Method for producing amino-group-containing compound, method for separating amino-group-containing compound, and device for producing amino-group-containing compound

Abstract

The purpose of one aspect of the present invention is to provide a method for producing an amino group-containing compound, which is capable of more easily removing an N-terminal protecting group-derived compound. A method for producing an amino-group-containing compound according to one aspect of the present invention comprises: a slug flow formation step for forming a slug flow of a hydrophobic layer and a hydrophilic layer; the hydrophobic layer is formed of a hydrophobic solution containing an amino group-containing compound to be recovered, an N-terminal protecting group-derived compound that is a compound derived from an N-terminal protecting group, and an organic solvent, and the hydrophilic layer is formed of an acidic aqueous solution, and the separation step recovers the hydrophobic solution by separating the hydrophobic layer from a slug flow.

Description

Technical Field

[0001] The present invention relates to a method for producing an amino-containing compound, a method for separating an amino-containing compound, and an apparatus for producing an amino-containing compound. Background Art

[0002] In order to use amino-containing compounds such as peptides as materials for cosmetics and pharmaceuticals, it is necessary to develop technologies for producing amino-containing compounds in large quantities at low cost. A known method for producing peptides in large quantities involves condensing an N-terminally protected amino acid residue with a peptide whose C-terminus is protected by a hydrophobic protecting group in a liquid phase to gradually extend the peptide chain. Because the hydrophobic protecting group is called a tag, this method is also known as liquid-phase tagging.

[0003] In liquid-phase labeling methods, for each condensation reaction of an amino acid residue, a capture agent formed by binding the protecting group deprotected from the peptide to a capture agent that captures the protecting group is removed by liquid separation and washing. For example, Patent Document 1 describes a method for producing a peptide, which includes the following steps: washing a reaction solution containing an N-unprotected C-protected peptide using water and / or a hydrophilic organic solvent under continuous flow in a flow reactor, followed by continuous flow separation using an oil-water separation unit to separate an organic layer containing the N-unprotected C-protected peptide, thereby purifying the N-unprotected C-protected peptide.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2020 / 218497 Summary of the Invention

[0007] Problems to be solved by the invention

[0008] Removal of N-terminal protective group derivatives, such as capture bodies, is a costly and time-consuming process in the production of amino-containing compounds. The technology described in Patent Document 1 has not yet been fully investigated regarding the removal efficiency of capture bodies. Therefore, there is a need for the development of technologies that can more easily remove N-terminal protective group derivatives.

[0009] One aspect of the present invention is to provide a method for producing an amino group-containing compound, which can more easily remove the N-terminal protective group-derived compound.

[0010] Solutions for solving problems

[0011] To solve the above-mentioned problems, a method for producing an amino-containing compound according to one aspect of the present invention includes a slug flow forming step and a separation step. The slug flow forming step forms a slug flow comprising a hydrophobic layer and a hydrophilic layer. The hydrophobic layer is formed from a hydrophobic solution containing the amino-containing compound to be recovered, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent. The hydrophilic layer is formed from an acidic aqueous solution. The separation step recovers the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

[0012] In one aspect of the present invention, a method for separating an amino-containing compound includes a slug flow forming step and a separation step. The slug flow forming step forms a slug flow comprising a hydrophobic layer and a hydrophilic layer, wherein the hydrophobic layer is formed from a hydrophobic solution containing the amino-containing compound to be recovered, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer is formed from an acidic aqueous solution. The separation step recovers the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

[0013] In one aspect of the present invention, an apparatus for producing an amino-containing compound includes a slug flow forming unit and a separation unit connected to the slug flow forming unit. The slug flow forming unit forms a slug flow comprising a hydrophobic layer and a hydrophilic layer. The hydrophobic layer is formed of a hydrophobic solution containing an amino-containing compound to be recovered, an N-terminal protecting group-derived compound, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, and an organic solvent. The hydrophilic layer is formed of an acidic aqueous solution. The separation unit recovers the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

[0014] Effects of the Invention

[0015] According to one aspect of the present invention, a method for producing an amino group-containing compound that can more easily remove an N-terminal protective group derivative compound can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a block diagram showing the structure of an apparatus for producing an amino group-containing compound according to one embodiment of the present invention.

[0017] Figure 2 This is a graph showing the results of HPLC of the hydrophilic layer obtained in Example 1-3. DETAILED DESCRIPTION

[0018] [Method for producing amino group-containing compounds]

[0019] The present inventors have discovered that by washing a hydrophobic solution containing an N-terminal protecting group derivative compound, which is a compound derived from an N-terminal protecting group that may be generated by deprotection of the N-terminal protecting group after a condensation reaction, in a slug flow using an acidic aqueous solution, at least a portion of the N-terminal protecting group derivative compound can be readily removed into the acidic aqueous solution. This allows, for example, improving the purity of the amino group-containing compound in the hydrophobic solution or removing more components that may interfere with other steps such as further amino acid condensation of the amino group-containing compound after washing, thereby completing the present invention.

[0020] Specifically, the method for producing an amino-containing compound according to one aspect of the present invention includes a slug flow forming step, wherein the slug flow forming step forms a slug flow comprising a hydrophobic layer and a hydrophilic layer. The hydrophobic layer is formed from a hydrophobic solution containing the amino-containing compound to be recovered, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent. The hydrophilic layer is formed from an acidic aqueous solution. The separation step recovers the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow. Hereinafter, the method for producing an amino-containing compound according to one aspect of the present invention may be simply referred to as the "production method according to one aspect of the present invention."

[0021] (Slug flow forming process)

[0022] The slug flow formation step is a step for forming a slug flow consisting of a hydrophobic layer formed by a hydrophobic solution and a hydrophilic layer formed by an acidic aqueous solution. The slug flow formation step forms a slug flow in which the hydrophobic and hydrophilic layers alternate along the flow direction. During the slug flow, at least a portion of the N-terminal protecting group derivative compound contained in the hydrophobic layer migrates to the adjacent hydrophilic layer, thereby reducing the number of capture bodies contained in the hydrophobic layer.

[0023] In this specification, "slug flow" refers to a flow in which, in at least a portion of the flow, alternating flows of hydrophobic layers and hydrophilic layers are formed along the flow direction. "Slug flow" includes not only the situation where alternating flows of hydrophobic layers and hydrophilic layers are formed in the entire flow path through which the flow passes, but also the situation where alternating flows of hydrophobic layers and hydrophilic layers are formed only in a portion of the flow path. For example, "slug flow" includes: alternating flows of hydrophobic layers and hydrophilic layers are formed from the starting point of the flow path where the hydrophobic layer and the hydrophilic layer are mixed to the end point of the flow path; alternating flows of hydrophobic layers and hydrophilic layers are formed only near the starting point; and alternating flows of hydrophobic layers and hydrophilic layers are formed discontinuously in the flow path. In "slug flow", as long as alternating flows of hydrophobic layers and hydrophilic layers are formed in at least a portion of the flow, parallel flows and turbulent flows formed by hydrophobic layers and hydrophilic layers may be formed locally.

[0024] (Hydrophobic solution)

[0025] The hydrophobic solution contains the amino-containing compound to be recovered, an N-terminal protective group derivative compound, and an organic solvent. The hydrophobic solution may further contain a capture agent for capturing the N-terminal protective group derivative compound. Without limitation, the reaction solution used in the condensation reaction for synthesizing the amino-containing compound can be used as the hydrophobic solution.

[0026] (Containing amino compounds)

[0027] In one aspect of the present invention, an amino group-containing compound is any compound having at least one of a primary and secondary amino group. Examples of amino group-containing compounds include amino acid monomers and peptides formed by peptide bonds between two or more amino acids. In peptides, any of the C-terminus and side chain termini of the peptide chain may have substituents such as protecting groups.

[0028] The amino-containing compound to be recovered may be the same as or different from the amino-containing compound produced as the target product in the production method according to one aspect of the present invention. Without limitation, the amino-containing compound to be recovered may be a precursor of the target product, the amino-containing compound, such as a compound having a portion of the peptide sequence of the target product, the amino-containing compound.

[0029] The amino-containing compound produced or recovered in the production method according to one aspect of the present invention may be a peptide. The number of amino acids constituting the peptide can be appropriately set according to the intended use of the peptide to be produced, for example, two or more. Furthermore, the production method according to one aspect of the present invention can be preferably used to produce peptides composed of a larger number of amino acids, more preferably to produce amino-containing compounds composed of three or more, and even more preferably five or more, amino acids bonded together.

[0030] The amino acid residue sequence of the peptide is not particularly limited, and the N-terminal residue of the peptide can be lysine (Lys) or proline (Pro). When the N-terminal residue is one of these amino acid residues, if the capture body is removed according to the conventional method, the hydrophobic solution containing the capture body in the cleaning solution may form a latex, and liquid separation may take a long time. However, according to one aspect of the present invention, the latex formation during the removal of the capture body is reduced, and liquid separation can be performed in a shorter time.

[0031] The C-terminus of the amino-containing compound to be recovered may be protected by a C-terminal protecting group. The C-terminal protecting group may be a C-terminal protecting group that can be used in liquid phase labeling. Examples of the C-terminal protecting group include the C-terminal protecting group represented by the following formula (1).

[0032]

[0033] In formula (1), m Qs each represent an oxygen atom. m R 1 Each independently represents a group represented by the following formula (A). 2 Each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group or a halogen atom. X represents the binding position to the C-terminus of the amino-containing compound. m represents an integer of 2 or 3. k represents an integer of 0 or more and (5-j) or less. m [QR 1 ] is substituted at the meta position relative to the substituent containing X. The total number of carbon atoms in the C-terminal protecting group represented by formula (1) is 40 or more and 60 or less.

[0034]

[0035] In formula (A), * represents the binding position. 1a 、R 1b 、R 1c 、R 1d and R 1e Each independently represents a hydrogen atom or an alkyl group. n1 represents an integer from 0 to 6. When n1 is 1 or more, the repeating unit in the bracket with n1 is an alkylene group. n2 represents an integer from 0 to 6. When n2 is 1 or more, the repeating unit in the bracket with n2 is an alkylene group. 1a 、R 1b 、R 1c and R 1d At least two of them are hydrogen atoms.

[0036] Examples of the C-terminal protecting group include protecting groups represented by any of the following formulae: wherein X represents the binding position to the C-terminus of the amino group-containing compound.

[0037]

[0038] The C-terminal protecting group is not limited to the above-mentioned protecting groups, and protecting groups known in the technical field can be used.

[0039] (N-terminal protective group derivative compound)

[0040] In one aspect of the present invention, the N-terminal protecting group derivative compound is a compound derived from the N-terminal protecting group that protects the N-terminal of the amino-containing compound. Examples of the N-terminal protecting group derivative compound include decomposition products produced by deprotecting the N-terminal protecting group of the N-terminal protecting amino-containing compound, thereby decomposing the N-terminal protecting group, and a capture body (hereinafter referred to as "capture body") formed by combining a capture agent with the decomposition product. Typically, the decomposition product and the capture body are generated as by-products in the manufacturing method involved in one aspect of the present invention. The decomposition product and the capture body sometimes reduce the yield and purity of the manufactured amino-containing compound, so it is sometimes desirable to remove at least a portion of them. In one aspect of the present invention, by easily removing at least a portion of the N-terminal protecting group derivative compound, it is possible to achieve cost reduction and shortening of the time required for the manufacturing method involved in one aspect of the present invention. The N-terminal protecting group derivative compound can be one type or a combination of multiple types.

[0041] One aspect of the present invention is particularly suitable for use when the N-terminal protective group-derived compound is a trapping agent. Specifically, one aspect of the present invention allows for better cleaning of the trapping agent. The production method of one aspect of the present invention facilitates removal of the trapping agent, effectively reducing costs and time.

[0042] The N-terminal protecting group is not particularly limited as long as it is a functional group that can be used to protect at least one of the primary and secondary amino groups of the amino-containing compound. Examples of the N-terminal protecting group include protecting groups having a fluorene skeleton such as a 9-fluorenylmethoxycarbonyl group (Fmoc group), a tert-butyloxycarbonyl group (Boc group), a benzyloxycarbonyl group (Cbz group), an allyloxycarbonyl (Alloc) group, an acetyl (Ac) group, and a trichloroacetyl group.

[0043] (decomposition products)

[0044] In one aspect of the present invention, the object of removal may be a decomposition product, which is a compound generated by decomposing the N-terminal protecting group of the N-terminal protected amino group-containing compound by deprotection.

[0045] As examples of the decomposition product, there can be cited dibenzofullerene (DBF) derived from an Fmoc group; CO2 and isobutylene derived from a Boc group; and toluene derived from a Cbz group.

[0046] (capture body)

[0047] In one aspect of the present invention, the target for removal may be a capture body. The capture body is a compound formed by binding a capture agent to a decomposition product derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound being recovered. Furthermore, the hydrophobic solution may contain, in addition to the capture body, the capture agent and the decomposition product derived from the N-terminal protecting group, separately and unbound.

[0048] The capture agent is a compound that forms a capture body by binding to the decomposition product. The capture agent can be appropriately selected based on the structure of the N-terminal protecting group and the structure of the decomposition product derived from the N-terminal protecting group. Examples of capture agents include secondary amines. For example, when the N-terminal protecting group is a protecting group having a fluorene skeleton, and thus the decomposition product has a fullerene skeleton, the capture agent is preferably a secondary amine from the perspective of efficiently capturing the decomposition product and removing the free decomposition product from the reaction system. Among the secondary amines, the capture agent is preferably at least one selected from the group consisting of morpholine, piperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, thiomorpholine dioxide, 4-methylpiperazine, 4-aminopiperidine, diethylamine and pyrrolidine.

[0049] The capture body is formed by the combination of the decomposition product and the capture agent. The structure of the capture body is determined by the N-terminal protecting group and the structure of the capture agent and is not particularly limited. Examples of capture bodies include compounds represented by any of the following formulas. These compounds are capture bodies formed by the combination of the decomposition product DBF and the capture agents morpholine, piperidine, pyrrolidine, 4-methylpiperazine, or diethylamine.

[0050]

[0051] (Organic Solvent)

[0052] In one aspect of the present invention, the organic solvent is not particularly limited as long as it renders the hydrophobic solution containing the organic solvent incompatible with the acidic aqueous solution. The organic solvent may be a known hydrophobic organic solvent that can be used as a reaction solvent in a peptide condensation reaction. In the production method according to one aspect of the present invention, when the condensation reaction and the removal of the N-terminal protecting group derivative compound are repeated, the organic solvent in the slug flow formation step is preferably the same as the reaction solvent in the condensation reaction from the perspective of improving operational simplicity and reducing the adverse effects of the condensation reaction and the removal of the N-terminal protecting group derivative compound.

[0053] As the example of organic solvent, ethers, acetates, halogenated hydrocarbons, aromatic hydrocarbons and hydrocarbons can be listed. As organic solvent, one can be used alone, or a variety of them can be mixed and used. In the organic solvent, from being easy to liquid separation and low cost, it is preferred to contain at least one selected from the group consisting of 4-methyltetrahydropyran (MTHP), cyclopentyl methyl ether (CPME), chloroform, ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, ethyl acetate, isopropyl acetate, dichloromethane, toluene, xylene, hexane, heptane and cyclohexane, further preferably containing at least one selected from the group consisting of 4-methyltetrahydropyran, cyclopentyl methyl ether and chloroform.

[0054] (Other ingredients in the hydrophobic solution)

[0055] The hydrophobic solution may contain other components in addition to the above-mentioned components. Examples of other components include condensing agents, activators, and catalysts useful in peptide condensation reactions, as well as byproducts of the condensation reaction and deprotecting agents. Specific examples of condensing agents, activators, catalysts, and deprotecting agents are described below.

[0056] In the prior art, when a reaction solution containing an N-terminal protecting group derivative compound, a condensing agent, an activating agent, and a deprotecting agent is subjected to a liquid separation and washing process for removing the N-terminal protecting group derivative compound, latex may be formed, making the liquid separation and washing process time-consuming. Therefore, a preliminary liquid separation and washing process for removing the condensing agent, activating agent, and deprotecting agent, respectively, may be required before the liquid separation and washing process. However, according to the production method of one aspect of the present invention, even if the hydrophobic solution contains any of the condensing agent, activating agent, and deprotecting agent as components other than the N-terminal protecting group derivative compound, the formation of latex is reduced, enabling liquid separation and washing to be performed in a shorter time. Furthermore, according to the production method of one aspect of the present invention, the preliminary liquid separation and washing process can be omitted, thereby enabling the removal of the N-terminal protecting group derivative compound with fewer liquid separation and washing processes.

[0057] (acidic aqueous solution)

[0058] The acidic aqueous solution forms a hydrophilic layer that extracts the N-terminal protecting group derivative compound from the hydrophobic layer in the slug flow. The acidic aqueous solution is not particularly limited as long as it contains an acid, and an example thereof is an aqueous solution containing Bronsted acid.

[0059] From the perspective of more efficiently removing N-terminal protecting group derivative compounds, the acidic aqueous solution is preferably an aqueous solution containing at least one Brønsted acid selected from the group consisting of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and citric acid. From the perspective of more efficiently removing N-terminal protecting group derivative compounds, the concentration of the Brønsted acid in the acidic aqueous solution is preferably 1.0 mol / L or higher, more preferably 2.0 mol / L or higher. Furthermore, from the perspective of reducing the decomposition of amino group-containing compounds contained in the hydrophobic layer, the concentration of the Brønsted acid in the acidic aqueous solution is preferably 12.0 mol / L or lower, more preferably 6.0 mol / L or lower, and even more preferably 4.0 mol / L or lower.

[0060] The pH of the acidic aqueous solution is not particularly limited, and may be, for example, less than 7.0. The pH of the acidic aqueous solution is preferably less than 5.0, and more preferably less than 3.0.

[0061] (Operational Conditions of Slug Flow Formation Process)

[0062] The method for forming a slug flow comprising a hydrophobic layer formed by a hydrophobic solution and a hydrophilic layer formed by an acidic aqueous solution is not particularly limited. Examples thereof include a method of introducing the hydrophobic solution and the acidic aqueous solution from separate flow paths into a confluence portion for mixing, and a method of forming a slug flow by external control such as a solenoid valve.

[0063] In the method of mixing at the confluence, the inner diameters of the inlet passages for the hydrophobic solution and the acidic aqueous solution, and the outlet passage for discharging the resulting slug flow, can be appropriately selected. From the perspective of obtaining a slug flow with stable formation of the hydrophilic and hydrophobic layers, the inner diameter is preferably 0.4 mm or greater, more preferably 0.6 mm or greater, and preferably 6.5 mm or less, more preferably 4.5 mm or less.

[0064] The flow rates of the hydrophobic solution and the acidic aqueous solution introduced into the confluence can be fixed or variably controlled by alternating the two solutions into the confluence. From the perspective of simplifying the slug flow formation process, the flow rates of the hydrophobic solution and the acidic aqueous solution are preferably fixed. From the perspective of stabilizing the length of a hydrophobic layer in the slug flow in the flow direction, i.e., the slug length, and improving process reproducibility, the flow rate of the hydrophobic solution is preferably 0.3 mL / min or greater, more preferably 1.0 mL / min or greater, preferably 10 mL / min or less, and more preferably 6.0 mL / min or less. From the perspective of shortening the slug length, increasing the contact area of each layer with the adjacent layer per unit volume, and further promoting the migration of the N-terminal protecting group derivative compound, the flow rate of the acidic aqueous solution is preferably 0.5 times or greater than the flow rate of the hydrophobic solution, more preferably 1.0 times or greater. Furthermore, from the perspective of reducing wastewater, the flow rate of the acidic aqueous solution is preferably 10 times or less than the flow rate of the hydrophobic solution, more preferably 4.0 times or less.

[0065] The slug flow forming step may include allowing the formed slug flow to flow within a tube. The length of the tube may be appropriately selected so that the slug flow resides within the tube for a desired period of time. To ensure that the N-terminal protecting group derivative compound is fully transferred to the hydrophilic layer, the residence time is preferably 3 seconds or longer, more preferably 5 seconds or longer. To ensure that the N-terminal protecting group derivative compound is fully removed and the production method according to one aspect of the present invention is completed in a shorter period of time, the residence time is preferably 500 seconds or shorter, more preferably 300 seconds or shorter. For example, the tube length is preferably 0.015 m or longer, more preferably 0.1 m or longer, preferably 20 m or shorter, and more preferably 10 m or shorter, but the length is not limited thereto. The inner diameter of the tube may be sufficient to maintain the slug flow within the tube. The inner diameter of the tube is preferably 0.4 mm or longer, more preferably 0.6 mm or longer, preferably 6.5 mm or shorter, and more preferably 4.5 mm or shorter, but the length is not limited thereto.

[0066] (Separation process)

[0067] The separation step is a step of recovering a hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow. In the production method according to one aspect of the present invention, the slug flow in which the N-terminal protecting group derivative compound is removed is less likely to produce fine latex, and the hydrophobic layer has a certain size, thereby easily separating the hydrophobic layer from the slug flow. The recovered hydrophobic solution contains the amino-containing compound to be recovered, but does not contain the N-terminal protecting group derivative compound contained in the hydrophobic solution used in the slug flow formation step, or the amount contained is smaller than the amount in the slug flow formation step.

[0068] The method for separating the hydrophobic layer is not particularly limited and can be intermittent separation or continuous separation. When performing intermittent separation, known methods can be used, for example, the slug flow introduced into the storage tank can be allowed to stand and the hydrophobic layer separated into the upper and lower hydrophobic layers (hydrophobic solution) and the hydrophilic layer (acidic aqueous solution) can be recovered. When performing continuous separation, known methods can be used, for example, the slug flow can be introduced into an oil-water separation membrane. In addition, for example, the slug flow can be converted into a parallel flow in which the upper and lower layers flow parallel to the flow, and the layer corresponding to the hydrophobic layer in the upper and lower layers can be recovered.

[0069] (Deprotection step)

[0070] The production method according to one aspect of the present invention may further include a deprotection step before the slug flow formation step. The deprotection step involves contacting an N-terminally protected amino-containing compound with a deprotecting agent and a capture agent in an organic solvent before the slug flow formation step, thereby forming the amino-containing compound and the capture body to be recovered. The deprotection step can produce a hydrophobic solution containing the amino-containing compound and the capture body.

[0071] In the deprotection step, the organic solvent and scavenger described in the slug flow formation step can be used, and therefore, their description will not be repeated. The N-terminally protected amino-containing compound is the target of recovery, i.e., an amino-containing compound whose N-terminus is protected by an N-terminal protecting group. The composition of the N-terminally protected amino-containing compound will be readily understood by those skilled in the art having referenced the above description of the amino-containing compound and the N-terminal protecting group.

[0072] The deprotecting agent is a compound that deprotects the N-terminal protecting group from the N-terminal protected amino compound. The deprotecting agent can be appropriately selected according to the N-terminal protecting group. Examples of deprotecting agents include organic bases such as 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), 1,4-diazabicyclo[2.2.2]octane (DABCO), triethylamine, and tributylamine; and inorganic bases such as potassium tert-butoxide and sodium tert-butoxide, but are not limited thereto. The deprotecting agent is preferably at least one selected from the group consisting of DBU, DBN, and DABCO, and more preferably DBU.

[0073] The method for contacting the N-terminally protected amino group-containing compound with the deprotecting agent and the trapping agent in an organic solvent is not particularly limited. For example, the deprotecting agent and the trapping agent may be added to the reaction solution containing the N-terminally protected amino group-containing compound in any order.

[0074] In another aspect of the present invention, a scavenger may not be used in the deprotection step. Specifically, the deprotection step may be a step in which the N-terminally protected amino-containing compound and a deprotecting agent are contacted in an organic solvent prior to the slug flow formation step to form the amino-containing compound and decomposition products to be recovered. The method of the deprotection step can be appropriately selected depending on the type of N-terminal protecting group.

[0075] (Condensation process)

[0076] In addition, the production method according to one aspect of the present invention may further include a condensation step prior to the deprotection step. The condensation step is a step in which an N-terminally protected amino acid is condensed with the N-terminus of an amino-containing compound precursor prior to the deprotection step to obtain an N-terminally protected amino-containing compound. The amino-containing compound precursor is a compound having a structure in which one residue of the N-terminal amino acid in the amino-containing compound has been removed. Furthermore, the N-terminally protected amino acid is any amino acid having an N-terminal protecting group bound to the amino group.

[0077] The condensation step can be performed by adding an N-terminal protected amino acid and a condensing agent, and optionally adding an activator and a catalyst, to a reaction solution in which an amino group-containing compound precursor is dissolved in an organic solvent.

[0078] As the condensing agent, a known compound that can be used for the amidation reaction can be used. Examples of the condensing agent include 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), O-(benzotriazol-1-yl)-1,1,3,3 -tetramethyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate (COMU), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl).

[0079] The activating agent may be a known compound that, in the presence of a condensing agent, derivatizes an amino acid into a corresponding active ester or symmetrical anhydride, thereby readily undergoing an amidation reaction. Examples of the activating agent include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt), 3-hydroxy-1,2,3-benzotriazine-4(3H)-one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboxylimide (HONb), pentafluorophenol, and ethyl cyano(hydroxyimino)acetate (Oxyma).

[0080] The catalyst may be a known compound that can catalyze the amidation reaction. Examples of the catalyst include dimethylaminopyridine (DMAP).

[0081] (An example of condensation step and deprotection step)

[0082] An example of the scheme of the condensation step and the deprotection step is shown below. In the following scheme, as the condensation step, an amino compound precursor H-AA having its C-terminus protected by an R group is 1 -OR was dissolved in MTHP / DMF (8 / 2) mixture at 30-40 v / w, and the N-terminal protective amino acid Fmoc-AA was added. 2 -OH (1.3 equivalents), condensing agent EDCI·HCl (1.3 equivalents) and activating agent Oxyma (0.1 equivalents) were stirred at room temperature for 1 hour. After the condensation reaction was confirmed by HPLC, morpholine (0.4 equivalents) was added as the active ester Fmoc-AA 2 -Ox capture agent, stirred at room temperature for 30 minutes. Then, as a deprotection step, capture agent morpholine (20.0 equivalents) and deprotection agent DBU (7.0 equivalents) were added, stirred at room temperature for 1 hour to carry out deprotection reaction, and an amino group-containing compound H-AA was obtained. 2 -AA 1 -OR. While not limiting, the reaction solution can then be transferred to a separatory funnel and washed and separated by adding 10% saline (25-30 v / w, twice). The organic layer can then be washed and separated by adding 2M hydrochloric acid (25-30 v / w, twice), followed by washing and separation, and then washed and separated with a 0.5M aqueous sodium bicarbonate solution (25-30 v / w). The organic layer can then be dried over an appropriate amount of sodium sulfate and filtered while rinsing with an appropriate amount of MTHP to obtain an amino acid condensate in the form of a solution.

[0083]

[0084] (Neutralization process)

[0085] In addition, the production method according to one aspect of the present invention may further include a neutralization step after the separation step. The neutralization step involves contacting the recovered hydrophobic solution with a base to neutralize the hydrophobic solution after the separation step. The neutralization step allows the reactivity of the hydrophobic solution in subsequent steps to be controlled by neutralizing the low-pH hydrophobic solution recovered in the separation step. Furthermore, the neutralization step facilitates the removal of any activating agent that may be present in the hydrophobic solution.

[0086] The neutralization step can be performed, for example, by forming a slug flow consisting of a hydrophobic layer formed by the hydrophobic solution and a hydrophilic layer formed by the alkaline aqueous solution. This method allows the neutralized hydrophobic solution to be easily separated from the slug flow, and any activator that may be present in the hydrophobic solution can be removed into the alkaline aqueous solution.

[0087] The alkaline aqueous solution is not particularly limited as long as it contains a base, and may be, for example, an aqueous solution containing a Brønsted base. From the perspective of achieving an appropriate pH value for the hydrophobic aqueous solution, the alkaline aqueous solution is preferably an aqueous solution containing at least one Brønsted base selected from the group consisting of sodium bicarbonate, sodium carbonate, potassium carbonate, and sodium hydroxide.

[0088] From the perspective of cleaning efficiency, the concentration of the Brønsted base in the alkaline aqueous solution is preferably 0.1 mol / L or higher, more preferably 0.5 mol / L or higher. Furthermore, from the perspective of inhibiting peptide degradation, the concentration of the Brønsted base in the alkaline aqueous solution is preferably 2.0 mol / L or lower, more preferably 1.0 mol / L or lower. The pH of the alkaline aqueous solution can be, for example, from 8 to 12, but is not limited thereto.

[0089] In the neutralization step, when a slug flow of the hydrophobic layer and the hydrophilic layer is formed, the operating conditions can be the same as those in the above-mentioned slug flow forming step.

[0090] In addition, the neutralization step can be performed using a known method for neutralizing a hydrophobic solution. For example, a base can be added to the hydrophobic solution or the hydrophobic solution can be introduced into a basic column to allow the hydrophobic solution to contact the base and thereby be neutralized. Examples of the base include tertiary amines such as triethylamine and diisopropylethylamine. Examples of the basic column include a column filled with DIAION TM (Mitsubishi Chemical Corporation) and the like, and columns filled with inorganic bases such as solid sodium bicarbonate.

[0091] (A series of repeated steps)

[0092] A production method according to one aspect of the present invention may include repeating a series of steps sequentially comprising an optional condensation step, an optional deprotection step, a slug flow formation step, a separation step, and an optional neutralization step. Repeating this series of steps allows condensation of an amino acid with the N-terminus of an amino-containing compound, thereby extending the amino-containing compound. The number of times the series of steps is repeated is not particularly limited and may be determined based on the number of amino acid condensations.

[0093] When a series of steps are repeated, the amino-containing compound to be recovered in one cycle can be used as an amino-containing compound precursor in the condensation step of the next cycle. Furthermore, when a series of steps are repeated, the amino-containing compound to be recovered in the final cycle can be the amino-containing compound produced as the target product by the production method according to one aspect of the present invention.

[0094] (Extraction process)

[0095] The production method according to one aspect of the present invention may further include an extraction step after the separation step. The extraction step is a step for extracting the amino-containing compound to be recovered from the hydrophobic solution recovered in the separation step. The amino-containing compound can be extracted using any method used to separate and produce the amino-containing compound from a hydrophobic solution. Examples include, but are not limited to, extraction and washing, crystallization, and chromatography.

[0096] The extraction step may include deprotecting the C-terminal protecting group of the amino-containing compound. Deprotection of the C-terminal protecting group can be performed using known methods, such as treatment with trifluoroacetic acid (TFA). TFA treatment can also be performed with other molecules, such as water, thioanisole, 1,2-ethanedithiol, phenol, and triisopropylsilane.

[0097] [Method for separating amino compounds]

[0098] It should be noted that, for the sake of convenience, descriptions of steps or components having the same functions as those described above will be omitted.

[0099] The present invention also encompasses a method for separating amino-containing compounds. One aspect of the present invention relates to a method for separating amino-containing compounds, comprising a slug flow forming step and a separation step. The slug flow forming step forms a slug flow comprising a hydrophobic layer and a hydrophilic layer. The hydrophobic layer is formed from a hydrophobic solution containing the amino-containing compound to be recovered, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, and an organic solvent. The hydrophilic layer is formed from an acidic aqueous solution. The separation step separates the hydrophobic layer from the slug flow to recover the hydrophobic solution containing the amino-containing compound to be recovered. Furthermore, the method for separating amino-containing compounds according to one aspect of the present invention may further comprise any of a deprotection step, a condensation step, a neutralization step, and an extraction step.

[0100] [Apparatus for producing amino compounds]

[0101] One aspect of the present invention relates to an apparatus for producing an amino-containing compound. The apparatus for producing an amino-containing compound according to one aspect of the present invention includes a slug flow forming unit, wherein the slug flow forming unit forms a slug flow comprising a hydrophobic layer and a hydrophilic layer. The hydrophobic layer is formed from a hydrophobic solution containing an amino-containing compound, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, i.e., an N-terminal protecting group derivative compound, and an organic solvent. The hydrophilic layer is formed from an acidic aqueous solution. The separation unit separates the hydrophobic layer from the slug flow. The apparatus for producing an amino-containing compound may hereinafter be simply referred to as a "production apparatus."

[0102] The production apparatus includes a slug flow forming section and a separation section. Furthermore, the production apparatus may further include any of a deprotection section, a condensation section, a neutralization section, and an extraction section. The slug flow forming section, the separation section, the deprotection section, the condensation section, the neutralization section, and the extraction section are components for carrying out the aforementioned slug flow forming step, the separation step, the deprotection step, the condensation step, and the extraction step, respectively.

[0103] (One embodiment of the present manufacturing apparatus)

[0104] Reference Figure 1 One embodiment of the present production apparatus will be described. Figure 1 1 is a block diagram showing the structure of an amino compound production apparatus 10 according to one embodiment of the present invention. Figure 1 As shown, the manufacturing apparatus 10 includes a slug flow forming portion 20 and a separation portion 30. The slug flow forming portion 20 is connected to the separation portion 30.

[0105] The slug flow forming section 20 is configured to form a slug flow consisting of a hydrophobic layer and a hydrophilic layer. The hydrophobic layer is formed from a hydrophobic solution containing an amino-containing compound, a compound derived from an N-terminal protecting group that protects the N-terminus of the amino-containing compound, and an organic solvent; the hydrophilic layer is formed from an acidic aqueous solution. The slug flow forming section 20 includes a hydrophobic solution tank 21, an acidic aqueous solution tank 22, a mixing section 23, and a flow section 24. The hydrophobic solution tank 21, the acidic aqueous solution tank 22, and the flow section 24 are each connected to the mixing section 23.

[0106] The hydrophobic solution tank 21 is configured to store a hydrophobic solution and introduce the hydrophobic solution into the mixing unit 23. In this embodiment, the hydrophobic solution tank 21 is a combination of a tank storing the hydrophobic solution and a pump connected to the tank.

[0107] The acidic aqueous solution tank 22 stores the acidic aqueous solution and introduces the acidic aqueous solution into the mixing unit 23. In this embodiment, the acidic aqueous solution tank 22 is a combination of a tank storing the acidic aqueous solution and a pump connected to the tank.

[0108] In this embodiment, the hydrophobic solution tank 21 and the acidic aqueous solution tank 22 are independently controlled to introduce the stored solutions into the mixing section 23 at a fixed flow rate. However, in one aspect of the present invention, the hydrophobic solution tank 21 and the acidic aqueous solution tank 22 may be jointly controlled to introduce the stored solutions into the mixing section 23 at a variable flow rate.

[0109] The mixing section 23 is configured to introduce a hydrophobic solution and an acidic aqueous solution to form a slug flow. In this embodiment, a T-type mixer is used as the mixing section 23. In the T-type mixer, the hydrophobic solution and the acidic aqueous solution are introduced through two opposing inlet passages, respectively, and the slug flow is discharged through the remaining discharge passage. This configuration allows friction between the inner wall of the T-type mixer and the inner wall of the tube to form a circulation flow within each of the hydrophobic and hydrophilic layers in the slug flow, further promoting the migration of the N-terminal protective group derivative compound.

[0110] In one aspect of the present invention, the mixing section 23 is not limited to a T-type mixer; any type of mixer capable of forming a slug flow may be used. Examples of the mixing section 23 include a Y-type mixer, a spiral mixer, and a static mixer. Furthermore, as described above, methods for forming a slug flow using external control such as a solenoid valve may also be used.

[0111] The flow passage 24 is a structure for allowing the slug flow formed in the mixing section to flow. In this embodiment, a PFA (tetrafluoroethylene / perfluoroalkyl vinyl ether copolymer) tube having an inner diameter of 1.59 mm and a length of 1 m is used as the flow passage 24 .

[0112] In one aspect of the present invention, the flow passage 24 is not limited to the PFA tube described above, and any member that allows slug flow to flow through may be used. When a tube is used as the flow passage 24, the material, inner diameter, and length of the tube may be appropriately selected.

[0113] The separation section 30 is connected to the slug flow forming section and is used to separate the hydrophobic layer from the slug flow. In this embodiment, a storage tank is used as the separation section 30. The storage tank has a slug flow inlet connected to the flow passage 24 and openable and closable discharge ports located at the upper and lower portions, respectively. In this embodiment, with the upper and lower discharge ports closed, the slug flow is introduced into the storage tank from the inlet. The introduction is stopped when a predetermined amount of hydrophobic and hydrophilic layers have accumulated. After a predetermined period of quiescence, the hydrophobic and hydrophilic layers are discharged from the discharge ports, achieving separation of the hydrophobic layers.

[0114] In one aspect of the present invention, the separation unit 30 is not limited to the above-mentioned storage tank, and may be a member utilizing either batch separation or continuous separation. An example of the separation unit 30 is an oil-water separation membrane.

[0115] 〔Summarize〕

[0116] As can be understood from the above description, the method for producing an amino-containing compound according to the first aspect of the present invention includes a slug flow forming step and a separation step, wherein the slug flow forming step forms a slug flow comprising a hydrophobic layer and a hydrophilic layer, wherein the hydrophobic layer is formed from a hydrophobic solution containing the amino-containing compound to be recovered, a compound derived from an N-terminal protecting group for protecting the N-terminus of the amino-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer is formed from an acidic aqueous solution, and the separation step recovers the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

[0117] In the method for producing an amino-containing compound according to the second aspect of the present invention, in addition to the configuration of the method for producing an amino-containing compound according to the first aspect, the N-terminal protecting group derivative compound is a capture body formed by binding a capture agent to a decomposition product derived from the N-terminal protecting group.

[0118] The method for producing an amino-containing compound involved in the third aspect of the present invention is based on the configuration of the method for producing an amino-containing compound involved in the first or second aspect, wherein the organic solvent comprises at least one selected from the group consisting of 4-methyltetrahydropyran, cyclopentyl methyl ether, chloroform, diethyl ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, ethyl acetate, isopropyl acetate, dichloromethane, toluene, xylene, hexane, heptane and cyclohexane.

[0119] The method for producing an amino group-containing compound according to the fourth aspect of the present invention is based on the configurations of the methods for producing an amino group-containing compound according to the first to third aspects, wherein the organic solvent contains at least one selected from the group consisting of 4-methyltetrahydropyran, cyclopentyl methyl ether, and chloroform.

[0120] The method for producing an amino-containing compound according to the fifth aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the first to fourth aspects, wherein the acidic aqueous solution is an aqueous solution containing at least one Bronsted acid selected from the group consisting of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid and citric acid.

[0121] The method for producing an amino-containing compound according to the sixth aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the first to fifth aspects, wherein the concentration of the Bronsted acid contained in the acidic aqueous solution is greater than or equal to 1.0 mol / L and less than or equal to 12.0 mol / L.

[0122] The method for producing an amino-containing compound according to a seventh aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the first to sixth aspects, wherein the slug flow forming step includes: mixing the hydrophobic solution having a flow rate of 0.3 mL / min or more with the acidic aqueous solution having a flow rate of 1.0 times or more and 10 times or less the flow rate of the hydrophobic solution to form the slug flow.

[0123] The method for producing an amino-containing compound involved in the eighth aspect of the present invention, in addition to the configuration of the method for producing an amino-containing compound involved in the second aspect and any one of the third to seventh aspects cited from the second aspect, further includes a deprotection step, wherein, before the slug flow forming step, the N-terminally protected amino-containing compound to be recovered, i.e., the amino-containing compound whose N-terminus is protected by the above-mentioned N-terminal protecting group, is brought into contact with a deprotecting agent for deprotecting the N-terminal protecting group from the N-terminally protected amino-containing compound and the capturing agent in the organic solvent to form the amino-containing compound and the capturing body, wherein the N-terminal protecting group is a protecting group having a fluorene skeleton and the capturing agent is a secondary amine.

[0124] The method for producing an amino-containing compound according to the ninth aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the first to eighth aspects, further comprising a neutralization step, wherein the recovered hydrophobic solution is contacted with a base after the separation step to neutralize the hydrophobic solution.

[0125] The method for producing an amino-containing compound according to the tenth aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the first to ninth aspects, wherein the amino-containing compound to be recovered is a peptide formed by bonding two or more amino acids.

[0126] The method for producing an amino-containing compound according to the eleventh aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the first to tenth aspects, wherein the C-terminus of the amino-containing compound to be recovered is protected by a C-terminal protecting group represented by the following formula (1):

[0127]

[0128] [wherein, m Qs represent oxygen atoms, m Rs represent oxygen atoms, 1 are independently a group represented by the following formula (A),

[0129]

[0130] (where * represents the binding position, R 1a 、R 1b 、R 1c 、R 1d and R 1e Each independently represents a hydrogen atom or an alkyl group, n1 represents an integer from 0 to 6, when n1 is 1 or more, the repeating unit shown in the bracket with n1 is an alkylene group, n2 represents an integer from 0 to 6, when n2 is 1 or more, the repeating unit shown in the bracket with n2 is an alkylene group, wherein R 1a 、R 1b 、R 1c and R 1d At least two of them are hydrogen atoms),

[0131] k R 2 Each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group or a halogen atom, X represents the binding position to the C-terminus of the amino-containing compound, m represents an integer of 2 or 3, k represents an integer of 0 or more and (5-j) or less, m [QR 1 ] is substituted at the meta position relative to the substituent containing X, and the total number of carbon atoms is 40 or more and 60 or less].

[0132] The method for producing an amino-containing compound according to the 12th aspect of the present invention is based on the configuration of the method for producing an amino-containing compound according to any one of the above-mentioned aspects 1 to 11, and further includes an extraction step, wherein the extraction step extracts the recovery object, i.e., the amino-containing compound, from the recovered hydrophobic solution after the separation step.

[0133] The method for separating an amino-containing compound according to the 13th aspect of the present invention comprises a slug flow forming step and a separation step, wherein the slug flow forming step forms a slug flow comprising a hydrophobic layer and a hydrophilic layer, the hydrophobic layer being formed from a hydrophobic solution containing the amino-containing compound to be recovered, a compound derived from an N-terminal protecting group for protecting the N-terminus of the amino-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer being formed from an acidic aqueous solution, and the separation step recovering the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

[0134] The apparatus for producing an amino-containing compound according to the fourteenth aspect of the present invention comprises a slug flow forming section and a separation section connected to the slug flow forming section, wherein the slug flow forming section forms a slug flow comprising a hydrophobic layer and a hydrophilic layer, the hydrophobic layer being formed of a hydrophobic solution containing an amino-containing compound to be recovered, a compound derived from an N-terminal protecting group for protecting the N-terminus of the amino-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer being formed of an acidic aqueous solution, and the separation section recovering the hydrophobic solution containing the amino-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

[0135] [Appendix]

[0136] The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0137] Example

[0138] Next, the present invention will be described in further detail with reference to examples. However, these examples do not limit the scope of the present invention.

[0139] [Manufacturing Example]

[0140] In the following preparation examples, hydrophobic solutions containing peptides and capture bodies used in subsequent examples were prepared. In these preparation examples, compounds X, Y, and Z represented by the following formulas (X), (Y), and (Z), respectively, were used as C-terminal protecting groups (tags) for amino-containing compounds.

[0141]

[0142] <Production Example 1> Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4)

[0143] Preparation Example 1-1: Synthesis of H-Leu-OTagX (1-1)

[0144] Compound X (5.62 g, 6.78 mmol) was dissolved in 211.1 mL of a MTHP / acetonitrile (8 / 2) mixture. Fmoc-Leu-OH (3.35 g, 9.49 mmol), EDCI·HCl (1.82 g, 9.49 mmol), and DMAP (0.083 g, 0.678 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Subsequently, morpholine (0.236 mL, 2.71 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, morpholine (11.8 mL, 136 mmol) and DBU (7.08 mL, 47.4 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, and 10% brine (140 mL x 2) was added for separation and washing. Furthermore, 2 M hydrochloric acid (140 mL x 2) was added to the organic layer for separation and washing, followed by separation and washing with 0.5 M aqueous sodium bicarbonate solution (140 mL). The organic layer was dried over an appropriate amount of sodium sulfate and then filtered while being rinsed with an appropriate amount of MTHP to obtain an amino acid condensate H-Leu-OTagX (1-1) as a solution.

[0145] Preparation Example 1-2: Synthesis of H-Ile-Leu-OTagX (1-2)

[0146] To the solution of H-Leu-OTagX (1-1) obtained above, 42.1 mL of acetonitrile, 3.11 g (8.81 mmol) of Fmoc-Ile-OH, 1.69 g (8.81 mmol) of EDCI·HCl and 0.289 g (2.03 mmol) of Oxyma were added and stirred at room temperature for 1 hour. Subsequently, 0.236 mL (2.71 mmol) of morpholine was added and stirred at room temperature for 30 minutes. Subsequently, 11.8 mL (136 mmol) of morpholine and 7.08 mL (47.4 mmol) of DBU were added and stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, 10% brine (140 mL × 2 times) was added, and the mixture was separated and washed. Furthermore, 2 M hydrochloric acid (140 mL × 2 times) was added to the organic layer and the mixture was separated and washed, and then 0.5 M aqueous sodium bicarbonate solution (140 mL) was used for separation and washing. The organic layer was dried over an appropriate amount of sodium sulfate and then filtered while being rinsed with an appropriate amount of MTHP to obtain an amino acid condensate H-Ile-Leu-OTagX (1-2) as a solution.

[0147] Preparation Example 1-3: Synthesis of H-Tyr(tBu)-Ile-Leu-OTagX (1-3)

[0148] Using H-Ile-Leu-OTagX (1-2) as the amino acid condensate and Fmoc-Tyr (tBu) -OH as the amino acid to be condensed, the same operations as in Production Example 1-2 were performed to obtain the amino acid condensate H-Tyr (tBu) -Ile-Leu-OTagX (1-3) in the form of a solution.

[0149] Preparation Example 1-4: Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTagX (1-4)

[0150] The same procedures as in Preparation Example 1-2 were performed, except that H-Tyr(tBu)-Ile-Leu-OTagX(1-3) was used as the amino acid condensate and Fmoc-Pro-OH was used as the amino acid to be condensed, and no liquid separation was performed, to obtain a peptide (H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4)) as a hydrophobic solution. The hydrophobic solution also contained a capture body formed by binding morpholine to dibenzofullerene (DBF) derived from Fmoc.

[0151] <Production Example 2> Synthesis of H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-7)

[0152] Preparation Example 2-1: Synthesis of H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-5)

[0153] The hydrophobic solution obtained in Preparation Example 1-4 was washed twice with 10% saline, twice with 2M hydrochloric acid, and once with a 0.5M aqueous sodium bicarbonate solution to obtain a hydrophobic solution. Subsequently, the same procedures as in Preparation Example 1-2 were performed, except that the obtained hydrophobic solution was used instead of the amino acid condensate solution and Fmoc-Arg(Pbf)-OH was used as the amino acid to be condensed, to obtain the amino acid condensate H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-5) in the form of a solution.

[0154] Preparation Example 2-2: Synthesis of H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-6) H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-5) was used as the amino acid condensate, and Fmoc-Arg(Pbf)-OH was used as the amino acid to be condensed. The same operations as in Preparation Example 1-2 were performed except for this, to obtain the amino acid condensate H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-6) in the form of a solution.

[0155] Preparation Example 2-3: Synthesis of H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-7)

[0156] Using H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-6) as the amino acid condensate and Fmoc-Lys(Boc)-OH as the amino acid to be condensed, the same procedures as in Preparation Example 1-2 were followed, except that only 10% saline (60 mL) was used for the separation and washing, and the subsequent drying, rinsing, and filtration were omitted. A peptide H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-7) was obtained as a hydrophobic solution. This hydrophobic solution also contained a capture body formed by binding morpholine to dibenzofullerene (DBF) derived from Fmoc.

[0157] <Production Example 3> Synthesis of H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-10)

[0158] Preparation Example 3-1: Synthesis of H-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-8)

[0159] The hydrophobic solution obtained in Preparation Example 2-3 was further washed twice with 2M hydrochloric acid and once with a 0.5M aqueous sodium bicarbonate solution to obtain a hydrophobic solution. Subsequently, the same procedures as in Preparation Example 1-2 were performed, except that the obtained hydrophobic solution was used instead of the amino acid condensate solution and Fmoc-Asn(Trt)-OH was used as the amino acid to be condensed, to obtain an amino acid condensate H-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-8) in the form of a solution.

[0160] Preparation Example 3-2: Synthesis of H-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-9)

[0161] Using H-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-8) as the amino acid condensate and using Fmoc-Glu(OtBu)-OH as the amino acid to be condensed, the same operations as in Production Example 1-2 were performed to obtain the amino acid condensate H-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-9) in the form of a solution.

[0162] Preparation Example 3-3: Synthesis of H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-10)

[0163] H-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-9) was used as the amino acid condensate, Fmoc-Tyr(tBu)-OH was used as the amino acid to be condensed, and as the liquid separation and washing, only the liquid separation and washing with 10% saline (60 mL) was performed, and the subsequent drying, rinsing and filtration were omitted. Otherwise, the same operations as in Preparation Example 1-2 were performed to obtain the peptide H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-10) in the form of a hydrophobic solution. The hydrophobic solution also contains a capture body formed by bonding morpholine to dibenzofullerene (DBF) derived from Fmoc.

[0164] <Production Example 4> Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTagY (4-4)

[0165] Preparation Example 4-1: Synthesis of H-Leu-OTagY (4-1)

[0166] 2.00 g (3.03 mmol) of compound Y was dissolved in 60.0 mL of a MTHP / acetonitrile (8 / 2) mixture, and 1.60 g (4.54 mmol) of Fmoc-Leu-OH, 0.87 g (4.54 mmol) of EDCI·HCl, and 0.037 g (0.303 mmol) of DMAP were added, followed by stirring at room temperature for 2 hours. Subsequently, 0.158 mL (1.82 mmol) of morpholine was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, 5.27 mL (60.5 mmol) of morpholine and 3.16 mL (21.2 mmol) of DBU were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, and 10% brine (60 mL) was added for separation and washing. Furthermore, 2 M hydrochloric acid (60 mL × 2 times) was added to the organic layer for separation and washing, and then 0.5 M aqueous sodium bicarbonate solution (60 mL) was used for separation and washing. The organic layer was dried over an appropriate amount of sodium sulfate and then filtered while being rinsed with an appropriate amount of MTHP to obtain an amino acid condensate H-Leu-OTagY (4-1) as a solution.

[0167] Preparation Example 4-2: Synthesis of H-Ile-Leu-OTagY (4-2)

[0168] To the solution of H-Leu-OTagY (4-1) obtained above, 12.0 mL of acetonitrile, 1.39 g (3.93 mmol) of Fmoc-Ile-OH, 0.754 g (3.93 mmol) of EDCI·HCl, and 0.129 g (0.908 mmol) of Oxyma were added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 0.105 mL (1.21 mmol) of morpholine was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, 5.27 mL (60.5 mmol) of morpholine and 3.16 mL (21.2 mmol) of DBU were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, and 10% brine (60 mL) was added to separate and wash the mixture. Furthermore, 2 M hydrochloric acid (60 mL × 2 times) was added to the organic layer, and the mixture was separated and washed, and then 0.5 M aqueous sodium bicarbonate solution (60 mL) was used for separation and washing. The organic layer was dried over an appropriate amount of sodium sulfate and then filtered while being rinsed with an appropriate amount of MTHP to obtain an amino acid condensate H-Ile-Leu-OTagY (4-2) as a solution.

[0169] Preparation Example 4-3: Synthesis of H-Tyr(tBu)-Ile-Leu-OTagY (4-3)

[0170] The same operation as in Production Example 4-2 was performed except that H-Ile-Leu-OTagY (4-2) was used as the amino acid condensate and Fmoc-Tyr (tBu) -OH was used as the amino acid to be condensed, to obtain the amino acid condensate H-Tyr (tBu) -Ile-Leu-OTagY (4-3) in the form of a solution.

[0171] Preparation Example 4-4: Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTagY (4-4)

[0172] Using H-Tyr(tBu)-Ile-Leu-OTagY(4-3) as the amino acid condensate and Fmoc-Pro-OH as the amino acid to be condensed, the same procedures as in Preparation Example 4-2 were followed except that only 10% saline (60 mL) was used for the separation and washing, and the subsequent drying, rinsing, and filtration were omitted to obtain a peptide (H-Pro-Tyr(tBu)-Ile-Leu-OTagY(4-4)) as a hydrophobic solution. This hydrophobic solution also contained a capture body formed by morpholine bound to dibenzofullerene (DBF) derived from Fmoc.

[0173] <Production Example 5> Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTagZ (5-4)

[0174] Preparation Example 5-1: Synthesis of H-Leu-OTagZ (5-1)

[0175] Compound Z 2.00 g (2.19 mmol) was dissolved in 70 mL of a THF / acetonitrile (8 / 2) mixture, and Fmoc-Leu-OH 1.16 g (3.28 mmol), EDCI·HCl 0.63 g (3.28 mmol) and DMAP 0.027 g (0.219 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Subsequently, morpholine 0.114 mL (1.31 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, morpholine 3.81 mL (43.8 mmol) and DBU 2.29 mL (15.3 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After removing the solvent from the reaction solution using an evaporator, acetonitrile 100 mL was added, and the mixture was stirred for 30 minutes. The precipitated solid was filtered off to obtain the amino acid condensate H-Leu-OTagZ (5-1) as a solid.

[0176] Preparation Example 5-2: Synthesis of H-Ile-Leu-OTagZ (5-2)

[0177] The H-Leu-OTagZ (5-1) obtained above was dissolved in THF 48 mL, acetonitrile 12.0 mL, Fmoc-Ile-OH 1.01 g (2.85 mmol), EDCI·HCl 0.546 g (2.85 mmol) and Oxyma 0.093 g (0.657 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Subsequently, morpholine 0.076 mL (0.876 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, morpholine 3.81 mL (43.8 mmol) and DBU 2.29 mL (15.3 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After removing the solvent from the reaction solution using an evaporator, acetonitrile 100 mL was added and the mixture was stirred for 30 minutes. The precipitated solid was filtered out to obtain the amino acid condensate H-Ile-Leu-OTagZ (5-2) as a solid.

[0178] Preparation Example 5-3: Synthesis of H-Tyr(tBu)-Ile-Leu-OTagZ (5-3)

[0179] H-Ile-Leu-OTagZ (5-2) was used as the amino acid condensate, and Fmoc-Tyr (tBu) -OH was used as the amino acid to be condensed. The same operation as in Production Example 5-2 was performed, and H-Tyr (tBu) -Ile-Leu-OTagZ (5-3) was obtained in the form of a solution.

[0180] Preparation Example 5-4: Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTagZ (5-4)

[0181] Using H-Tyr(tBu)-Ile-Leu-OTagZ(5-3) as the amino acid condensate, Fmoc-Pro-OH as the amino acid to be condensed, and a mixed solution of MTHP / acetonitrile (8 / 2) as the solvent, a 10% saline (60 mL) separation wash was performed, and the subsequent solvent removal and filtration were omitted. The same procedures as in Preparation Example 5-3 were performed to obtain a peptide (H-Pro-Tyr(tBu)-Ile-Leu-OTagZ(5-4)) as a hydrophobic solution. The hydrophobic solution also contained a capture body formed by morpholine bound to dibenzofullerene (DBF) derived from Fmoc.

[0182] [Example]

[0183] In the following examples, the hydrophobic solutions obtained in the production examples were subjected to cleaning using slug flows formed under various conditions, and the capture body removal rate in the hydrophobic layer after separation was measured.

[0184] <Device>

[0185] The following apparatus was used in the Examples.

[0186] Flow reactor: A reactor connected to a PFA tube (1.59 mm inner diameter, FLON INDUSTRY) and a PFA connector "PFA-220-6" (1 / 8 inch outer diameter, Swagelok)

[0187] T-type mixer: PFA ferrule tee "PFA-220-3" (outer diameter 1 / 8 inch, Swagelok), stainless steel ferrule tee "SS-200-3" (outer diameter 1 / 8 inch, Swagelok)

[0188] Pump: Diaphragm pump "QI-100-TT-PS" (TACMINA)

[0189] <Method for measuring removal rate>

[0190] Compound Y pre-added to the hydrophobic solution was used as an internal standard. The ratio of the area value of the capture body peak to the area value of the internal standard was measured before and after slug flow cleaning by HPLC, and the capture body removal rate was calculated using the following formula.

[0191] Residual rate (%) = ratio after cleaning / ratio before cleaning × 100

[0192] Removal rate (%) = 100 - residual rate (%)

[0193] Liquid chromatography (HPLC) conditions:

[0194] Column: InertSustain C18 (3 μm, 4.6×125 mm)

[0195] Mobile phase A: 0.1% trifluoroacetic acid (TFA) in water, mobile phase B: THF

[0196] Eluent: Measurement was performed according to the gradient conditions of mobile phase A / mobile phase B shown in Table 1.

[0197] Flow rate: 1.0 mL / min

[0198] Column temperature: 40°C

[0199] Detector: UV-visible spectrophotometer (λ=220nm)

[0200] Table 1

[0201]

[0202] <Example 1> Slug Flow Cleaning Including Preliminary Cleaning (Study of Conditions for Stable Slug Flow Formation)

[0203] Example 1-1: Cleaning using chloroform solution

[0204] The hydrophobic solution obtained in Preparation Example 1-4 was pre-washed twice with 10% saline and once with 2M hydrochloric acid to obtain a chloroform solution containing 0.04 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4). The chloroform solution and 2M hydrochloric acid aqueous solution were introduced into a T-type mixer (SS-200-3) using a diaphragm pump at flow rates of 0.34 mL / min and 0.37 mL / min, respectively, and merged to form a slug flow (a flow in which the hydrophobic layer formed by the chloroform solution and the hydrophilic layer formed by the hydrochloric acid aqueous solution alternately flow along the flow direction). The slug flow discharged from the T-type mixer was passed through a PFA tube (inner diameter 1.59 mm, length 2 m, residence time 354 seconds) and discharged into a beaker. The discharged slug flow quickly separated into two layers: the hydrophobic layer and the hydrophilic layer. The hydrophobic layer was recovered to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) in the form of a solution.

[0205] The length of the liquid plug of the hydrophobic layer formed in the slug flow is in the range of 1 to 6 cm.

[0206] Example 1-2: Cleaning using MTHP solution

[0207] The hydrophobic solution obtained in Preparation Example 1-4 was pre-washed twice with 10% saline and once with 2M hydrochloric acid to obtain a MTHP solution containing 0.04 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4). The same procedures as in Example 1-1 were followed, except that the resulting MTHP solution was used instead of the chloroform solution, to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) as a solution. In this example, the slug flow discharged into the beaker rapidly separated into two layers: a hydrophobic layer and a hydrophilic layer.

[0208] The length of the liquid plug of the hydrophobic layer formed in the slug flow is in the range of 1 to 20 cm.

[0209] The hydrophobic solution obtained in Preparation Example 1-4 was pre-washed twice with 10% saline and once with 2M hydrochloric acid to obtain an MTHP solution containing 0.04 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4). The same procedures as in Example 1-1 were followed, except that the MTHP solution obtained was used instead of the chloroform solution, and the flow rates of the MTHP solution and 2M hydrochloric acid aqueous solution were set to 3.0 mL / min and 3.0 mL / min, respectively (the residence time of the slug flow in the PFA tube was 45 seconds). H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) was obtained as a solution. In this example, the slug flow discharged into the beaker rapidly separated into two layers: a hydrophobic layer and a hydrophilic layer.

[0210] The liquid plug of the hydrophobic layer formed in the slug flow is 1 cm long and stable.

[0211] The results of Example 1 are shown in Table 2. As shown in Table 2, in Examples 1-1 to 1-3, especially in Example 1-3, a slug flow of the hydrophobic solution and the acidic aqueous solution was stably formed. In addition, the results of HPLC of the hydrophilic layer obtained in Example 1-3 are shown in Table 2. Figure 2 .Depend on Figure 2 The captured body peak (6.3 min) shown in FIG. 3 shows that the captured body was removed to the hydrophilic layer by using slug flow to wash the hydrophobic solution. Figure 2 There is no peptide peak in the middle (17.6 min), which also shows that the target product (ie, peptide) has not moved to the hydrophilic layer.

[0212] Table 2

[0213]

[0214] <Example 2> Slug flow cleaning without preliminary cleaning

[0215] Example 2-1:

[0216] The MTHP / acetonitrile (8 / 2) mixed solution containing 0.02 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) obtained in Preparation Example 1-4 and a 2M aqueous hydrochloric acid solution were introduced into a T-type mixer (PFA-220-3) using a diaphragm pump at a flow rate of 3.0 mL / min and 3.0 mL / min, respectively, and merged to form a slug flow. The slug flow discharged from the T-type mixer was discharged into a beaker through a PFA tube (inner diameter 1.59 mm, length 2 m, residence time 45 seconds). The discharged slug flow was quickly separated into two layers, a hydrophobic layer and a hydrophilic layer. The hydrophobic layer was recovered to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) in the form of a solution.

[0217] The capture agent removal rate in the obtained solution was 89.16%. In addition, the pH of the hydrophilic layer after washing was about 1, and the deprotecting agent DBU and the capture agent morpholine contained in the hydrophobic solution before washing were removed from the hydrophobic layer.

[0218] Example 2-2:

[0219] The same operation as in Example 2-1 was performed except that the flow rate of the 2M hydrochloric acid aqueous solution was set at 4.5 mL / min to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) as a solution. The capture body removal rate was 90.16%.

[0220] Example 2-3:

[0221] The same operation as in Example 2-1 was carried out except that the flow rate of the 2M hydrochloric acid aqueous solution was set at 6.0 mL / min to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) as a solution. The capture body removal rate was 92.56%.

[0222] Example 2-4:

[0223] The same operation as in Example 2-1 was performed except that the flow rate of the 2M hydrochloric acid aqueous solution was set at 10.0 mL / min to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) as a solution. The capture body removal rate was 96.71%.

[0224] Example 2-5:

[0225] H-Pro-Tyr(tBu)-Ile-Leu-OTag (1-4) was obtained as a solution by the same operation as in Example 2-3 except that the hydrochloric acid concentration was changed to 4 M. The capture body removal rate was 94.94%.

[0226] Example 2-6:

[0227] H-Pro-Tyr(tBu)-Ile-Leu-OTag (1-4) was obtained as a solution by the same operation as in Example 2-3 except that the hydrochloric acid concentration was changed to 6 M. The capture body removal rate was 97.38%.

[0228] Example 2-7:

[0229] The same operation as in Example 2-3 was carried out except that the length of the PFA tube was changed to 4 m to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (1-4) as a solution. The capture body removal rate was 93.69%.

[0230] Example 2-8:

[0231] The same operation as in Example 2-3 was carried out except that the length of the PFA tube was changed to 1 m to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (1-4) as a solution. The capture body removal rate was 96.14%.

[0232] Example 2-9:

[0233] The same procedures as in Example 2-3 were followed except that a chloroform / acetonitrile (8 / 2) mixed solution was used instead of a MTHP / acetonitrile (8 / 2) mixed solution as the organic solvent to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (1-4) as a solution. The capture removal rate was 51.10%.

[0234] Example 2-10:

[0235] The same procedures as in Example 2-3 were followed, except that a CPME / acetonitrile (8 / 2) mixed solution was used instead of a MTHP / acetonitrile (8 / 2) mixed solution as the organic solvent, to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (1-4) as a solution. The capture removal rate was 98.35%.

[0236] The results of Example 2 are shown in Table 3. As shown in Table 2, the capture bodies can be removed at a high removal rate by using a slug flow to clean the hydrophobic solution with an acidic aqueous solution. Furthermore, the results of Example 2 show that even when the peptide solution obtained by the condensation reaction is subjected to a slug flow to clean the solution with an acidic aqueous solution without preliminary cleaning, latex is not formed in the slug flow, and the discharged liquid quickly separates into two layers: a hydrophilic layer and a hydrophobic layer. This shows that by using the production method according to one aspect of the present invention, the capture bodies can be removed with a small number of liquid separation washes.

[0237] Table 3

[0238]

[0239] <Example 3> Slug Flow Cleaning of a Peptide Solution Containing Tag X

[0240] The hydrophobic solution obtained in Preparation Example 1-4 was pre-washed once with 10% saline to obtain an MTHP solution containing 0.02 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4). A chloroform solution and a 2M hydrochloric acid aqueous solution were introduced into a T-type mixer (PFA-220-3) using a diaphragm pump at flow rates of 3.0 mL / min and 6.0 mL / min, respectively, and merged to form a slug flow (a flow formed by alternating flow of the hydrophobic layer formed by the chloroform solution and the hydrophilic layer formed by the hydrochloric acid aqueous solution along the flow direction). The slug flow discharged from the T-type mixer was passed through a PFA tube (inner diameter 1.59 mm, length 1 m) and discharged into a beaker. The discharged slug flow quickly separated into two layers, a hydrophobic layer and a hydrophilic layer. The hydrophobic layer was recovered to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) in the form of a solution. The capture body removal rate was 92.16%.

[0241] <Example 4> Slug Flow Cleaning of a Peptide Solution Containing Tag Y

[0242] The MTHP solution containing 0.02 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagY(4-4) obtained in Preparation Example 4-4 and a 2M aqueous hydrochloric acid solution were introduced into a T-type mixer (PFA-220-3) using a diaphragm pump at flow rates of 3.0 mL / min and 6.0 mL / min, respectively, and merged to form a slug flow. The slug flow discharged from the T-type mixer was discharged into a beaker through a PFA tube (inner diameter 1.59 mm, length 1 m). The discharged slug flow was quickly separated into two layers, a hydrophobic layer and a hydrophilic layer. The hydrophobic layer was recovered to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagY(4-4) in the form of a solution. The capture body removal rate was 91.66%.

[0243] <Example 5> Slug Flow Cleaning of a Peptide Solution Containing Tag Z

[0244] The MTHP solution containing 0.02 mmol / mL of H-Pro-Tyr(tBu)-Ile-Leu-OTagZ(5-4) obtained in Preparation Example 5-4 and a 2M aqueous hydrochloric acid solution were introduced into a T-type mixer (PFA-220-3) using a diaphragm pump at flow rates of 3.0 mL / min and 6.0 mL / min, respectively, and merged to form a slug flow. The slug flow discharged from the T-type mixer was discharged into a beaker through a PFA tube (inner diameter 1.59 mm, length 1 m). The discharged slug flow was quickly separated into two layers, a hydrophobic layer and a hydrophilic layer. The hydrophobic layer was recovered to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagZ(5-4) in the form of a solution. The capture body removal rate was 88.22%.

[0245] <Example 6> Study on the Generation of Peptide Decomposition Products

[0246] Furthermore, the tBu group contained as a Tyr side chain protecting group in the peptides used in Examples 3 to 5 may be deprotected under acidic conditions. Therefore, washing in a slug flow using an acidic aqueous solution may generate peptide degradation products resulting from the deprotection of the tBu group.

[0247] Therefore, peptides H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4), H-Pro-Tyr(tBu)-Ile-Leu-OTagY(4-4), and H-Pro-Tyr(tBu)-Ile-Leu-OTagZ(5-4) bound to various tags (Compounds X to Z) were subjected to slug flow cleaning and then quantitatively analyzed by HPLC. The production rate of each peptide degradation product was determined by calculating the ratio of the area of the peptide degradation product to the total area of the peptide H-Pro-Tyr(tBu)-Ile-Leu-OTag and the various peptide degradation products H-Pro-Tyr-Ile-Leu-OTag.

[0248] [Experimental Results]

[0249] The experimental results of peptide degradation product generation are shown in Table 4. As shown in Table 4, when the peptides bound to the tag compound X or Y were subjected to slug flow cleaning using an acidic aqueous solution, the generation of peptide degradation products was reduced compared to the peptide bound to the compound Z.

[0250] Table 4

[0251]

[0252] <Example 7> Slug Flow Cleaning of a 7-Residue Peptide

[0253] The MTHP solution containing 0.02 mmol / mL of H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-7) obtained in Preparation Example 2-3 and a 2M aqueous hydrochloric acid solution were introduced into a T-type mixer (PFA-220-3) at a flow rate of 3.0 mL / min and 6.0 mL / min, respectively, using a diaphragm pump and merged to form a slug flow. The slug flow discharged from the T-type mixer was discharged into a beaker through a PFA tube (inner diameter 1.59 mm, length 2 m). The discharged slug flow was quickly separated into two layers, a hydrophobic layer and a hydrophilic layer. The hydrophobic layer was recovered to obtain H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-7) in the form of a solution. The capture body removal rate was 83.86%.

[0254] <Example 8> Slug Flow Cleaning of a 10-Residue Peptide

[0255] The MTHP solution containing 0.02 mmol / mL of H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-10) obtained in Preparation Example 3-3 and a 2M aqueous hydrochloric acid solution were introduced into a T-type mixer (PFA-220-3) using a diaphragm pump at flow rates of 3.0 mL / min and 6.0 mL / min, respectively, and merged to form a slug flow. The slug flow discharged from the T-type mixer was discharged into a beaker through a PFA tube (inner diameter 1.59 mm, length 2 m). The discharged slug flow quickly separated into two layers: a hydrophobic layer and a hydrophilic layer. The hydrophobic layer was recovered to obtain H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-10) as a solution. The capture body removal rate was 78.12%.

[0256] <Example 9> Washing with hydrochloric acid aqueous solution followed by slug flow washing with sodium bicarbonate aqueous solution

[0257] The hydrophobic layers containing the peptide H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) obtained in Examples 2-1 to 2-8 were combined, and the resulting hydrophobic solution (pH 1.97) and 0.5M sodium bicarbonate aqueous solution were introduced into a T-type mixer (PFA-220-3) using a diaphragm pump at flow rates of 3.0 mL / min and 6.0 mL / min, respectively, and merged to form a slug flow (a flow formed by alternating flow of the hydrophobic layer formed by the hydrophobic solution and the hydrophilic layer formed by the sodium bicarbonate aqueous solution along the flow direction). The slug flow discharged from the T-type mixer was discharged into a beaker through a PFA tube (inner diameter 1.59 mm, length 2 m). The discharged slug flow was quickly separated into two layers, the hydrophobic layer and the hydrophilic layer. The hydrophobic layer was recovered to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTagX(1-4) in the form of a solution (pH 8.15). The removal rate of the activator Oxyma was measured in the same manner as for the capture body. The removal rate of the activator was 85.20%.

[0258] Industrial applicability

[0259] The present invention can be used, for example, to produce amino group-containing compounds such as peptides.

[0260] Description of Reference Numerals

[0261] 10 Manufacturing equipment

[0262] 20 Slug flow formation section

[0263] 21 Hydrophobic solution tank

[0264] 22 Acidic aqueous solution tank

[0265] 23 Mixing Section

[0266] 24 Circulation Department

[0267] 30 Separation

Claims

1. A method for producing an amino compound, comprising a slug flow forming step and a separation step. The slug flow forming step forms a slug flow having a hydrophobic layer and a hydrophilic layer, wherein the hydrophobic layer is formed of a hydrophobic solution containing an amino group-containing compound to be recovered, a compound derived from an N-terminal protecting group for protecting the N-terminus of the amino group-containing compound, i.e., an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer is formed of an acidic aqueous solution. The separation step recovers the hydrophobic solution containing the amino group-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

2. The method for producing an amino-containing compound according to claim 1, wherein The N-terminal protecting group derivative compound is a capturing body formed by combining a capturing agent with a decomposition product derived from the N-terminal protecting group.

3. The method for producing an amino-containing compound according to claim 1 or 2, wherein The organic solvent includes at least one selected from the group consisting of 4-methyltetrahydropyran, cyclopentyl methyl ether, chloroform, diethyl ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, ethyl acetate, isopropyl acetate, dichloromethane, toluene, xylene, hexane, heptane, and cyclohexane.

4. The method for producing an amino-containing compound according to claim 1 or 2, wherein The organic solvent includes at least one selected from the group consisting of 4-methyltetrahydropyran, cyclopentyl methyl ether, and chloroform.

5. The method for producing an amino-containing compound according to claim 1 or 2, wherein The acidic aqueous solution is an aqueous solution containing at least one Bronsted acid selected from the group consisting of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and citric acid.

6. The method for producing an amino-containing compound according to claim 1 or 2, wherein The concentration of the Bronsted acid contained in the acidic aqueous solution is 1.0 mol / L or more and 12.0 mol / L or less.

7. The method for producing an amino-containing compound according to claim 1 or 2, wherein The slug flow forming step includes mixing the hydrophobic solution having a flow rate of 0.3 mL / min or more and the acidic aqueous solution having a flow rate of 1.0 times or more and 10 times or less the flow rate of the hydrophobic solution to form the slug flow.

8. The method for producing an amino-containing compound according to claim 2, wherein It also includes a deprotection step, In the deprotection step, before the slug flow forming step, the N-terminally protected amino-containing compound to be recovered, wherein the N-terminus of the amino-containing compound is protected by the N-terminal protecting group, is brought into contact with a deprotecting agent for deprotecting the N-terminal protecting group from the N-terminally protected amino-containing compound and the capturing agent in the organic solvent to form the amino-containing compound and the capturing body. The N-terminal protecting group is a protecting group having a fluorene skeleton, The capture agent is a secondary amine.

9. The method for producing an amino group-containing compound according to any one of claims 1, 2 and 8, wherein It also includes the neutralization process, The neutralization step neutralizes the hydrophobic solution by bringing the recovered hydrophobic solution into contact with a base after the separation step.

10. The method for producing an amino group-containing compound according to any one of claims 1, 2 and 8, wherein The amino compound to be recovered is a peptide formed by the combination of two or more amino acids.

11. The method for producing an amino group-containing compound according to any one of claims 1, 2 and 8, wherein The C-terminus of the amino-containing compound to be recovered is protected by a C-terminal protecting group represented by the following formula (1): [Where, The m Qs represent oxygen atoms, m R 1 are independently a group represented by the following formula (A), (Where, * indicates the binding position, R 1a 、R 1b 、R 1c 、R 1d and R 1e Each independently represents a hydrogen atom or an alkyl group, n1 represents an integer of 0 or more and 6 or less. When n1 is 1 or more, the repeating unit in the brackets with n1 is an alkylene group. n2 represents an integer of 0 or more and 6 or less. When n2 is 1 or more, the repeating unit in the parentheses with n2 is an alkylene group. Among them, R 1a 、R 1b 、R 1c and R 1d At least two of them are hydrogen atoms), k R 2 are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group or a halogen atom, X represents the binding position to the C-terminus of the amino-containing compound, m represents an integer of 2 or 3, k represents an integer greater than or equal to 0 and less than or equal to (5-j), m [QR 1 ] is substituted at the meta position relative to the substituent containing X, The total carbon number is 40 or more and 60 or less].

12. The method for producing an amino group-containing compound according to any one of claims 1, 2 and 8, wherein It also includes the extraction process, The extraction step extracts the amino group-containing compound to be recovered from the hydrophobic solution recovered after the separation step.

13. A method for separating an amino compound, comprising a slug flow forming step and a separation step. The slug flow forming step forms a slug flow having a hydrophobic layer and a hydrophilic layer, wherein the hydrophobic layer is formed of a hydrophobic solution containing an amino group-containing compound to be recovered, a compound derived from an N-terminal protecting group for protecting the N-terminus of the amino group-containing compound, i.e., an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer is formed of an acidic aqueous solution. The separation step recovers the hydrophobic solution containing the amino group-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

14. A production apparatus for an amino compound, comprising a slug flow forming portion and a separation portion connected to the slug flow forming portion. The slug flow forming portion forms a slug flow of a hydrophobic layer and a hydrophilic layer, wherein the hydrophobic layer is formed of a hydrophobic solution containing an amino group-containing compound to be recovered, a compound derived from an N-terminal protecting group for protecting the N-terminus of the amino group-containing compound, namely an N-terminal protecting group derivative compound, and an organic solvent, and the hydrophilic layer is formed of an acidic aqueous solution. The separation unit recovers the hydrophobic solution containing the amino group-containing compound to be recovered by separating the hydrophobic layer from the slug flow.

Images