Continuous production method of peptides

Abstract

A method for producing a peptide, comprising the following steps (1), (A), (2) and (B). (1) The step of producing an N-protected C-protected peptide in which each of the N-terminal amino group and the C-terminal is protected by a protecting group, a side-chain functional group may also be protected by a protecting group and at least one of the C-terminal and the side-chain functional group is protected by a pseudo solid-phase protecting group (i.e., an N-protected C-protected peptide) by a condensation reaction in a flow reactor; (A) the step of subjecting a reaction solution containing the N-protected C-protected peptide to washing and oil-water separation in the flow reactor to separate an organic layer containing the N-protected C-protected peptide; (2) the step of subjecting the organic layer containing the N-protected C-protected peptide to a reaction for removing the protecting group for the N-terminal amino group in the flow reactor to produce a C-protected peptide in which the N-terminal amino group is not protected, the C-terminal is protected by the protecting group, the side-chain functional group may also be protected by the protecting group and at least one of the C-terminal and the side-chain functional group is protected by the pseudo solid-phase protecting group (i.e., an N-unprotected C-protected peptide); and (B) the step of subjecting a reaction solution containing the N-unprotected C-protected peptide to washing and oil-water separation in the flow reactor to separate an organic layer containing the N-unprotected C-protected peptide.

Description

[Technical field]

[0001] The present invention relates to a method for continuous production of peptides and is useful in the field of peptide synthesis. [Background technology]

[0002] Peptide synthesis is classified into solid-phase and liquid-phase methods depending on the form of C-protected peptide (C-protected amino acid in the dipeptide-forming reaction).

[0003] In the solid-phase method, peptide elongation is carried out with the C-terminus of the peptide (or amino acid) bound to a solid support, and the target peptide is cleaved from the solid support in the final stage. Therefore, excess or unreacted residual reagents and by-products can be easily removed by washing the solid support. For this reason, the solid-phase method is used in the industrial production of peptide drugs. However, since the reaction is limited to the surface of the solid support, there are challenges in terms of scale-up and reactivity.

[0004] In contrast, the liquid phase method can be easily scaled up and has relatively good reactivity, so it can be a means to solve the above problems of the solid phase method. However, the liquid phase method has the problem that it is necessary to remove residual reagents and by-products at each step of the condensation reaction and deprotection reaction, which complicates the manufacturing process and increases the time required to manufacture the final drug. Furthermore, even at a small scale for research purposes, the liquid phase method has the problem that the manufacturing process is complicated and takes a long time to manufacture.

[0005] In order to solve the problems of the liquid phase method as described above, it has been considered to use a production method using a pseudo solid-phase protecting group (Patent Documents 1 to 7), which allows isolation and purification to be performed by solid-liquid separation (i.e., crystallization). However, when using an isolation and purification method by crystallization, the process becomes discontinuous, making it difficult to produce peptides continuously.

[0006] On the other hand, a production method using a pseudo-solid-phase protecting group has been found, which performs the reaction in a uniform liquid phase, changes the solvent composition after the reaction, and performs isolation and purification only by extraction and washing without requiring solid-liquid separation (Patent Documents 8 to 12). In peptide synthesis, by protecting the C-terminus and / or side chain functional group of an amino acid or peptide with a pseudo-solid-phase protecting group, the lipid solubility and solubility in organic solvents (particularly non-polar organic solvents) of the intermediate peptide obtained in each step of the peptide elongation reaction are significantly improved, and purification can be performed only by extraction and washing. Therefore, according to this method, complicated and time-consuming operations such as solid-liquid separation are not required, and the speed is improved and efficiency and productivity are significantly improved.

[0007] The above-mentioned production method using a pseudo solid-phase protecting group is a very useful technique that combines the advantages of both solid-phase and liquid-phase reactions, and has attracted attention from an industrial point of view. However, when the purification step is carried out in a discontinuous flow by batch processing each time, the purification takes time, which is an issue that remains, and further improvement is desired.

[0008] Incidentally, the above-mentioned liquid-phase peptide synthesis process is a synthesis process that has been widely used in the field of organic synthesis, called "batch synthesis." In recent years, in the field of organic synthesis, "flow synthesis" utilizing flow reactors (especially flow microreactors) has also been studied. "Flow synthesis" is said to have advantages such as shortening the synthesis time and easiness of scale-up. For example, in the field of peptide synthesis, a continuous synthesis device for peptide synthesis utilizing a flow reactor has been proposed, which has an oil-water separation means for separating and removing the aqueous phase components that remain unreacted after synthesis (Patent Document 13). In addition, the use of flow reactors in peptide synthesis has been studied for short-chain peptides with four or fewer amino acid residues, but the target compounds were limited (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2000-44493 A [Patent Document 2] International Publication No. 2006 / 104166 [Patent Document 3] International Publication No. 2007 / 034812 [Patent Document 4] International Publication No. 2007 / 122847 [Patent Document 5] International Publication No. 2010 / 113939 [Patent Document 6] International Publication No. 2010 / 104169 [Patent Document 7] International Publication No. 2011 / 078295 [Patent Document 8] International Publication No. 2012 / 029794 [Patent Document 9] International Publication No. 2016 / 140232 [Patent Document 10] International Publication No. 2003 / 018188 [Patent Document 11] International Publication No. 2017 / 038650 [Patent Document 12] International Publication No. 2019 / 009317 [Patent Document 13] JP 2007-186452 A [Non-patent literature]

[0010] [Non-Patent Document 1] S. Fuse et al., Angew.Chem.Int. Ed.2014,53,851-855 Summary of the Invention [Problem to be solved by the invention]

[0011] A major concern when applying flow synthesis in a flow reactor is a phenomenon called "clogging." If the product does not dissolve sufficiently in the solvent and solids such as crystals are formed, the flow reactor will become clogged and the intended purpose cannot be achieved. This is also due to the type of protecting group used during synthesis, but when the target peptide is particularly long, sufficient solubility in the solvent cannot necessarily be expected, so this becomes a major concern when applying to peptide synthesis. Patent Document 13 discloses a continuous synthesis apparatus for peptide synthesis using a flow reactor, which has an oil-water separation means for separating and removing unreacted aqueous phase components after synthesis. However, no case of actually synthesizing peptides has been disclosed, and the scope of application, such as the maximum chain length of peptides that can be synthesized, has not been clarified. As mentioned above, Non-Patent Document 1 is known as an example of actually synthesizing peptides using a flow reactor, but it was studied on short-chain peptides with four or fewer amino acid residues, and the target compounds were limited. In addition, in order to establish a continuous synthesis, it is essential to remove residual raw materials, residual reagents, and reaction impurities (by-products) when excessively used so as not to affect the next reaction. In the case of Patent Document 13 and Non-Patent Document 1, it is considered that removing these impurities is also difficult. Furthermore, when synthesizing a peptide having a pseudo solid-phase protecting group, the product does not separate into layers because it has a structural feature that has a surfactant effect, and it was not clear whether the product peptide (especially when the peptide is elongated to a long chain) can be separated into an organic layer as originally intended by the oil-water separation method disclosed in Patent Document 13. [Means for solving the problem]

[0012] The present inventors have found a method for synthesizing a desired peptide by combining peptide chain elongation in a flow reactor (with a continuous flow if necessary) using peptides protected with a pseudo solid-phase protecting group so that the peptides maintain their solubility in a solvent even when the peptides become long chains and can easily remove reaction impurities, washing of the products in the flow reactor at each step to remove unreacted substances and by-products, and layer separation by an oil-water separation means, using peptides protected with a pseudo solid-phase protecting group. Surprisingly, they found that regardless of the peptide chain length, the flow reactor does not become clogged, and the products do not form micelles like surfactants, but are layered in the organic layer in the oil-water separation means, and thus the peptides can be synthesized and purified very efficiently (continuously as a whole production flow if necessary), and have completed the present invention. The present invention is as follows.

[0013] [1] A method for producing a peptide, comprising the following steps (A) and / or (B): (A) a step of purifying the N-protected C-protected peptide by washing a reaction solution containing an N-protected C-protected peptide (N-protected C-protected peptide) in which the N-terminal amino group and the C-terminus are protected by protecting groups, and the side chain functional groups may be further protected by protecting groups, and in which the C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group, with water and / or a hydrophilic organic solvent in a flow reactor in a continuous flow, and then separating the organic layer containing the N-protected C-protected peptide by an oil-water separation means in a continuous flow; (B) A process for purifying the N-unprotected C-protected peptide by washing a reaction solution containing a C-protected peptide (N-unprotected C-protected peptide) in which the N-terminal amino group is not protected, the C-terminus is protected by a protecting group, and a side chain functional group may be further protected by a protecting group, and in which the C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group, with water and / or a hydrophilic organic solvent in a flow reactor in a continuous flow, and then separating the organic layer containing the N-unprotected C-protected peptide by an oil-water separation means in a continuous flow.

[0014] [2] A method for producing a peptide, comprising the following steps (1), (A), (2), and (B): (1) a C-protected amino acid (C-protected amino acid) whose C-terminus is protected by a protecting group, and whose side chain functional groups may be further protected by a protecting group, and whose C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group, or a C-protected peptide (C-protected peptide) whose C-terminus is protected by a protecting group, and whose side chain functional groups may be further protected by a protecting group, and whose C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group; an N-protected amino acid (N-protected amino acid) in which the C-terminus is not protected, the N-terminal amino group is protected by a protecting group, and the side chain functional group may be further protected by a protecting group, or an N-protected peptide (N-protected peptide) in which the C-terminus is not protected, the N-terminal amino group is protected by a protecting group, and the side chain functional group may be further protected by a protecting group; Condensing agent; and a soluble organic solvent, introducing the peptide into a flow reactor and carrying out a condensation reaction in a continuous flow in the flow reactor to obtain an N-protected C-protected peptide (N-protected C-protected peptide) in which the N-terminal amino group and the C-terminus are protected by protecting groups and the side chain functional groups may be further protected by protecting groups, and in which at least one of the C-terminus or the side chain functional groups is protected by a pseudo solid-phase protecting group; (A) a step of purifying the N-protected C-protected peptide by washing a reaction solution containing the N-protected C-protected peptide in a flow reactor with water and / or a hydrophilic organic solvent in a continuous flow, and then separating the organic layer containing the N-protected C-protected peptide using an oil-water separation means in a continuous flow; (2) introducing the organic layer containing the N-protected C-protected peptide into a flow reactor in a continuous flow and removing the protecting group of the N-terminal amino group in the continuous flow in the flow reactor to obtain a C-protected peptide (N-unprotected C-protected peptide) in which the N-terminal amino group is not protected, the C-terminus is protected by a protecting group, and the side chain functional groups may be further protected by a protecting group, and at least one of the C-terminus or the side chain functional groups is protected by a pseudo solid-phase protecting group; (B) A process for purifying the N-unprotected C-protected peptide by washing the reaction solution containing the N-unprotected C-protected peptide in a flow reactor with a continuous flow of water and / or a hydrophilic organic solvent, and then separating the organic layer containing the N-unprotected C-protected peptide using an oil-water separation means in a continuous flow. [3] A method for producing the peptide according to [2] above, comprising carrying out steps (1), (A), (2), and (B) in that order. [4] A method for producing the peptide described in [2] above, comprising carrying out steps (2), (B), (1), and (A) in that order. [5] The method for producing a peptide according to any one of the above [1] to [4], wherein the reaction solution forms a slug flow during washing with water and / or a hydrophilic organic solvent in the flow reactor. [6] The method for producing a peptide according to any one of [1] to [5] above, wherein the oil-water separation means is either a continuous layer separation means having a configuration including a filter or a gravity-type continuous layer separation means. [7] The method for producing a peptide according to any one of [1] to [6], wherein the protecting group for the amino group is a 9-fluorenylmethyloxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group. [8] The method for producing a peptide according to [7], wherein the protecting group for the amino group is a 9-fluorenylmethyloxycarbonyl group. [9] A method for producing a peptide according to any one of [1] to [8], further comprising a step of removing all protecting groups after obtaining an organic layer containing the N-protected C-protected peptide obtained in step (A) or the N-unprotected C-protected peptide obtained in step (B).

[10] The method for producing a peptide according to any one of [1] to [9], wherein the produced peptide has 5 to 100 amino acid residues (more preferably, 5 to 50).

[11] The pseudo solid-phase protecting group is (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine); 3,4,5-Tri(2',3'-dihydrophytyloxy)benzyl alcohol; 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol; 3,4,5-Tri(octadecyloxy)cyclohexanemethanol; [Bis-(4-docoxy-phenyl)-methyl]-amine; 3,4,5-Tri(octadecyloxy)benzyl alcohol; 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy)benzyl alcohol; 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 2-Docosyloxy-9-(4-chlorophenyl)-9-fluorenol; 2-Docosyloxy-9-(4-chlorophenyl)-9-bromofluorene; 2,7-didoxy-9-(4-chlorophenyl)-9-bromofluorene; 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene; 1,12-Bis-[12-(2'-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane; 1,12-Bis-[12-(2'-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane; 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol; 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene; 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)cyclohexylmethoxy)-9-fluorenol; 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)cyclohexylmethoxy)-9-bromofluorene; 3,5-Didoxybenzyl alcohol; 2,4-Didoxybenzyl alcohol; 2,4-Bisoctadecyloxybenzyl alcohol; 3-Didocosylaminobenzyl alcohol; 3-Diphytylaminobenzyl alcohol; N-(2',3'-dihydrophytyl)-N-(3-hydroxymethylphenyl)acetamide; N-Triacontyl-N-(3-hydroxymethylphenyl)acetamide; 3-(Aminomethyl)-N,N-didocosylaniline;

[0015] [ka]

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] [ka]

[0020] [ka]

[0021] [ka]

[0022] [ka]

[0023] [ka]

[0024] [ka]

[0025] and

[0026] [ka]

[0027] (wherein TIPS is a triisopropylsilyl group, and TBDPS stands for tert-butyldiphenylsilyl group. The method for producing the peptide according to any one of [1] to

[10] , selected from the following:

[12] The pseudo solid-phase protecting group is (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine); 3,4,5-Tri(2',3'-dihydrophytyloxy)benzyl alcohol; 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol; and The method for producing the peptide according to any one of [1] to

[10] , wherein the peptide is selected from 3,4,5-tri(octadecyloxy)cyclohexanemethanol. Effect of the Invention

[0028] According to the present invention, by using a peptide chain protected by a pseudo solid-phase protecting group, the peptide can maintain its solubility in a solvent even when the peptide becomes a long chain, and can easily remove reaction impurities. In addition, by combining and utilizing the continuous flow of peptide chain elongation in a flow reactor, washing of the product in each step in the flow reactor to remove unreacted substances and by-products, and liquid separation by oil-water separation means, the purification and isolation process, which has been a problem in the liquid-phase peptide synthesis method, can be greatly simplified, and synthesis and purification can be made continuous as a whole peptide production flow. In addition, according to the present invention, it is possible to shorten the post-treatment operation time in the peptide elongation stage (i.e., the condensation step and / or the deprotection step) while taking advantage of the advantage of peptide synthesis by the pseudo solid-phase method that the product can be easily purified and isolated by oil-water separation. As described above, the present invention provides a new method for producing peptides that improves on the drawbacks of the conventional method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The amino acids that are the building blocks of the peptides produced by the present invention are compounds having an amino group and a carboxy group in the same molecule, and may be natural or unnatural amino acids, and may be L-, D- or racemic. Furthermore, the building blocks are not limited to amino acids, but may also be other compounds that can be used in peptide synthesis (hereinafter referred to as amino acid analogs). Those skilled in the art can appropriately select such amino acid analogs and produce or purchase them according to known methods or methods similar thereto.

[0030] The steps (1), (A), (2), and (B) in the above-mentioned [1] and [2] of the present invention will be described in detail below.

[0031] [About flow reactors] The flow reactors used in steps (1), (A), (2), and (B) of the present invention are not particularly limited and can be appropriately selected depending on the intended reaction (function). In addition, one or more flow reactors can be connected together to carry out the reaction. As the flow reactor, a flow microreactor requiring a micro effect is preferable, but a flow reactor not requiring a micro effect is also included.

[0032] The flow reactor used in the reaction may be made of, for example, metal, Teflon, glass, or silicon. From the viewpoints of compatibility with reagents, thermal conductivity, cost, etc., metal and Teflon are preferred.

[0033] The amounts of reagents, reaction temperature, and residence time during the reaction can be appropriately selected according to the reaction.

[0034] The pumps and mixers used in the flow reactor are not limited and any pumps and mixers known in the art can be used.

[0035] [Regarding step (1)] This step comprises the steps of: 1) providing a solution in a soluble organic solvent containing a C-protected amino acid (C-protected amino acid) whose C-terminus is protected with a protecting group, and whose side chain functional group may be further protected with a protecting group, and whose C-terminus or at least one of the side chain functional groups is protected with a pseudo solid-phase protecting group, or a C-protected peptide (C-protected peptide) whose C-terminus is protected with a protecting group, and whose side chain functional group may be further protected with a protecting group, and whose C-terminus or at least one of the side chain functional groups is protected with a pseudo solid-phase protecting group; The condensation reaction can be carried out by introducing into a flow reactor, separately, simultaneously, or after appropriately mixing any two or more of the following solutions, a soluble organic solvent solution containing an N-protected amino acid (N-protected amino acid) in which the side chain functional group may be further protected by a protecting group, or an N-protected peptide (N-protected peptide) in which the C-terminus is not protected, the N-terminal amino group is protected by a protecting group, and the side chain functional group may be further protected by a protecting group, and 3) a soluble organic solvent solution containing a condensation agent, into the flow reactor, and mixing them in a continuous flow in the flow reactor to allow the condensation reaction to proceed. Furthermore, a soluble organic solvent solution containing a condensation promoter may be appropriately mixed.

[0036] Here, the C-protected amino acid may be obtained by discontinuous batch synthesis, or may be purchased or obtained separately by other methods. The C-protected peptide may be one obtained by carrying out step (B) of the present invention, or may be one obtained by discontinuous batch synthesis, or may be one purchased or obtained separately by other methods. Among these, the one obtained by carrying out step (B) of the present invention is preferable.

[0037] In addition, the N-terminal amino group of the C-protected amino acid or C-protected peptide may be unprotected, or may be substituted with one or more substituents. In this case, examples of the substituent include those generally used in amino groups (e.g., alkyl groups having 1 to 6 carbon atoms), and substituents that activate the reaction with the N-protected amino acid or N-protected peptide. It is preferable that the N-terminal amino group of the C-protected amino acid or C-protected peptide is not protected.

[0038] (Reaction conditions for step (1)) As described above, this step (1) can be carried out by introducing 1) a solution containing a C-protected amino acid or a C-protected peptide, 2) a solution containing an N-protected amino acid or an N-protected peptide, and 3) a solution containing a condensing agent into a flow reactor, either separately, simultaneously, or after appropriately mixing any two or more of the solutions, and mixing them in a continuous flow in the flow reactor to allow the condensation reaction to proceed.

[0039] (C-protected amino acids / C-protected peptides)

[0040] (Pseudo Solid Phase Protecting Groups) A pseudo-solid-phase protecting group used for protecting the C-terminus (C-protection) of a C-protected amino acid or C-protected peptide or for protecting a side chain functional group as necessary is a "protecting group that allows a reaction to be carried out in a homogeneous liquid phase, and allows the product to be isolated and purified only by oil-water separation after the reaction by changing the solvent composition. The pseudo-solid-phase protecting group is not particularly limited and may be appropriately selected from those known to those skilled in the art, including those known in the art.

[0041] Among these, preferred pseudo solid-phase protecting groups include branched chain-containing aromatic compounds described in WO 2012 / 029794, and further branched chain-containing saturated carbocyclic compounds thereof. Specifically, they are represented by the following formula (I), and include a specific benzyl compound (in formula (I), X and Z are both hydrogen atoms, and R1 is a hydrogen atom); a specific diphenylmethane compound (in formula (I), X is a hydrogen atom, R1 is a hydrogen atom, k is 1, and Z is a group represented by formula (a) (in formula (I), R2 is a hydrogen atom, and m is 0); and a specific fluorene compound (in formula (I), X is a phenyl group, k is 1, Z is a group represented by formula (a) (in formula (I), m is 0), and R2 together with R1 represents a single bond to form a fluorene ring together with ring A). Formula (I):

[0042] [ka]

[0043] [In the formula, Ring A represents a benzene ring or a cyclohexane ring; Each of the k Q's independently represents a single bond, or -O-, -S-, -C(=O)O-, -C(=O)NH-, -NH-, or -NR a - indicates; k R a and -NR a -R in a each independently represents an organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300; aa or -S(0)2R aa (In the formula, R aa represents a hydrogen atom, an optionally substituted aliphatic hydrocarbon group, an optionally substituted aromatic hydrocarbon group, or an optionally substituted aromatic heterocyclic group); k represents an integer of 1 to 4; R1 is a hydrogen atom, or when Z is a group represented by the following formula (a), R1 may represent a single bond together with R2 to form a fluorene ring together with ring B: Ring A is R1, k QR a In addition to C(X)(Y)Z, further halogen atoms, C optionally substituted with one or more halogen atoms 1-6 C which may be substituted with an alkyl group and one or more halogen atoms 1-6 It may have a substituent selected from the group consisting of alkoxy groups; X represents a hydrogen atom or a phenyl group; Y represents a hydroxyl group or an -NHR group (R represents a hydrogen atom, an alkyl group, or an aralkyl group); and Z is a hydrogen atom or a group of formula (a):

[0044] [ka]

[0045] (In the formula, * indicates a bond position; Ring B represents a benzene ring or a cyclohexane ring; m represents an integer of 0 to 4; m Q's each independently represent a single bond, or -O-, -S-, -C(=O)O-, -C(=O)NH-, -NH-, or -NR b - indicates; m R b and -NR b -R in b each independently represents an organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 or more and 300 or less; R2 may represent a hydrogen atom or, together with R1, represent a single bond to form a fluorene ring together with ring A; and Ring B has m QR b In addition to R2, a halogen atom, C optionally substituted with one or more halogen atoms 1-6C which may be substituted with an alkyl group and one or more halogen atoms 1-6 ) which may have a substituent selected from the group consisting of alkoxy groups; R a and R b The organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains being 3 or more, and a total number of carbon atoms being 14 to 300, is represented by the formula (b):

[0046] [ka]

[0047] (In the formula, * indicates the position of a bond to an adjacent atom; R3 and R4 are each independently a hydrogen atom or C 1-4 represents an alkyl group; X1 is a single bond, C 1-4 It represents an alkylene group or an oxygen atom. However, R3 and R4 cannot both be hydrogen atoms. ) is a group having three or more identical or different divalent groups represented by the following formula:

[0048] The compound represented by formula (I) of the present invention and the compound to be protected are bonded by a condensation reaction between the hydroxyl group or NHR group, which is the Y group, and the carboxyl group or the like at the C-terminus of the compound to be protected.

[0049] In the present specification, the "alkyl group" represented by R in the above formula (I) is 1-30 Alkyl groups are preferred, and C 1-10 Alkyl groups, more preferably C 1-6 It is an alkyl group. Preferred specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc., and methyl and ethyl are particularly preferred.

[0050] In the present specification, the "aralkyl group" represented by R in the above formula (I) is7-30 Aralkyl groups are preferred, and C 7-20 Aralkyl groups, more preferably C 7-16 Aralkyl group (C 6-10 Aryl-C 1-6 Preferred specific examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, α-naphthylmethyl, 1-(α-naphthyl)ethyl, 2-(α-naphthyl)ethyl, 1-(α-naphthyl)propyl, β-naphthylmethyl, 1-(β-naphthyl)ethyl, 2-(β-naphthyl)ethyl, 1-(β-naphthyl)propyl, and the like, with benzyl being particularly preferred.

[0051] R is a hydrogen atom, C 1-6 Alkyl group or C 7-16 An aralkyl group is preferred, a hydrogen atom, methyl, ethyl or benzyl is more preferred, and a hydrogen atom is particularly preferred.

[0052] In this specification, the "halogen atom" in the above formula (I) is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[0053] In this specification, "ring A" in the above formula (I) represents a benzene ring or a cyclohexane ring, either of which is preferable.

[0054] In the above formula (I) of this specification, "ring B" represents a benzene ring or a cyclohexane ring, either of which is preferable.

[0055] In the present specification, R in the above formula (I) a , R b The "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300" represented by the formula (1) is an organic group having at least one aliphatic hydrocarbon group having one or more branched chains in its molecular structure, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300. The "branched chain" in the "aliphatic hydrocarbon group having one or more branched chains" refers to a linear or branched saturated aliphatic hydrocarbon group, and 1-6 Alkyl groups are preferred, C 1-4 An alkyl group is more preferred, and a methyl group or an ethyl group is even more preferred. The "branched chain" may also be substituted with one or more halogen atoms. The "aliphatic hydrocarbon group" in "aliphatic hydrocarbon group having one or more branched chains" is a linear, saturated or unsaturated aliphatic hydrocarbon group having a C2-C 300 Alkyl groups (preferably C3-C 100 Alkyl groups, more preferably C3-C 60 Alkyl group), C2-C 300 Alkenyl groups (preferably C3-C 100 Alkenyl groups, more preferably C3-C 60 alkenyl group) or C2-C 300 Alkynyl groups (preferably C3-C 100 Alkynyl groups, more preferably C3-C 60 alkynyl group). The position of the "aliphatic hydrocarbon group having one or more branched chains" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branches of 3 or more, and a total number of carbon atoms of 14 to 300" is not particularly limited, and may be present at a terminal (monovalent group) or at another position (for example, a divalent group). Specific examples of the "aliphatic hydrocarbon group having one or more branched chains" include branched isomers such as propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group (lauryl group), tridecyl group, myristyl group, cetyl group, stearyl group, arachyl group, behenyl group, oleyl group, linolyl group, and lignoceryl group, and include monovalent groups having one or more branched chains and divalent groups derived therefrom. Preferred are 3,7,11-trimethyldodecyl group, 3,7,11,15-tetramethylhexadecyl group (hereinafter sometimes referred to as 2,3-dihydrophytyl group), 2,2,4,8,10,10-hexamethylundecane-5-yl group, and groups represented by the formula:

[0056] [ka]

[0057] (In the formula, * indicates the bonding position to Q.

[0058] When there are multiple "aliphatic hydrocarbon groups having one or more branched chains" in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branches of 3 or more, and a total number of carbon atoms of 14 to 300", each of the "aliphatic hydrocarbon groups having one or more branched chains" may be the same or different.

[0059] In the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branches being 3 or more, and a total number of carbon atoms being 14 to 300", the moieties other than the "aliphatic hydrocarbon group having one or more branched chains" can be set arbitrarily. For example, it may have moieties such as -O-, -S-, -CO-, -NH-, -COO-, -OCONH-, -CONH-, -NHCO-, and hydrocarbon groups (monovalent or divalent groups). Examples of the "hydrocarbon group" include aliphatic hydrocarbon groups, aromatic aliphatic hydrocarbon groups, monocyclic saturated hydrocarbon groups, and aromatic hydrocarbon groups, and specifically, for example, monovalent groups such as alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, and aralkyl groups, and divalent groups derived therefrom are used. Examples of the "alkyl group" include C 1-6 Preferred are alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc. Examples of the "alkenyl group" include, for example, 2-6 Alkenyl groups are preferred, and examples thereof include vinyl, 1-propenyl, allyl, isopropenyl, butenyl, and isobutenyl. Examples of the "alkynyl group" include, for example, 2-6 Alkynyl groups are preferred, such as ethynyl, propargyl, 1-propynyl, etc. Examples of the "cycloalkyl group" include, for example, C 3-6Cycloalkyl groups are preferred, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. An "aryl group" is, for example, C 6-14 Aryl groups are preferred, such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, and 2-anthryl. 6-10 An aryl group is more preferable, and a phenyl group is particularly preferable. Examples of the "aralkyl group" include C 7-20 Aralkyl groups are preferred, and examples thereof include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl. Among these, C 7-16 Aralkyl group (C 6-10 Aryl-C 1-6 An alkyl group is more preferable, and benzyl is particularly preferable. The "hydrocarbon group" may be substituted with a substituent selected from a halogen atom (a chlorine atom, a bromine atom, a fluorine atom, an iodine atom), an oxo group, and the like.

[0060] The compound represented by formula (I) has k QR a In this case, Q is a single bond, or -O-, -S-, -C(=O)O-, -C(=O)NH- or -NH-, preferably -O-. k QR a The groups may be the same or different.

[0061] In the compounds of formula (I), R a , R b In the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300", the total number of carbon atoms is 14 or more, preferably 16 or more, and more preferably 18 or more. a , R bThe total number of carbon atoms in the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total number of branched chains of 3 or more" represented by the formula (I) is 300 or less, preferably 200 or less, and more preferably 160 or less. a , R b In the "organic group having at least one aliphatic hydrocarbon group having one or more branches, and the total number of branches being 3 or more," the total number of branches is 3 or more, preferably 4 or more, more preferably 8 or more, and even more preferably 10 or more. The larger the total number of branches, the more the compound protected by the compound of the present invention becomes an oily product with good solubility in various organic solvents, even if the peptide chain is long.

[0062] R a , R b As the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300", there is an organic group represented by the formula (b):

[0063] [ka]

[0064] (In the formula, * indicates the bond position to the adjacent atom; R3 and R4 are each independently a hydrogen atom or C 1-4 represents an alkyl group; X1 is a single bond, C 1-4 It represents an alkylene group or an oxygen atom. However, R3 and R4 cannot both be hydrogen atoms.) having three or more identical or different divalent groups, for example, groups represented by any of the following formulas (c) to (e) are preferred.

[0065] The number of carbon atoms, the number of repeating units (m1, n0 to n9) and the like in the definitions of each symbol in formulae (c) to (e) are shown for convenience, and can be appropriately changed within the above-mentioned definitions so that the total number of carbon atoms is 14 or more (preferably 16 or more, more preferably 18 or more) and 300 or less (preferably 200 or less, more preferably 160 or less). Formulae (c) to (e) will be explained in order below.

[0066] Equation (c) is as follows:

[0067] [ka]

[0068] [In the formula, * indicates the bond position with Q; R5 and R6 together represent a hydrogen atom or together represent =O; n0 represents an integer from 2 to 40; n0 R7 and R8 are each independently a hydrogen atom or C 1-4 represents an alkyl group; Each of n0 X2 is independently a single bond or C 1-4 represents an alkylene group; and R9 is a hydrogen atom or C 1-4 represents an alkyl group; R 10 is C 1-4 An alkyl group or a group of formula (I'):

[0069] [ka]

[0070] (In the formula, * indicates the bond position; Ring A' represents a benzene ring or a cyclohexane ring; The other symbols are as defined above. Here, the ring A' is, in addition to R, Q, and C(X)(Y)Z, further includes a halogen atom, a C(X)(Y)(Y) optionally substituted with one or more halogen atoms.1-6 C which may be substituted with an alkyl group and one or more halogen atoms 1-6 It may have a substituent selected from the group consisting of alkoxy groups. However, when R7 and R8 are not both hydrogen atoms and n0 is 2, R9 is C 1-4 represents an alkyl group.

[0071] In the group of formula (c), R5 and R6 are both hydrogen atoms; n0 is an integer from 2 to 40; n0 R7 and R8 are each independently a hydrogen atom, a methyl group, or an ethyl group; Each of the n0 X2's is independently a single bond, a methylene group, or an ethylene group; and R9 is preferably a hydrogen atom, a methyl group, or an ethyl group (provided that R7 and R8 are not both hydrogen atoms and n0 is 2, R9 represents a methyl or ethyl group).

[0072] More preferred groups of formula (c) are branched isomers having 14 to 160 carbon atoms, such as myristyl, cetyl, stearyl, arachyl and behenyl groups, and having a total branched chain number of 3 or more. Among these, 2,3-dihydrophytyl, 3,7,11-trimethyldodecyl and 2,2,4,8,10,10-hexamethyl-5-dodecanoyl groups are particularly preferred.

[0073] Equation (d) is as follows:

[0074] [ka]

[0075] (In the formula, * indicates the bond position with Q; m1 OR 11represents a hydroxyl group substituted with a group represented by formula (c') or a hydroxyl group substituted with a group having a polyalkylene glycol group having a total number of branched chains of 3 or more (e.g., a polypropylene glycol group, a polyneopentyl glycol group); m1 represents an integer of 1 to 3. The group represented by the above formula (c') is as follows: * is the same as the description of the group represented by formula (c) above, except that it indicates the bonding position to O, not the bonding position to Q.

[0076] In the group of formula (d), R 11 is a branched isomer having 14 to 30 carbon atoms, such as a myristyl group, a cetyl group, a stearyl group, an arachyl group, or a behenyl group, and more preferably a group having a total branch chain number of 3 or more, and among these, a 2,3-dihydrophytyl group or a 3,7,11-trimethyldodecyl group is particularly preferred.

[0077] Equation (e) is as follows:

[0078] [ka]

[0079] (In the formula, * indicates the bond position with Q; n1 represents an integer from 1 to 10; n2 represents an integer from 1 to 10; n1 R 15 and R 16 are each independently a hydrogen atom or C 1-4 represents an alkyl group; n1 X3 is a single bond or C 1-4 Represents an alkylene group; n2 R 17 and R 18 are each independently a hydrogen atom or C 1-4 represents an alkyl group; n2 X5 are single bonds or C 1-4 Represents an alkylene group; X4 is a single bond or C1-4 represents an alkylene group; and R 12 , R 13 , R 14 , R 19 , R 20 and R 21 are each independently a hydrogen atom or C 1-4 Indicates an alkyl group. However, R 15 and R 16 , and / or R 17 and R 18 are not both hydrogen atoms and n1+n2 is 2, then R 12 , R 13 and R 14 Two or more of the following are independently selected: C 1-4 Indicates an alkyl group or R 19 , R 20 and R 21 Two or more of the following are independently selected: C 1-4 It indicates an alkyl group.

[0080] In the group of formula (e), n1 is an integer from 1 to 5; n2 is an integer from 1 to 5; n1 R 15 and R 16 are each independently a hydrogen atom, a methyl group, or an ethyl group; n1 X3 each represents a single bond, a methylene group, or an ethylene group; n2 R 17 and R 18 are each independently a hydrogen atom, a methyl group, or an ethyl group; n2 X5 each represents a single bond, a methylene group, or an ethylene group; X4 is a single bond, a methylene group, or an ethylene group; and R 12 , R 13 , R 14 , R 19 , R 20 and R 21 are each independently a hydrogen atom or C 1-4 More preferably, the group is an alkyl group (provided that R15 and R 16 , and / or R 17 and R 18 are not both hydrogen atoms and n1+n2 is 2, then R 12 , R 13 and R 14 Two or more of the following are independently selected: C 1-4 Indicates an alkyl group or R 19 , R 20 and R 21 Two or more of the following are independently selected: C 1-4 indicates an alkyl group).

[0081] Particularly preferred groups of formula (e) include n1 is an integer from 1 to 5; n2 is an integer from 1 to 5; n1 R 15 and R 16 are each independently a hydrogen atom or a methyl group; n1 X3 each represents a single bond or a methylene group; n2 R 17 and R 18 are each independently a hydrogen atom or a methyl group; n2 X5 each represents a single bond or a methylene group; X4 is a single bond or a methylene group; and R 12 , R 13 , R 14 , R 19 , R 20 and R 21 is a methyl group (provided that R 15 and R 16 , and / or R 17 and R 18 but neither are hydrogen atoms.

[0082] R a , R bThe "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branches of 3 or more, and a total number of carbon atoms of 14 or more and 300 or less" represented by the formula (c) to (e) above may be a group having 3 or more groups in which X1 in the formula (b) is an oxygen atom, that is, a group containing a polyalkylene glycol group such as a polypropylene glycol group or a polyneopentyl glycol group, the total number of branched chains of which is 3 or more.

[0083] R a , R b Specific examples of the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300" shown in the following formula include the following groups. * represents a bonding position, n3 in the formula represents an integer of 3 or more, and n4 can be appropriately set so that the total number of carbon atoms in the group is 14 or more and 300 or less.

[0084] [ka]

[0085] Also, R a , R b Other embodiments of the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300" shown in the following formula include the following groups. * indicates the bond position.

[0086] [ka]

[0087] In the formula, n5 to n9 can be appropriately set so that the total number of carbon atoms in each group is 14 or more and 300 or less.

[0088] R a , R bPreferred specific examples of the "organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total number of carbon atoms of 14 to 300" include the following groups: 3,7,11,15-tetramethylhexadecyl group; 3,7,11-trimethyldodecyl group; 2,2,4,8,10,10-Hexamethyl-5-dodecanoyl group; 3,4,5-tri(3',7',11',15'-tetramethylhexadecyloxy)benzyl group; 3,5-Di(3',7',11',15'-tetramethylhexadecyloxy)benzyl group; Formula (f):

[0089] [ka]

[0090] (In the formula, * is the bonding position with Q, and n 10 is 23 to 34, and R 10 is a group represented by formula (I'). Formula (g):

[0091] [ka]

[0092] (In the formula, * is the bonding position with Q, and n 11 is 1 to 10; Formula (h):

[0093] [ka]

[0094] (In the formula, * is the bonding position with Q, and n 12 is 2 to 10. Formula (i):

[0095] [ka]

[0096] (In the formula, * is the bonding position with Q, and n 13 and n 14 are each independently 1 to 10; Formula (j):

[0097] [ka]

[0098] (In the formula, * is the bonding position with Q, and n 15 is 2 to 20.

[0099] More preferably, the following compounds are included: 2,4-Di(2',3'-dihydrophytyloxy)benzyl alcohol; 3,5-Di(2',3'-dihydrophytyloxy)benzyl alcohol; 4-(2',3'-dihydrophytyloxy)benzyl alcohol; 1-[(2-chloro-5-(2',3'-dihydrophytyloxy)phenyl)]-1-phenylmethanamine; 3,4,5-Tri(2',3'-dihydrophytyloxy)benzyl alcohol; 3,4,5-Tri(2',3'-dihydrophytyloxy)benzylamine; 4-(2',3'-dihydrophytyloxy)benzylamine; 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol (MTBPhy-OH); 4-(2',3'-dihydrophytyloxy)-2-methoxybenzyl alcohol; 4-(2',3'-dihydrophytyloxy)-2-methoxybenzylamine; 4-(2',3'-dihydrophytyloxy)-2-methylbenzyl alcohol; 4-(2',3'-dihydrophytyloxy)-2-methylbenzylamine; 2,2,4,8,10,10-Hexamethyl-5-dodecanoic acid (4-hydroxymethyl)phenylamide; 4-(3,7,11-trimethyldodecyloxy)benzyl alcohol; 2-(3,7,11-trimethyldodecyloxy)-9-phenylfluoren-9-ol; formula:

[0100] [ka]

[0101] (In the formula, n 16 represents 23 or 34. formula:

[0102] [ka]

[0103] (In the formula, n 17 represents 23 or 34. formula:

[0104] [ka]

[0105] (In the formula, n 18 represents 5 to 7; and formula:

[0106] [ka]

[0107] The method for producing the pseudo solid-phase protecting group is not particularly limited, but it can be produced from a raw material compound according to a method known per se (see

[0128] to

[0154] of International Publication No. 2012 / 029794) or a method similar thereto. In addition, the compound used as the raw material compound, for example, the group R 2 or R 4 The corresponding halides and the like are commercially available or can be produced according to a method known per se or a method analogous thereto.

[0108] Other preferred pseudo-solid-phase protecting groups in the present invention include those described in WO 2016 / 140232, WO 2003 / 018188, WO 2017 / 038650, and WO 2019 / 009317. Among these, WO 2017 / 038650 describes a benzyl compound whose end is modified with a silyl-based protecting group as a pseudo-solid-phase protecting group, and these pseudo-solid-phase protecting groups are also included in the present invention.

[0109] In addition to the above, pseudo solid-phase protecting groups that can be used in the present invention also include pseudo solid-phase protecting groups described in JP 2000-44493 A, WO 2006 / 104166 A, WO 2007 / 034812 A, WO 2007 / 122847 A, WO 2010 / 113939 A, WO 2010 / 104169 A, WO 2011 / 078295 A, and the like.

[0110] More specifically, an example of the pseudo solid-phase protecting group described above is a group that contains a pseudo solid-phase protecting group (e.g., a benzyl compound, a diphenylmethane compound, a fluorene compound, or a saturated carbocyclic compound thereof) that is soluble in a halogen-based solvent or an ether-based solvent and insoluble in a polar solvent and has a molecular weight of 300 or more, and that can be condensed with a carboxy group at the C-terminus.

[0111] One embodiment of the pseudo solid-phase protecting group that is soluble in the above halogen-based solvents or ether-based solvents and insoluble in polar solvents and has a molecular weight of 300 or more is a compound represented by the following formula (II). Among these, those having a molecular weight of 400 or more are preferred. Formula (II):

[0112] [ka]

[0113] [In the formula, Ring A represents a benzene ring or a cyclohexane ring; R1 is a hydrogen atom or b is a group represented by the following formula (a'), it may be taken together with R3 to represent a single bond to form a fluorene ring together with ring A and ring B: p R2s each independently represent an organic group having an aliphatic hydrocarbon group; p represents an integer of 1 to 4; In addition to p OR2, ring A further includes a halogen atom, a C optionally substituted with a halogen atom, 1-6 C optionally substituted with alkyl and halogen atoms 1-6 may have a substituent selected from the group consisting of alkoxy groups; R a represents a hydrogen atom or a phenyl group which may be substituted by a halogen atom; and R b is a hydrogen atom, or a group represented by the formula (a'):

[0114] [ka]

[0115] (In the formula, * indicates a bond position; Ring B represents a benzene ring or a cyclohexane ring; r represents an integer of 0 to 4; R4 each independently represents an organic group having an aliphatic hydrocarbon group; R3 may represent a hydrogen atom or, together with R1, represent a single bond to form a fluorene ring together with ring A and ring B; and Ring B may further include, in addition to r OR4, a halogen atom, a C optionally substituted with a halogen atom, 1-6 C optionally substituted with alkyl and halogen atoms 1-6 and optionally having a substituent selected from the group consisting of alkoxy groups; Y represents a hydroxy group, NHR (R represents a hydrogen atom, an alkyl group, or an aralkyl group), or a halogen atom.

[0116] The pseudo-solid-phase protecting group represented by the above formula (II) is bonded to a compound to be protected. That is, the pseudo-solid-phase protecting group in which Y is a hydroxyl group, -NHR group, or halogen atom protects the compound by condensing with the C-terminus of an amino acid or peptide, etc.

[0117] In the present specification, the "alkyl group" represented by R in the above formula (II) is a straight or branched C 1-30 Alkyl groups are preferred, and C 1-10 Alkyl groups, more preferably C 1-6 It is an alkyl group. Preferred specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc., and methyl and ethyl are particularly preferred.

[0118] In the present specification, the "aralkyl group" represented by R in the above formula (II) is 7-30 Aralkyl groups are preferred, and C 7-20 Aralkyl groups, more preferably C 7-16 Aralkyl group (C 6-10 Aryl-C 1-6 Preferred specific examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl, with benzyl being particularly preferred.

[0119] R is a hydrogen atom, C 1-6 Alkyl group or C 7-16 An aralkyl group is preferred, a hydrogen atom, methyl, ethyl or benzyl is more preferred, and a hydrogen atom is particularly preferred.

[0120] In the present specification, the "halogen atom" in the above formula (II) is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. In the present specification, the "halogen atom" represented by Y is preferably a chlorine atom, a bromine atom, or an iodine atom, and more preferably a bromine atom.

[0121] In this specification, "ring A" in the above formula (II) represents a benzene ring or a cyclohexane ring, either of which is preferred.

[0122] In this specification, "ring B" in the above formula (II) represents a benzene ring or a cyclohexane ring, either of which is preferable.

[0123] In this specification, the "organic group having an aliphatic hydrocarbon group" represented as R2 or R4 in the above formula (II) is a monovalent organic group having an aliphatic hydrocarbon group in its molecular structure.

[0124] The "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group" is a linear or branched, saturated or unsaturated aliphatic hydrocarbon group, preferably an aliphatic hydrocarbon group having 5 or more carbon atoms, more preferably an aliphatic hydrocarbon group having 5 to 60 carbon atoms, further preferably an aliphatic hydrocarbon group having 5 to 30 carbon atoms, and particularly preferably an aliphatic hydrocarbon group having 10 to 30 carbon atoms. The site of the "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group" is not particularly limited, and may be present at a terminal (monovalent group) or at another site (for example, a divalent group).

[0125] Examples of "aliphatic hydrocarbon groups" include monovalent groups such as an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, and an alkynyl group, and divalent groups derived therefrom. Preferred examples include monovalent groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, an octyl group, a decyl group, a dodecyl group, a lauryl group, a tridecyl group, a myristyl group, a cetyl group, a stearyl group, an arachyl group, a behenyl group, an oleyl group, and an isostearyl group, and divalent groups derived therefrom.

[0126] The moieties other than the "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group" can be set arbitrarily. For example, the linker may have moieties such as -O-, -S-, -COO-, -OCONH-, and -CONH-, as well as a hydrocarbon group (monovalent group or divalent group). Examples of the "hydrocarbon group" include an aliphatic hydrocarbon group, an aromatic aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, and an aromatic hydrocarbon group. Specifically, for example, monovalent groups such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, and an aralkyl group, and divalent groups derived therefrom, are used. Examples of the "alkyl group" include C 1-6 Preferred are alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc. Examples of the "alkenyl group" include, for example, 2-6 Alkenyl groups are preferred, and examples thereof include vinyl, 1-propenyl, allyl, isopropenyl, butenyl, and isobutenyl. Examples of the "alkynyl group" include, for example, 2-6 Alkynyl groups are preferred, such as ethynyl, propargyl, 1-propynyl, etc. Examples of the "cycloalkyl group" include, for example, C 3-6 Preferred are cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. An "aryl group" is, for example, C 6-14Aryl groups are preferred, such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, and 2-anthryl. 6-10 An aryl group is more preferable, and a phenyl group is particularly preferable. Examples of the "aralkyl group" include C 7-20 Aralkyl groups are preferred, and examples thereof include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl. Among these, C 7-16 Aralkyl group (C 6-10 Aryl-C 1-6 An alkyl group is more preferable, and benzyl is particularly preferable. The "hydrocarbon group" may be substituted with a substituent selected from a halogen atom (a chlorine atom, a bromine atom, a fluorine atom, an iodine atom), an alkyl group having 1 to 6 carbon atoms which may be substituted with one or more halogen atoms, an oxo group, and the like.

[0127] The "organic group having an aliphatic hydrocarbon group" constituting the OR2 group or OR4 group in the above formula (II) may have multiple "aliphatic hydrocarbon groups" due to branching, etc. When the "organic group having an aliphatic hydrocarbon group" has multiple "aliphatic hydrocarbon groups", they may be the same or different.

[0128] In the "organic group having an aliphatic hydrocarbon group" represented by R2 or R4 in the above formula (II), the lower limit of the total number of carbon atoms is preferably 5, more preferably 10, even more preferably 12, even more preferably 14, especially preferably 16, and particularly preferably 20. On the other hand, in the "organic group having an aliphatic hydrocarbon group" represented by R2 or R4, the upper limit of the total number of carbon atoms is preferably 200, more preferably 150, even more preferably 120, even more preferably 100, especially preferably 80, especially preferably 60, especially more preferably 40, and most preferably 30. Even when the peptide chain becomes long, the compound protected by the compound of the present invention becomes an oily product with good solubility in various organic solvents.

[0129] Specific preferred examples of the "OR2" group or the "OR4" group include dodecyloxy, cetyloxy, octadecyloxy, docosyloxy, docosyloxy-dodecyloxy, triacontyloxy, etc. There are a total of p or r "OR2" groups or "OR4" groups (p is an integer of 1 to 4, and r is an integer of 0 to 4), p is preferably 2 or 3, and r is preferably an integer of 0 to 2.

[0130] Preferable specific examples of the substituent that may be present in ring A or ring B in the above formula (II) include C 1-6 Alkoxy groups (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.) 1-4 alkoxy group), optionally substituted with one or more halogens 1-6 Alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.) 1-6 C substituted with halogens such as alkyl groups, trifluoromethyl, and trichloromethyl 1-6 alkyl group), or halogen atom, among which C 1-6 Alkoxy groups are preferred.

[0131] A preferred embodiment of the pseudo solid-phase protecting group represented by the above formula (II) is that in formula (II), Y is a hydroxy group; R1 is a hydrogen atom; R2 and / or R4 is an aliphatic hydrocarbon group having 5 to 60 carbon atoms; p is an integer from 1 to 3; The compound is one in which r is an integer of 0 to 2.

[0132] Another preferred embodiment of the pseudo solid-phase protecting group represented by the above formula (II) is Y is a hydroxy group; R a , R b and R1 are both hydrogen atoms; R2 is an aliphatic hydrocarbon group having 5 to 60 carbon atoms; The compound is one in which p is an integer of 1 to 3.

[0133] Another preferred embodiment of the pseudo solid-phase protecting group represented by the above formula (II) is Y is a hydroxy group; R a , R b and R1 are both hydrogen atoms; R2 is an alkyl group having 10 to 40 carbon atoms; The compound has p=2 or 3.

[0134] Another preferred embodiment of the pseudo solid-phase protecting group represented by the above formula (II) is Y is a hydroxy group; R a , R b and R1 are both hydrogen atoms; R2 is an alkyl group having 12 to 30 carbon atoms; The compound has p=2 or 3.

[0135] Another preferred embodiment of the pseudo solid-phase protecting group represented by the above formula (II) is Y is a hydroxy group; R a , R b and R1 are both hydrogen atoms; R2 is a benzyl group having 1 to 3 alkoxy groups having 12 to 30 carbon atoms; The compound is one in which p is an integer of 1 to 3.

[0136] Another preferred embodiment of the pseudo solid-phase protecting group represented by the above formula (II) is Y is a hydroxy group; R a , R b and R1 are both hydrogen atoms; R2 is a cyclohexylmethyl group having 1 to 3 alkoxy groups having 12 to 30 carbon atoms; The compound is one in which p is an integer of 1 to 3.

[0137] As described above, preferred examples of the pseudo-solid-phase protecting group of the present invention that is soluble in halogen-based solvents or ether-based solvents and insoluble in polar solvents and has a molecular weight of 300 or more include the following pseudo-solid-phase protecting groups.

[0138] 2,3,4-Trioctadecanoylbenzohydrol; [Phenyl(2,3,4-trioctadecyloxyphenyl)methyl]amine; 4,4'-Didoxybenzohydrol; Di(4-docoxyphenyl)methylamine; 4,4-Di(12-docosoxydodecyloxy)benzohydrol; Amino-bis[4-(12-dodecoxydodecyloxy)phenyl]methane; N-benzyl-[bis(4-docosyloxyphenyl)]methylamine; (4-Methoxy-phenyl)-[4-(3,4,5-tris-octadecyloxy-cyclohexylmethoxy)-phenyl]-methanol; {(4-Methoxy-phenyl)-[4-(3,4,5-tris-octadecyloxy-cyclohexylmethoxy)-phenyl]-methyl}-amine; [Bis-(4-doxoxy-phenyl)-methyl]-amine, and (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine) (NH2-Dpm(OPhy));

[0139] 4-(12'-docosyloxy-1'-dodecyloxy)benzyl alcohol; 4-(12'-docosyloxy-1'-dodecyloxy)-2-methoxybenzyl alcohol; 4-(12'-docosyloxy-1'-dodecyloxy)-2-methoxybenzylamine; 2-(12'-docosyloxy-1'-dodecyloxy)-4-methoxybenzyl alcohol; 2-(12'-docosyloxy-1'-dodecyloxy)-4-methoxybenzylamine; 2-Docosyloxy-4-methoxybenzyl alcohol; 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy)benzyl alcohol; 2-[3',5'-di(docosyloxy)benzyloxy]-4-methoxybenzyl alcohol; 2-Methoxy-4-[2',2',2'-tris(octadecyloxymethyl)ethoxy)benzyl alcohol; 2-Methoxy-4-[2',2',2'-tris(octadecyloxymethyl)ethoxy]benzylamine; 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 2-Methoxy-4-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 4-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 3,5-Dimethoxy-4-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; N-(4-hydroxymethyl-3-methoxyphenyl)-3,4,5-tris(octadecyloxy)cyclohexylcarboxamide; N-(5-hydroxymethyl-2-methoxyphenyl)-3,4,5-tris(octadecyloxy)cyclohexylcarboxamide; N-(4-hydroxymethylphenyl)-3,4,5-tris(octadecyloxy)cyclohexylcarboxamide; 1,22-bis[12-(4-hydroxymethyl-3-methoxyphenoxy)dodecyloxy]docosane; or 1,22-bis[12-(2-hydroxymethyl-5-methoxyphenoxy)dodecyloxy]docosane;

[0140] 2,4-Di(2',3'-dihydrophytyloxy)benzyl alcohol; 3,5-Di(2',3'-dihydrophytyloxy)benzyl alcohol; 4-(2',3'-dihydrophytyloxy)benzyl alcohol; 1-[(2-chloro-5-(2',3'-dihydrophytyloxy)phenyl)]-1-phenylmethanamine; 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl alcohol (TOBPhy-OH); 3,4,5-Tri(2',3'-dihydrophytyloxy)benzylamine; 4-(2',3'-dihydrophytyloxy)benzylamine; 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol (MTBPhy-OH); 4-(2',3'-dihydrophytyloxy)-2-methoxybenzyl alcohol; 4-(2',3'-dihydrophytyloxy)-2-methoxybenzylamine; 4-(2',3'-dihydrophytyloxy)-2-methylbenzyl alcohol; 4-(2',3'-dihydrophytyloxy)-2-methylbenzylamine; 2,2,4,8,10,10-Hexamethyl-5-dodecanoic acid (4-hydroxymethyl)phenylamide; 4-(3,7,11-trimethyldodecyloxy)benzyl alcohol; 2-(3,7,11-trimethyldodecyloxy)-9-phenylfluoren-9-ol;

[0141] 3,4,5-Tri(octadecyloxy)benzyl alcohol; 3,4,5-Tri(octadecyloxy)cyclohexanemethanol (TOC-OH); 2,4-Di(docosyloxy)benzyl alcohol; 4-Methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl alcohol; 4-Methoxy-2-[3',4',5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 2-Methoxy-4-[3',4',5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 4-[3',4',5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 3,5-Dimethoxy-4-[3',4',5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol; 2,4-Di(dodecyloxy)benzyl alcohol; 3,4,5-Tri(octadecyloxy)benzylamine; Bis(4-docosyloxyphenyl)methanol; Bis(4-docosyloxyphenyl)methylamine; 2-(12-docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-bromofluorene.

[0142] [Bis-(4-docoxy-phenyl)-methyl]-amine; 3,4,5-Tri(octadecyloxy)benzyl alcohol; 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy)benzyl alcohol; and 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol.

[0143] Among the above, the following pseudo solid-phase protecting groups are more preferred. (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine) (NH2-Dpm(OPhy)); 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl alcohol (TOBPhy-OH); 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol (MTBPhy-OH); 3,4,5-Tri(octadecyloxy)cyclohexanemethanol (TOC-OH); [Bis-(4-docoxy-phenyl)-methyl]-amine; 3,4,5-Tri(octadecyloxy)benzyl alcohol; 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy)benzyl alcohol; and 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol.

[0144] In addition to the above, the following pseudo solid-phase protecting groups are also preferred. (Pseudo solid-phase protecting groups described in WO 2010 / 104169) 2-Docosyloxy-9-(4-chlorophenyl)-9-fluorenol; 2-Docosyloxy-9-(4-chlorophenyl)-9-bromofluorene; 2,7-didoxy-9-(4-chlorophenyl)-9-bromofluorene; 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene; 1,12-Bis-[12-(2'-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane; 1,12-Bis-[12-(2'-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane; 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol; 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene; 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)cyclohexylmethoxy)-9-fluorenol; 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)cyclohexylmethoxy)-9-bromofluorene.

[0145] (The following pseudo solid-phase protecting groups described in WO 2010 / 104169): 2-Docosyloxy-9-(4-chlorophenyl)-9-fluorenol; 2-Docosyloxy-9-(4-chlorophenyl)-9-bromofluorene; 2,7-didoxy-9-(4-chlorophenyl)-9-bromofluorene; 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene; 1,12-Bis-[12-(2'-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane; 1,12-Bis-[12-(2'-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane; 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol; 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene; 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)cyclohexylmethoxy)-9-fluorenol; 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)cyclohexylmethoxy)-9-bromofluorene.

[0146] (Pseudo solid-phase protecting groups described in WO 2007 / 122847) 3,5-Didoxybenzyl alcohol; 2,4-Didoxybenzyl alcohol.

[0147] (Pseudo solid-phase protecting groups described in WO 2007 / 034812) 2,4-Bisoctadecyloxybenzyl alcohol.

[0148] (The following pseudo solid-phase protecting groups described in WO 2016 / 140232): 3-Didocosylaminobenzyl alcohol; 3-Diphytylaminobenzyl alcohol; N-(2',3'-dihydrophytyl)-N-(3-hydroxymethylphenyl)acetamide; N-Triacontyl-N-(3-hydroxymethylphenyl)acetamide; 3-(Aminomethyl)-N,N-didocosylaniline.

[0149] (Pseudo solid-phase protecting groups described in WO 2019 / 009317)

[0150] [ka]

[0151] [ka]

[0152] [ka]

[0153] [ka]

[0154] [ka]

[0155] [ka]

[0156] [ka]

[0157] [ka]

[0158] [ka]

[0159] [ka]

[0160] [ka]

[0161] Among the pseudo solid-phase protecting groups explained above, particularly preferred examples include the following pseudo solid-phase protecting groups. (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine) (NH2-Dpm(OPhy)); 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl alcohol (TOBPhy-OH); 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol (MTBPhy-OH); and 3,4,5-Tri(octadecyloxy)cyclohexanemethanol (TOC-OH).

[0162] The method for producing these pseudo solid-phase protecting groups is not particularly limited, and they can be produced from raw material compounds according to methods known per se (see JP 2000-44493 A, WO 2006 / 104166 A, WO 2007 / 034812 A, WO 2007 / 122847 A, WO 2010 / 113939 A, WO 2010 / 104169 A, WO 2011 / 078295 A, etc.) or methods equivalent thereto.

[0163] (C-terminal protecting group) Examples of the C-terminal protecting group include, in addition to the pseudo solid-phase protecting group described above, an ester type protecting group, an amide type protecting group, a hydrazide type protecting group, and the like.

[0164] As the ester-type protecting group, a substituted or unsubstituted alkyl ester or a substituted or unsubstituted aralkyl ester is preferably used. As the substituted or unsubstituted alkyl ester, a methyl ester, an ethyl ester, a tert-butyl ester, a cyclohexyl ester, a trichloroethyl ester, a phenacyl ester, etc. are preferably used. As the substituted or unsubstituted aralkyl ester, a benzyl ester, a p-nitrobenzyl ester, a p-methoxybenzyl ester, a diphenylmethyl ester, a 9-fluorenylmethyl (Fm) ester, a 4-picolyl (Pic) ester, etc. are preferably used.

[0165] Preferred examples of amide-type protecting groups include unsubstituted amides, primary amides such as N-methylamide, N-ethylamide, and N-benzylamide, and secondary amides such as N,N-dimethylamide, pyrrolidinylamide, and piperidinylamide.

[0166] As the hydrazide type protecting group, unsubstituted hydrazide, N-phenylhydrazide, N,N'-diisopropylhydrazide, and the like are preferably used.

[0167] Among these, ester-type protecting groups such as t-butyl esters and substituted or unsubstituted benzyl esters, which are stable under the deprotection conditions of the protecting group for the N-terminal amino group, are preferably used, and substituted or unsubstituted benzyl esters are particularly preferably used because they are relatively easy to synthesize.

[0168] (Protective group for side chain functional group) The amino acids or peptides used in the present invention often have functional groups such as amino, carboxy, and hydroxy groups that are subject to dehydration condensation reactions in addition to the amino or carboxy groups involved in the formation of peptide bonds. These functional groups are called side chain functional groups, in distinction from the amino and carboxy groups that form the peptide bonds of the main chain. The side chain functional groups do not necessarily need to be protected as long as they do not impair the essence of the present invention, but are preferably protected with an appropriate protecting group in order to prevent undesirable side reactions during the formation of peptide bonds by dehydration condensation reactions and during the deprotection reaction of the N-terminal amino group.

[0169] The protecting group of the side chain functional group is subject to certain restrictions in combination with the protecting group of the N-terminal amino group, similar to the above-mentioned protecting group of the C-terminus. That is, the protecting group of the side chain functional group is required to be retained until the desired amino acid sequence is completed without being removed even under the conditions for removing the protecting group of the N-terminus amino group (e.g., Fmoc group). The protecting group is not particularly limited as long as the side chain functional group does not cause undesirable side reactions during the formation of peptide bonds by dehydration condensation reaction and the deprotection reaction of the N-terminus amino group.

[0170] The protecting group for the side chain functional group is not particularly limited as long as it is stable under the deprotection conditions of the protecting group (temporary protecting group) for the N-terminal amino group, and examples of the protecting groups include those described in Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1985) and Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons (1999).

[0171] When the side chain functional group is a carboxy group, examples of the protecting group for the C-terminus include the same protecting groups as those mentioned above.

[0172] When the side chain functional group is a carboxy group, it may be protected by the above-mentioned pseudo solid-phase protecting group, and this embodiment is also included in the scope of the present invention.

[0173] When the side chain functional group is an amino group, examples of the protecting group include a urethane type protecting group, an acyl type protecting group, and a sulfonyl type protecting group.

[0174] Examples of urethane-type protecting groups include a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl (Boc) group, a benzyloxycarbonyl (Cbz) group, etc., and preferably a methoxycarbonyl group, an ethoxycarbonyl group, a Boc group, etc. Among them, the Boc group is particularly preferably used because it can be selectively deprotected under mild acidic conditions.

[0175] As the acyl-type protecting group, for example, a formyl group, an acetyl group, a trifluoroacetyl group, etc. are preferably used.

[0176] As the sulfonyl-type protecting group, for example, a p-toluenesulfonyl (Ts) group, a p-tolylmethanesulfonyl group, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, and the like are preferably used.

[0177] Similarly, for side chain functional groups other than those mentioned above, those that are stable under the deprotection conditions of the protecting group (temporary protecting group) for the N-terminal amino group (eg, Fmoc group, etc.) can be selected and used.

[0178] For example, when the functional group on the peptide is a hydroxy group (including a phenolic hydroxy group), examples of protecting groups include alkyl-type protecting groups, alkoxyalkyl-type protecting groups, acyl-type protecting groups, and alkylsilyl-type protecting groups.

[0179] Examples of the alkyl-type protecting group include a methyl group, an ethyl group, and a tert-butyl group.

[0180] Examples of alkoxyalkyl-type protecting groups include a methoxymethyl group (MOM group), a 2-tetrahydropyranyl group (THP group), and an ethoxyethyl group (EE group).

[0181] Examples of acyl-type protecting groups include an acetyl group, a pivaloyl group, and a benzoyl group.

[0182] Examples of alkylsilyl-type protecting groups include a trimethylsilyl group (TMS group), a triethylsilyl group (TES group), a tert-butyldimethylsilyl group (TBS group or TBDMS group), a triisopropylsilyl group (TIPS group), and a tert-butyldiphenylsilyl group (TBDPS group).

[0183] Other functional groups can also be protected with protective groups commonly used in the art. For example, the guanidino group of arginine can be protected with a p-toluenesulfonyl group, a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf) group, etc. The amide groups of asparagine and glutamine, and the imidazole group of histidine can be protected with a trityl group, a benzyloxymethyl group, etc. The SH group of cysteine ​​can be protected with a trityl group. Also, the indole group of tryptophan can be protected with a Boc group, a formyl group, etc.

[0184] Although the protecting groups for the functional groups on the peptide have been described above, those skilled in the art can carry out this step by appropriately selecting a protecting scheme in the art (e.g., Fmoc / tBu strategy, tBu / Bzl strategy, Bzl / tBu strategy, etc.) that is selected along with the overall synthetic strategy when carrying out the present invention. Among these, the Fmoc / tBu strategy is preferred.

[0185] After the desired peptide bond is formed, the side chain functional group may be deprotected as necessary.

[0186] (N-protected amino acids / N-protected peptides)

[0187] (Protective group for N-terminal amino group) The protecting group (temporary protecting group) of the N-terminal amino group of the acid component used in the condensation step in the present invention is, for example, a 9-fluorenylmethyloxycarbonyl group (hereinafter also referred to as an Fmoc group), a tert-butoxycarbonyl group (hereinafter also referred to as a Boc group), or a benzyloxycarbonyl group (hereinafter also referred to as a Cbz group (or Z group)), preferably an Fmoc group or a Boc group. Most preferably, it is an Fmoc group. Although the Cbz group may also be used in peptide synthesis, blowing in hydrogen gas using metal powder during catalytic reduction during deprotection is time-consuming in a flow reactor, and therefore, in the case of synthesizing longer peptides, the Fmoc group or the Boc group is more preferable. Of these, the Boc group may be preferable for sequences that do not contain Cys, Met, etc., but the Fmoc group is particularly preferable since it can be used universally in peptide synthesis regardless of the sequence, and while it is essential to remove the reaction residue derived from the Fmoc group generated during deprotection, in the present invention, the Fmoc group is particularly preferable from the viewpoint that this residue can be easily removed by using this pseudo solid-phase protecting group.

[0188] Suitable side chain functionalities include the same protecting groups as those mentioned above for C-protected amino acids / C-protected peptides.

[0189] The C-protected amino acids, C-protected peptides, N-protected amino acids, and N-protected peptides used in this step can be appropriately synthesized by those skilled in the art starting from known compounds according to methods known in the art or the methods described herein.

[0190] (Condensing agents, etc.)

[0191] This step is carried out using a condensing agent, a condensation promoter, etc. under peptide synthesis conditions generally used in the field of peptide chemistry.

[0192] Condensation agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-ethyl-N'-3-dimethylaminopropylcarbodiimide and its hydrochloride (EDC·HCl), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), [O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU), 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU), and O-benzotriazol-N,N,N',N'-tetramethyluronium Examples of the condensing agent include hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine-N-methylmorpholine (DMT-MM), and N'-cyclooctyl-N,N-dimethylurea (COMU). Among these, preferred is a water-soluble condensing agent, and more preferred is 2-chloro-4,6-dimethoxy-1,3,5-triazine-N-methylmorpholine (DMT-MM) or N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl).

[0193] The amount of the condensing agent used is, for example, 1 to 10 mol, and preferably 1 to 5 mol, per mol of the C-protected amino acid or C-protected peptide.

[0194] Examples of the condensation promoter include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1,2,3-triazole-5-carboxylic acid ethyl ester (HOCt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), [O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), ethyl 2-cyano-2-hydroxyiminoacetate (Oxyma), and the like, preferably HOBt and Oxyma.

[0195] The amount of the condensation promoter used is preferably 0.05 mol or more, more preferably 0.9 mol or more, and preferably 1.5 mol or less, more preferably 1.1 mol or less, per mol of the C-protected amino acid or C-protected peptide.

[0196] In this step, a particularly preferred combination of a condensation agent and a condensation promoter is N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt) or ethyl 2-cyano-2-hydroxyiminoacetate (Oxyma).

[0197] The above condensing agents and condensation promoters include those for activating the N-terminus as well as those for activating the C-terminus.

[0198] (Solvent)

[0199] The solvent used is a soluble organic solvent and is not particularly limited as long as it can dissolve each reaction component. A solvent that does not affect the reaction is preferable. Since the higher the solubility in the solvent, the better the reactivity can be expected, it is preferable to select a solvent in which each reaction component has a high solubility. In addition, since the liquids are separated by an oil-water separation means in a later step, a solvent that is not miscible with water is preferable. Specifically, ethers such as diethyl ether, tetrahydrofuran (THF), 1,4-dioxane, methyl-t-butyl ether, and cyclopentyl methyl ether (CPME) are preferable; acetate esters such as ethyl acetate and isopropyl acetate; halogenated hydrocarbons such as chloroform and dichloromethane; aromatic hydrocarbons such as toluene and xylene; and hydrocarbons such as hexane, heptane, and cyclohexane are preferable. Two or more of these solvents may be mixed in an appropriate ratio and used. From the viewpoint of being able to expect a good extraction operation and being industrially usable, tetrahydrofuran (THF), ethyl acetate, isopropyl acetate, chloroform, dichloromethane, cyclopentyl methyl ether (CPME), and toluene are preferred, tetrahydrofuran (THF), ethyl acetate, isopropyl acetate, cyclopentyl methyl ether (CPME), and toluene are more preferred, tetrahydrofuran (THF), ethyl acetate, isopropyl acetate, cyclopentyl methyl ether (CPME) are even more preferred, and tetrahydrofuran (THF), chloroform, isopropyl acetate, and cyclopentyl methyl ether (CPME) are even more preferred. Mixed solvents in which a polar organic solvent (dimethylformamide (DMF), etc.) is added to these solvents are also acceptable.

[0200] A preferred concentration of the solution containing a C-protected amino acid or a C-protected peptide, or the solution containing an N-protected amino acid or an N-protected peptide is 3 to 1000 times diluted with the solvent.

[0201] (Reaction conditions)

[0202] When a flow reactor is used in step (1), the mixing conditions, flow rate (flow velocity), and temperature control conditions of each solution can be appropriately selected. For the solution mixing conditions, it is preferable to use a T-shaped mixer or a cross-shaped mixer. The flow rate (flow velocity) varies depending on the piping, and appropriate conditions can be selected as appropriate. The temperature control condition is preferably 5 to 50°C.

[0203] [Regarding Process (A)] This step is carried out by introducing the reaction solution containing the N-protected C-protected peptide and water and / or a hydrophilic organic solvent into a flow reactor separately, simultaneously, or after mixing, and then washing with a continuous flow, and then subjecting the mixture to an oil-water separation means with a continuous flow to separate an "aqueous layer or hydrophilic organic solvent layer" from an organic layer, thereby separating the organic layer containing the N-protected C-protected peptide. This allows efficient purification and isolation of the N-protected C-protected peptide for the subsequent step.

[0204] Here, the N-protected C-protected peptide may be one obtained by carrying out step (1) of the present invention, or may be one obtained by discontinuous batch synthesis, or may be one purchased or obtained separately by other methods. Among these, the one obtained by carrying out step (1) of the present invention is preferred.

[0205] Water and / or a hydrophilic organic solvent can be used to remove and wash unreacted materials and by-products during extraction. A hydrophilic organic solvent can also be used instead of water.

[0206] The water also includes a mixed solution of water and salt. Neutral solutions are preferred. Specific examples of hydrophilic organic solvents include nitriles such as acetonitrile and propionitrile, ketones such as acetone, methyl ethyl ketone and 2-butanone, amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP), and sulfoxides such as dimethyl sulfoxide (DMSO). From the viewpoint of aiding solubility while not affecting layer separation, acetonitrile, N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP) are preferred. When only a hydrophilic organic solvent is used, for example, nitriles such as acetonitrile and propionitrile, and amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP) are preferred, and acetonitrile, N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP) are particularly preferred. In this case, by using an organic solvent that is not mixed with the hydrophilic organic solvent (for example, hydrocarbons such as hexane, heptane, cyclohexane, etc.; aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as chloroform and dichloromethane) as a reaction solvent, the hydrophilic organic solvent and the organic layer can be separated, and the organic layer can be separated. As the water and hydrophilic organic solvent, a mixed solvent of water and a hydrophilic organic solvent (acetonitrile, N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP)), or a mixed solvent of "a mixed solution of water and salt" and a hydrophilic organic solvent (acetonitrile, N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP)) is preferred.

[0207] Concentration conditions are preferably 5- to 100-fold dilution with respect to the substrate (N-protected, C-protected peptide).

[0208] Regarding washing, suitable conditions can be selected as appropriate with regard to the mixing method and washing time, but washing at a neutral pH is preferred, and a "mixture of water and salt" is preferred.

[0209] The oil-water separation means may be a continuous layer separation means having a structure including a filter or a gravity type continuous layer separation means. Any filter may be appropriately selected, and examples of the filter include a means having a structure including a filter such as a membrane filter. For example, a liquid-liquid separator system manufactured by Zaiput may be used. Gravity-type continuous layer separation means is a layer separation means that utilizes gravity, and is a continuous separating funnel that gradually removes liquid from the bottom and top layers of the separated layers. Examples include means that include a gravity settler and means that include an inclined plate. For example, Parkson's Lamella Gravity Settler can be mentioned.

[0210] When a flow reactor is used in step (A), the mixing conditions of each solution, the flow rate (flow velocity), and the temperature control conditions can be appropriately selected. Although not limited thereto, conditions in which a slug flow (a flow in which an organic layer and an aqueous layer are alternately repeated) is formed after the reaction solution containing the N-protected C-protected peptide and water and / or a hydrophilic organic solvent are introduced into the flow reactor are particularly preferred. Such conditions can be appropriately determined by a person skilled in the art.

[0211] [Regarding step (2)] This step is carried out by introducing an organic layer containing the N-protected C-protected peptide into a flow reactor in a continuous flow, and removing the protecting group of the N-terminal amino group with a continuous flow in the flow reactor to obtain a C-protected peptide (N-unprotected C-protected peptide) in which the N-terminal amino group is unprotected, the C-terminus is protected by a protecting group, and the side chain functional groups may be further protected by a protecting group, and in which the C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group.

[0212] The N-unprotected C-protected peptide may be one obtained by carrying out step (A) of the present invention, or may be one obtained by discontinuous batch synthesis, or may be one purchased or obtained separately by other methods. Among these, the one obtained by carrying out step (A) of the present invention is preferred.

[0213] The removal (deprotection) of the N-terminal protecting group of the N-protected C-protected peptide can be carried out according to a method known in the field of peptide synthesis. For example, deprotection can be carried out by treating with a base when the protecting group is an Fmoc group, by treating with an acid when the protecting group is a Boc group, by catalytic reduction when the protecting group is a Cbz group, etc. Deprotection is carried out by introducing a solution of N-protected and C-protected peptides in a solvent that does not affect the reaction into the appropriate flow reactor. Depending on the type of protecting group to be removed, a solution of a suitable deprotection reagent (organic base, acid, catalyst for catalytic reduction, etc.) in a solvent that does not affect the reaction, or a suspension in a solvent that does not affect the reaction, is also introduced into the flow reactor. When catalytic reduction is performed, hydrogen gas is also introduced into the flow reactor. The reaction components can be introduced into the flow reactor separately, simultaneously, or after mixing any two or more reaction components appropriately, and then mixed in a continuous flow in the flow reactor to allow the deprotection reaction to proceed.

[0214] The organic base that can be used for removing the Fmoc group is not particularly limited, and examples thereof include secondary amines such as diethylamine, piperidine, and morpholine, and tertiary amines such as diisopropylethylamine, dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN). The amount of the organic base used is, for example, 1 to 100 moles, preferably 1 to 10 moles, relative to 1 mole of the N-protected C-protected peptide.

[0215] More preferably, the removal of the Fmoc group is carried out by treatment with a non-nucleophilic organic base in a halogenated or ethereal solvent, and the deprotection is carried out in a solvent that does not affect the reaction.

[0216] Examples of the non-nucleophilic base include 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), among which DBU and DBN are preferred, and DBU is more preferred. The amount of the non-nucleophilic base used is preferably 0.8 equivalents or more, more preferably 1 equivalent or more, and preferably 5 equivalents or less, more preferably 3 equivalents or less, relative to the reaction substrate (N-Fmoc-protected C-protected amino acid or N-Fmoc-protected C-protected peptide).

[0217] The halogen-based solvent or the ether-based solvent may be a mixed solvent of two or more kinds. The halogen-based solvent or the ether-based solvent is preferably chloroform, dichloromethane, tetrahydrofuran (THF) or cyclopentyl methyl ether (CPME). These can be used in a mixture with N,N-dimethylformamide (DMF). In addition, propylene carbonate can be used as a solvent, and when propylene carbonate is used, a mixed solvent with N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP) is preferable.

[0218] The above-mentioned Fmoc group removal reaction is more preferably carried out in the presence of 3-mercaptopropionic acid, thiomalic acid, and cysteine, and particularly preferably in the presence of thiomalic acid.

[0219] When the temporary protecting group at the N-terminus is an Fmoc group, an excess amount of an organic base used in deprotection may adversely affect the reaction product during post-treatment such as solvent distillation, and therefore a neutralization step by addition of an acid may be incorporated prior to the next step.

[0220] The acid that can be used to remove the Boc group is not particularly limited, and may be mineral acids such as hydrogen chloride, sulfuric acid, nitric acid, etc., carboxylic acids such as formic acid, trifluoroacetic acid (TFA), sulfonic acids such as methanesulfonic acid, p-toluenesulfonic acid, etc., or a mixture thereof. Examples of the mixture include hydrogen bromide / acetic acid, hydrogen chloride / dioxane, and hydrogen chloride / acetic acid. Among them, when an acid that is not an aqueous solution, such as formic acid or methanesulfonic acid, is used in a non-aqueous system, it is possible to selectively remove the Boc group while leaving behind a pseudo-solid-phase protecting group, which is a protecting group of a carboxy group that is hydrolyzed under acidic conditions. In particular, sulfonic acids that are liquid at room temperature and water-soluble, such as methanesulfonic acid, are suitable because they can rapidly proceed with the reaction at room temperature with a relatively small amount of use when used in a non-aqueous system. The amount of the acid used is, for example, 1 to 100 moles, preferably 1 to 10 moles, per mole of the N-protected C-protected amino acid or N-protected C-protected peptide.

[0221] The catalyst that can be used for removing the Cbz group is not particularly limited, and examples thereof include palladium, etc. The amount of the catalyst used is, for example, 1 part by weight or more, preferably 5 parts by weight or more, and for example, 50 parts by weight or less, preferably 30 parts by weight, relative to 100 parts by weight of the N-protected C-protected peptide.

[0222] Examples of the solvent that does not affect the reaction include halogenated solvents such as chloroform, dichloromethane, 1,2-dichloroethane, etc.; aromatic hydrocarbons such as toluene, xylene, etc.; ether solvents such as diethyl ether, cyclopentyl methyl ether (CPME), tetrahydrofuran (THF), 1,4-dioxane, etc.; and mixtures thereof, and preferably chloroform, dichloromethane, or tetrahydrofuran (THF).

[0223] When a flow reactor is used in step (2), the mixing conditions of each solution, the flow rate (flow velocity), and the temperature control conditions can be appropriately selected. As for the mixing conditions of the solution, it is preferable to use a T-shaped mixer or a cross-shaped mixer. The flow rate (flow velocity) varies depending on the piping, and appropriate conditions can be selected as appropriate. The temperature control condition is preferably 5 to 50°C.

[0224] [Regarding Process (B)] This step is carried out by introducing the reaction solution containing the N-unprotected C-protected peptide and water and / or a hydrophilic organic solvent into a flow reactor separately, simultaneously, or after mixing, and then washing with a continuous flow, and then subjecting the mixture to an oil-water separation means with a continuous flow to separate an "aqueous layer or hydrophilic organic solvent layer" from an organic layer, thereby separating the organic layer containing the N-unprotected C-protected peptide. This allows efficient purification and isolation of the N-unprotected C-protected peptide for the subsequent step.

[0225] Here, the N-unprotected C-protected peptide may be one obtained by carrying out step (2) of the present invention, or may be one obtained by discontinuous batch synthesis, or may be one purchased or obtained separately by other methods. Among these, the one obtained by carrying out step (2) of the present invention is preferred.

[0226] Water and / or hydrophilic organic solvents can be used to remove and wash unreacted materials and by-products during extraction. Various hydrophilic organic solvents can be used instead of water.

[0227] The water also includes a mixed solution of water and sodium carbonate or potassium carbonate, or a mixed solution of water and table salt. Specific examples of hydrophilic organic solvents include nitriles such as acetonitrile and propionitrile, ketones such as acetone, methyl ethyl ketone and 2-butanone, amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP), and sulfoxides such as dimethyl sulfoxide (DMSO). From the viewpoint of aiding solubility while not affecting layer separation, acetonitrile, N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP) are preferred. When only a hydrophilic organic solvent is used, for example, nitriles such as acetonitrile and propionitrile, and amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP) are preferred, and acetonitrile, N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP) are particularly preferred. In this case, by using an organic solvent that is not mixed with the hydrophilic organic solvent (for example, hydrocarbons such as hexane, heptane, cyclohexane, etc.; aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as chloroform and dichloromethane) as a reaction solvent, the hydrophilic organic solvent and the organic layer can be separated, and the organic layer can be separated. As the water and hydrophilic organic solvent, a mixed solvent of water and a hydrophilic organic solvent (acetonitrile, N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP)), or any of "a mixed solution of water and sodium carbonate," "a mixed solution of water and potassium carbonate," or "a mixed solution of water and salt," and a mixed solvent of a hydrophilic organic solvent (acetonitrile, N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP)) is preferred.

[0228] Concentration conditions are preferably 2- to 100-fold dilution with respect to the substrate (N-unprotected, C-protected peptide).

[0229] Regarding the washing, suitable conditions can be selected as appropriate with regard to the mixing method and washing time, with washing in a basic solution being preferred.

[0230] In this step, washing can be performed multiple times (e.g., twice) to enhance the washing effect. For example, the first wash can be performed with a basic solvent such as an aqueous sodium carbonate solution, and the second wash can be performed with a neutral solvent such as saline. Those skilled in the art can appropriately select washing conditions such as the number of washes and washing solvent depending on the type of peptide produced, the type of reagent used, etc.

[0231] It is preferable to carry out a base treatment after removal of the Fmoc group, the Boc group, or the Cbz group, since this facilitates the progress of the condensation reaction in the next step. The base treatment can be any treatment that can free the N-terminal amino group of the peptide, and specific examples of the base treatment include washing with an aqueous sodium carbonate solution or an organic base.

[0232] The oil-water separation means may be a continuous layer separation means including a filter or a gravity type continuous layer separation means. Any filter may be selected as appropriate, and examples of such means include a means including a filter such as a membrane filter. For example, a liquid-liquid separator system from Zaiput may be used. The gravity type continuous layer separation means is a layer separation means that utilizes gravity, and is a continuous separating funnel that gradually removes liquid from the bottom and top layers of the separated layers. Examples of such means include a means including a gravity settler and a means including an inclined plate. For example, Parkson's Lamella Gravity Settler may be used.

[0233] When a flow reactor is used in step (B), the mixing conditions of each solution, the flow rate (flow velocity), and the temperature control conditions can be appropriately selected. Although not limited thereto, conditions in which a slug flow (a flow in which an organic layer and an aqueous layer are alternately repeated) is formed after the reaction solution containing the N-unprotected C-protected peptide and the water and / or hydrophilic organic solvent are introduced into the flow reactor are particularly preferred. Such conditions can be appropriately determined by a person skilled in the art.

[0234] The N-unprotected C-protected peptide obtained in step (B) can be further used as "C-protected peptide (C-protected peptide) in which the C-terminus is protected by a protecting group and the side chain functional groups may be further protected by a protecting group, at least one of which is a pseudo solid-phase protecting group" in step (1) and subjected to the cycle of steps (1), (A), (2) and (B) of the present invention, and the peptide chain can be elongated by appropriately repeating this cycle. In addition, when the N-unprotected C-protected peptide obtained in step (B) has a target peptide sequence, the final product peptide can be obtained by further removing the C-protecting group and the protecting group of the side chain functional group. All of these embodiments are within the scope of the present invention. This deprotection step can be appropriately carried out by a method known per se or by referring to the method for removing a protecting group described in this specification.

[0235] When applying the present invention, there is no particular upper limit to the target peptide chain length (number of amino acid residues), but this number of amino acid residues is preferably 100 or less, more preferably 50 or less, even more preferably 30 or less, and particularly preferably 20 or less. There is also no particular lower limit, but this number of amino acid residues is preferably 5 or more, more preferably 6 or more.

[0236] The above describes in detail each embodiment of steps (1), (A), (2) and (B) of the present invention. Steps (1), (A), (2) and (B) can be carried out continuously (continuously) using a flow reactor. This is one of the preferred embodiments. However, it is not necessary to carry out the steps in the order of step (1) → step (A) → step (2) → step (B), and it is also possible to start from an intermediate step (e.g., step (2)) depending on the raw material compound used. In this case, the raw material compound may be obtained, for example, by batch synthesis, and may not necessarily be obtained by carrying out the steps of the present invention. Such other embodiments are also included within the scope of the present invention.

[0237] (Removal of pseudo solid-phase protecting groups) The removal of the pseudo solid-phase protecting group is carried out after the above step (A) or the above step (B), thereby obtaining the final target peptide whose C-terminus is -COOH (for example, when Y in the above formula (I) or formula (II) is a hydroxyl group or a halogen atom) or -CONHR (for example, when Y in the above formula (I) or formula (II) is an NHR group).

[0238] Among the pseudo-solid-phase protecting groups described in JP 2000-44493 A, WO 2006 / 104166, WO 2007 / 034812, WO 2007 / 122847, WO 2010 / 113939, WO 2010 / 104169, WO 2011 / 078295, WO 2012 / 029794, WO 2016 / 140232, WO 2003 / 018188, WO 2017 / 038650, WO 2019 / 009317, etc., 2,4-O-alkyl type or 2,4,6-O-alkyl type pseudo-solid-phase protecting groups can selectively remove only the pseudo-solid-phase protecting group. Examples of the reagent used for selective deprotection of the pseudo solid-phase protecting group include acids (e.g., trifluoroacetic acid (hereinafter referred to as TFA), hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid), fluorine-substituted alcohols (e.g., trifluoroethanol (hereinafter referred to as TFE), hexafluoroisopropanol (hereinafter referred to as HFIP)), and the like, among which TFA, TFE, and HFIP are preferred. Examples of the solvent used for deprotection include chloroform, dichloromethane, 1,2-dichloroethane, and mixed solvents thereof. The concentration of the acid used for deprotection of the pseudo solid-phase protecting group is, for example, 0.1 w / v% to 5 w / v%, which is a weak acid treatment, and the concentration of the fluorine-substituted alcohol used for deprotection of the pseudo solid-phase protecting group is, for example, 10 w / v% to 100 w / v%.

[0239] On the other hand, it is also possible to remove the pseudo solid-phase protecting group derived from an aromatic compound (a compound having a pseudo solid-phase protecting group) in which Y in the formula (I) or the formula (II) is a hydroxy group, an -NHR group, or a halogen atom, together with the protecting groups of other side chains in the peptide. In that case, a conventional method used in the field, particularly in peptide synthesis, is used, but a method of adding an acid or the like is preferably adopted. As the acid, TFA, hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, and the like are used. Among them, TFA is particularly preferred. The amount of the acid used is appropriately set depending on the type of acid used, and an appropriate amount for removing the pseudo solid-phase protecting group is used. The amount of the acid used is preferably 3 moles or more, more preferably 5 moles or more, and preferably 100 moles or less, more preferably 50 moles or less, per mole of N-protected C-protected peptide or N-unprotected C-protected peptide. In addition to the use of these, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, BF3·etherate, and the like can also be added as a source of a further strong acid.

[0240] The protecting group of the side chain functional group can be appropriately removed according to a method commonly used in the art or a method for removing a protecting group described in this specification, depending on the type of the protecting group.

[0241] The reaction temperature is not particularly limited as long as the reaction proceeds, but is preferably 0° C. to 50° C., more preferably 0° C. to 30° C. The reaction time is, for example, 0.5 to 24 hours.

[0242] [Independent use of Process (A) / Process (B)] The present invention relates to a method for producing a peptide comprising carrying out the steps (1), (A), (2) and (B) described in detail above, and if necessary, a condensation step corresponding to step (1) or a deprotection step corresponding to step (2) may be carried out under conventional batch method conditions, and then the purification / separation step of the reaction solution containing the obtained product may be carried out by the method of step (A) or step (B) of the present invention, whereby the N-protected C-protected peptide or the N-unprotected C-protected peptide can be efficiently purified and isolated and efficiently subjected to another reaction step. Thus, a method for producing a peptide comprising independently utilizing step (A) and / or step (B) of the present invention is also included within the scope of the present invention. EXAMPLES

[0243] The present invention will be described in more detail below with reference to examples, but these examples do not limit the scope of the present invention in any way. Furthermore, unless otherwise specified, the reagents, devices, and materials used in the present invention are commercially available. Furthermore, when amino acids and the like are represented by abbreviations in this specification, each representation is based on the abbreviations according to the IUPAC-IUB Commission on Biochemical Nomenclature or the commonly used abbreviations in the relevant field.

[0244] Preparation Example 1: Reaction with pseudo solid-phase protecting groups Using Fmoc-Leu-OH as a raw material and 4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine (hereinafter referred to as NH2-Dpm(OPhy)) as a pseudo solid-phase protecting group, Fmoc-Leu-NH-Dpm(OPhy) was synthesized according to a standard method (see International Publication No. 2010 / 113939 and International Publication No. 2012 / 029794), and further, a de-Fmoc reaction was carried out using a base to obtain H-Leu-NH-Dpm(OPhy).

[0245] Example 1: Synthesis of a two-residue peptide (H-Tyr(tBu)-Leu-NH-Dpm(OPhy))

[0246] Equipment used (flow reactor, mixer, pump, oil-water separator) Flow reactor: PFA / PTFE tubing (inner diameter 1.0 mm or 1.6 mm) [Swagelok] Mixer: T-shaped mixer union tee SS-100-3 (1 / 16 inch OD) or SS-200-3 (1 / 8 inch OD) [Swagelok] Pump: Plunger pump YMCU-22 [YMC], Syringe pump YSP-301 [YMC], or Diaphragm pump Q-10-6T-P-M49 [Takumina] Oil-water separation membrane device: SEP-10 [Zaiput] Oil / water separation membrane: OB-2000-S-10 (hydrophobic membrane with pore size of 1.0 μm) [Zaiput]

[0247] Step (1): Condensation reaction A chloroform solution (Solution-1) containing 0.10 mmol / ml of H-Leu-NH-Dpm(OPhy) obtained in Production Example 1 and a dimethylformamide (DMF) solution (Solution-2) containing 0.11 mmol / ml of Fmoc-Tyr(tBu)-OH and 0.04 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at a flow rate of 0.20 ml / min using a pump (plunger pump YMCU-22 (syringe pump YSP-301 or diaphragm pump Q-10-6T-P-M49 can also be used)) and mixed using a T-shaped mixer (union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.05 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) was pumped at a flow rate of 0.40 ml / min and mixed with the mixture of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixture was then reacted for 10 min in a PFA tube (inner diameter 1.0 mm, length 10 m) to obtain a Fmoc-Tyr(tBu)-Leu-NH-Dpm(OPhy) solution.

[0248] Step (A): Extraction after condensation reaction The Fmoc-Tyr(tBu)-Leu-NH-Dpm(OPhy) solution obtained in step (1) above was pumped at a flow rate of 0.25 ml / min, and the 20 wt% NaCl aqueous solution was pumped at a flow rate of 0.17 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 2 m) for 10 min to inactivate the EDC·HCl remaining in the condensation reaction and to select it into the aqueous layer. The mixture was then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0249] Step (2): Fmoc removal reaction The organic layer (Solution-1) containing 0.10 mmol / ml of Fmoc-Tyr(tBu)-Leu-NH-Dpm(OPhy) obtained in the above step (A) was pumped at a flow rate of 0.56 ml / min, and a DMF solution (Solution-2) containing 1.10 mmol / ml of thiomalic acid and 3.30 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was pumped at a flow rate of 0.20 ml / min, respectively, and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixed solution was reacted for 10 min in a PFA tube (inner diameter 1.0 mm, length 10 m) to obtain a H-Tyr(tBu)-Leu-NH-Dpm(OPhy) solution. Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct.

[0250] Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction A chloroform solution (Solution-1) containing 0.10 mmol / ml of H-Tyr(tBu)-Leu-NH-Dpm(OPhy) obtained in the above step (2) was pumped at a flow rate of 0.34 ml / min, and a 5.0 wt% Na2CO3 aqueous solution (Solution-2) containing 0.33 mmol / ml of acetic acid was pumped at a flow rate of 0.37 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic and aqueous layers are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm, length 2 m) for 6 min to select the fulvene adduct generated during the Fmoc removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0251] Step (B)-2: Washing with a sodium chloride solution after washing with a sodium carbonate solution The H-Tyr(tBu)-Leu-NH-Dpm(OPhy) solution (Solution-1) obtained in the above step (B)-1 was pumped at a flow rate of 0.40 ml / min, and the 20 wt% NaCl aqueous solution (Solution-2) was pumped at a flow rate of 0.27 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic and aqueous layers are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm, length 2 m) for 6 min to select the fulvene adducts that could not be completely selected in the above step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0252] Example 2: Synthesis of 6-residue peptide (H-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-NH-Dpm(OPhy)) Equipment used (flow microreactor, coil tube reactor, tube, mixer, oil-water separation means) Flow Microreactor: R2 Plus [Vapourtec] Coil Tube Reactor: Coil Tube Reactor (PFA material, 1 / 8 inch OD, 1 / 16 inch ID, 5ml or 10ml capacity) [Vapourtec] or Coil Tube Reactor (PFA material, 1 / 8 inch OD, 1 / 16 inch ID, 5ml or 10ml capacity) [Idex] Tubing: Tubing (PFA material, 1 / 8 inch OD, 0.020 inch ID) [Idex] or Tubing (PFA material, 1 / 8 inch OD, 1 / 16 inch ID) [Idex] Mixer: T-shaped mixer (ETFE, 0.020 inch ID) [Idex], cross mixer (ETFE, 0.020 inch ID) [Idex], or T-shaped mixer (stainless steel, 1 / 8 inch OD) [Swagelok] Oil-water separation membrane device: SEP-10 [Zaiput] Oil-water separation membrane: Hydrophobic membrane with pore size of 1.0 μm or 0.5 μm [Zaiput]

[0253] Step (2): Fmoc removal reaction A solution of 2.2 g of Fmoc-Leu-NH-Dpm(OPhy) obtained in Production Example 1 dissolved in 37 ml of chloroform was introduced into one side of a T-shaped mixer (manufactured by Idex, made of ETFE material, inner diameter 0.020 inches) at a flow rate of 0.83 ml / min. At the same time, a solution of DBU and thiomalic acid dissolved in DMF (2.4 mol / l and 0.79 mol / l, respectively) was introduced into the other side of the T-shaped mixer at a flow rate of 0.28 ml / min. The two liquids were mixed at room temperature, and then passed through a coil tube reactor (manufactured by Vapourtec, made of PFA material, capacity 10 ml) from the outlet of the T-shaped mixer at room temperature to carry out the Fmoc removal reaction. The reaction mixture was introduced from the outlet of the coil tube reactor into one side of the next T-shaped mixer (manufactured by Idex, made of ETFE material, inner diameter 0.020 inch), and at the same time, a chloroform solution of acetic acid (2.1 mol / l) was introduced from the other side at a flow rate of 0.093 ml / min. After mixing the two liquids at room temperature, the mixture was passed from the outlet of the T-shaped mixer into a coil tube reactor (manufactured by Vapourtec, made of PFA material, capacity 5 ml) at room temperature to quench the Fmoc deprotection reaction.

[0254] Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction The quenched mixture obtained in the above step (2) was introduced from the outlet of the coil tube reactor into one side of a T-shaped mixer (manufactured by Swagelok, stainless steel material, outer diameter 1 / 8 inch) and at the same time, 7.5 w / w% Na2CO3 solution was introduced from the other side at a flow rate of 0.80 ml / min, and the two liquids were mixed at room temperature, and then passed through the outlet of the T-shaped mixer into a coil tube reactor (Idex tube, PFA material, outer diameter 1 / 16 inch, inner diameter 0.020 inch, capacity 10 ml) at room temperature for washing. A liquid separator consisting of Zaiput SEP-10 and a Zaiput hydrophobic membrane (pore size 1.0 μm) was installed at the outlet of the coil tube reactor, and the washing mixture was passed through it. The discharged liquid from the liquid separator was separated into an organic layer and an aqueous layer, and the aqueous layer was collected in a measuring cylinder (54 ml).

[0255] Step (B)-2: Washing with a sodium chloride solution after washing with a sodium carbonate solution The organic layer obtained in the above step (B)-1 was introduced into one side of a T-shaped mixer (manufactured by Swagelok, stainless steel material, outer diameter 1 / 8 inch). A solution of 20 w / w% NaCl aqueous solution and DMF mixed at 6:4 was introduced from the other side of the T-shaped mixer at a flow rate of 0.88 ml / min, and the two liquids were mixed at room temperature, and then the mixture was passed through a coil tube reactor (Idex tube, PFA material, outer diameter 1 / 16 inch, inner diameter 0.020 inch, capacity 5 ml) from the outlet of the T-shaped mixer at room temperature for washing. A liquid separator consisting of Zaiput SEP-10 with a Zaiput hydrophobic membrane (pore size 0.5 μm) was installed at the outlet of the coil tube reactor, and the washing mixture was passed through it. The discharged liquid from the liquid separator was separated into an organic layer and an aqueous layer, and each was collected in a measuring cylinder, and 68 ml of the organic layer containing H-Leu-NH-Dpm(OPhy) and 46 ml of the aqueous layer were obtained.

[0256] Step (1): Condensation reaction 25 ml of the organic layer of H-Leu-NH-Dpm(OPhy) obtained in the above step (B)-2 was introduced into one side of a cross-shaped mixer (manufactured by Idex, made of ETFE material, inner diameter 0.020 inches) at a flow rate of 0.68 ml / min. At the same time, a solution of Fmoc-Tyr(tBu)-OH and HOBt dissolved in DMF (0.50 mol / l and 0.10 mol / l, respectively) was introduced into the other side at a flow rate of 0.065 ml / min. Furthermore, a solution of EDC·HCl in chloroform (0.033 mol / l) was introduced into the remaining side at a flow rate of 0.042 ml / min. The three liquids were mixed at room temperature, and then passed through the outlet of the cross-shaped mixer into a coil tube reactor (manufactured by Vapourtec, made of PFA material, capacity 10 ml) heated to 40°C to carry out the condensation reaction.

[0257] Step (A): Extraction after condensation reaction The reaction mixture obtained in the above step (1) was introduced from the outlet of the coil tube reactor into one side of the next T-shaped mixer (manufactured by Idex, ETFE material, inner diameter 0.020 inches), and at the same time, 20 w / w% NaCl aqueous solution was introduced from the other side at a flow rate of 0.12 ml / min. The two liquids were mixed at room temperature, and then passed through the outlet of the T-shaped mixer into a coil tube reactor (manufactured by Vapourtec, PFA material, capacity 5 ml) at room temperature for washing. A liquid separator consisting of Zaiput's SEP-10 and a Zaiput's hydrophobic membrane (pore size 1.0 μm) was installed at the outlet of the coil tube reactor, and the washing mixture was passed through it. The discharged liquid from the liquid separator was separated into an organic layer and an aqueous layer, and each was collected in a measuring cylinder, and 44 ml of the organic layer containing Fmoc-Tyr(tBu)-Leu-NH-Dpm(OPhy) and 45 ml of the aqueous layer were obtained.

[0258] The Fmoc-Tyr(tBu)-Leu-NH-Dpm(OPhy) obtained above was further subjected to the Fmoc removal reaction step (2), step (B)-1, and step (B)-2 to obtain H-Tyr(tBu)-Leu-NH-Dpm(OPhy). The above steps (1), (A), (2), (B)-1, and (B)-2 were then repeated, with Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, and Fmoc-Ile-OH being used in that order as N-protected amino acids to obtain the protected peptide H-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-NH-Dpm(OPhy). LC / MS M + m / z 1688.3

[0259] Example 3: Synthesis of a 5-residue peptide (H-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy))

[0260] Equipment used (flow reactor, mixer, pump, oil-water separator) The same equipment as in Example 1 was used.

[0261] Step (1): Condensation reaction (2-residue peptide) A chloroform solution (Solution-1) containing 0.10 mmol / ml of H-Leu-NH-Dpm(OPhy) obtained in Production Example 1 and a DMF solution (Solution-2) containing 0.35 mmol / ml of Fmoc-Lys(Boc)-OH and 0.09 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.50 ml / min and 0.21 ml / min, respectively, using a pump (plunger pump YMCU-22 (syringe pump YSP-301 or diaphragm pump Q-10-6T-P-M49 can also be used)) and mixed using a T-shaped mixer (union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.54 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) was pumped at a flow rate of 0.13 ml / min and mixed with the mixture of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted in a PFA tube (inner diameter 1.0 mm) to obtain a Fmoc-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0262] Step (A): Extraction after condensation reaction (2-residue peptide) The Fmoc-Lys(Boc)-Leu-NH-Dpm(OPhy) solution obtained in step (1) above was pumped at a flow rate of 0.84 ml / min, and the 20 wt% NaCl aqueous solution was pumped at a flow rate of 0.56 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm) to inactivate the N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) remaining in the condensation reaction and to separate it into the aqueous layer. The oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm) was used to separate the two, and the permeated organic layer was collected.

[0263] Step (2): Fmoc removal reaction (two-residue peptide) The organic layer (Solution-1) containing 0.06 mmol / ml of Fmoc-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (A) was pumped at a flow rate of 0.73 ml / min, and a DMF solution (Solution-2) containing 0.67 mmol / ml of thiomalic acid and 2.02 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was pumped at a flow rate of 0.35 ml / min, and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixed solution was reacted in a PFA tube (inner diameter 1.0 mm) to obtain a H-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0264] Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc deprotection reaction (2-residue peptide) A chloroform solution containing H-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (2) (Solution-1) was pumped at a flow rate of 1.08 ml / min, and a 5.0 wt% Na2CO3 aqueous solution containing 0.26 mmol / ml of acetic acid (Solution-2) was pumped at a flow rate of 0.80 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct generated during the Fmoc removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0265] Step (B)-2: Washing with aqueous sodium chloride solution after washing with aqueous sodium carbonate solution (two-residue peptide) The H-Lys(Boc)-Leu-NH-Dpm(OPhy) solution (Solution-1) obtained in the above step (B)-1 was pumped at a flow rate of 1.08 ml / min, and the 20 wt% NaCl aqueous solution (Solution-2) was pumped at a flow rate of 0.88 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct that could not be completely selected in the above step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0266] Step (1): Condensation reaction (three-residue peptide) A chloroform solution (Solution-1) containing 0.05 mmol / ml of H-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (B)-2 and a dimethylformamide (DMF) solution (Solution-2) containing 0.18 mmol / ml of Fmoc-Glu(OtBu)-OH and 0.05 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.50 ml / min and 0.21 ml / min, respectively, using a pump (plunger pump YMCU-22 (syringe pump YSP-301 or diaphragm pump Q-10-6T-P-M49 can also be used)) and mixed using a T-shaped mixer (union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.27 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) was pumped at a flow rate of 0.13 ml / min and mixed with the mixture of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted in a PFA tube (inner diameter 1.0 mm) to obtain a Fmoc-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0267] Step (A): Extraction after condensation reaction (three-residue peptide) The Fmoc-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution obtained in step (1) above was pumped at a flow rate of 0.84 ml / min, and the 20 wt% NaCl aqueous solution was pumped at a flow rate of 0.56 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm) to inactivate the EDC·HCl remaining in the condensation reaction and to separate it into the aqueous layer. The oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm) was used to separate the two, and the permeated organic layer was collected.

[0268] Step (2): Fmoc removal reaction (three-residue peptide) The organic layer (Solution-1) containing 0.03 mmol / ml of Fmoc-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (A) was pumped at a flow rate of 0.73 ml / min, and a DMF solution (Solution-2) containing 0.26 mmol / ml of thiomalic acid and 0.79 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was pumped at a flow rate of 0.35 ml / min, and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixed solution was reacted in a PFA tube (inner diameter 1.0 mm) to obtain a H-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0269] Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc deprotection reaction (three-residue peptide) A chloroform solution (Solution-1) containing H-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (2) was pumped at a flow rate of 1.08 ml / min, and a 5.0 wt% Na2CO3 aqueous solution (Solution-2) containing 0.10 mmol / ml of acetic acid was pumped at a flow rate of 0.80 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct generated during the Fmoc removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0270] Step (B)-2: Washing with aqueous sodium chloride solution after washing with aqueous sodium carbonate solution (three-residue peptide) The H-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution (Solution-1) obtained in the above step (B)-1 was pumped at a flow rate of 1.08 ml / min, and the 20 wt% NaCl aqueous solution (Solution-2) was pumped at a flow rate of 0.88 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct that could not be completely selected in the above step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected to obtain the following. H-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPy) LC-MS M + m / z 1303.3

[0271] Step (1): Condensation reaction (four-residue peptide) A chloroform solution (Solution-1) containing 0.04 mmol / ml of H-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (B)-2 and a dimethylformamide (DMF) solution (Solution-2) containing 0.15 mmol / ml of Fmoc-Ala-OH·HO and 0.04 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.50 ml / min and 0.21 ml / min, respectively, using a pump (plunger pump YMCU-22 (syringe pump YSP-301 or diaphragm pump Q-10-6T-P-M49 can also be used)) and mixed using a T-shaped mixer (union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.23 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) was pumped at a flow rate of 0.13 ml / min and mixed with the mixture of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted in a PFA tube (inner diameter 1.0 mm) to obtain a Fmoc-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0272] Step (A): Extraction after condensation reaction (four-residue peptide) The Fmoc-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution obtained in the above step (1) was pumped at a flow rate of 0.84 ml / min, and the 20 wt% NaCl aqueous solution was pumped at a flow rate of 0.56 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The slug flow (a flow in which organic and aqueous layers alternate) that followed the joining was passed through a PFA tube (inner diameter 1.6 mm) to inactivate the EDC·HCl remaining in the condensation reaction and to separate it into the aqueous layer. The oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm) was then used to separate the two, and the permeated organic layer was collected.

[0273] Step (2): Fmoc removal reaction (four-residue peptide) The organic layer (Solution-1) containing 0.03 mmol / ml of Fmoc-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (A) was pumped at a flow rate of 0.73 ml / min, and a DMF solution (Solution-2) containing 0.27 mmol / ml of thiomalic acid and 0.82 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was pumped at a flow rate of 0.35 ml / min, and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixed solution was reacted in a PFA tube (inner diameter 1.0 mm) to obtain a H-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0274] Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc deprotection reaction (four-residue peptide) A chloroform solution (Solution-1) containing H-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (2) was pumped at a flow rate of 1.08 ml / min, and a 5.0 wt% Na2CO3 aqueous solution (Solution-2) containing 0.11 mmol / ml of acetic acid was pumped at a flow rate of 0.80 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct generated during the Fmoc removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0275] Step (B)-2: Washing with aqueous sodium chloride solution after washing with aqueous sodium carbonate solution (four-residue peptide) The H-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution (Solution-1) obtained in the above step (B)-1 was pumped at a flow rate of 1.08 ml / min, and the 20 wt% NaCl aqueous solution (Solution-2) was pumped at a flow rate of 0.88 ml / min, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct that could not be completely selected in the above step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected to obtain the following. H-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPy)LC-MS M + m / z 1374.4

[0276] Step (1): Condensation reaction (5-residue peptide) A chloroform solution (Solution-1) containing 0.02 mmol / ml of H-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (B)-2 and a dimethylformamide (DMF) solution (Solution-2) containing 0.06 mmol / ml of Fmoc-Glu(OtBu)-OH and 0.02 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.50 ml / min and 0.21 ml / min, respectively, using a pump (plunger pump YMCU-22 (syringe pump YSP-301 or diaphragm pump Q-10-6T-P-M49 can also be used)), and mixed using a T-shaped mixer (union T SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.10 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC·HCl) was pumped at a flow rate of 0.13 ml / min and mixed with the mixture of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted in a PFA tube (inner diameter 1.0 mm) to obtain a Fmoc-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0277] Step (A): Extraction after condensation reaction (5-residue peptide) The Fmoc-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution obtained in the above step (1) was pumped at a flow rate of 0.84 ml / min, and 20 wt% NaCl aqueous solution was pumped at a flow rate of 0.56 ml / min, respectively, and the two were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic and aqueous layers were alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to inactivate the EDC·HCl remaining in the condensation reaction and to select it into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0278] Step (2): Fmoc removal reaction (five-residue peptide) The organic layer (Solution-1) containing 0.10 mmol / ml of Fmoc-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (A) was pumped at a flow rate of 0.73 ml / min, and a DMF solution (Solution-2) containing 0.08 mmol / ml of thiomalic acid and 0.25 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was pumped at a flow rate of 0.35 ml / min, respectively, and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixed solution was reacted in a PFA tube (inner diameter 1.0 mm) to obtain a H-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution.

[0279] Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc deprotection reaction (5-residue peptide) A chloroform solution (Solution-1) containing H-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) obtained in the above step (2) was pumped at a flow rate of 1.08 ml / min, and a 5.0 wt% Na2CO3 aqueous solution (Solution-2) containing 0.03 mmol / ml of acetic acid was pumped at a flow rate of 0.80 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct generated during the Fmoc removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected.

[0280] Step (B)-2: Washing with aqueous sodium chloride solution after washing with aqueous sodium carbonate solution (5-residue peptide) The H-Glu(OtBu)-Ala-Glu(OtBu)-Lys(Boc)-Leu-NH-Dpm(OPhy) solution (Solution-1) obtained in the above step (B)-1 was pumped at a flow rate of 1.08 ml / min, and the 20 wt% NaCl aqueous solution (Solution-2) was pumped at a flow rate of 0.88 ml / min, and the two solutions were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm) to select the fulvene adduct that could not be completely selected in the above step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm), and the permeated organic layer was collected. LC / MS M + m / z 1559.2

[0281] The devices used in the following Examples 4 to 11 are as follows: Equipment used (flow reactor, mixer, pump, oil-water separator) Pumps: Plunger pump YMCU-22 [YMC], Syringe pump YSP-301 [YMC], Diaphragm pump Q-10-6T-P-M49 [Takumina] Flow reactor: PFA / PTFE tube (inner diameter 1.0 mm or 1.6 mm) [available from Swagelok, YMC, etc.); SUS piping (inner diameter 1.0 mm) [GL Sciences] Mixer: T-shaped mixer: Union Tee SS-100-3 (outer diameter 1 / 16 inch) or SS-200-3 (outer diameter 1 / 8 inch) [Swagelok], T-shaped micro mixer (inner diameter 1.0 mm) [Sankoh Seiki Kogyo] Oil-water separation membrane device: SEP-10 [Zaiput] Oil / water separation membrane: OB-2000-S-10 (hydrophobic membrane with pore size 1.0 μm), OB-900-S-10 (hydrophobic membrane with pore size 0.5 μm) [Zaiput]

[0282] Example 4: 15-residue peptide ( Contains D-amino acids)(H-Glu(OtBu)-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy)

[0283] Step (B)-1: Washing with an aqueous sodium carbonate solution after the Fmoc removal reaction / Step (B)-2: Washing with an aqueous sodium chloride solution after washing with an aqueous sodium carbonate solution A chloroform solution containing 0.08 mmol / ml of H-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy prepared by the batch method (Solution-1) was mixed with 5.0 wt% Na2CO3 aqueous solution and dimethylformamide (DMF) in a volume ratio of 8:2 (Solution-2) at 0.80 ml / min using a diaphragm pump. The two streams were joined using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch) at a flow rate of 1000 rpm, and the resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 10.0 m) for 11 min to separate the fulvene adduct produced during the Fmoc removal reaction into the aqueous layer, followed by separation using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm). The organic layer was then mixed with a 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 (Solution 3) using a diaphragm pump at a flow rate of 0.88 ml / min in a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which the organic layer and aqueous layer alternate) was then passed through a PFA tube (inner diameter 1.6 m). The mixture was passed through a tubing (5.0 mm, length 5.0 m) for 5 minutes to remove the fulvene adducts that could not be completely removed in step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-900-S-10; hydrophobic membrane with a pore size of 0.5 μm) to recover the organic layer containing H-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. LC / MS M+ m / z 2734.1 *) TOBPhy represents the 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl group.

[0284] Step (1): Condensation reaction / Step (A): Extraction after condensation reaction A chloroform solution (solution-1) containing 0.04 mmol / ml of H-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained by successively carrying out the above steps (B)-1 and (B)-2, and a dimethylformamide (DMF) solution (solution-2) containing 0.14 mmol / ml of Fmoc-Ala-OH and 0.04 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.500 and 0.208 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.23 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) was fed at a flow rate of 0.133 ml / min and mixed with the mixed solution of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 18 min in a PFA tube (inner diameter 1.6 mm, length 7.5 m). Furthermore, a 20 wt% NaCl aqueous solution (Solution-4) was fed at 0.56 ml / min using a diaphragm pump and mixed with the mixed solution of Solutions-1, 2, and 3 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 minutes to inactivate the EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer. The organic layer containing Fmoc-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy was then recovered by separation using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm).

[0285] Step (2): Fmoc removal reaction / Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction / Step (B)-2: Washing with aqueous sodium chloride solution after washing with aqueous sodium carbonate solution An organic layer (solution-1) containing 0.03 mmol / mL of Fmoc-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained by successively carrying out the above steps (1) and (A) and a dimethylformamide (DMF) solution (solution-2) containing 0.33 mmol / mL of thiomalic acid and 0.98 mmol / mL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.732 and 0.346 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc-removal reaction solution was mixed with 5.0 wt% Na2CO3 aqueous solution and dimethylformamide (DMF) in a volume ratio of 8:2 (solution-3), which was then mixed with the reaction solution in a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch) at a flow rate of 0.80 ml / min using a diaphragm pump. The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 10.0 m) for 11 min to separate the fulvene adduct generated during the Fmoc-removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm).The organic layer was then mixed with a 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 (Solution-4) using a diaphragm pump at a flow rate of 0.88 ml / min in a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which the organic layer and the aqueous layer alternate) was then passed through a PFA tube (inner diameter 1.6 mm, The solution was passed through a tube (length: 5.0 m) for 5 minutes to remove fulvene adducts that could not be completely removed in step (B)-1 into the aqueous layer, and then the mixture was separated using an oil-water separation membrane (OB-900-S-10; hydrophobic membrane with a pore size of 0.5 μm) to recover the organic layer containing H-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. LC / MS M + m / z 2804.9

[0286] Step (1): Condensation reaction / Step (A): Extraction after condensation reaction A chloroform solution (solution-1) containing 0.03 mmol / ml of H-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained by successively carrying out the above-mentioned step (2), step (B)-1, and step (B)-2, and a dimethylformamide (DMF) solution (solution-2) containing 0.11 mmol / ml of Fmoc-Glu(OtBu)-OH and 0.03 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.500 and 0.208 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.23 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) was fed at a flow rate of 0.133 ml / min and mixed with the mixed solution of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixture was then reacted for 18 min in a PFA tube (inner diameter 1.6 mm, length 7.5 m). Furthermore, a 20 wt% NaCl aqueous solution (Solution-4) was fed at 0.56 ml / min using a diaphragm pump and mixed with the mixed solution of Solutions-1, 2, and 3 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 min to inactivate the EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer. The organic layer containing Fmoc-Glu(OtBu)-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy was then recovered by separation using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm).

[0287] Step (2): Fmoc removal reaction / Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction / Step (B)-2: Washing with aqueous sodium chloride solution after washing with aqueous sodium carbonate solution The organic layer (solution-1) containing 0.02 mmol / ml of Fmoc-Glu(OtBu)-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained by successively carrying out the above steps (1) and (A) and 0.26 mmol / ml of thiomalic acid and 0.78 mmol / ml of 1,8-diazabicyclo[5.4 Dimethylformamide (DMF) solutions (solution-2) containing 0.0-7-undecene (DBU) were pumped at flow rates of 0.732 and 0.346 ml / min using a plunger pump (a syringe pump or diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was converted to a fulvene adduct by reacting it with thiomalic acid. This Fmoc-removal reaction solution was mixed with 5.0 wt% Na2CO3 aqueous solution and dimethylformamide (DMF) in a volume ratio of 8:2 (solution-3) and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch) at a flow rate of 0.80 ml / min using a diaphragm pump. The mixed solution became a slug flow (a flow in which organic and aqueous layers alternately repeat) and was passed through a PFA tube (inner diameter 1.6 mm, length 10.0 m) for 11 min to separate the fulvene adducts generated during the Fmoc-removal reaction into the aqueous layer, and then separated using an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm).The organic layer was then mixed with a 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 (Solution-4) using a diaphragm pump at a flow rate of 0.88 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which the organic layer and the aqueous layer alternate) was then passed through a PFA tube (inner diameter 1.6 mm, length 5.0 mm) and separated into 100 mL of water. The mixture was passed through a 300 suction column (300 mm) for 5 min to remove the fulvene adducts that could not be completely removed in step (B)-1 into the aqueous layer, and then separated using an oil-water separation membrane (OB-900-S-10; a hydrophobic membrane with a pore size of 0.5 μm) to recover the organic layer containing H-Glu(OtBu)-Ala-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. LC / MS M + m / z 2991.18

[0288] Example 5: Synthesis of a two-residue peptide (H-Asn(Trt)-Leu-NH-Dpm(OPhy))

[0289] Step (1): Condensation reaction To a chloroform solution (Solution-1) containing 0.03 mmol / ml of H-Leu-NH-Dpm(OPhy) obtained in Preparation Example 1, 1.50 eq. of 1-hydroxybenzotriazole (HOBt) anhydride, Fmoc-Asn(Trt)-OH, and N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) were added to obtain Fmoc-Asn(Trt)-Leu-NH-Dpm(OPhy). Then, 1.50 eq. of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) and thiomalic acid were added to quench the excess active ester.

[0290] Step (A): Extraction after condensation reaction The solution containing 0.03 mmol / ml of Fmoc-Asn(Trt)-Leu-NH-Dpm(OPhy) obtained in the above step (1) (Solution-1) and 20 wt% NaCl aqueous solution (Solution-2) were fed at 1.500 and 1.50 ml / min using a plunger pump and a diaphragm pump, respectively, and were joined using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the joining was passed through a PFA tube (inner diameter 1.6 mm, length 7.5 m) for 5 min to inactivate EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer, and then separated with a separatory funnel to recover the organic layer.

[0291] Step (2): Fmoc removal reaction / Step (B)-3: Washing with aqueous sodium chloride solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.04 mmol / ml of Fmoc-Asn(Trt)-Leu-NH-Dpm(OPhy) obtained in the above step (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.44 mmol / ml of thiomalic acid and 1.60 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.700 and 0.300 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with 20.0 wt% NaCl aqueous solution using a diaphragm pump at a flow rate of 0.50 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 2.0 m) for 3 min to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer containing H-Asn(Trt)-Leu-NH-Dpm(OPhy) was then recovered by separation using a separatory funnel.

[0292] Step (2): Fmoc removal reaction / Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.04 mmol / mL of Fmoc-Asn(Trt)-Leu-NH-Dpm(OPhy) obtained in the above step (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.44 mmol / mL of thiomalic acid and 1.60 mmol / mL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.700 and 0.300 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with a 5.0 wt% Na2CO3 aqueous solution (Solution-3) using a diaphragm pump at a flow rate of 0.50 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The mixed solution, which became a slug flow (a flow in which organic and aqueous layers alternately repeat), was passed through a PFA tube (inner diameter 1.6 mm, length 2.0 m) for 3 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer was then separated using a separatory funnel to recover the organic layer.

[0293] Step (B)-2: Washing with a sodium chloride solution after washing with a sodium carbonate solution The organic layer (solution-1) obtained in step (B)-1 and a liquid (solution-2) obtained by mixing 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 were mixed using a plunger pump and a diaphragm pump at flow rates of 1.500 and 1.50 ml / min, respectively, in a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the mixture was mixed was passed through a PFA tube (inner diameter 1.6 mm, length 7.5 m) for 5 min to select the fulvene adduct that could not be completely selected in step (B)-1 into the aqueous layer, and then the organic layer containing H-Asn(Trt)-Leu-NH-Dpm(OPhy) was recovered by separation using a separatory funnel. LC / MS M + m / z 1245.9

[0294] Example 6: Synthesis of two-residue peptide (H-Asp(OtBu)-Leu-NH-Dpm(OPhy))

[0295] Step (1): Condensation reaction / Step (A): Extraction after condensation reaction A chloroform solution (Solution-1) containing 0.10 mmol / ml of H-Leu-NH-Dpm (OPhy) obtained in Production Example 1 and a dimethylformamide (DMF) solution (Solution-2) containing 0.35 mmol / ml of Fmoc-Asp (OtBu)-OH and 0.09 mmol / ml of ethyl 2-cyano-2-hydroxyiminoacetate (Oxyma) were pumped at flow rates of 0.500 and 0.208 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.55 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) was fed at a flow rate of 0.133 ml / min and mixed with the mixed solution of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 9 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Furthermore, a 20 wt% NaCl aqueous solution (Solution-4) was fed at 0.56 ml / min using a diaphragm pump and mixed with the mixed solution of Solutions-1, 2, and 3 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 min to inactivate the EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer. The organic layer containing Fmoc-Asp(OtBu)-Leu-NH-Dpm(OPhy) was then recovered by separation in a separatory funnel.

[0296] Step (2): Fmoc removal reaction / Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.09 mmol / ml of Fmoc-Asp(OtBu)-Leu-NH-Dpm(OPhy) obtained by successively carrying out the above steps (1) and (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.91 mmol / ml of thiomalic acid and 2.74 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.732 and 0.346 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with a 5.0 wt% Na2CO3 aqueous solution (Solution-3) using a diaphragm pump at a flow rate of 0.80 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 5 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer was then separated using a separatory funnel to recover the organic layer.

[0297] Step (B)-2: Washing with a sodium chloride solution after washing with a sodium carbonate solution The organic layer (Solution-1) obtained in step (B)-1 and a mixture (Solution-2) of 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 were mixed using a plunger pump and a diaphragm pump at flow rates of 1.500 and 1.50 ml / min, respectively, in a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The slug flow (a flow in which organic and aqueous layers are alternately repeated) after the mixture was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 3 min to select the fulvene adducts that could not be completely selected in step (B)-1 into the aqueous layer, and the organic layer containing H-Asp(OtBu)-Leu-NH-Dpm(OPhy) was then recovered by separation using a separatory funnel. LC / MS 2M + m / z 2120.7

[0298] Example 7: Synthesis of a two-residue peptide (H-Ala-Leu-NH-Dpm(OPhy))

[0299] Step (1): Condensation reaction / Step (A): Extraction after condensation reaction A chloroform solution (Solution-1) containing 0.10 mmol / ml of H-Leu-NH-Dpm(OPhy) obtained in Production Example 1 and a dimethylformamide (DMF) solution (Solution-2) containing 0.35 mmol / ml of Fmoc-Ala-OH and 0.09 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.500 and 0.208 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.55 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) was fed at a flow rate of 0.133 ml / min and mixed with the mixed solution of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 9 min in a SUS pipe (inner diameter 1.0 mm, length 10.0 m) while keeping the temperature in a water bath at 43°C. Furthermore, a 20 wt% NaCl aqueous solution (Solution-4) was fed at 0.56 ml / min using a diaphragm pump and mixed with the mixed solution of Solutions-1, 2, and 3 using a T-shaped mixer (Sanko Seiki Kogyo Micromixer; inner diameter 1.0 mm). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a stainless steel pipe (inner diameter 1.0 mm, length 7.0 m) for 4 min to inactivate the EDC.HCl remaining in the condensation reaction and to remove it into the aqueous layer. The organic layer containing Fmoc-Ala-Leu-NH-Dpm (OPhy) was then recovered by separation in a separatory funnel.

[0300] Step (2): Fmoc removal reaction / Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.09 mmol / ml of Fmoc-Ala-Leu-NH-Dpm (OPhy) obtained by the continuous implementation of the above steps (1) and (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.92 mmol / ml of thiomalic acid and 2.75 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.732 and 0.346 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 7 min in a SUS pipe (inner diameter 1.0 mm, length 10.0 m) while keeping the temperature in a water bath at 43°C. Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with a 5.0 wt% K2CO3 aqueous solution (Solution-3) using a diaphragm pump at a flow rate of 0.80 ml / min using a T-shaped mixer (a micromixer manufactured by Sanko Seiki Kogyo Co., Ltd.; inner diameter 1.0 mm). The mixed solution, which became a slug flow (a flow in which organic and aqueous layers alternately repeat), was passed through a SUS pipe (inner diameter 1.0 mm, length 7.0 m) for 3 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer was then separated using a separatory funnel to recover the organic layer.

[0301] Step (B)-2: Washing with a sodium chloride solution after washing with a sodium carbonate solution The organic layer (Solution-1) obtained in step (B)-1 and a mixture (Solution-2) of 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 were mixed using a plunger pump and a diaphragm pump at flow rates of 1.000 and 1.00 ml / min, respectively, in a T-shaped mixer (Micromixer manufactured by Sanko Seiki Kogyo Co., Ltd.; inner diameter 1.0 mm). The slug flow (a flow in which organic and aqueous layers are alternately repeated) after the mixture was passed through a SUS pipe (inner diameter 1.0 mm, length 7.0 m) for 3 min to select the fulvene adducts that could not be completely selected in step (B)-1 into the aqueous layer, and the organic layer containing H-Ala-Leu-NH-Dpm (OPhy) was recovered by separation using a separatory funnel. LC / MS 2M + m / z 1920.6

[0302] Example 8: Synthesis of a two-residue peptide (H-Cys(Trt)Lys(Boc)-NH-Dpm(OPhy))

[0303] Step (1): Condensation reaction (preparation by batch method) To a cyclopentyl methyl ether (CPME) solution (Solution-1) containing 0.06 mmol / ml of H-Lys(Boc)-NH-Dpm(OPhy) obtained in the same manner as in Production Example 1, 1.50 eq. of 1-hydroxybenzotriazole (HOBt) anhydride, Fmoc-Cys(Trt)-OH, and N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) were added to obtain Fmoc-Cys(Trt)-Leu-NH-Dpm(OPhy). Then, 1.50 eq. of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) and mercaptopropionic acid were added to quench the excess active ester.

[0304] Step (A): Extraction after condensation reaction The solution containing 0.06 mmol / ml of Fmoc-Cys(Trt)-Lys(Boc)-NH-Dpm(OPhy) obtained in the above step (1) (Solution-1) and 20 wt% NaCl aqueous solution (Solution-2) were fed at 1.500 and 1.50 ml / min using a plunger pump and a diaphragm pump, respectively, and were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). After joining, the liquid became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) and was passed through a PFA tube (inner diameter 1.6 mm, length 7.5 m) for 5 min to inactivate EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer, and then separated with a separatory funnel to recover the organic layer.

[0305] Step (2): Fmoc removal reaction / Step (B)-3: Washing with aqueous sodium chloride solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.06 mmol / ml of Fmoc-Cys(Trt)-Lys(Boc)-NH-Dpm(OPhy) obtained in the above step (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.72 mmol / ml of mercaptopropionic acid and 1.44 mmol / ml of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.700 and 0.300 ml / min using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and reacted for 8 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with mercaptopropionic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with 20.0 wt% NaCl aqueous solution using a diaphragm pump at a flow rate of 0.50 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and the resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer containing H-Cys(Trt)-Lys(Boc)-NH-Dpm(OPhy) was then recovered by separation using a separatory funnel. LC / MS M + m / z 1350.0

[0306] Example 9: Synthesis of a two-residue peptide (H-Gly-Glu(OtBu)-OMTBPhy)

[0307] Step (1): Condensation reaction / Step (A): Extraction after condensation reaction A chloroform solution (Solution-1) containing 0.06 mmol / ml of H-Glu(OtBu)-OMTBPhy obtained by a method similar to that of Production Example 1 and a dimethylformamide (DMF) solution (Solution-2) containing 0.21 mmol / ml of Fmoc-Gly-OH and 0.05 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.500 and 0.208 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.32 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) was fed at a flow rate of 0.133 ml / min and mixed with the mixed solution of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 9 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Furthermore, a 20 wt% NaCl aqueous solution (Solution-4) was fed at 0.56 ml / min using a diaphragm pump and mixed with the mixed solution of Solutions-1, 2, and 3 using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 min to inactivate the EDC.HCl remaining in the condensation reaction and to remove it into the aqueous layer. The organic layer containing Fmoc-Gly-Glu(OtBu)-OMTBPhy was then separated in a separatory funnel and recovered. *) MTBPhy refers to the 2-(3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy)-4-methoxybenzyl group.

[0308] Step (2): Fmoc removal reaction / Step (B)-1: Washing with aqueous sodium carbonate solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.05 mmol / mL of Fmoc-Gly-Glu(OtBu)-OMTBPhy obtained by successively carrying out the above steps (1) and (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.52 mmol / mL of thiomalic acid and 1.57 mmol / mL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.732 and 0.346 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with a 5.0 wt% Na2CO3 aqueous solution (Solution-3) using a diaphragm pump at a flow rate of 0.80 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 5 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer was then separated using a separatory funnel to recover the organic layer.

[0309] Step (B)-2: Washing with a sodium chloride solution after washing with a sodium carbonate solution The organic layer (solution-1) obtained in step (B)-1 and a liquid (solution-2) obtained by mixing 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 were mixed using a plunger pump and a diaphragm pump at flow rates of 1.500 and 1.50 ml / min, respectively, in a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the mixture was mixed was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 3 minutes to select the fulvene adduct that could not be completely selected in step (B)-1 into the aqueous layer, and the organic layer containing H-Gly-Glu(OtBu)-OMTBPhy was then separated in a separatory funnel to recover the organic layer. LC / MS M + m / z 1376.1

[0310] Example 10: Synthesis of a two-residue peptide (H-Tyr(tBu)-Phe-OTOC)

[0311] Step (1): Condensation reaction / Step (A): Extraction after condensation reaction A chloroform solution (Solution-1) containing 0.06 mmol / ml of H-Phe-OTOC obtained by the same method as in Production Example 1 and a dimethylformamide (DMF) solution (Solution-2) containing 0.22 mmol / ml of Fmoc-Tyr(tBu)-OH and 0.06 mmol / ml of 1-hydroxybenzotriazole (HOBt) anhydride were pumped at flow rates of 0.500 and 0.208 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), and mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). A chloroform solution (Solution-3) containing 0.34 mmol / ml of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) was fed at a flow rate of 0.133 ml / min and mixed with the mixed solution of Solutions-1 and 2 using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 9 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Furthermore, a 20 wt% NaCl aqueous solution (Solution-4) was fed at 0.56 ml / min using a diaphragm pump and mixed with the mixed solution of Solutions-1, 2, and 3 using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 min to inactivate the EDC.HCl remaining in the condensation reaction and to remove it into the aqueous layer. The organic layer containing Fmoc-Tyr(tBu)-Phe-OTOC was then recovered by separation in a separatory funnel. *)TOC stands for 3,4,5-tri(octadecyloxy)cyclohexylmethyl group.

[0312] Step (2): Fmoc removal reaction / Step (B)-1: Washing with potassium carbonate solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.05 mmol / mL of Fmoc-Tyr(tBu)-Phe-OTOC obtained by successively carrying out the above steps (1) and (A) and a dimethylformamide (DMF) solution (Solution-2) containing 0.55 mmol / mL of thiomalic acid and 1.66 mmol / mL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.732 and 0.346 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 7 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with a 5.0 wt% K2CO3 aqueous solution (Solution-3) using a diaphragm pump at a flow rate of 0.80 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch). The resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 5 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer was then separated using a separatory funnel to recover the organic layer.

[0313] Step (B)-2: Washing with an aqueous solution of sodium chloride after washing with an aqueous solution of potassium carbonate The organic layer (solution-1) obtained in step (B)-1 and a liquid (solution-2) obtained by mixing 20.0 wt% NaCl aqueous solution and dimethylformamide (DMF) in a volume ratio of 6:4 were mixed using a plunger pump and a diaphragm pump at flow rates of 1.500 and 1.50 ml / min, respectively, in a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). The liquid that became a slug flow (a flow in which organic layer and aqueous layer are alternately repeated) after the mixture was mixed was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 3 minutes to select the fulvene adduct that could not be completely selected in step (B)-1 into the aqueous layer, and the organic layer containing H-Tyr(tBu)-Phe-OTOC was then recovered by separation using a separatory funnel. LC / MS M + m / z 1286.0

[0314] Example 11: 16-residue peptide ( Contains D-amino acids )(H-Leu-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy)

[0315] Step (1): Condensation reaction To a chloroform solution (solution-1) containing 0.03 mmol / ml of H-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy prepared by the batch method, 1.50 eq. of 1-hydroxybenzotriazole (HOBt) anhydride, Fmoc-Glu(OtBu)-OH, and N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) were added to obtain Fmoc-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. Then, 1.50 eq. each of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) and thiomalic acid were added to quench the excess active ester.

[0316] Step (A): Extraction after condensation reaction A solution containing 0.03 mmol / ml of Fmoc-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained in the above step (1) (Solution-1) and a 20 wt% NaCl aqueous solution (Solution-2) were pumped at 1,500 and 1.50 ml / min using a plunger pump and a diaphragm pump, respectively, and the flows were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternate) and was passed through a PFA tube (inner diameter 1.6 mm, length 7.5 m) for 5 min to inactivate the EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer. The organic layer containing Fmoc-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy was then recovered by separation using an oil-water separation membrane (OB-900-S-10; hydrophobic membrane with a pore size of 0.5 μm).

[0317] Step (2): Fmoc removal reaction / Step (B)-3: Washing with aqueous sodium chloride solution after Fmoc removal reaction The organic layer (solution-1) containing 0.03 mmol / mL of Fmoc-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained in the above step (A) and a dimethylformamide (DMF) solution (solution-2) containing 0.40 mmol / mL of thiomalic acid and 1.45 mmol / mL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were pumped at flow rates of 0.700 and 0.300 ml / min, respectively, using a plunger pump (a syringe pump or a diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and reacted in a PFA tube (inner diameter 1.0 mm, length 10.0 m) for 7 min. Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with 20.0 wt% NaCl aqueous solution at a flow rate of 0.50 ml / min using a diaphragm pump in a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and the resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 min to remove the fulvene adduct generated during the Fmoc removal reaction and into the aqueous layer, and then the organic layer was separated using a separatory funnel to recover the organic layer. Furthermore, the obtained organic layer was passed through an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm) to remove the small amount of water remaining in the organic layer, thereby obtaining an organic layer containing H-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. LC / MS M 2+ m / z 1460.0

[0318] Step (1): Condensation reaction To a chloroform solution (solution-1) containing 0.03 mmol / ml of H-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained by successively carrying out the above-mentioned step (2) and step (B)-3, 1.50 eq. of 1-hydroxybenzotriazole (HOBt) anhydride, Fmoc-Lys(Boc)-OH, and N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) were added to obtain Fmoc-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. Then, 1.50 eq. each of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) and thiomalic acid were added to quench the excess active ester.

[0319] Step (A): Extraction after condensation reaction A solution containing 0.03 mmol / ml of Fmoc-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained in the above step (1) (Solution-1) and a 20 wt% NaCl aqueous solution (Solution-2) were pumped at 1,500 and 1,50 ml / min using a plunger pump and a diaphragm pump, respectively, and the flows were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternately flow) and was passed through a PFA tube (inner diameter 1.6 mm, length 7.5 m) for 5 min to inactivate the EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer. The organic layer containing Fmoc-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy was then recovered by separation using an oil-water separation membrane (OB-900-S-10; hydrophobic membrane with a pore size of 0.5 μm).

[0320] Step (2): Fmoc removal reaction / Step (B)-3: Washing with aqueous sodium chloride solution after Fmoc removal reaction The organic layer (Solution-1) containing 0.04 mmol / mL of Fmoc-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained in the above step (A), 0.50 mmol / mL of thiomalic acid and 1.80 mmol / mL of 1,8-diazabicyclo[5.4.0]-7 Dimethylformamide (DMF) solutions (Solution-2) containing 1-undecene (DBU) were pumped at flow rates of 0.700 and 0.300 ml / min using a plunger pump (a syringe pump or diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 8 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc deprotection reaction solution was mixed with a 20.0 wt % NaCl aqueous solution at a flow rate of 0.50 ml / min using a diaphragm pump in a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and the resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 minutes to remove the fulvene adduct produced during the Fmoc deprotection reaction and into the aqueous layer. The organic layer was then recovered by separation in a separatory funnel. Furthermore, the obtained organic layer was passed through an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm) to remove the small amount of water remaining in the organic layer, thereby obtaining an organic layer containing H-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. LC / MS M 2+ m / z 1574.0

[0321] Step (1): Condensation reaction To a chloroform solution (solution-1) containing 0.04 mmol / ml of H-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained by successively carrying out the above-mentioned step (2) and step (B)-3, 1.50 eq. of 1-hydroxybenzotriazole (HOBt) anhydride, Fmoc-Leu-OH, and N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) were added to obtain Fmoc-Leu-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. Then, 1.50 eq. each of N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) and thiomalic acid were added to quench the excess active ester.

[0322] Step (A): Extraction after condensation reaction A solution containing 0.03 mmol / ml of Fmoc-Leu-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained in the above step (1) (solution-1) and a 20 wt% NaCl aqueous solution (solution-2) were pumped at 1,500 and 1.50 ml / min using a plunger pump and a diaphragm pump, respectively, and the flows were joined using a T-shaped mixer (Union Tee SS-200-3; outer diameter 1 / 8 inch). After merging, the liquid became a slug flow (a flow in which organic and aqueous layers alternate) and was passed through a PFA tube (inner diameter 1.6 mm, length 7.5 m) for 5 min to inactivate the EDC.HCl remaining in the condensation reaction and to select it into the aqueous layer. The organic layer containing Fmoc-Leu-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy was then recovered by separation using an oil-water separation membrane (OB-900-S-10; hydrophobic membrane with a pore size of 0.5 μm).

[0323] Step (2): Fmoc removal reaction / Step (B)-3: Washing with aqueous sodium chloride solution after Fmoc removal reaction The organic layer (solution-1) containing 0.05 mmol / ml of Fmoc-Leu-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy obtained in the above step (A) and 0.63 mmol / ml of thiomalic acid and 2.26 mmol / ml of 1,8-diazabicyclo[5.4.0 A dimethylformamide (DMF) solution (Solution-2) containing 7-undecene (DBU) was pumped at a flow rate of 0.700 and 0.300 ml / min using a plunger pump (a syringe pump or diaphragm pump can also be used), mixed using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and then reacted for 8 min in a PFA tube (inner diameter 1.0 mm, length 10.0 m). Dibenzofulvene (DBF) generated during the reaction was reacted with thiomalic acid to convert it to a fulvene adduct. This Fmoc removal reaction solution was mixed with a 20.0 wt % NaCl aqueous solution using a diaphragm pump at a flow rate of 0.50 ml / min using a T-shaped mixer (Union Tee SS-100-3; outer diameter 1 / 16 inch), and the resulting slug flow (a flow in which organic and aqueous layers alternate) was passed through a PFA tube (inner diameter 1.6 mm, length 5.0 m) for 7 minutes to remove the fulvene adduct produced during the Fmoc removal reaction and into the aqueous layer. The organic layer was then separated using a separatory funnel to recover the organic layer. Furthermore, the obtained organic layer was passed through an oil-water separation membrane (OB-2000-S-10; hydrophobic membrane with a pore size of 1.0 μm) to remove the small amount of water remaining in the organic layer, thereby obtaining an organic layer containing H-Leu-Lys(Boc)-Glu(OtBu)-(D)Pro-Pro-Gln(Trt)-Ala-Ala-(D)Pro-Pro-Ile-Pro-Gln(Trt)-Ala-Ala-Leu-OTOBPhy. LC / MS M 2+ m / z 1630.6 [Industrial Applicability]

[0324] The present invention relates to a method for continuous production of peptides and is useful in the field of peptide synthesis.

[0325] This application is based on patent application No. 2019-084652 filed in Japan (filing date: April 25, 2019), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a peptide, comprising the following step (A) and / or step (B): (A) a step of purifying the N-protected C-protected peptide by washing a reaction solution containing an N-protected C-protected peptide (N-protected C-protected peptide) in which the N-terminal amino group and the C-terminus are protected by protecting groups, the side chain functional groups may be further protected by protecting groups, and at least one of the C-terminus or the side chain functional groups is protected by a pseudo solid-phase protecting group with water and / or a hydrophilic organic solvent in a flow reactor in a continuous flow, and then separating the solution by an oil-water separation means in a continuous flow to separate the organic layer containing the N-protected C-protected peptide; (B) a step of purifying the N-unprotected C-protected peptide by washing a reaction solution containing a C-protected peptide (N-unprotected C-protected peptide) in which the N-terminal amino group is not protected, the C-terminus is protected with a protecting group, a side chain functional group may be further protected with a protecting group, and at least one of the C-terminus or side chain functional groups is protected with a pseudo solid-phase protecting group with water and / or a hydrophilic organic solvent in a flow reactor in a continuous flow, and then separating the organic layer containing the N-unprotected C-protected peptide by an oil-water separation means in a continuous flow; Here, the pseudo solid-phase protecting group in the above steps (A) and (B) is (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine); 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl alcohol; 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol; and 3,4,5-tri(octadecyloxy)cyclohexanemethanol The term "a" refers to a portion of a compound selected from the group consisting of the above, excluding a bond formed by chemical reaction of the compound with at least one of the C-terminus or side chain functional groups.

2. In step (A), washing with a continuous flow of water and / or a hydrophilic organic solvent in a flow reactor is carried out under concentration conditions that result in a 5- to 100-fold dilution with respect to the substrate (N-protected C-protected peptide); In step (B), washing with a continuous flow of water and / or a hydrophilic organic solvent in a flow reactor is carried out under concentration conditions that result in a 2- to 100-fold dilution with respect to the substrate (N-unprotected C-protected peptide); A method for producing the peptide according to claim 1.

3. A method for producing a peptide, comprising the following steps (1), (A), (2), and (B): (1) A C-protected amino acid (C-protected amino acid) in which the C-terminus is protected by a protecting group, the side chain functional group may be further protected by a protecting group, and the C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group, or a C-protected peptide (C-protected peptide) in which the C-terminus is protected by a protecting group, the side chain functional group may be further protected by a protecting group, and the C-terminus or at least one of the side chain functional groups is protected by a pseudo solid-phase protecting group; an N-protected amino acid (N-protected amino acid) in which the C-terminus is not protected, the N-terminal amino group is protected with a protecting group, and the side chain functional group may be further protected with a protecting group, or an N-protected peptide (N-protected peptide) in which the C-terminus is not protected, the N-terminal amino group is protected with a protecting group, and the side chain functional group may be further protected with a protecting group; 1 to 10 moles of a condensing agent per mole of C-protected amino acid or C-protected peptide; and a soluble organic solvent, introducing the peptide into a flow reactor and carrying out a condensation reaction in a continuous flow in the flow reactor to obtain an N-protected C-protected peptide (N-protected C-protected peptide) in which the N-terminal amino group and the C-terminus are protected by protecting groups, the side chain functional groups may be further protected by protecting groups, and at least one of the C-terminus or the side chain functional groups is protected by a pseudo solid-phase protecting group by N-terminal extension; (A) a step of purifying the N-protected C-protected peptide by washing a reaction solution containing the N-protected C-protected peptide in a flow reactor with water and / or a hydrophilic organic solvent in a continuous flow, and then separating the organic layer containing the N-protected C-protected peptide using an oil-water separation means in a continuous flow; (2) introducing the organic layer containing the N-protected C-protected peptide into a flow reactor in a continuous flow and removing the protecting group of the N-terminal amino group in the continuous flow in the flow reactor to obtain a C-protected peptide (N-unprotected C-protected peptide) in which the N-terminal amino group is not protected, the C-terminus is protected by a protecting group, and the side chain functional groups may be further protected by a protecting group, and at least one of the C-terminus or the side chain functional groups is protected by a pseudo solid-phase protecting group; (B) a step of purifying the N-unprotected C-protected peptide by washing the reaction solution containing the N-unprotected C-protected peptide in a flow reactor with water and / or a hydrophilic organic solvent in a continuous flow, and then separating the organic layer containing the N-unprotected C-protected peptide by an oil-water separation means in a continuous flow; Here, the pseudo solid-phase protecting group in the above steps (1) and (2) is (4',4'-bis(2,3-dihydrophytyloxy)phenyl)methylamine); 3,4,5-tri(2',3'-dihydrophytyloxy)benzyl alcohol; 2-[3,4,5-tri(2',3'-dihydrophytyloxy)benzyloxy]-4-methoxybenzyl alcohol; and 3,4,5-tri(octadecyloxy)cyclohexanemethanol The term "a" refers to a portion of a compound selected from the group consisting of the above, excluding a bond formed by chemical reaction of the compound with at least one of the C-terminus or side chain functional groups.

4. In step (A), washing with a continuous flow of water and / or a hydrophilic organic solvent in a flow reactor is carried out under concentration conditions that result in a 5- to 100-fold dilution with respect to the substrate (N-protected C-protected peptide); In step (B), washing with a continuous flow of water and / or a hydrophilic organic solvent in a flow reactor is carried out under concentration conditions that result in a 2- to 100-fold dilution with respect to the substrate (N-unprotected C-protected peptide); A method for producing the peptide according to claim 3.

5. The method for producing the peptide according to claim 3 or 4, wherein steps (1), (A), (2), and (B) are carried out in this order.

6. The method for producing the peptide according to claim 3 or 4, wherein steps (2), (B), (1) and (A) are carried out in this order.

7. The method for producing a peptide according to any one of claims 1 to 6, wherein the oil-water separation means is either a continuous layer separation means having a configuration including a filter, or a Gravity-type continuous layer separation means.

8. 8. The method for producing a peptide according to any one of claims 1 to 7, wherein the protecting group for the amino group is a 9-fluorenylmethyloxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group.

9. The method for producing a peptide according to claim 8, wherein the protecting group for the amino group is a 9-fluorenylmethyloxycarbonyl group.

10. The method for producing the peptide according to any one of claims 1 to 9, further comprising a step of removing all protecting groups after obtaining the organic layer containing the N-protected C-protected peptide obtained in step (A) or the N-unprotected C-protected peptide obtained in step (B).

11. The method for producing a peptide according to claim 1 or 3, wherein the peptide produced has 5 to 100 amino acid residues.

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