. The crude product was dissolved in ethyl acetate, washed with 10% K2CO3 aqueous solution, water, and saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature.A semi-pure tripeptide represented by HL-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu was obtained (Compound 6t).

[0380] (Step 2: Synthesis of Pentapeptide (Compound 7c))

[0381] Using a syringe pump, a dichloromethane solution (flow rate: 1.20 mL / min) of an N-terminal protected amino acid represented by Cbz-L-Glu(Ot-Bu)-OH (compound 1e; 0.324 M, 1.1 molar equivalent) and N,N-diisopropylethylamine (0.389 M, 1.3 molar equivalent) and a dichloromethane solution (flow rate: 2.00 mL / min) of thionyl chloride (0.233 M, 1.3 molar equivalent) were introduced into a T-type mixer at 20° C. The obtained mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of amino acid N-carboxylic anhydride represented by L-Glu(Ot-Bu)-NCA (compound 3e; 0.324M, 1.1 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of N,N-diisopropylethylamine (0.233M, 1.3 molar equivalent) and N-methylimidazole (0.194M, 1.1 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into a reaction tube 3 (inner diameter: 0.800 mm, length: 2122 mm, volume: 1067 μL, reaction time: 10 seconds). After reaching a steady state after about 90 seconds, the obtained mixture was poured into a dichloromethane solution of a semi-pure tripeptide (compound 6t; 1.0 molar equivalent) at room temperature for 106 seconds. After stirring at room temperature for 10 minutes, 1M hydrochloric acid (3.5 mL) was added for quenching. Water was further added, the aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated NaHCO3 aqueous solution and saturated brine, dried with sodium sulfate, filtered, and concentrated in vacuo at room temperature to obtain a semi-pure pentapeptide (compound 7b; SEQ ID NO: 2). The crude product was recrystallized using ethyl acetate. At room temperature, 10 wt % palladium on carbon (25 wt %) was added to a methanol solution containing the obtained pentapeptide represented by Cbz-L-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (compound 7b; 1.0 molar equivalent). The obtained mixture was stirred at room temperature under a hydrogen atmosphere. After stirring for 3 hours, the obtained mixture was filtered through cerium silica and concentrated in vacuo at room temperature.The crude product was dissolved in ethyl acetate, washed with 1M NaOH aqueous solution, water, and saturated brine, dried over magnesium sulfate, filtered, and concentrated in vacuo at room temperature to obtain a semi-pure pentapeptide represented by HL-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (Compound 7c; SEQ ID NO: 3).

[0382] (Step 3: Synthesis of Heptapeptide (Compound 8b))

[0383] Using a syringe pump, a dichloromethane solution (flow rate: 1.20 mL / min) of an N-terminal protected amino acid represented by Cbz-Gly-OH (compound 1f; 0.324 M, 1.1 molar equivalent) and N,N-diisopropylethylamine (0.389 M, 1.3 molar equivalent) and a dichloromethane solution (flow rate: 2.00 mL / min) of thionyl chloride (0.233 M, 1.3 molar equivalent) were introduced into a T-type mixer at 20° C. The obtained mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. The obtained mixture and a dichloromethane solution of amino acid N-carboxylic anhydride represented by L-Asp(Ot-Bu)-NCA (compound 3e; 0.324M, 1.1 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture, a dichloromethane solution of N,N-diisopropylethylamine (0.233M, 1.3 molar equivalent) and N-methylimidazole (0.194M, 1.1 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture was introduced into a reaction tube 3 (inner diameter: 0.800 mm, length: 2122 mm, volume: 1067 μL, reaction time: 10 seconds). After reaching a steady state after about 90 seconds, the obtained mixture was poured into a dichloromethane solution of a semi-pure pentapeptide (compound 7c; 1.0 molar equivalent) at room temperature for 33 seconds. After stirring at room temperature for 10 minutes, 1M hydrochloric acid (2 mL) was added for quenching. Water was further added, the aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated NaHCO3 aqueous solution and saturated saline, dried with sodium sulfate, filtered, and concentrated in vacuo at room temperature to obtain a semi-pure heptapeptide (compound 8a; SEQ ID NO: 4). The crude product was recrystallized using ethyl acetate and methanol. At room temperature, 10 wt % palladium on carbon (25 wt %) was added to a methanol solution containing the obtained semi-pure heptapeptide represented by Cbz-Gly-L-Asp(Ot-Bu)-L-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (Compound 8a; 1.0 molar equivalent). The obtained mixture was stirred at room temperature under a hydrogen atmosphere. After stirring for 2 hours, the obtained mixture was filtered through cerium silica and concentrated in vacuo at room temperature.The crude product was dissolved in a mixed solution of ethyl acetate and methanol, washed with 1M NaOH aqueous solution, water, and saturated saline, dried over magnesium sulfate, filtered, and concentrated in vacuo at room temperature to obtain a semi-pure heptapeptide represented by H-Gly-L-Asp(Ot-Bu)-L-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (Compound 8b; SEQ ID NO: 5).

[0384] (Step 4: Synthesis of octapeptide (Compound 9b))

[0385] Using a syringe pump, an N-terminal protected amino acid represented by Boc-L-Lys(Boc)-OH (compound 1g; 0.333M, 1.3 molar equivalents), an acetonitrile solution of N,N-diisopropylethylamine (0.333M, 1.3 molar equivalents) and N-methylmorpholine (0.333M, 1.3 molar equivalents) (flow rate: 1.20 mL / min), and an acetonitrile solution of methyl chloroformate (0.240M, 1.5 molar equivalents) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C. The resulting mixture was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. After reaching a steady state after about 60 seconds, the resulting mixture was poured into an acetonitrile / dimethylformamide solution of a semi-pure heptapeptide (compound 8b) at room temperature for 20 seconds. After stirring at room temperature for 20 minutes, 1M hydrochloric acid (2 mL) was added to the reaction mixture to quench. Water was further added, the aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated NaHCO3 aqueous solution and saturated saline, dried with sodium sulfate, filtered, and concentrated in vacuo at room temperature to obtain a semi-pure protected octapeptide (Compound 9a; SEQ ID NO: 6). The crude product was recrystallized using methanol. At room temperature, trifluoroacetic acid was added to an acetonitrile / dimethylformamide solution containing the obtained semi-pure octapeptide (Compound 9a; 1.0 molar equivalent) represented by Boc-L-Lys(Boc)-Gly-L-Asp(t-Bu)-L-Glu(t-Bu)-L-Glu(t-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu, and stirred at room temperature. After stirring for 2.5 hours, the resulting mixture was filtered through cerium silicate and concentrated in vacuo at room temperature to obtain a semi-pure heptapeptide (Compound 9b; SEQ ID NO: 7). By using only one silica gel column chromatography, a beef flavor enhancing peptide (Compound 9b) as an octapeptide represented by HL-Lys-Gly-L-Asp-L-Glu-L-Glu-L-Ser-L-Leu-L-Ala-OH was obtained with a high purity of 98%.

[0386] [Chemical formula 47]

[0387]

[0388]

[0389] [Chemical formula 48]

[0390]

[0391] Cbz-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (Compound 6s):

[0392] HPLC conditions: COSMOSIL 5C 18 -AR-II 4.6mm ID×150mm, CH3CN+0.1% formic acid / H2O+0.1% formic acid, 0-20 minutes: 30-100%, 20-25 minutes: 100%, 25-27 minutes: 100-30%, 27-30 minutes: 30%, flow rate 1.0mL / min, detection wavelength 254nm, temperature 40℃, retention time: 14.7 minutes. 685.3mg, 1.28mmol, 82%, HPLC purity: 94%.

[0393] Colorless amorphous solid, 1 H NMR (400MHz, CDCl3): δ 7.38-7.33(m, 5H), 6.93(d, J=7.8Hz, 1H), 6.55(d, J=6.4Hz, 1H), 5.96(d, J=4.8Hz, 1H), 5.12(s, 2H), 4.49-4.39(m, 2H), 4.25(brs, 1H), 3.85-3.83(m, 1H), 3.43(dd, J=7.2, 8.8Hz, 1H),1.70-1.51(m, 3H), 1.46(s, 9H), 1.35(d, J=7.2Hz, 3H), 1.18(s, 9H), 0.93(d, J=6.8Hz, 3H), 0.92(d, J=6.0Hz, 3H) ppm; HRMS(ESI): calcd for [C 28 H 45 N3O7+H] + 536.3330, found 536.3330, [C 28 H 45 N3O7+Na] + 558.3150, found 558.3152.

[0394] [Chemical formula 49]

[0395]

[0396] Cbz-L-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (compound 7b):

[0397] HPLC conditions: COSMOSIL 5C 18 -AR-II 4.6mm ID×150mm, CH3CN+0.1% formic acid / H2O+0.1% formic acid, 0-20 minutes: 50-100%, 20-25 minutes: 100%, 25-30 minutes: 50%, flow rate 1.0mL / min, detection wavelength 254nm, temperature 40℃, retention time: 14.5 minutes.

[0398] 295.1 mg, 0.326 mmol, 52%, HPLC purity: 93%,

[0399] 69.5 mg, 0.076 mmol, 12%, HPLC purity: 95%.

[0400] Colorless amorphous solid, 1 H NMR (400MHz, CDCl3): δ 7.82(d, J=4.4Hz, 1H), 7.36-7.31(m, 5H), 7.11(d, J=8.0Hz, 2H), 6.81(d, J=7.6Hz, 1H), 5.79(d, J=5.6Hz, 1H),5.16(d, J=12.4Hz, 1H), 5.08(d, J=12.4Hz, 1H), 4.53-4.47(m, 1H), 4.43-4.34(m,2H), 4.21-4.13(m, 2H), 3.85(dd, J=3.7, 8.8Hz, 1H), 3.52(dd, J=4.8, 8.8Hz,1H), 2.53-2.29(m, 4H), 2.19-1.91(m, 4H), 1.78-1.61(m, 3H), 1.45(s, 9H), 1.44(s, 9H), 1.43(s, 9H), 1.35(d, J=7.3Hz, 3H), 1.17(s, 9H), 0.92(d, J=6.4Hz,3H), 0.90(d, J=6.4Hz, 3H) ppm; HRMS(ESI): calcd for [C 46 H 75 N5O 13 +H] + 906.5434, found 906.5432, [C 46 H 75 N5O 13 +Na] + 928.5254, found 928.5254.

[0401] [Chemical formula 50]

[0402]

[0403] Cbz-L-Gly-L-Asp(Ot-Bu)-L-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (compound 8a):

[0404] HPLC conditions: COSMOSIL 5C 18 -AR-II 4.6mm ID×150mm, CH3CN+0.1% formic acid / H2O+0.1% formic acid, 0-20 minutes: 50-100%, 20-25 minutes: 100%, 25-30 minutes: 50%, flow rate 1.0mL / min, detection wavelength 254nm, temperature 40℃, retention time: 14.5 minutes.

[0405] 135.6 mg, 0.120 mmol, 64%, HPLC purity: 90%.

[0406] Colorless amorphous solid, 1 H NMR (400MHz, DMSO-d6): δ 8.18(d, J=7.6Hz, 1H), 8.11(d,J=6.8Hz, 1H), 7.97-7.94(m, 2H), 7.87(d, J=7.6Hz, 1H), 7.74(d, J=8.8 Hz, 1H),7.48(brt, J=6.0Hz, 1H), 7.35-7.32(m, 5H), 5.02(s, 2H), 4.63-4.5(m, 1H), 4.40-4.21(m, 4H), 4.11-4.08(m, 1H), 3.64(d, J=6.0Hz, 2H), 3.46-3.41(m, 1H), 3.33 (overlapping with H2O, 1H), 2.64 (dd, J=4.8, 16.0Hz, 1H), 2.50 (overlapping with DMSO, 1H), 2.28-2.14 (m, 4H), 1.87 (brs, 2H), 1.78-1.70 (m, 2H), 1.66-1.59 (m, 1H), 1.47-1.34 (m,38H), 1.23 (d, J=7.2Hz, 3H), 1.09 (s, 9H), 0.87 (d, J=6.8Hz, 3H), 0.84 (d, J=6.4Hz, 3H) ppm; HRMS (ESI): calcd for [C 56 H91 N7O 17 +H] + 1134.6544, found 1134.6542,[C 56 H 91 N7O 17 +Na] + 1156.6364, found 1156.6363.

[0407] [Chemical formula 51]

[0408]

[0409] Boc-L-Lys(Boc)-Gly-L-Asp(Ot-Bu)-L-Glu(Ot-Bu)-L-Glu(Ot-Bu)-L-Ser(t-Bu)-L-Leu-L-Ala-Ot-Bu (compound 9a):

[0410] HPLC conditions: COSMOSIL 5C 18 -AR-II 4.6mm ID×150mm, CH3CN+0.1% formic acid / H2O+0.1% formic acid, 0-20 minutes: 50-100%, 20-25 minutes: 100%, 25-30 minutes: 50%, flow rate 1.0mL / min, detection wavelength 254nm, temperature 40℃, retention time: 15.9 minutes.

[0411] 76.3 mg, 0.058 mmol, 55%, HPLC purity: 86%.

[0412] Colorless amorphous solid, 1H NMR (400MHz, DMSO-d6): δ 8.11(d, J=6.8Hz, 1H), 8.10(d,J=8.4Hz, 1H), 8.04(brt, J=5.2Hz, 1H), 7.95(d, J=6.8Hz, 1H), 7.94(d, J=6.8Hz,1H), 7.86(d, J=8.0Hz, 1H), 7.74(d, J=8.8Hz, 1H), 6.91(d, J=7.2Hz, 1H), 6.75(brt, J=4.8Hz, 1H), 4.65-4.59(m, 1H), 4.38-4.21(m, 4H), 4.11-4.07(m, 3.88-3.83(m, 1H), 3.77-3.65(m, 2H), 3.51-3.40(m, 2H), 2.90-2.85(m, 2H), 2.66(dd, J=5.2, 16.0Hz, 1H), 2.43(dd, J=8.2, 16.0Hz, overlaps with DMSO, 1H), 2.28-2.13(m, 4H), 1.92-1.83(m, 2H), 1.77-1.69(m, 2H), 1.66-1.58(m, 1H), 1.47-1.33(m,62H), 1.24(d, J=7.2Hz, 3H), 1.09(s, 9H), 0.87(d, J=6.4Hz, 3H), 0.84(d, J=6.8Hz, 3H) ppm; HRMS(ESI): calcd for [C 64 H 113 N9O 20 +Na] + 1350.7994, found 1350.7993.

[0413] [Chemical formula 52]

[0414]

[0415] HL-Lys-L-Gly-L-Asp-L-Glu-L-Glu-L-Ser-L-Leu-L-Ala-OH·TFA (compound 9b):

[0416] Chromatographic conditions (reverse phase): Biotage Sfar Bio C18 Duo 25g, 0.1% TFA in H2O:0.1% TFAin CH3CN=100:0-70:30, flow rate 5mL / min, detection wavelength 215nm and Biotage Sfar Bio C18 Duo 10g, 0.1% TFA in H2O:0.1% TFA in CH3CN=100:0-70:30, flow rate 2mL / min, detection wavelength 215nm.

[0417] HPLC conditions: COSMOSIL 5C 18 -AR-II 4.6mm ID×150mm, CH3CN+0.1%TFA / H2O+0.1%TFA, 0-15 minutes: 0-20%, 15-20 minutes: 20%, 20-25 minutes: 0%, flow rate 1.0mL / min, detection wavelength 215nm, temperature 40℃, retention time: 13.1 minutes.

[0418] 36.4 mg, 0.034 mmol, 85% (total yield 13%), HPLC purity: 98%.

[0419] Colorless amorphous solid, 1 H NMR (400 MHz, D2O): δ 4.79 (overlapping with H2O, 1H), 4.44 (t, J=6.0 Hz, 1H), 4.41-4.33 (m, 4H), 4.08 (t, J=6.8 Hz, 1H), 4.04 (s, 2H), 3.85 (d, J=6.0 Hz, 2H), 3.01 (t, J=8.0 Hz, 2H), 2.98-2.94 (m, 1H), 2.89 (dd, J=6.8, 17.2 Hz,1H), 2.51-2.48 (m, 4H), 2.18-2.11 (m, 2H), 2.06-1.98 (m, 2H), 1.96-1.92 (m, 2H),1.76-1.63(m, 5H), 1.53-1.47(m, 2H), 1.42(d, J=7.2Hz, 3H), 0.93(d, J=5.6Hz,3H), 0.88(d, J=6.0Hz, 3H) ppm; 13C NMR (100 MHz, D2O, using CH3CN as internal standard): δ 176.1, 176.0, 175.2, 173.2, 173.1, 172.32, 172.27, 171.5, 170.5, 169.8, 169.4, 161.9 (q, J C-F =35.2Hz), 115.5(q, J C-F =290.8Hz), 60.0, 54.6, 52.5, 52.3, 52.1, 51.3,49.1, 47.7, 41.5, 38.8, 38.1, 34.4, 29.4, 29.0, 25.4, 25.0, 24.9, 23.3, 21.3,20.3, 19.7, 15.2 ppm; HRMS(ESI): calcd for [C 34 H 57 N9O 16 +H] + 848.3996, found848.3998, [C 34 H 57 N9O 16 +Na] + 870.3815, found 870.3815.

[0420] [Example 9]

[0421] Synthesis of tripeptides (compounds epi-6i-1, epi-6i-2, epi-6o-1 and epi-6i-2)

[0422] Next, each diastereomer of the tripeptide (Compounds 6i and 6o) obtained in Example 6 was synthesized by the following method (Table 6).

[0423] Using a syringe pump, N-terminal protected amino acid represented by Fmoc-Cys(t-Bu)-OH (compound 1h) or N-terminal protected amino acid represented by Cbz-Ala-OH (compound 1i) (0.300M, 1.00 molar equivalent) and dichloromethane (flow rate: 1.20 mL / min) of N,N-diisopropylethylamine (0.360M, 1.2 molar equivalent) and dichloromethane solution (flow rate: 2.00 mL / min) of thionyl chloride (0.216M, 1.20 molar equivalent) were introduced into a T-type mixer at 20° C. The obtained mixture 1 was introduced into a reaction tube 1 (inner diameter: 0.800 mm, 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. The obtained mixture 1 and a dichloromethane solution of amino acid N-carboxylic anhydride represented by Phe-NCA (compound 3g) or amino acid N-carboxylic anhydride represented by Cys(Bn)-NCA (compound 3h) (0.300M, 1.00 molar equivalent) (flow rate: 1.20 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. The obtained mixture 2 was introduced into a reaction tube 2 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The obtained mixture 2 and a dichloromethane solution of N,N-diisopropylethylamine (0.216 M, 1.2 molar equivalent) and N-methylimidazole (0.180 M, 1.00 molar equivalent) (flow rate: 2.00 mL / min) were introduced into a T-type mixer at 20°C using a syringe pump. At the same temperature, the obtained mixture 3 was introduced into a reaction tube 3 (inner diameter: 0.800 mm, reaction time: 10 seconds or 15 seconds). Using a syringe pump, the obtained mixture 3 and a dichloromethane solution of a C-terminal protected amino acid represented by HL-Ala-Ot-Bu (compound 5c; 0.360 M, 1.20 molar equivalent) were introduced into a T-type mixer at 20°C (flow rate: 1.20 mL / min). At the same temperature, the obtained mixture 4 was introduced into a reaction tube 4 (inner diameter: 0.800 mm, length: 2519 mm, volume: 1267 μL, reaction time: 10 seconds). After reaching a steady state after 90 seconds, the reacted mixture 4 was poured into 1 M hydrochloric acid (1.5 mL) and dichloromethane (8 mL) at room temperature for 25 seconds. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 aqueous solution and saturated saline, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The residue was purified by recrystallization or washing, and identified by HPLC-UV analysis using a chiral column. The results are shown in Figure 2 and Figure 3 The purification conditions, yield and yield are shown in Table 6 (Examples 9-1 to 9-4). Figure 2and Figure 3 The results show that the compound obtained by the method of the present invention does not contain a separately synthesized diastereomer, and diastereomerization is very difficult to occur in the method of the present invention. In addition, since cysteine ​​is known to be an amino acid that easily undergoes diastereomerization, it is obvious that the method of the present invention can also be applied to amino acids that easily undergo diastereomerization.

[0424] [Chemical formula 53]

[0425]

[0426] Fmoc-D-Cys(t-Bu)-L-Phe-L-Ala-Ot-Bu (compound epi-6i-1):

[0427] White solid, melting point 152-156℃, IR(neat): 3283, 1732, 1644, 1550, 1226, 1149,758, 741 cm -1 ; [α] 24 D = -0.061 (c 0.95, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.2Hz, 2H), 7.58(d, J=7.2Hz, 2H), 7.39(dd, J=7.2, 7.2Hz, 2H), 7.30(dd, J=7.2,7.2Hz, 2H), 7.26-7.13(m, 5H), 6.86(d, J=6.4Hz, 1H), 6.47(d, J=7.2Hz, 1H),5.72(brs, 1H), 4.72-4.67(m, 1H), 4.40-4.28(m, 4H), 4.20(t, J=6.8Hz, 1H), 3.15(dd, J=6.4, 13.6Hz, 1H), 3.07(dd, J=6.8, 13.6Hz, 1H), 2.94(dd, J=6.0, 12.8Hz,1H), 2.76(dd, J=6.8, 12.8Hz, 1H), 1.42(s, 9H), 1.31(d, J=8.4Hz, 12H) ppm; 13CNMR(100MHz, CDCl3): δ 171.6, 170.2, 169.8, 156.1, 143.9, 143.8, 141.4, 136.3,129.5, 128.7, 127.8, 127.2, 125.3, 120.1, 82.1, 67.5, 55.2, 54.5, 49.0, 47.2,43.3, 38.2, 31.0, 30.9, 28.0, 18.5 ppm; HRMS(ESI): calcd for [C 38 H 47 N3O6S1+Na] + 696.3078, found 696.3080.

[0428] [Chemical formula 54]

[0429]

[0430] Fmoc-L-Cys(t-Bu)-D-Phe-L-Ala-Ot-Bu (compound epi-6i-2):

[0431] White solid, melting point 125-127℃, IR(neat): 3352, 1731, 1657, 1524, 1235, 1151,758, 741 cm -1 ; [α] 24 D = -7.5 (c 0.84, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.76(d, J=7.2Hz, 2H), 7.58(d, J=7.2Hz, 2H), 7.40(dd, J=7.2, 7.2Hz, 2H), 7.31-7.19(m,7H), 6.86(d, J=6.8Hz, 1H), 6.39(d, J=6.4Hz, 1H), 5.73(brs, 1H), 4.72-4.69(m,1H), 4.42-4.19(m, 5H), 3.15(dd, J=6.4, 13.2Hz, 1H), 3.07(dd, J=7.6, 13.2Hz,1H), 2.97(dd, J=6.0, 12.8Hz, 1H), 2.78(dd, J=7.2, 12.8Hz, 1H), 1.41(s, 9H), 1.32(s, 9H), 1.21(d, J=7.2Hz, 3H) ppm; 13C NMR (100MHz, CDCl3): δ 171.9, 170.2,169.6, 156.1, 143.9, 143.8, 141.4, 136.5, 129.5, 128.8, 127.9, 127.2, 125.3,120.1, 82.1, 67.5, 55.3, 54.6, 48.8, 47.2, 43.3, 38.3, 31.0, 30.8, 18.3 ppm; HRMS(ESI): calcd for [C 38 H 47 N3O6S1+Na] + 696.3078, found 696.3077.

[0432] [Chemical formula 55]

[0433]

[0434] Cbz-D-Ala-L-Cys(Bn)-L-Ala-Ot-Bu (compound epi-6o-1):

[0435] Yellow oil, IR(neat): 3287, 1732, 1645, 1541, 1508, 1234, 1148, 698 cm -1 ; [α] 24 D = +11.1 (c 1.16, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.32-7.22(m, 10H), 7.05(d, J=6.4Hz, 1H), 6.95(d, J=7.6Hz, 1H), 5.52(d, J=7.6Hz, 1H), 5.13(d, J=12.4Hz, 1H), 5.05(d, J=12.4Hz, 1H), 4.57(d, J=6.0Hz, 1H), 4.42-4.36(m, 1H), 4.25-4.22(m, 1H), 3.74(s, 2H), 2.89(dd, J=4.0, 13.2Hz, 1H), 2.73(dd, J=6.4, 13.2Hz,1H), 1.44(s, 9H), 1.37(d, J=7.2Hz, 3H), 1.35(d, J=7.6Hz, 3H) ppm; 13C NMR (100MHz, CDCl3): δ 172.5, 171.6, 169.5, 156.1, 138.1, 136.2, 129.2, 128.7,128.6, 128.3, 128.2, 127.3, 82.1, 67.2, 52.2, 50.9, 49.1, 36.6, 33.7, 28.0,18.7, 18.3 ppm; HRMS(ESI): calcd for [C 28 H 37 N3O6S1+Na] + 566.2295, found 566.2295.

[0436] [Chemical formula 56]

[0437]

[0438] Cbz-L-Ala-D-Cys(Bn)-L-Ala-Ot-Bu (compound epi-6o-2):

[0439] Yellow oil, IR(neat): 3296, 1732, 1717, 1647, 1540, 1523, 1241, 1150, 698cm -1 ; [α] 24 D =1.25(c 1.41, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.33-7.22(m, 10H), 7.06(d, J=6.4Hz, 1H), 6.90(d, J=7.6Hz, 1H), 5.46(d, J=6.4Hz, 1H), 5.43(d, J=12.4Hz, 1H), 5.04(d, J=12.4Hz, 1H), 4.60(d, J=6.4Hz, 1H), 4.44-4.37(m, 1H), 4.21-4.14(m, 1H), 3.73(s, 2H), 2.95(dd, J=5.2, 14.0Hz, 1H), 2.74(dd, J=6.4,14.0Hz, 1H), 1.44(s, 9H), 1.38(d, J=6.8Hz, 3H), 1.35(d, J=7.2Hz, 3H) ppm; 13CNMR (100MHz, CDCl3): δ 172.6, 171.9, 169.5, 156.2, 138.2, 136.2, 129.2, 128.74, 128.67, 128.4, 128.2, 127.3, 82.1, 67.2, 52.1, 51.1, 49.1, 36.5, 33.3, 28.1,18.3, 18.2 ppm; HRMS(ESI): calcd for [C 28 H 37 N3O6S1+Na] + 566.2295, found 566.2292.

[0440] [Comparative Example 1]

[0441] Sequential synthesis of tripeptide (compound epi-6a-1)

[0442] Next, a tripeptide (compound epi-6a-1) was synthesized by extending the amino acid residues one by one according to the conventional technique. Compound epi-6a-1 is a diastereomer of the tripeptide (compound 6a) obtained in Example 4.

[0443] At -20°C, add isobutyl chloroformate (1.0 molar equivalent) to a dichloromethane solution of an N-terminal protected amino acid represented by Fmoc-L-Phe-OH (compound 1a; 1.0 molar equivalent) and N-methylmorpholine (1.1 molar equivalent). After stirring at -20°C for 1 minute, add a C-terminal protected amino acid represented by HL-Ala-Ot-Bu (1.1 molar equivalent) and N,N-diisopropylethylamine (1.1 molar equivalent) at -20°C. After stirring at room temperature for 15 minutes, pour the resulting mixture into dichloromethane and 1M hydrochloric acid at the same temperature. Extract the aqueous layer with dichloromethane, wash the organic layer with saturated NaHCO3 aqueous solution and saturated brine, dry it with magnesium sulfate, filter it, and concentrate it in vacuo at room temperature. It is necessary to purify the residue by silica gel column chromatography. An N-terminally protected dipeptide represented by Fmoc-L-Phe-L-Ala-Ot-Bu was obtained (Compound S3). The purification conditions, amount and yield are shown in Table 6 (Comparative Example 1-1).

[0444] At 0°C, 1,8-diazabicyclo[5.4.0]undec-7-ene (1.0 molar equivalent) was added to a dichloromethane solution of compound S3 (1.0 molar equivalent). The reaction was monitored by thin layer chromatography. After the reaction was completed, the obtained solution was purified by silica gel column chromatography. The N-terminal unprotected dipeptide represented by HL-Phe-L-Ala-Ot-Bu (compound S5) was obtained. The purification conditions, yield and yield are shown in Table 6 (Comparative Example 1-2).

[0445] At -20°C, isobutyl chloroformate (1.1 molar equivalent) was added to a dichloromethane solution of an N-terminal protected amino acid represented by Fmoc-D-Phe-OH (1.0 molar equivalent) and N-methylmorpholine (1.1 molar equivalent). After stirring at -20°C for 1 minute, an N-terminal unprotected dipeptide represented by HL-Phe-L-Ala-Ot-Bu (compound S5; 1.1 molar equivalent) and N,N-diisopropylethylamine (1.1 molar equivalent) were added at -20°C. After stirring at room temperature for 15 minutes, the resulting mixture was poured into dichloromethane and 1M hydrochloric acid at the same temperature. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 aqueous solution and saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The residue needs to be purified by silica gel column chromatography. The obtained tripeptide represented by Fmoc-D-Phe-L-Phe-L-Ala-Ot-Bu (compound epi-6a-1) was separated by silica gel column chromatography, the crude product was purified by recrystallization, and each was identified by HPLC-UV analysis using a chiral column. The results are shown in Figure 4 The purification conditions, yield and yield are shown in Table 6 (Comparative Examples 1-3).

[0446] [Chemical formula 57]

[0447]

[0448] Fmoc-L-Phe-L-Ala-Ot-Bu (Compound S3: Org. Biomol. Chem. 2022, 20, 3303-3310.):

[0449] White solid, 1H NMR (400MHz, CDCl3): δ 7.69(d, J=7.2Hz, 2H), 7.51-7.46(m,2H), 7.35-7.31(m, 2H), 7.24-7.12(m, 7H), 7.02(d, J=6.8Hz, 1H), 5.96(d, , 1.29(d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.7, 170.8, 156.0, 143.8, 143.7, 141.2, 136.5, 129.4,128.5, 127.6, 127.0, 126.9, 125.15, 125.07, 119.9, 81.8, 67.1, 56.0, 48.7,47.0, 38.8, 27.9, 18.3 ppm.

[0450] [Chemical formula 58]

[0451]

[0452] HL-Phe-L-Ala-Ot-Bu (Compound S5: ACS Catal. 2018, 8, 2181-2187.):

[0453] Colorless oil, 1 H NMR (400MHz, CDCl3): δ 7.78(d, J=7.6Hz, 1H), 7.33-7.29(m, 2H), 7.25-7.21(m, 3H), 4.50-4.42(m, 1H), 3.62(dd, J=3.6, 9.6Hz, 1H), 3.24(dd, J=3.6, 14.0Hz, 1H), 2.72(dd, J=9.2, 14.0Hz, 1H), 1.46(s, 11H), 1.36(d, J=7.6Hz,3H) ppm; 13C NMR (100MHz, CDCl3): δ 173.8, 172.2, 137.8, 129.4, 128.7, 126.8, 81.8, 56.3, 48.3, 41.0, 28.0, 18.6 ppm.

[0454] [Chemical formula 59]

[0455]

[0456] Fmoc-D-Phe-L-Phe-L-Ala-Ot-Bu (compound epi-6a-1):

[0457] White solid, melting point 97-101℃, IR(neat): 3283, 1732, 1644, 1541, 1245, 1224,1148, 757, 741 cm -1 ; [α] 24 D = +5.20 (c 2.92, CHCl3); 1 H NMR (400MHz, CDCl3): δ 7.72-7.70 (m, 2H), 7.48(dd, J=8.0, 8.0Hz, 2H), 7.34(dd, J=7.2, 8.0Hz, 2H), 7.25-6.96(m, 14H), 5.94(d, J=8.4Hz, 1H), 4.84-4.81(m, 1H), 4.52(d, J=7.2Hz, 1H),4.38-4.31(m, 2H), 4.19-4.08(m, 2H), 3.05-2.87(m, 4H), 1.39(s, 9H), 1.26(d, J=7.2Hz, 3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.6, 171.2, 170.3, 156.0, 143.9,143.8, 141.3, 136.5, 136.4, 129.5, 128.6, 127.7, 127.1, 127.0, 125.2, 125.1,120.0, 81.9, 67.2, 56.4, 54.2, 48.9, 47.1, 38.8, 38.3, 28.0, 18.3 ppm; HRMS(ESI): calcd for [C 40 H 43 N3O6+Na] +684.3044, found 684.3047.

[0458] [Comparative Example 2]

[0459] Sequential synthesis of tripeptide (compound epi-6a-2)

[0460] A tripeptide (epi-6a-2) was synthesized by extending the amino acid residues one by one in the same manner as in Comparative Example 1. Compound epi-6a-2 is a diastereomer of the tripeptide (Compound 6a) obtained in Example 4.

[0461] At -20°C, isobutyl chloroformate (1.0 molar equivalent) was added to a dichloromethane solution of an N-terminal protected amino acid represented by Fmoc-D-Phe-OH (1.0 molar equivalent) and N-methylmorpholine (1.1 molar equivalent). After stirring at -20°C for 1 minute, a C-terminal protected amino acid represented by HL-Ala-Ot-Bu (1.1 molar equivalent) and N,N-diisopropylethylamine (1.1 molar equivalent) were added at -20°C. After stirring at room temperature for 15 minutes, the resulting mixture was poured into dichloromethane and 1M hydrochloric acid at the same temperature. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with a saturated aqueous NaHCO3 solution and saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. A step of purifying the residue by silica gel column chromatography was required. The N-terminal protected dipeptide represented by Fmoc-D-Phe-L-Ala-Ot-Bu (compound S4) was obtained. The purification conditions, yield and yield are shown in Table 6 (Comparative Example 2-1).

[0462] At 0°C, 1,8-diazabicyclo[5.4.0]undec-7-ene (1.0 molar equivalent) was added to a dichloromethane solution of compound S4 (1.0 molar equivalent). The reaction was monitored by thin layer chromatography. After the reaction was completed, the obtained solution was purified by silica gel column chromatography. The N-terminal unprotected dipeptide represented by HD-Phe-L-Ala-Ot-Bu (compound S6) was obtained. The purification conditions, yield and yield are shown in Table 6 (Comparative Example 2-2).

[0463] At -20°C, isobutyl chloroformate (1.1 molar equivalent) was added to a dichloromethane solution of an N-terminal protected amino acid represented by Fmoc-L-Phe-OH (1.0 molar equivalent) and N-methylmorpholine (1.1 molar equivalent). After stirring at -20°C for 1 minute, an N-terminal unprotected dipeptide represented by HD-Phe-L-Ala-Ot-Bu (1.1 molar equivalent) and N,N-diisopropylethylamine (1.1 molar equivalent) were added at -20°C. After stirring at room temperature for 15 minutes, the resulting mixture was poured into dichloromethane and 1M hydrochloric acid at the same temperature. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 aqueous solution and saturated brine, dried with magnesium sulfate, filtered, and concentrated in vacuo at room temperature. The residue needs to be purified by silica gel column chromatography. The obtained tripeptide (compound epi-6a-2) represented by Fmoc-L-Phe-D-Phe-L-Ala-Ot-Bu was analyzed by silica gel column chromatography, and the crude product was purified by recrystallization and identified by HPLC-UV analysis using a chiral column. The results are shown in Figure 4 The purification conditions, yield and yield are shown in Table 6 (Comparative Example 2-3).

[0464] [Chemical formula 60]

[0465]

[0466] Fmoc-D-Phe-L-Ala-Ot-Bu (Compound S4):

[0467] White solid, melting point 133-135℃, IR(neat): 3299, 1732, 1716, 1655, 1540, 1450,1253, 1147, 757, 741 cm -1 ; [α] 24 D = +18.5 (c 2.38, CHCl3); 1H NMR (400MHz, CDCl3): δ7.75(d, J=7.6Hz, 2H), 7.53(dd, J=7.2, 7.2Hz, 2H), 7.38(dd, J=7.2, 7.2Hz, 2H), 7.31-7.20(m, 7H), 6.32(d, J=4.8Hz, 1H), 5.55(d, J=7.2Hz, 1H), 4.47-4.31(m,4H), 4.18(t, J=7.2Hz, 1H), 3.15-3.01(m, 2H), 1.41(s, 9H), 1.20(d, J=7.2Hz,3H) ppm; 13 C NMR (100MHz, CDCl3): δ 171.8, 170.1, 155.9, 143.9, 143.8, 141.4,136.5, 129.5, 128.8, 127.8, 127.2, 125.2, 120.1, 82.2, 67.2, 56.3, 48.7,47.2, 39.1, 28.0, 18.4 ppm; HRMS(ESI): calcd for [C 31 H 34 N2O5+Na] + 537.2360, found537.2363.

[0468] [Chemical formula 61]

[0469]

[0470] HD-Phe-L-Ala-Ot-Bu (Compound S6: ACS Catal. 2018, 8, 2181-2187.):

[0471] Colorless oil, 1 H NMR (400MHz, CDCl3): δ 7.66(d, J=7.6Hz, 1H), 7.34-7.30(m, 2H), 7.26-7.22(m, 3H), 4.50-4.43(m, 1H), 3.59(dd, J=4.4, 10.0Hz, 1H), 3.30(dd, J=4.0, 13.6Hz, 1H), 2.66(dd, J=9.6, 13.6Hz, 1H), 1.47(s, 11H), 1.36(d, J=7.2Hz,3H) ppm; 13C NMR (100MHz, CDCl3): δ 173.9, 172.3, 138.3, 129.4, 128.8, 126.9, 81.9, 56.8, 48.4, 41.2, 28.1, 18.7 ppm.

[0472] [Chemical formula 62]

[0473]

[0474] Fmoc-L-Phe-D-Phe-L-Ala-Ot-Bu (compound epi-6a-2):

[0475] Colorless solid, melting point 138-142℃, IR(neat): 3299, 1732, 1716, 1655, 1540, 1450,1253, 1147, 757, 741 cm -1 ; [α] 24 D = +1.85 (c 3.19, CHCl3); 1 H NMR (400MHz, CDCl3): δ7.74(d, J=7.6Hz, 2H), 7.51(dd, J=7.6, 7.6Hz, 2H), 7.38(dd, J=7.6, 7.6Hz, 2H), 7.29-7.13(m, 10H), 7.24(d, J=6.8Hz, 2H), 6.65-6.60(m, 2H), 5.67(d, J=7.6Hz,1H), 4.71(d, J=6.4Hz, 1H), 4.40-4.30(m, 3H), 4.24-4.12(m, 2H), 3.04-3.02(m,3H), 2.80(dd, J=8.0, 13.2Hz, 1H), 1.38(s, 9H), 1.15(d, J=7.2Hz, 3H) ppm; 13 CNMR(100MHz, CDCl3): δ 171.8, 170.9, 169.7, 156.1, 143.8, 141.4, 136.4, 129.5,128.8, 128.7, 127.8, 127.2, 127.1, 125.3, 125.2, 120.1, 82.0, 67.3, 56.7,54.2, 48.7, 47.2, 38.6, 38.2, 28.0, 18.2 ppm; HRMS(ESI): calcd for [C 40 H43 [N3O6+Na] + 684.3044, found 684.3046.

[0476] [Table 6]

[0477]

Claims

1. A method for preparing a di-residue extended peptide represented by the following general formula (6): [Chemical formula 1] In formula (6), R 1 represents a hydrogen atom or an alkyl group; R 4 , R 8 and R 10 The same or different, represents a hydrogen atom or a methyl group; R 2 , R 5 , R 6 , R 9 and R 11 Same or different, represents the side chain of the amino acid residue; R 2 , R 5 , R 6 , R 9 and R 11 They may form a ring together with the adjacent nitrogen atom through the carbon atom to which they are bonded or not; R 3 , R 7 and R 12 are the same or different and represent a hydrogen atom or a substituted or unsubstituted monovalent organic group; 1 represents a hydrogen atom, an amino protecting group, a label or a solid phase carrier; P 2 represents a hydrogen atom, a protecting group of a carboxyl group, a label or a solid phase carrier; n1 represents an integer greater than 0, and when n1 is an integer greater than 2, n1 R 1 , R 2 and R 3 Optional the same or different; n2 represents an integer greater than 0, when n2 is an integer greater than 2, n2 R 10 , R 11 and R 12 Optional same or different, The preparation method comprises: a reaction step of reacting a first amino acid or peptide represented by the following general formula (1), a halogenating agent, a tertiary amine represented by the following general formula (3), an amino acid N-carboxylic anhydride (NCA) represented by the following general formula (4), and a second amino acid or peptide represented by the following general formula (5), [Chemical formula 2] In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , P 1 and n1 and R 1 , R 2 , R 3 , R4 , R 5 , P 1 and n1 are the same, [Chemical formula 3] In formula (3), R 13 , R 14 , R 15 are the same or different and represent substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or, R 13 , R 14 , R 15 Two or more of the groups are connected to form a ring with or without one or more heteroatoms or substituents, [Chemical formula 4] In formula (4), R 6 and R 7 With the R 6 and R 7 same, [Chemical formula 5] In formula (5), R 8 , R 9 , R 10 , R 11 , R 12 , P 2 and n2 and the R 8 , R 9 , R 10 , R 11 , R 12 , P 2 The same as n2.

2. The preparation method according to claim 1, wherein The reaction process comprises: (A) a reaction step of reacting a first amino acid or peptide represented by the general formula (1), a halogenating agent, a tertiary amine represented by the general formula (3), and an amino acid N-carboxylic anhydride (NCA) represented by the general formula (4); and (B) A reaction step of reacting the reaction mixture obtained in the step (A) with the second amino acid or peptide represented by the general formula (5).

3. The preparation method according to claim 2, wherein The step (A) comprises: (A1) a reaction step of reacting a first amino acid or peptide represented by the general formula (1), a halogenating agent, and a tertiary amine represented by the general formula (3); and (A2) A reaction step of reacting the reaction mixture obtained in the step (A1) with the amino acid N-carboxylic anhydride (NCA) represented by the general formula (4).

4. The preparation method according to claim 1, wherein The halogenating agent is thionyl represented by the following general formula (2): [Chemical formula 6] In formula (2), X's are the same or different and represent a halogen atom.

5. The preparation method according to claim 1, wherein The tertiary amine includes an aliphatic amine and / or a heterocyclic aromatic amine.

6. The preparation method according to claim 1, wherein The reaction temperature of the reaction process is -80~100°C.

7. The preparation method according to claim 1, wherein The reaction time of the reaction step is less than 60 minutes.

8. The preparation method according to any one of claims 1 to 7, wherein The reaction process is carried out by a flow method.

9. The preparation method according to claim 8, wherein: The reaction process is performed by a microfluidic method.

10. An acylated NCA represented by the following general formula (8): [Chemical formula 7] In formula (8), R 1 represents a hydrogen atom, or a substituted or unsubstituted monovalent organic group; R 4 Represents a hydrogen atom or a methyl group; R 2 , R 5 and R 6 Same or different, represents the side chain of amino acid residues; R 2 , R 5 and R 6 They may form a ring together with the adjacent nitrogen atom through the carbon atom to which they are bonded or not; R 3 and R 7 represents a hydrogen atom, or a substituted or unsubstituted monovalent organic group; P 1 represents a hydrogen atom, an amino protecting group, a label or a solid phase carrier; n1 represents an integer greater than 0, and when n1 is an integer greater than 2, n1 R 1 ~R 3 Optional same or different.

11. A peptide represented by the following general formula (9): [Chemical formula 8] In formula (9), R 16 , R 17 and R 18 Same or different, represents the side chain of the amino acid residue; R 16 , R 17 and R 18 Through the carbon atom to which they are bonded, they may form a ring together with the adjacent nitrogen atom or not; 3 represents a protecting group for a hydrogen atom or an amino group; P 4 It represents a protecting group for a hydrogen atom or a carboxyl group.

12. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

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